The CD8 glycoprotein is expressed by thymocytes, mature T cells and natural killer (NK) cells and has been implicated in the recognition of monomorphic determinants on major histocompatibility complex (MHC) Class I antigens, and in signal transduction during the course of T-cell activation. Both human and rodent CD8 antigens are comprised of two distinct polypeptide chains, alpha and beta. The majority of monoclonal antibodies (mAb) reactive with the human CD8 antigen bind the CD8 alpha chain, while a single mAb, T8/<em>2</em>T8-5H7, has been identified which binds to the CD8 alpha/beta heterodimer. While the two chains of CD8 have been presumed to be coordinately expressed in normal T cells, this is not always the case. Northern blot analysis of a panel of T-cell leukemias and normal cells demonstrate that CD8 alpha and CD8 beta are not invariably co-transcribed and phenotypic analysis of fresh and interleukin <em>2</em> (IL-<em>2</em>) expanded peripheral blood mononuclear cells (PBMC) confirm that the CD8 alpha and CD8 beta chains are differentially expressed at the cell surface. Four distinct subpopulations of CD8+ cells have been identified based on the expression of CD8 alpha/alpha or CD8 alpha/beta complexes: (1) T-cell receptor (TcR) alpha beta+ T cells which are CD8 alpha+/beta+; (<em>2</em>) TcR alpha beta+ T cells which are CD8 alpha+/beta-; (3) TcR gamma <em>delta</em>+ T cells which are CD8 alpha+/beta- and (4) natural killer (NK) cells which are CD8 alpha+/beta-. We also demonstrate the down-regulation of the CD8 alpha/beta hetero<em>dimers</em> from the surface of a CD8+ T-cell clone following treatment with phorbol myristate acetate (PMA) while CD8 alpha/alpha homo<em>dimers</em> remain on the cell surface. This observation demonstrates that a) a CD8+ T-cell clone can express both CD8 alpha/alpha homo<em>dimers</em> and CD8 alpha/beta hetero<em>dimers</em> and b) these two complexes do not have identical biological properties. Together, these data suggest that CD8 alpha/alpha and CD8 alpha/beta <em>dimers</em> may not subserve identical functions. The differential contribution of these two CD8 complexes should be considered in models of T-cell-mediated cytotoxicity and T-cell activation.

Low voltage-activated (LVA), T-type, calcium channels mediate diverse biological functions and are inhibited by Gbetagamma <em>dimers</em>, yet the molecular events required for channel inhibition remain unknown. Here, we identify protein kinase A (PKA) as a molecular switch that allows Gbeta(<em>2</em>)gammax <em>dimers</em> to effect voltage-independent inhibition of Ca(v)3.<em>2</em> channels. Inhibition requires phosphorylation of Ser(1107), a critical serine residue on the II-III loop of the channel pore protein. S1107A prevents inhibition of unitary currents by recombinant Gbeta(<em>2</em>)gamma(<em>2</em>) <em>dimers</em> but does not disrupt <em>dimer</em> binding nor change its specificity. Gbetagamma <em>dimers</em> released upon receptor activation also require PKA activity for their inhibitory actions. Hence, dopamine inhibition of Ca(v)3.<em>2</em> whole cell current is precluded by Gbetagamma-scavenger proteins or a peptide that blocks PKA catalytic activity. Fittingly, when used alone at receptor-selective concentrations, <em>D</em>(1) or <em>D</em>(<em>2</em>) agonists do not elicit channel inhibition yet together synergize to inhibit Ca(v)3.<em>2</em> channel currents. We propose that a dual-receptor regulatory mechanism is used by dopamine to control Ca(v)3.<em>2</em> channel activity. This mechanism, for example, would be important in aldosterone producing adrenal glomerulosa cells where channel dysregulation would lead to overproduction of aldosterone and consequent cardiac, renal, and brain target organ damage.

The metal-in<em>d</em>uce<em>d</em> assembly of a <em>d</em>esigne<em>d</em> pepti<em>d</em>e-base<em>d</em> rubre<em>d</em>oxin mo<em>d</em>el is <em>d</em>escribe<em>d</em>. The C16C19-GGY pepti<em>d</em>e has the sequence Ac-K(IEALEGK)(<em>2</em>)(CEACEGK)(IEALEGK)GGY-ami<em>d</em>e in which the presence of the Cys-X-X-Cys metal bin<em>d</em>ing <em>d</em>omain of rubre<em>d</em>oxin was use<em>d</em> to place cysteine resi<em>d</em>ues at the hy<em>d</em>rophobic "a" an<em>d</em> "<em>d</em>" positions upon formation of a homo<em>dimer</em>ic alpha-helical coile<em>d</em>-coil. Circular <em>d</em>ichroism spectroscopy shows that the apopepti<em>d</em>e exists as a ran<em>d</em>om coil an<em>d</em> assembles into a coile<em>d</em>-coil in the presence of C<em>d</em>(<em>2</em>+). Metal bin<em>d</em>ing is monitore<em>d</em> by the appearance of a new LMCT ban<em>d</em> at <em>2</em>38 nm. UV-Vis titrations an<em>d</em> SDS-PAGE experiments are use<em>d</em> to show that this <em>d</em>esigne<em>d</em> metalloprotein exists as a metal-bri<em>d</em>ge<em>d</em> coile<em>d</em>-coil <em>dimer</em>.

The crystal structure of an unusual monomeric cytochrome c' from Rhodopseudomonas palustris (RPCP) has been determined at <em>2</em>.3 A resolution. RPCP has the four-helix (helices A, B, C and <em>D</em>) bundle structure similar to <em>dimer</em>ic cytochromes c'. However the amino acid composition of the surface of helices A and B in RPCP is remarkably different from that of the <em>dimer</em>ic cytochromes c'. This surface forms the <em>dimer</em> interface in the latter proteins. RPCP has seven charged residues on this surface contrary to the <em>dimer</em>ic cytochromes c', which have only two or three charged groups on the corresponding surface. Moreover, hydrophobic residues on this surface of RPCP are two to three times fewer than in <em>dimer</em>ic cytochromes c'. As a result of the difference in amino acid composition, the A-B surface of RPCP is rather hydrophilic compared with <em>dimer</em>ic cytochromes c'. We thus suggest that RPCP is monomeric in solution because of the hydrophilic nature of the A-B surface. The amino acid composition of the A-B surface is similar to that of Rhodobacter capsulatus cytochrome c' (RCCP), which is an equilibrium admixture of monomer and <em>dimer</em>. The charge distribution of the A-B surface in RCCP, however, is considerably different from that of RPCP. <em>D</em>ue to the difference, RCCP can form <em>dimers</em> by both ionic and hydrophobic interactions. These <em>dimers</em> are quite different from those in proteins which form strong <em>dimers</em> such as in Chromatium vinosum, Rhodospirillum rubrum, Rhodospirillum molischianum and Alcaligenes. Cytochrome c' can be classified into two types. Type 1 cytochromes c' have hydrophobic A-B surfaces and they are globular. The A-B surface of type <em>2</em> cytochromes c' is hydrophilic and they take a monomeric or flattened <em>dimer</em>ic form.

An improved method for the preparation of the heme octapeptide acetyl-MP8, obtained by proteolysis of horse heart cytochrome c, is described. AcMP8 obeys Beer's law at pH 7.0 in aqueous solution up to a concentration of 3 x 10(-)(5) M. The self-association constant measured at <em>2</em>5 degrees C (log K(<em>D</em>) = 4.04) is an order of magnitude lower than that for MP8, reflecting the role of the N-acetyl protecting group in abolishing intermolecular coordination. However, AcMP8 does form pi-stacked <em>dimers</em> in aqueous solution with increasing ionic strength. A more weakly packed pi-pi <em>dimer</em> reaches a maximum abundance at approximately 3 M ionic strength, but a more tightly packed <em>dimer</em> is favored at &mgr;>> 3 M. An equilibrium model based on charge neutralization by specific binding of Na(+) ions gives a total molecular charge of 3- for AcMP8 at pH 7.0 and a self-association constant log K(<em>D</em>) = 4.<em>2</em>0. AcMP8 exhibits six spectroscopically active pH-dependent transitions. The Glu-<em>2</em>1 c-terminal carboxylate binds to the heme iron at low pH (pK(a) = <em>2</em>.1) but is substituted by His-18 (pK(a) = 3.1<em>2</em>) as the pH increases. The two heme propanoic acid substituents ionize with pK(a)'s of 4.95 and 6.1. This is followed by ionization of iron-bound water with a pK(a) = 9.59, <em>D</em>eltaH = 48 +/- 1 kJ mol(-)(1), and <em>DeltaS</em> = -<em>2</em><em>2</em> +/- 3 J K(-)(1) mol(-)(1). The electronic spectra indicate that AcMP8 is predominantly in the S = (5)/(<em>2</em>) state at pH 7.0, while the hydroxo complex at pH 10.5 corresponds to an equilibrium mixture of S = (5)/(<em>2</em>) and S = (1)/(<em>2</em>) states at <em>2</em>5 degrees C. In the final transition, His-18 ionizes to form the S = (1)/(<em>2</em>) histidinate complex with a pK(a) of 1<em>2</em>.71. AcMP8 is relatively stable under alkaline conditions, <em>dimer</em>izing slowly at high pH (k = <em>2</em>.59 +/- 0.14 M(-)(1) s(-)(1)) to form a high-spin &mgr;-oxo-bridged species. The pH-dependent behavior of AcMP8 in the presence of excess 3-cyanopyridine, however, is markedly different. At low pH, AcMP8 simultaneously binds the exogenous ligand and the Glu-<em>2</em>1 c-terminal carboxylate with a pK(a) < <em>2</em>. His-18 replaces the carboxylate ligand at higher pH (pK(a) = <em>2</em>.60), and both heme propanoic acid groups ionize with a mean pK(a) = 5.10. Unlike AcMP8.OH(-), the axial histidine of the 3-CNPy complex ionizes at near neutral pH (pK(a) = 7.83), prior to being replaced by OH(-) (pK(a) = 10.13). The sixth transition in the AcMP8/3-CNPy system produces the bis(hydroxo) complex (pK(a)>> 13).

The activating protein-1 (AP-1) transcription factor complex is a heterogeneous entity, composed in mammalian cells of <em>dimers</em> chosen from a group of at least eight proteins belonging to three families: jun, fos, and activating transcription factor (ATF). The AP-1 complexes participate in diverse biological processes that include cell proliferation, survival, and differentiation. These seemingly contrasting functions have been attributed to the intensity and duration of the signals provided by AP-1, but the biological consequences of changing composition of the AP-1 complex have not been fully explored. Here, we show that functional AP-1 is required for 1,<em>2</em>5-dihydroxyvitamin <em>D</em>3 (1,<em>2</em>5<em>D</em>)-induced monocytic differentiation, and that the composition of the AP-1 protein complex that binds TRE, its cognate <em>D</em>NA element, changes as cells differentiate. In HL60 cells in an early stage of differentiation, the principal AP-1 components detected by gel shift analysis include c-jun, ATF-<em>2</em>, fos-B, fra-1, and fra-<em>2</em>. In cells with a more established monocytic phenotype, the demonstrable AP-1 components are c-jun, ATF-<em>2</em>, jun-B, and fos-B. Following the addition of 1 nmol/L of 1,<em>2</em>5<em>D</em>, the cellular content of each of these four proteins markedly increased in a sustained manner, whereas the increases in c-fos, fra-1, fra-<em>2</em>, and jun-<em>D</em> were minimal, if any. Small increases in mRNA levels encoding all AP-1 component proteins, except c-fos, were also noted. These findings provide a basis for the previously found participation of the c-Jun N-terminal kinase pathway in 1,<em>2</em>5<em>D</em>-induced differentiation of myeloid leukemia cells, and direct attention to jun-B and fos-B as new cellular therapeutic targets, that may promote replicative quiescence associated with differentiation of malignant cells.

Notch is a single-pass transmembrane receptor that mediates cell fate choice in various species and developmental contexts. The Notch signal is transduced by its intracellular domain, which acts as a transcriptional activator, and is released from the plasma membrane by proteolytic cleavages. This process is initiated by intercellular association of the epidermal growth factor (EGF) repeats between Notch and the DSL (<em>Delta</em>, Serrate, Lag-<em>2</em>) ligands but the detailed mechanism is yet to be clarified. Here we demonstrate that Notch1 can form homo<em>dimer</em>s, which is achieved by its EGF motifs. The Notch1 <em>dimer</em> formation increased in response to ligand presentation and HES1 promoter was stimulated, implying that receptor homo<em>dimer</em>ization is an important initial step in Notch signal transduction. EGF motifs also serve as a protection against proteases, including TNF-alpha converting enzyme, which prevents Notch1 from ligand-independent activation. Multiple functions of the Notch EGF motifs, such as the prevention of constitutive activation, reciprocal interaction with the ligands and lateral interaction for homo<em>dimer</em>ization, appear to constitute crucial elements of the Notch signaling system.

Heterogeneity of <em>D</em>NA repair has been observed at different levels of genomic organization, including chromatin domains, expressed genes and <em>D</em>NA strands. If heterogeneity also existed intragenically, it could reveal fine details of the excision repair mechanism in vivo. Here we measure the frequency of UV-induced cyclobutane pyrimidine <em>dimers</em> at individual nucleotides within defined portions of two Escherichia coli genes, lacl and lacZ, at various times after irradiation. Two domains of differential repair rates were apparent, with repair being slow at nucleotides adjacent to the transcription start sites. In lacZ, the domain of faster repair began 3<em>2</em> bases downstream of the transcription start site and required the mfd gene. Since mfd codes for a transcription-repair coupling factor, this transcription-coupled repair system evidently becomes operative downstream of the initiation complex region in vivo. Unexpectedly, however, (1) an mfd mutation reduced repair in the downstream domain even when transcription was at a very low level and (<em>2</em>) induction of lacZ transcription with isopropyl-beta-<em>D</em>-thiogalactoside overcame this reduction. Evidently, the Mfd transcription-repair coupling factor is required for basal levels of strand-specific repair in this gene, but induced levels of repair are related to transcription through another mechanism.

Broad spectrum assays which measure a range of fibrinogen/fibrin derivatives (F<em>D</em>Ps) in serum have become an established means of identifying activation of blood coagulation and/or fibrinolysis, such as occurs in disseminated intravascular coagulation (<em>D</em>IC). There is considerable interest in the application of these assays to the diagnosis of other hypercoagulable states, such as recurrent deep venous thrombosis and myocardial infarction. In recent years, more sensitive and specific F<em>D</em>P assays (e.g. for fragment E, fragment E neoantigen, <em>D</em>-<em>dimer</em>, fragment <em>D</em> neoantigen, fibrinopeptide A and fibrin fragment beta 15-4<em>2</em>) have been devised, some of which allow measurement in plasma of F<em>D</em>Ps without interference from fibrinogen or certain of its derivatives. It was predicted that these assays would both avoid the possibility of artifacts introduced as a consequence of serum preparation and improve detection of hypercoagulable states. In the light of these expectations we have reviewed data published on the use of assays to detect clinical hypercoagulability, giving prominence to assays of crosslinked fibrin derivatives and nothing particularly certain studies that have compared the performance of different assays on the same samples. The accumulating evidence indicates that all of the assays are adequate for detection of <em>D</em>IC. The same cannot be said for other hypercoagulable states. Here much variation is evident between different studies of similar patients in the ability of a particular marker to discriminate between a normal control group and patients determined to be hypercoagulable by an independent method. This variability would seem to be a function of patient group heterogeneity and selection, as assays that detect different antigenic determinants produce results on the same plasma samples that are well correlated. It appears that the precise antigenic determinant does not critically affect detection of hypercoagulability. Additionally, some studies have indicated that use of serum need not introduce artifacts. <em>D</em>espite there being no other obvious advantage, the convenience of some of the plasma assays may well encourage their widespread use. Assays have also been developed for measuring activation fragments of coagulation proteins (e.g. prothrombin fragment F1 + <em>2</em> and protein C activation peptide) and for proteinase inhibitor complexes (e.g. thrombin-antithrombin complex) generated during activation of coagulation. The latter assays have been useful in providing a biochemical definition of a 'prethrombotic state'.

p97/VCP associated with Ufd1-Npl4 is considered a key player in ER-associated degradation (ERAD). RNA interference (RNAi) of one component of the Ufd1-Npl4 hetero<em>dimer</em> destabilizes the VCP-Ufd1-Npl4 complex inducing proteasome-dependent degradation of the other component and releasing free VCP. In contrast to RNAi of VCP, RNAi of Ufd1 or Npl4 depleting approximately 90% of the VCP-Ufd1-Npl4 complexes does not induce unfolded protein response, indicating that the Ufd1-Npl4 <em>dimer</em> is not involved in the regulation of ER function by VCP. RNAi of Ufd1 or Npl4 is associated with a <em>2</em>-fold increase in the levels of polyubiquitinated proteins, which form dispersed aggregates often associated with calnexin-positive structures. However, contrary to the effects of proteasome inhibition, RNAi of Ufd1 or Npl4 does not induce an accumulation of alpha-TCR and <em>delta</em>-CD3, two ERAD substrates overexpressed in HeLa cells. Instead, a 60-70% decrease in their levels is observed. The decrease in alpha-TCR levels is associated with a 50% decrease of its half-life. Upregulation of the putative channel forming protein, derlin-1, may contribute to the increased degradation of ERAD substrates. To explain our findings, we propose a model, where association of emerging ERAD substrates with VCP-Ufd1-Npl4 is not required for their degradation but has a regulatory role.

OBJECTIVE

To investigate the hypothesis that day/night patterns of prothrombotic activity differ between patients with obstructive sleep apnea (OSA) and individuals with no OSA.

METHODS

Prothrombotic markers' day/night rhythms recorded over one <em>2</em>4-h period.

METHODS

General clinical research center.

METHODS

38 untreated OSA patients as verified by polysomnography (apnea-hypopnea index>> or = 10/h sleep) and <em>2</em><em>2</em> non-OSA controls.

RESULTS

Blood samples were collected every <em>2</em> h to measure plasma levels of fibrinolysis-inhibiting plasminogen activator inhibitor (PAI)-1 and the primary fibrin degradation product <em>D</em>-<em>dimer</em>. <em>D</em>ay/night variation in hemostasis factors was examined using a cosinor analysis. Mesor (mean) PAI-1 over the <em>2</em>4-h period was higher (P = 0.015), and mesor of <em>D</em>-<em>dimer</em> was lower (P = 0.001) in patients with OSA than in the non-OSA controls. These group differences stayed significant when controlling for age and gender. After further adjustment for body mass index, mean arterial pressure, and smoking, the relationship between OSA and PAI-1 became non-significant, but the relationship between OSA and <em>D</em>-<em>dimer</em> continued to be significant (P = 0.006). In the fully adjusted analysis, the amplitude (peak) for <em>D</em>-<em>dimer</em> was lower in OSA patients than in non-OSA controls (P = 0.048). The acrophase (time of the peak) for PAI-1 and <em>D</em>-<em>dimer</em> did not significantly differ between groups.

CONCLUSIONS

The relatively higher average level of PAI-1 and lower average level of <em>D</em>-<em>dimer</em> across the <em>2</em>4-h in OSA patients might reflect decreased fibrinolytic capacity and fibrin degradation, respectively. The findings provide some evidence for a prothrombotic state in OSA, but were only partially independent of metabolic variables.

Electron transfer between the Rieske iron-sulfur protein (Fe(<em>2</em>)S(<em>2</em>)) and cytochrome c(1) was studied using the ruthenium <em>dimer</em>, Ru(<em>2</em>)<em>D</em>, to either photoreduce or photooxidize cytochrome c(1) within 1 micros. Ru(<em>2</em>)<em>D</em> has a charge of +4, which allows it to bind with high affinity to the cytochrome bc(1) complex. Flash photolysis of a solution containing beef cytochrome bc(1), Ru(<em>2</em>)<em>D</em>, and a sacrificial donor resulted in reduction of cytochrome c(1) within 1 micros, followed by electron transfer from cytochrome c(1) to Fe(<em>2</em>)S(<em>2</em>) with a rate constant of 90,000 s(-1). Flash photolysis of reduced beef bc(1), Ru(<em>2</em>)<em>D</em>, and a sacrificial acceptor resulted in oxidation of cytochrome c(1) within 1 micros, followed by electron transfer from Fe(<em>2</em>)S(<em>2</em>) to cytochrome c(1) with a rate constant of 16,000 s(-1). Oxidant-induced reduction of cytochrome b(H) was observed with a rate constant of <em>2</em>50 s(-1) in the presence of antimycin A. Electron transfer from Fe(<em>2</em>)S(<em>2</em>) to cytochrome c(1) within the Rhodobacter sphaeroides cyt bc(1) complex was found to have a rate constant of 60,000 s(-1) at <em>2</em>5 degrees C, while reduction of cytochrome b(H) occurred with a rate constant of 1000 s(-1). <em>D</em>ouble mutation of Ala-46 and Ala-48 in the neck region of the Rieske protein to prolines resulted in a decrease in the rate constants for both cyt c(1) and cyt b(H) reduction to <em>2</em>5 s(-1), indicating that a conformational change in the Rieske protein has become rate-limiting.

Pepti<em>d</em>e inhibitors of phage lamb<em>d</em>a site-specific recombination were previously isolate<em>d</em> by screening synthetic combinatorial pepti<em>d</em>e libraries. These inhibitors cause the accumulation of complexes between the recombinase an<em>d</em> the Holli<em>d</em>ay junction interme<em>d</em>iate of several highly <em>d</em>ivergent tyrosine recombinases. Pepti<em>d</em>e WRWYCR an<em>d</em> its <em>d</em>-amino aci<em>d</em> <em>d</em>erivative bin<em>d</em> to the center of protein-free junctions an<em>d</em> prevent their resolution either by site-specific recombinases or by junction resolvases or helicases. With lesser affinity, the pepti<em>d</em>es also bin<em>d</em> to branche<em>d</em> DNA molecules that mimic replication forks. The pepti<em>d</em>es are bacterici<em>d</em>al to both gram-positive an<em>d</em> gram-negative bacteria, presumably because they can interfere with DNA repair an<em>d</em> with chromosome <em>dimer</em> resolution by the XerC an<em>d</em> XerD tyrosine recombinases. In or<em>d</em>er to test the correspon<em>d</em>ence between their mechanism in vivo an<em>d</em> in vitro, we have teste<em>d</em> an<em>d</em> shown pepti<em>d</em>e wrwycr's ability to inhibit the excision of several prophages (lamb<em>d</em>a, P<em>2</em><em>2</em>, Gifsy-1, Gifsy-<em>2</em>, Fels-1, Fels-<em>2</em>) an<em>d</em> to trap Holli<em>d</em>ay junction interme<em>d</em>iates of phage lamb<em>d</em>a site-specific recombination in vivo. In a<em>d</em><em>d</em>ition, we foun<em>d</em> that the pepti<em>d</em>e inhibits replication of the Salmonella prophage Fels-1 while integrate<em>d</em> in the chromosome. These fin<em>d</em>ings further support the propose<em>d</em> mechanistic basis for the antimicrobial activity of the pepti<em>d</em>e an<em>d</em> its use as a tool to <em>d</em>issect stran<em>d</em> exchange-<em>d</em>epen<em>d</em>ent DNA repair within cells.

Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(<em>2</em>+) ions, of the (alphabetagamma<em>delta</em>)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits. The gamma subunit is also known to interact with the <em>delta</em> subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(<em>2</em>+) ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1<em>2</em>37) subunits interact. The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1<em>2</em>37 from an alpha-gamma <em>dimer</em> that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (<em>D</em>asgupta, M., Honeycutt, T., and Blumenthal, <em>D</em>. K. (1989) J. Biol. Chem. <em>2</em>64, 17156-17163), we tested the influence of Ca(<em>2</em>+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(<em>2</em>+). The results herein support the existence of a Ca(<em>2</em>+)-sensitive communication network among the <em>delta</em>, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator. The similarity of such a Ca(<em>2</em>+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK.

Mitochondria of <em>D</em>rosophila melanogaster undergo Ca(<em>2</em>+)-induced Ca(<em>2</em>+) release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of <em>D</em>rosophila is not permeable to sucrose and appears to be selective for Ca(<em>2</em>+) and H(+). We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-4<em>2</em>3, and by Mg(<em>2</em>+)/A<em>D</em>P; (ii) that expression of human cyclophilin <em>D</em> in mitochondria of <em>D</em>rosophila S<em>2</em>R(+) cells sensitizes the mCrC to Ca(<em>2</em>+) but does not increase its apparent size; and (iii) that purified <em>dimers</em> of <em>D</em>. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca(<em>2</em>+) and thiol oxidants and inhibited by Mg(<em>2</em>+)/γ-imino ATP. These findings indicate that the mCrC is the PTP of <em>D</em>. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species.

Aldehyde dehydrogenases are isolated as <em>dimers</em> or tetramers but have essentially identical structures. The homotetramer (AL<em>D</em>H1 or AL<em>D</em>H<em>2</em>) is a <em>dimer</em> of <em>dimers</em> (A-B + C-<em>D</em>). In the tetrameric enzyme, Ser500 from subunit "<em>D</em>" interacts with Arg84, a conserved residue, from subunit "A". In the <em>dimer</em>ic AL<em>D</em>H3 form, the interaction cannot exist. It has been proposed that the formation of the tetramer is prevented by the presence of a C-terminal tail in AL<em>D</em>H3 that is not present in AL<em>D</em>H1 or <em>2</em>. To understand the forces that maintain the tetramer, deletion of the tail in AL<em>D</em>H3, addition of different tails in AL<em>D</em>H1, and mutations of different residues located in the <em>dimer</em>-<em>dimer</em> interface were made. Gel filtration of the recombinantly expressed enzymes demonstrated that no change in their oligomerization occurred. Urea denaturation showed a diminution to the stability of the AL<em>D</em>H1 mutants. The K(m) for propionaldehyde was similar to that of the wild-type enzyme, but the K(m) for NA<em>D</em> was altered. A double mutant of <em>D</em>80G and S8<em>2</em>A produced an enzyme with the ability to form <em>dimers</em> and tetramers in a protein-concentration-dependent manner. Though stable, this <em>dimer</em>ic form was inactive. The tetramer exhibited 10% activity compared with the wild type. Sequence alignment demonstrated that the hydrophobic surface area is increased in the tetrameric enzymes. The hydrophobic surface seems to be the main force that drives the formation of tetramers. The results indicated that residues 80 and 8<em>2</em> are involved in maintaining the tetramer after its assembly but the C-terminal extension contributes to the overall stability of the assembled protein.

Nitroalkane oxi<em>d</em>ase from Fusarium oxysporum catalyzes the oxi<em>d</em>ation of nitroalkanes to al<em>d</em>ehy<em>d</em>es with pro<em>d</em>uction of nitrite an<em>d</em> hy<em>d</em>rogen peroxi<em>d</em>e. The enzyme has a molecular weight of 47 955 +/- 39, as <em>d</em>etermine<em>d</em> by MALDI-TOF mass spectrometry; un<em>d</em>er non<em>d</em>enaturing con<em>d</em>itions, the aggregation state of the enzyme is best <em>d</em>escribe<em>d</em> by a tetramer-<em>dimer</em> self-associating mo<em>d</em>el, with an association constant of (8.5 +/- 4.4) x 10(6) M-1 (pH 7.0 an<em>d</em> 4 <em>d</em>egreesC). The amino aci<em>d</em> composition an<em>d</em> the N-terminal amino aci<em>d</em> sequence <em>d</em>o not match any known protein or open rea<em>d</em>ing frame. The inactive 5-nitrobutyl-1,5-<em>d</em>ihy<em>d</em>roflavin foun<em>d</em> in the enzyme as purifie<em>d</em> was converte<em>d</em> to FAD, allowing characterization of the active FAD-containing enzyme. With nitroethane as substrate, the Vmax an<em>d</em> Km values are 655 +/- 45 min-1 an<em>d</em> <em>2</em>.9 +/- 0.5 mM at pH 8.0 an<em>d</em> 30 <em>d</em>egreesC, respectively. One mole of FAD per mole of monomer enzyme is require<em>d</em> for catalysis. No activity can be <em>d</em>etecte<em>d</em> with amino aci<em>d</em>s or alpha-hy<em>d</em>roxy aci<em>d</em>s as substrates. Reversible removal of the FAD cofactor yiel<em>d</em>s inactive enzyme. The properties of the FAD cofactor in nitroalkane oxi<em>d</em>ase are within the range <em>d</em>escribe<em>d</em> for other oxi<em>d</em>ases. The UV-visible absorbance spectrum of the active enzyme shows maxima at 446, 384, an<em>d</em> <em>2</em>74 nm; the extinction coefficient at 446 nm is 11.7 mM-1 cm-1. The neutral form of the flavin semiquinone, with maxima at 536 an<em>d</em> 34<em>2</em> nm, is kinetically stabilize<em>d</em>. The UV-visible absorbance spectrum of the re<em>d</em>uce<em>d</em> enzyme is typical of the anionic form of a flavin, with a peak centere<em>d</em> at 335 nm. The affinity of the enzyme for sulfite is low (K<em>d</em> value of 13.8 +/- 0.9 mM at pH 7.0 an<em>d</em> <em>2</em>5 <em>d</em>egreesC); this result, along with the stabilization of the neutral flavin semiquinone, suggests the presence of a weak positive charge near the N(1)-C(<em>2</em>)=O of FAD. The re<em>d</em>uction potential of the enzyme is -367 mV. Benzoate an<em>d</em> phenylacetic aci<em>d</em> are competitive inhibitors, with Kis values of 5.1 +/- 0.6 an<em>d</em> 13.1 +/- <em>2</em>.3 mM, respectively. Bin<em>d</em>ing of benzoate to nitroalkane oxi<em>d</em>ase results in spectral changes similar to those observe<em>d</em> with <em>d</em>-amino aci<em>d</em> oxi<em>d</em>ase. The absorbance spectrum of the flavin boun<em>d</em> to nitroalkane oxi<em>d</em>ase is pH-<em>d</em>epen<em>d</em>ent, with a pKa value of 8.4.

<AbstractText>The objectives of this study were to analyze the clinical features of coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) and evaluate the diagnosis and treatment.</AbstractText><AbstractText>A retrospective analysis of the clinical manifestation and auxiliary examination of 19 patients with COVI<em>D</em>-19 from the Liyuan Hospital intensive care unit (ICU) between January 16, <em>2</em>0<em>2</em>0 and February <em>2</em>0, <em>2</em>0<em>2</em>0 was undertaken.</AbstractText><AbstractText>There were 11 male and 8 female cases among the patients. The median (range) age was 73 (38-91) years. Of these patients, 8 (4<em>2</em>.1%) had died and the median duration from ICU admission to death was <em>2</em> (interquartile range (IQR): 1-10.75) days. Seven of these 8 patients had underlying diseases. The auxiliary examination showed fever (68.4%), dry cough (15.8%), dyspnea (10.5%), and diarrhea (5.3%). All 19 cases showed ground-glass changes on chest computed tomography. Serum hypersensitive C-reactive protein (hs-CRP) and serum amylase A (SAA) were clearly increased in all of the cases. Among the 19 cases, there were 16 (84.<em>2</em>%) cases in which the total number of lymphocytes decreased, 1<em>2</em> cases (63%) had reduced liver function, and 11 cases (58%) had deviant results for fibrinogen (FIB) and <em>D</em>-<em>dimer</em>, in particular, the <em>D</em>-<em>dimer</em> level was significantly higher in the non-survivors compared with the survivors.</AbstractText><AbstractText>There were more men than women among critically ill patients. All of the cases showed ground-glass changes on chest computed tomography and the vast majority of patients displayed fever and dry cough. The clinical laboratory indices change significantly, especially the <em>D</em>-<em>dimer</em> level among non-survivors.</AbstractText>

Venous thromboembolism (VTE) occurs frequently in patients with cancer and contributes to elevated morbidity and mortality. Risk factors for the occurrence of VTE events in patients with cancer have been investigated in numerous clinical studies. For now more than 10 years, the Vienna Cancer and Thrombosis Study (CATS) has focused on the identification of parameters predictive of future VTE occurrence. CATS has contributed to new findings, which may help identify patients at high risk of developing VTE, by means of biomarkers (such as <em>D</em>-<em>dimer</em>, prothrombin fragment 1+<em>2</em>, soluble P-selectin, platelet count, coagulation factor VIII activity, thrombin generation potential, etc.). The association of tissue factor bearing microparticles and the mean platelet volume with the risk of VTE was also elaborately investigated in the framework of CATS. More recently CATS has researched clinical and clinicopathologic parameters which contribute to identification of patients at risk of VTE. The type of cancer is one of the most important risk factor for VTE occurrence. Also the stage of cancer and the histological grade of a tumor have been found to be associated with the occurrence of cancer-related VTE. In further investigations, venous diseases including a history of previous VTE, a history of superficial thrombophlebitis and the presence of varicose veins, have been associated with the risk of VTE in CATS.

OBJECTIVE

This study investigated the connection among HER-<em>2</em> gene amplification, HER-<em>2</em> protein expression, and markers of tumor angiogenesis and oxygenation in patients with operable, invasive breast tumors.

METHODS

From 1988 to 1995, 4<em>2</em>5 patients with metastatic breast cancer were enrolled in a study of high-dose chemotherapy with autologous transplant. Primary tumor blocks were obtained and evaluated using immunohistochemistry (IHC) staining of vessels with von Willebrand factor antibody. Mean microvessel densities (MVD) were determined by counting von Willebrand factor stained cells in three separate "vascular hot spots" using image analysis. Tumor samples were also stained for HER-<em>2</em> by IHC, HER-<em>2</em> gene amplification by fluorescence in situ hybridization, carbonic anhydrase 9 by IHC, and vascular endothelial growth factor (VEGF) by IHC. Plasma from 36 patients with primary tumor samples had VEGF (R&D Systems, MN) and d-dimer (American Diagnostica, Greenwich, CT) levels determined.

RESULTS

There was a significant positive correlation between HER-<em>2</em> gene amplification and both maximum and average MVD (Spearman coefficient = 0.51 and 0.50; P = 0.03 and 0.05, respectively). There was an inverse correlation with HER-<em>2</em> gene amplification and expression of the tumor hypoxia marker CA-9 (chi(<em>2</em>) P = 0.0<em>2</em>). The level of HER-<em>2</em> gene amplification correlated with plasma d-dimer levels (Spearman coefficient = 0.43; P = 0.0<em>2</em>1). Interestingly, tumors with HER-<em>2</em> by IHC had decreased amounts of VEGF staining (chi(<em>2</em>) = 5.81; P = 0.01). There was no correlation between HER-<em>2</em> by IHC and MVD or d-dimer. Of all of the variables examined, only average (P = 0.0016) and maximum MVD (P = 0.01<em>2</em>8) predicted disease-free survival (Cox univariate model).

CONCLUSIONS

HER-<em>2</em>-amplified breast cancers have increased amounts of angiogenesis, decreased amounts of hypoxia, and increased markers of fibrin degradation. These findings have prognostic, predictive, and therapeutic implications in breast cancer treatment.

The polycystic ovary syndrome (PCOS) is associated with an increased risk of cardiovascular disease (CV<em>D</em>). Insulin resistance (IR), hyperandrogenism, and dyslipidemia are well-known cardiovascular risk factors in PCOS. Impaired fibrinolysis could also contribute to the development of CV<em>D</em> in PCOS. Global fibrinolytic capacity (GFC) is a recently developed method, which is reflected by the amount of generated <em>D</em>-<em>dimer</em> when the fibrinolysis of a freeze-dried fibrin clot is stopped by introducing aprotinin. GFC is sensitive to all the factors involved in the process of fibrinolysis. We evaluated whether women with PCOS have any alterations in the GFC and other essential hemostatic parameters. Fifty-nine nonobese, normal glucose-tolerant women with PCOS (age, <em>2</em><em>2</em>.9 +/- 4.4 yr; body mass index, <em>2</em>3.0 +/- <em>2</em>.4 kg/m(<em>2</em>) ) and <em>2</em>3 age- and body mass index-matched healthy controls participated. We measured GFC and triglycerides; total cholesterol; H<em>D</em>L-cholesterol (H<em>D</em>L-C); lipoprotein-a; prothrombin time; partial thromboplastin time; thrombin time; antithrombin III; factors II, V, VII, and X; fibrinogen; plasminogen; antiplasmin; and <em>D</em>-<em>dimer</em>. Serum glucose and insulin (at baseline and during a 75-g <em>2</em>-h oral glucose tolerance test) were also measured, and IR was assessed by homeostatic model assessment. GFC was significantly lower in the PCOS group, compared with the control group (<em>2</em>.49 +/- 1.6 vs. 5.95 +/- <em>2</em>.43 microg/ml, P < 0.001). All the other coagulation and fibrinolysis parameters were comparable between the two groups. The PCOS group had lower H<em>D</em>L-C and higher IR values. GFC was correlated with testosterone and free testosterone negatively and with H<em>D</em>L-C positively. There was no correlation between GFC and any of the IR parameters. Our results suggest that women with PCOS have impaired fibrinolysis, as reflected by the decreased GFC. This impairment is not related to the IR and may increase the risk of CV<em>D</em> in PCOS.

The Drosophila Notch gene product is a transmembrane protein that functions as a receptor of intercellular signals in several Drosophila developmental processes. Two other transmembrane proteins, encoded by the genes <em>Delta</em> and Serrate, genetically and molecularly behave as Notch ligands. All these proteins share the presence of epidermal growth factor (EGF)-like repeats in their extracellular domain. The Notch protein has 36 EGF-like repeats, <em>2</em> of which, numbers 11 and 1<em>2</em>, are required for the interaction with the <em>Delta</em> and Serrate ligands. We have isolated and molecularly characterized a Notch mutation in its <em>Delta</em>- and Serrate-binding domain that behaves genetically as both a Notch antimorphic and a loss-of-function mutation. This mutation, NM1, carries a Glu->>Val substitution in the Notch EGF repeat 1<em>2</em>. The NM1 allele interacts with other Notch alleles such as Abruptex and split and with mutations in the Notch-ligand genes <em>Delta</em> and Serrate. The basis for the genetic antimorphism of NM1 seems to reside in the titration of Notch wild-type products into NM1/N+ nonfunctional <em>dimers</em> and/or the titration of <em>Delta</em> products into nonfunctional ligand-receptor complexes.

There are few extensive studies about clinicopathological findings of spontaneous canine babesiosis caused by a large form of the parasite found in Europe. To further characterize and describe clinicopathological findings in dogs affected with this large form of Babesia in northeastern Italy, we evaluated <em>2</em>3 Italian dogs with canine babesiosis by means of clinical history, physical examination, hematological, biochemical, hemostatic tests, serum electrophoresis and urinalysis. Seventeen dogs (74%) had recently traveled on a hunting trip (within 5-15 days of being presented to the clinic) to Bosnia and Herzegovina (n=7), to Croatia (n=8) and to Hungary (n=<em>2</em>). The duration of clinical signs ranged from 1 to 5 days prior to the arrival at the clinic. The main clinical signs were dehydration (100%), apathy (74%), anorexia or decrease appetite (70%) and fever (68%). The anemia was present in 74% of the dogs and classified as mild (35%), moderate (59%) and severe (6%). In all cases, the anemia was normocytic and normochromic. Only three dogs presented erythrocyte regeneration. Seventy percent of dogs had hemolytic anemia and 30% had non-hemolytic anemia. Sixty-nine percent of dogs showed leucopenia and 74% neutropenia. Leucocitosis, due to mature neutrophilia and lymphocytosis, was present in one dog. Activated lymphocytes were noted in 61% of dogs. In all dogs, thrombocytopenia and an elevated hyperfibrinogenemia were present. Significant prolonged activated partial thromboplastin time (aPTT) was only found in one case. In four dogs, both plasma fibrinogen/fibrin degradation products (F<em>D</em>Ps) and <em>D</em>-<em>Dimer</em> were increased. Antithrombin (AT) was slightly decreased in 11 of the <em>2</em>3 dogs. In the majority of cases, mild elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinekinase (CK), total bilirubin and lactic acid and decrease of total iron and total iron binding capacity (TIBC) were present. In conclusion, the main clinicopathological findings were a mild to severe thrombocytopenia, a mild to moderate hemolytic anemia, neutropenia and hyperfibrinogenemia.

The T-cell antigen receptor is a multisubunit complex composed of seven transmembrane chains (alpha beta gamma <em>delta</em> epsilon zeta <em>2</em>). Subunit interactions within this complex were defined by analyzing the subunit composition of partial complexes. These partial complexes were observed in mutant and tumor T cells that fail to synthesize one or more of the receptor chains or in fibroblasts transfected with genes encoding T-cell antigen receptor chains. In addition, partial complexes were generated by immunoprecipitation with antibodies that cause selective dissociation of T-cell antigen receptor chains. The alpha and beta chains were found to form a disulfide-linked <em>dimer</em> in the absence of any of the other chains. The gamma, <em>delta</em>, and epsilon chains were also efficiently associated in the absence of a complete hetero<em>dimer</em>. Complexes composed only of <em>delta</em> epsilon or gamma epsilon could be observed. Both these <em>dimers</em>, as well as the gamma <em>delta</em> epsilon trimer, could form stable complexes with alpha beta, even in the absence of zeta <em>2</em>. The zeta <em>2</em> <em>dimer</em> could bind directly to alpha beta. In the absence of a complete clonotypic hetero<em>dimer</em>, zeta <em>2</em> was not stably associated with gamma <em>delta</em> epsilon. These observations suggest a model in which alpha beta interacts directly with the gamma <em>delta</em> epsilon trimer and zeta <em>2</em>, with less-direct interaction between the latter two.