The natriuretic peptides are hormones that can stimulate natriuretic, diuretic, and vasorelaxant activity in vivo, presumably through the activation of two known cell surface receptor guanylyl cyclases (ANPR-A and ANPR-B). Although atrial natriuretic peptide (ANP) and, to a lesser extent, brain natriuretic peptide (BNP) are efficient activators of the ANPR-A guanylyl cyclase, neither hormone can significantly stimulate ANPR-B. A member of this hormone family, C-type natriuretic peptide (CNP), potently and selectively activated the human ANPR-B guanylyl cyclase. CNP does not increase guanosine 3',5'-monophosphate accumulation in cells expressing human ANPR-A. The affinity of CNP for ANPR-B is 50- or 500-fold higher than ANP or BNP, respectively. This ligand-receptor pair may be involved in the regulation of fluid homeostasis by the central nervous system.

Normal cells contain p53 protein in a latent state that can be activated for sequence-specific transcription by low levels of UV radiation without an increase in protein levels. Microinjection of cells with an antibody specific to the C-terminal negative regulatory domain can activate the function of p53 as a specific transcription factor in the absence of irradiation damage, suggesting that posttranslational modification of a negative regulatory domain in vivo is a rate-limiting step for p53 activation. Small peptides derived from the negative regulatory domain of p53 have been used as biochemical tools to distinguish between allosteric and steric mechanisms of negative regulation of p53 tetramer activity. Presented is the development of a highly specific peptide activation system that is consistent with an allosteric mechanism of negative regulation and that forms a precedent for the synthesis of novel low molecular mass modifiers of the p53 response.

BACKGROUND

Recent animal studies showed that osteocalcin action is related to not only bone metabolism but also glucose metabolism and fat mass. We investigated the relationship between two bone formation markers, serum osteocalcin and bone-specific alkaline phosphatase, and glucose metabolism, serum adiponectin, and the amount of fat mass as well as atherosclerosis parameters in men and postmenopausal women with type 2 diabetes.

METHODS

A total of 179 men and 149 postmenopausal women were recruited consecutively, and radiographic and biochemical characteristics were collected. Brachial-ankle pulse wave velocity (baPWV) and intima-media thickness (IMT) were evaluated as the parameters of atherosclerosis.

RESULTS

Multiple regression analysis adjusted for age, duration of diabetes, body mass index, and serum creatinine showed that osteocalcin negatively correlated with fasting plasma glucose and hemoglobin A(1c) in both men and postmenopausal women (P < 0.05) and with percent fat, baPWV, and IMT in men (P < 0.05). Osteocalcin positively correlated with total adiponectin in postmenopausal women (P < 0.001). After additional adjustments for systolic blood pressure, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, hemoglobin A(1c), and Brinkmann index, osteocalcin still significantly and negatively correlated with baPWV and IMT in men. In contrast, osteocalcin did not correlate with fasting C-peptide, and bone-specific alkaline phosphatase did not correlate with any variable in either men or postmenopausal women.

CONCLUSIONS

Serum osteocalcin is associated with glucose and total adiponectin levels, fat mass, and atherosclerosis parameters in patients with type 2 diabetes, suggesting that osteocalcin is important for not only bone metabolism but also glucose and fat metabolism.

The far upstream element (FUSE) of the human c-myc proto-oncogene stimulates expression in undifferentiated cells. A FUSE-binding protein (FBP) is present in undifferentiated but not differentiated cells. Peptide sequences from the purified protein allowed cloning of cDNAs encoding FBP. Expression of FBP mRNA declined upon differentiation, suggesting transcriptional regulation of FBP. Features in the FBP cDNA suggest that FBP is also regulated by RNA processing, translation, and post-translational mechanisms. Both cellular and recombinant FBP form sequence-specific complexes with a single strand of FUSE. Transfection of FBP into human leukemia cells stimulated c-myc-promoter-driven expression from a reporter plasmid in a FUSE-dependent manner. Deletion and insertion mutagenesis of FBP defined a novel single-strand DNA-binding domain. Analysis of the primary and predicted secondary structure of the amino acid sequence reveals four copies of a reiterated unit comprised of a 30-residue direct repeat and an amphipathic alpha-helix separated by an 18- to 21-residue spacer. The third and fourth copies of this repeat-helix unit constitute the minimum single-stranded DNA-binding domain. To determine whether the FUSE site, in vivo, possesses single-strand conformation, and therefore could be bound by FBP, cells were treated with potassium permanganate (KMnO4) to modify unpaired bases. Modification of genomic DNA in vivo revealed hyperreactivity associated with single-stranded DNA in the FUSE sequence and protection on the strand that binds FBP in vitro. The role of single-stranded DNA and single-strand binding proteins in c-myc regulation is discussed.

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

An understanding of the relationship between the breadth and magnitude of T-cell epitope responses and viral loads is important for the design of effective vaccines. For this study, we screened a cohort of 46 subtype C human immunodeficiency virus type 1 (HIV-1)-infected individuals for T-cell responses against a panel of peptides corresponding to the complete subtype C genome. We used a gamma interferon ELISPOT assay to explore the hypothesis that patterns of T-cell responses across the expressed HIV-1 genome correlate with viral control. The estimated median time from seroconversion to response for the cohort was 13 months, and the order of cumulative T-cell responses against HIV proteins was as follows: Nef>> Gag>> Pol>> Env>> Vif>> Rev>> Vpr>> Tat>> Vpu. Nef was the most intensely targeted protein, with 97.5% of the epitopes being clustered within 119 amino acids, constituting almost one-third of the responses across the expressed genome. The second most targeted region was p24, comprising 17% of the responses. There was no correlation between viral load and the breadth of responses, but there was a weak positive correlation (r = 0.297; P = 0.034) between viral load and the total magnitude of responses, implying that the magnitude of T-cell recognition did not contribute to viral control. When hierarchical patterns of recognition were correlated with the viral load, preferential targeting of Gag was significantly (r = 0.445; P = 0.0025) associated with viral control. These data suggest that preferential targeting of Gag epitopes, rather than the breadth or magnitude of the response across the genome, may be an important marker of immune efficacy. These data have significance for the design of vaccines and for interpretation of vaccine-induced responses.

The novel technique electron capture dissociation (ECD) of electrospray generated [M + nH]n+ polypeptide cations produces rapid cleavage of the backbone NH-Ca bond to form c and z ions (in the modified notation of Roepstorff and Fohlman). The potential of the Fourier transform mass spectrometry equipped with ECD in structure analysis of O-glycosylated peptides in the 3 kDa range has been investigated. Totally, 85% of the available interresidue bonds were cleaved in five glycopeptides; more stable c ions accounted for 62% of the observed fragmentation. The c series provided direct evidence on the glycosylation sites in every case studied, with no glycan (GalNAc and dimannose) losses observed from these species. Less stable z ions supported the glycan site assignment, with minor glycan detachments. These losses, as well as the observed formation of even-electron z ions, are attributed to radical-site-initiated reactions. In favorable cases, complete sequence and glycan position information is obtained from a single-scan spectrum. The "mild" character of ECD supports the previously proposed non-ergodic (cleavage prior to energy randomization) mechanism, and the low internal energy increment of fragments.

A cDNA corresponding to the 2.6 kb NF-kappa B mRNA species present in a variety of lymphoid cell lines has been molecularly cloned. The deduced 607 amino acid sequence is identical to the sequence of the C-terminal region of 110 kd NF-kappa B protein. A 70 kd protein can be identified in lymphoid cells using antibodies raised against the C-terminal region of p110 NF-kappa B. Comparison of the two-dimensional tryptic peptide maps of the 70 kd protein expressed in cells and the in vitro translated product encoded by the cDNA display extensive homology. The 70 kd protein expressed in bacteria prevents sequence-specific DNA binding of p50-p65 NF-kappa B heterodimer, p50 homodimer, and c-rel. p70 also interferes with transactivation by c-rel and prevents its nuclear translocation. The 70 kd protein, predominantly found in lymphoid cells, is a new member of the I kappa B family of proteins and is referred to as I kappa B gamma.

BACKGROUND

Tandem mass spectrometry (MS/MS) identifies protein sequences using database search engines, at the core of which is a score that measures the similarity between peptide MS/MS spectra and a protein sequence database. The TANDEM application was developed as a freely available database search engine for the proteomics research community. To extend TANDEM as a platform for further research on developing improved database scoring methods, we modified the software to allow users to redefine the scoring function and replace the native TANDEM scoring function while leaving the remaining core application intact. Redefinition is performed at run time so multiple scoring functions are available to be selected and applied from a single search engine binary. We introduce the implementation of the pluggable scoring algorithm and also provide implementations of two TANDEM compatible scoring functions, one previously described scoring function compatible with PeptideProphet and one very simple scoring function that quantitative researchers may use to begin their development. This extension builds on the open-source TANDEM project and will facilitate research into and dissemination of novel algorithms for matching MS/MS spectra to peptide sequences. The pluggable scoring schema is also compatible with related search applications P3 and Hunter, which are part of the X! suite of database matching algorithms. The pluggable scores and the X! suite of applications are all written in C++.

BACKGROUND

Source code for the scoring functions is available from http://proteomics.fhcrc.org

The goal of this work was to develop a growth factor delivery system for use in wound healing that would provide localized release of heparin-binding growth factors in a biomimetic manner, such that release occurs primarily in response to cell-associated enzymatic activity during healing. A key element of the drug delivery system was a bi-domain peptide with an N-terminal transglutaminase substrate and a C-terminal heparin-binding domain, based on antithrombin III. The bi-domain peptide was covalently cross-linked to fibrin matrices during coagulation by the transglutaminase activity of factor XIIIa and served to immobilize heparin electrostatically to the matrix, which in turn immobilized the heparin-binding growth factor and slowed its passive release from the matrix. Basic fibroblast growth factor (bFGF) was considered as an example of a heparin-binding growth factor, and cell culture experimentation was performed in the context of peripheral nerve regeneration. A mathematical model was developed to determine the conditions where passive release of bFGF would be slow, such that active release could dominate. These conditions were tested in an assay of neurite extension from dorsal root ganglia to determine the ability of the delivery system to release bioactive growth factor in response to cell-mediated processes. The results demonstrated that bFGF, immobilized within fibrin containing a 500-fold molar excess of immobilized heparin relative to bFGF, enhanced neurite extension by up to about 100% relative to unmodified fibrin. A variety of control experiments demonstrate that all components of the release system are necessary and that the bi-domain peptide must be covalently bound to the fibrin matrix. The results thus suggest that these matrices could serve as therapeutic materials to enhance peripheral nerve regeneration through nerve guide tubes and may have more general usefulness in tissue engineering.

The betaA4 peptide, a major component of senile plaques in Alzheimer's disease (AD) brain, has been found in cerebrospinal fluid (CSF) and blood of both AD patients and normal subjects. Although betaA4 1-40 is the major form produced by cell metabolism and found in CSF, recent observations suggest that the long-tailed betaA4 1-42 plays a more crucial role in AD pathogenesis. Here, we established new monoclonal antibodies against the C-terminal end of betaA4 1-40 and 1-42, and used them for the specific Western blot detection. After optimizing the assay conditions, these antibodies detected low picogram amount of betaA4, and both betaA4 1-40 and 1-42 levels in CSF could be determined by direct loading of the samples. Blood levels of betaA4 1-40 and 1-42 were also determined by specific immunoprecipitation followed by Western blot detection. We found that CSF betaA4 1-42 level is lower in AD patients compared with non-demented controls, although there was a significant overlap between the groups. The level of betaA4 1-40 in CSF, and of betaA4 1-40 as well as betaA4 1-42 in plasma, were not different between AD patients and controls. Besides the 4-kDa full-length betaA4 band, we could also detect several N-terminal variants of betaA4 in CSF and plasma of both AD patients and controls. Two N-terminally truncated betaA4 species migrating at the position of 3.3 and 3.7 kDa were found in CSF, while 3.7- and 5-kDa forms were found in plasma. The relative abundance of these various species were considerably different in the CSF and plasma, suggesting that the cellular source and/or clearance of betaA4 is different in these two compartments.

Agouti-related protein (Agrp) is present in rat and human hypothalamus and is structurally related to agouti protein. Overexpression of either of these proteins results in obesity. However the effect of exogenous Agrp and its in vivo interaction with alpha-melanocyte stimulating hormone (alphaMSH), the likely endogenous melanocortin 3 and 4 receptor (M<em>C</em>3-R and M<em>C</em>4-R) agonist, have not been demonstrated. We report that 1 nmol of Agrp(83-132), a <em>C</em>-terminal fragment of Agrp, when administered intracerebroventricularly (I<em>C</em>V) into rats, increased food intake over a 24-h period (23.0+/-1.4 g saline vs 32.9+/-2.3 g Agrp, p<0.05). The hyperphagia was similar to that seen when 1 nmol of the synthetic M<em>C</em>3-R and M<em>C</em>4-R antagonist SHU9119 was given i.c.v. (19.6+/-1.8 g saline vs 32.5+/-1.7 g SHU9119, p<0.001). Both Agrp(83-132) and SHU9119 blocked the reduction in 1-h food intake of i.c.v. alphaMSH at the beginning of the dark phase. This effect occurred independently of whether the antagonists were administered simultaneously, or nine hours prior, to the alphaMSH. We have also shown Agrp(83-132) is an antagonist at the M<em>C</em>3-R and M<em>C</em>4-R, with similar inhibition of cAMP activation to that previously reported for the full length <em>peptide</em>. In conclusion, Agrp(83-132) administered i.c.v. increases feeding with long lasting effects and is able to inhibit the action of alphaMSH. This interaction may be mediated by the M<em>C</em>3-R and/or M<em>C</em>4-R.

A novel neuropeptide with 38 residues (PACAP38) was isolated from ovine hypothalamic tissues using the pituitary adenylate cyclase activation in rat pituitary cell cultures as a parameter of the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567-574, 1989). From the side fractions obtained during the purification of PACAP38, a shorter form peptide with 27 residues corresponding to the N-terminal 27 amino acids of PACAP38 and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP27 showed a biological activity of adenylate cyclase stimulation comparable to PACAP38. Moreover PACAP27 which shows a considerable homology with vasoactive intestinal polypeptide (VIP) demonstrated a similar vasodepressor activity as VIP, but the adenylate cyclase stimulating activity was about 1000 times greater than VIP.

Glycemic control is improved more after gastric bypass surgery (GBP) than after equivalent diet-induced weight loss in patients with morbid obesity and type 2 diabetes mellitus. We applied metabolomic profiling to understand the mechanisms of this better metabolic response after GBP. <em>C</em>irculating amino acids (AAs) and acylcarnitines (A<em>C</em>s) were measured in plasma from fasted subjects by targeted tandem mass spectrometry before and after a matched 10-kilogram weight loss induced by GBP or diet. Total AAs and branched-chain AAs (B<em>C</em>AAs) decreased after GBP, but not after dietary intervention. Metabolites derived from B<em>C</em>AA oxidation also decreased only after GBP. Principal components (P<em>C</em>) analysis identified two major P<em>C</em>s, one composed almost exclusively of A<em>C</em>s (P<em>C</em>1) and another with B<em>C</em>AAs and their metabolites as major contributors (P<em>C</em>2). P<em>C</em>1 and P<em>C</em>2 were inversely correlated with pro-insulin concentrations, the <em>C</em>-<em>peptide</em> response to oral glucose, and the insulin sensitivity index after weight loss, whereas P<em>C</em>2 was uniquely correlated with levels of insulin resistance (HOMA-IR). These data suggest that the enhanced decrease in circulating AAs after GBP occurs by mechanisms other than weight loss and may contribute to the better improvement in glucose homeostasis observed with the surgical intervention.

Increases in neuronal activity in response to tissue injury lead to changes in gene expression and prolonged changes in the nervous system. These functional changes appear to contribute to the hyperalgesia and spontaneous pain associated with tissue injury. This activity-dependent plasticity involves neuropeptides, such as dynorphin, substance P and calcitonin gene-related peptide, and excitatory amino acids, such as NMDA, which are chemical mediators involved in nociceptive processing. Unilateral inflammation in the hindpaw of the rat results in an increase in the expression of preprodynorphin and preproenkephalin mRNA in the spinal cord, which parallels the behavioral hyperalgesia associated with the inflammation. Cellular intermediate-early genes, such as c-fos, are also expressed in spinal cord neurons following inflammation and activation of nociceptors. Peripheral inflammation results in an enlargement of the receptive fields of many of these neurons. Dynorphin applied to the spinal cord also induces an enlargement of receptive fields. NMDA antagonists block the hyperexcitability produced by inflammation. A model has been proposed in which dynorphin, substance P and calcitonin gene-related peptide enhance excitability at NMDA receptor sites, leading first to dorsal horn hyperexcitability and then to excessive depolarization and excitotoxicity.

In mammalian small intestine, a H(+)-coupled peptide transporter is responsible for the absorption of small peptides arising from digestion of dietary proteins. Recently a cDNA clone encoding a H+/peptide cotransporter has been isolated from a rabbit intestinal cDNA library (Fei, Y.J., Kanai, Y., Nussberger, S., Ganapathy, V., Leibach, F.H., Romero, M.F., Singh, S.K., Boron, W. F., and Hediger, M. A. (1994) Nature 368, 563-566). Screening of a human intestinal cDNA library with a probe derived from the rabbit H+/peptide cotransporter cDNA resulted in the identification of a cDNA which when expressed in HeLa cells or in Xenopus laevis oocytes induced H(+)-dependent peptide transport activity. The predicted protein consists of 708 amino acids with 12 membrane-spanning domains and two putative sites for protein kinase C-dependent phosphorylation. The cDNA-induced transport process accepts dipeptides, tripeptides, and amino beta-lactam antibiotics but not free amino acids as substrates. The human H+/peptide cotransporter exhibits a high degree of homology (81% identity and 92% similarity) to the rabbit H+/peptide cotransporter. But surprisingly these transporters show only a weak homology to the H(+)-coupled peptide transport proteins present in bacteria and yeast. Chromosomal assignment studies with somatic cell hybrid analysis and in situ hybridization have located the gene encoding the cloned human H+/peptide cotransporter to chromosome 13 q33->>q34.

Apolipoprotein E (apoE) genotype has a major influence on the risk for Alzheimer disease (AD). Different apoE isoforms may alter AD pathogenesis via their interactions with the amyloid beta-peptide (Abeta). Mice lacking the lipid transporter ABCA1 were found to have markedly decreased levels and lipidation of apoE in the central nervous system. We hypothesized that if Abca1-/- mice were bred to the PDAPP mouse model of AD, PDAPP Abca1-/ mice would have a phenotype similar to that of PDAPP Apoe+/- and PDAPP Apoe-/- mice, which develop less amyloid deposition than PDAPP Apoe+/+ mice. In contrast to this prediction, 12-month-old PDAPP Abca -/- mice had significantly higher levels of hippocampal Abeta, and cerebral amyloid angiopathy was significantly more common compared with PDAPP Abca1+/+ mice. Amyloid precursor protein (APP) C-terminal fragments were not different between Abca1 genotypes prior to plaque deposition in 3-month-old PDAPP mice, suggesting that deletion of Abca1 did not affect APP processing or Abeta production. As expected, 3-month-old PDAPP Abca1-/- mice had decreased apoE levels, but they also had a higher percentage of carbonate-insoluble apoE, suggesting that poorly lipidated apoE is less soluble in vivo. We also found that 12-month-old PDAPP Abca1-/- mice had a higher percentage of carbonate-insoluble apoE and that apoE deposits co-localize with amyloid plaques, demonstrating that poorly lipidated apoE co-deposits with insoluble Abeta. Together, these data suggest that despite substantially lower apoE levels, poorly lipidated apoE produced in the absence of ABCA1 is strongly amyloidogenic in vivo.

Five polyheptapeptides (Ac-(Lys-Leu-Glu-Ala-Leu-Glu-Gly)n-Lys-amide, where n = 1-5) of 8, 15, 22, 29, and 36 residues were synthesized by the solid-phase method. The peptides were purified by reversed-phase high-performance liquid chromatography. The ability of these peptides to form two-stranded alpha-helical coiled-coils in benign medium (1.1 M KCCM-tropomyosin). The peptides TM-8, TM-15, and TM-22 were shown to be monomeric in both denaturant (8 M urea) and benign medium by gel-filtration high-performance liquid chromatography on TSK G2000 SW while peptides TM-29 and TM-36 were shown to be dimeric in benign medium both by gel-filtration and sedimentation equilibrium experiments. The CD spectra of the polyheptapeptides TM-8, TM-15, and TM-22 show large increases in molar ellipticity at 220 nm on the addition of trifluoroethanol (helix-inducing solvent) to the benign buffer. By comparison, the two-stranded polyheptapeptides (TM-29 and TM-36) and CM-tropomyosin do not show any increase in molar ellipticity at 220 nm. The helicity of polyheptapeptides increases with increasing chain length, with TM-36 having a value comparable with CM-tropomyosin [( theta]220 = -31,800 degrees and -32,200 degrees, respectively) which is considered to be essentially 100% alpha-helical. These small two-stranded alpha-helical coiled-coils are considerably more stable to temperature and urea denaturation than CM-tropomyosin. Whereas CM-tropomyosin is almost completely denatured in the presence of 6 M urea, TM-29 and TM-36 maintain 22 and 70% of their helicity, respectively. The 30% denaturation values (t30) are 74, 62, and 37 degrees C for TM-36, TM-29, and CM-tropomyosin, respectively, in benign medium (1.1 M KCCC for TM-36 and TM-29, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of tumor necrosis factor (TNF)-alpha action has recently been shown to reverse insulin resistance dramatically and to improve glycemic control in obese rodents. This double-blind study was designed to assess the effects of a recombinant-engineered human TNF-alpha-neutralizing antibody (CDP571) on glucose homeostasis in obese NIDDM patients. Glycemic control and insulin sensitivity were monitored in 21 NIDDM subjects for a 2-week run-in and then for 6 weeks after treatment in a randomized fashion with a single intravenous dose of either CDP571 (5 mg/kg) or an equivalent volume of normal saline. The prolonged half-life of the antibody ensured adequate plasma levels as measured throughout the study. Concentrations of fasting glucose (CDP571: 10.0 +/- 0.8, 10.1 +/- 0.8, 10.0 +/- 1.0; placebo: 8.5 +/- 0.6, 8.1 +/- 0.5, 8.7 +/- 0.8 mmol/l at baseline, day 1, and week 4, respectively), fasting serum insulin (CDP571: 21.2 +/- 2.8, 21.0 +/- 2.8, 24.8 +/- 3.3; placebo: 19.0 +/- 2.8, 20.8 +/- 2.9, 17.5 +/- 2.2 pmol/l, respectively), and C-peptide remained unaffected by the type of treatment throughout the study. The percentage rate of glucose clearance per minute (KITT) during intravenous insulin sensitivity tests was identical in the CDP571 and placebo groups at baseline and also at 1 and 4 weeks after treatment (mean +/- SE; CDP571: 1.33 +/- 0.21, 1.44 +/- 0.25, 1.26 +/- 0.18; placebo: 1.38 +/- 0.15, 1.47 +/- 0.20, 1.52 +/- 0.20; P = 0.85, 0.93, and 0.36, respectively). TNF-alpha neutralization over a period of 4 weeks had no effect on insulin sensitivity in obese NIDDM subjects.

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) proceeds in two steps. Initially, an immature virus with a spherical capsid shell consisting of uncleaved Gag polyproteins is formed. Extracellular proteolytic maturation causes rearrangement of the inner virion structure, leading to the conical capsid of the infectious virus. Using an in vitro assembly system, we show that the same HIV-1 Gag-derived protein can form spherical particles, virtually indistinguishable from immature HIV-1 capsids, as well as tubular or conical particles, resembling the mature core. The assembly phenotype could be correlated with differential binding of the protein to monoclonal antibodies recognizing epitopes in the HIV-1 capsid protein (CA), suggesting distinct conformations of this domain. Only tubular and conical particles were observed when the protein lacked spacer peptide SP1 at the C-terminus of CA, indicating that SP1 may act as a molecular switch, whose presence determines spherical capsid formation, while its cleavage leads to maturation.

The structure of a stable recombinant ectodomain of influenza hemagglutinin HA(2) subunit, EHA(2) (23-185), defined by proteolysis studies of the intact bacterial-expressed ectodomain, was determined to 1.9-A resolution by using x-ray crystallography. The structure reveals a domain composed of N- and C-terminal residues that form an N cap terminating both the N-terminal alpha-helix and the central coiled coil. The N cap is formed by a conserved sequence, and part of it is found in the neutral pH conformation of HA. The C-terminal 23 residues of the ectodomain form a 72-A long nonhelical structure ordered to within 7 residues of the transmembrane anchor. The structure implies that continuous alpha helices are not required for membrane fusion at either the N or C termini. The difference in stability between recombinant molecules with and without the N cap sequences suggests that additional free energy for membrane fusion may become available after the formation of the central triple-stranded coiled coil and insertion of the fusion peptide into the target membrane.

<em>C</em>erebral deposition of amyloid beta-<em>peptide</em> (Abeta) in the brain is an invariant feature of Alzheimer's disease (AD). A consistent protective effect of wine consumption on AD has been documented by epidemiological studies. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of wine-related polyphenols (myricetin, morin, quercetin, kaempferol (+)-catechin and (-)-epicatechin) on the formation, extension, and destabilization of beta-amyloid fibrils (fAbeta) at pH 7.5 at 37 degrees <em>C</em> in vitro. All examined polyphenols dose-dependently inhibited formation of fAbeta from fresh Abeta(1-40) and Abeta(1-42), as well as their extension. Moreover, these polyphenols dose-dependently destabilized preformed fAbetas. The overall activity of the molecules examined was in the order of: myricetin = morin = quercetin>> kaempferol>> (+)-catechin = (-)-epicatechin. The effective concentrations (E<em>C</em>50) of myricetin, morin and quercetin for the formation, extension and destabilization of fAbetas were in the order of 0.1-1 micro m. In cell culture experiments, myricetin-treated fAbeta were suggested to be less toxic than intact fAbeta, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Although the mechanisms by which these polyphenols inhibit fAbeta formation from Abeta, and destabilize pre-formed fAbetain vitro are still unclear, polyphenols could be a key molecule for the development of preventives and therapeutics for AD.

Cellular immune responses are likely to play a key role in determining the clinical outcome in acute infection with hepatitis C virus (HCV), but the dynamics of such responses and their relationship to viral clearance are poorly understood. In a previous study we have shown highly activated, multispecific cytotoxic T lymphocyte responses arising early and persisting in an individual who subsequently cleared the virus. In this study the HCV-specific CD8+ lymphocytes response has been similarly analyzed, using peptide-HLA class I tetramers, in a further nine individuals with documented acute HCV infection, six of whom failed to clear the virus. Significant populations of virus-specific CD8+ lymphocytes were detected at the peak of acute hepatic illness (maximally 3.5% of CD8+ lymphocytes). Frequencies were commonly lower than those seen previously and were generally not sustained. Early HCV-specific CD8+ lymphocytes showed an activated phenotype in all patients (CD38+ and HLA class II+), but this activation was short-lived. Failure to sustain sufficient numbers of activated virus-specific CD8+ lymphocytes may contribute to persistence of HCV.

The Gag polyprotein of retroviruses is sufficient for assembly and budding of virus-like particles from the host cell. In the case of human immunodeficiency virus (HIV), Gag contains the domains matrix, capsid (CA), nucleocapsid (NC) and p6 which are separated by the viral proteinase inside the nascent virion, leading to morphological maturation to yield an infectious virus. In the mature virus, CA forms a capsid shell surrounding the ribonucleoprotein core consisting of NC and the genomic RNA. To define requirements for particle assembly and functional contributions of individual domains, we expressed domains of HIV Gag in Escherichia coli and purified the products to near homogeneity. In vitro assembly of CA, with or without the C-terminally adjacent spacer peptide, yielded tubular structures with a diameter of approximately 55 nm and heterogeneous length. Efficient particle formation required high protein concentration, high salt and neutral to alkaline pH. In contrast, in vitro assembly of CA-NC occurred at a 20-fold lower protein concentration and in low salt, but required addition of RNA. These results suggest that hydrophobic interactions of capsid proteins are sufficient for particle formation while the RNA-binding nucleocapsid domain may concentrate and align structural proteins on the viral genome.