Human T-cell-mediated autoimmune diseases are genetically linked to particular alleles of MHC class II genes. Susceptibility to pemphigus vulgaris (PV), an autoimmune disease of the skin, is linked to a rare subtype of HLA-DR4 (DRB1*0402, 1 of 22 known DR4 subtypes). The PV-linked DR4 subtype differs from a rheumatoid arthritis-associated DR4 subtype (DRB1*0404) only at three residues (DR beta 67, 70, and 71). The disease is caused by autoantibodies against desmoglein 3 (DG), and T cells are thought to trigger the autoantibody production against this keratinocyte adhesion molecule. Based on the DRB1*0402 binding motif, seven candidate peptides of the DG autoantigen were identified. T cells from four PV patients with active disease responded to one of these DG peptides (residues 190-204); two patients also responded to DG-(206-220). T-cell clones specific for DG-(190-204) secreted high levels of interleukins 4 and 10, indicating that they may be important in triggering the production of DG-specific autoantibodies. The DG-(190-204) peptide was presented by the disease-linked DRB1*0402 molecule but not by other DR4 subtypes. Site-directed mutagenesis of DRB1*0402 demonstrated that selective presentation of DG-(190-204), which carries a positive charge at the P4 position, was due to the negatively charged residues of the P4 pocket (DR beta 70 and 71). DR beta 71 has a negative charge in DRB1*0402 but a positive charge in other DR4 subtypes, including the DR4 subtypes linked to rheumatoid arthritis. The charge of the P4 pocket in the DR4 peptide binding site therefore appears to be a critical determinant of MHC-linked susceptibility to PV and rheumatoid arthritis.

Tertiary interactions that allow RNA to fold into intricate three-dimensional structures are being identified, but little is known about the thermodynamics of individual interactions. Here we quantify the tertiary structure contributions of individual hydrogen bonds in a "ribose zipper" motif of the recently crystallized Tetrahymena group I intron P4-P6 domain. The 2'-hydroxyls of P4-P6 nucleotides C109/A184 and A183/G110 participate in forming the "teeth" of the zipper. These four nucleotides were substituted in all combinations with their 2'-deoxy and (separately) 2'-methoxy analogues, and thermodynamic effects on the tertiary folding DeltaG degrees ' were assayed by the Mg2+ dependence of electrophoretic mobility in nondenaturing gels. The 2'-deoxy series showed a consistent trend with an average contribution to the tertiary folding DeltaG degrees' of -0.4 to -0.5 kcal/mol per hydrogen bond. Contributions were approximately additive, reflecting no cooperativity among the hydrogen bonds. Each "tooth" of the ribose zipper (comprising two hydrogen bonds) thus contributes about -1.0 kcal/mol to the tertiary folding DeltaG degrees'. Single 2'-methoxy substitutions destabilized folding by approximately 1 kcal/mol, but the trend reversed with multiple 2'-methoxy substitutions; the folding DeltaG degrees' for the quadruple 2'-methoxy derivative was approximately unchanged relative to wild-type. On the basis of these data and on temperature-gradient gel results, we conclude that entropically favorable hydrophobic interactions balance enthalpically unfavorable hydrogen bond deletions and steric clashes for multiple 2'-methoxy substitutions. Because many of the 2'-deoxy derivatives no longer have the characteristic hydrogen-bond patterns of the ribose zipper motif but simply have individual long-range ribose-base or ribose-ribose hydrogen bonds, we speculate that the energetic value of -0.4 to -0.5 kcal/mol per tertiary hydrogen bond may be more generally applicable to RNA folding.

Synchrotron X-ray-dependent hydroxyl radical footprinting was used to probe the folding kinetics of the P4-P6 domain of the Tetrahymena group I ribozyme, which forms a stable, closely packed tertiary structure. The 160-nt domain folds independently at a similar rate (approximately 2 s(-1)) as it does in the ribozyme, when folding is measured in 10 mM sodium cacodylate and 10 mM MgCl(2). Surprisingly, tertiary interactions around a three-helix junction (P5abc) within the P4-P6 domain fold at least 25 times more rapidly (k>>/= 50 s(-1)) in isolation, than when part of the wild-type P4-P6 RNA. This difference implies that long-range interactions in the P4-P6 domain can interfere with folding of P5abc. P4-P6 was observed to fold much faster at higher ionic strength than in 10 mM sodium cacodylate. Analytical centrifugation was used to measure the sedimentation and diffusion coefficients of the unfolded RNA. The hydrodynamic radius of the RNA decreased from 58 to 46 A over the range of 0-100 mM NaCl. We propose that at low ionic strength, the addition of Mg(2+) causes the domain to collapse to a compact intermediate where P5abc is trapped in a non-native structure. At high ionic strength, the RNA rapidly collapses to the native structure. Faster folding most likely results from a different average initial conformation of the RNA in higher salt conditions.

The x-ray crystal structure of succinyl-CoA synthetase (SCS) from Escherichia coli has been determined by the method of multiple isomorphous replacement to a resolution of 2.5 A. Crystals of SCS are tetragonal with a space group of P4(3)22 and unit cell dimensions of a = b = 98.47 A and c = 400.6 A. One molecule of SCS (142 kDa) is contained in the asymmetric unit. The current model has been refined to a conventional R factor of 21.6% with root mean square deviations from ideal stereochemistry of 0.022 A for bond lengths and 3.25 degrees for bond angles. The quaternary organization of the E. coli enzyme is an alpha 2 beta 2 heterotetramer. In this tetramer, the alpha-subunits interact only with the beta-subunits, whereas the beta-subunits interact to form the dimer of alpha beta-dimers. The two active site pockets are located at regions of contact between alpha- and beta-subunits. One molecule of coenzyme A is bound to each alpha-subunit at a typical nucleotide-binding motif, and His-246 of each alpha-subunit is phosphorylated. This phosphohistidine, a catalytic intermediate, is stabilized by two helix dipoles (the "power" helices), one from each of the two subunit types. A short segment of the beta-subunit from one alpha beta-dimer is in close proximity to the CoA-binding site of the other alpha beta-dimer, providing a possible rationale for the overall tetrameric structure.

The signaling apparatus that controls bacterial chemotaxis is composed of a core complex containing chemoreceptors, the histidine autokinase CheA, and the coupling protein CheW. Site-specific spin labeling and pulsed dipolar ESR spectroscopy (PDS) have been applied to investigate the structure of a soluble ternary complex formed by Thermotoga maritima CheA (TmCheA), CheW, and receptor signaling domains. Thirty-five symmetric spin-label sites (SLSs) were engineered into the five domains of the CheA dimer and CheW to provide distance restraints within the CheA:CheW complex in the absence and presence of a soluble receptor that inhibits kinase activity (Tm14). Additional PDS restraints among spin-labeled CheA, CheW, and an engineered single-chain receptor labeled at six different sites allow docking of the receptor structure relative to the CheA:CheW complex. Disulfide cross-linking between selectively incorporated Cys residues finds two pairs of positions that provide further constraints within the ternary complex: one involving Tm14 and CheW and another involving Tm14 and CheA. The derived structure of the ternary complex indicates a primary site of interaction between CheW and Tm14 that agrees well with previous biochemical and genetic data for transmembrane chemoreceptors. The PDS distance distributions are most consistent with only one CheW directly engaging one dimeric Tm14. The CheA dimerization domain (P3) aligns roughly antiparallel to the receptor-conserved signaling tip but does not interact strongly with it. The angle of the receptor axis with respect to P3 and the CheW-binding P5 domains is bound by two limits differing by approximately 20 degrees . In one limit, Tm14 aligns roughly along P3 and may interact to some extent with the hinge region near the P3 hairpin loop. In the other limit, Tm14 tilts to interact with the P5 domain of the opposite subunit in an interface that mimics that observed with the P5 homologue CheW. The time domain ESR data can be simulated from the model only if orientational variability is introduced for the P5 and, especially, P3 domains. The Tm14 tip also binds beside one of the CheA kinase domains (P4); however, in both bound and unbound states, P4 samples a broad range of distributions that are only minimally affected by Tm14 binding. The CheA P1 domains that contain the substrate histidine are also broadly distributed in space under all conditions. In the context of the hexagonal lattice formed by trimeric transmembrane chemoreceptors, the PDS structure is best accommodated with the P3 domain in the center of a honeycomb edge.

We attempted to define the role of tumor suppressor genes in the pathogenesis of human bladder cancer through a combined molecular genetic and immunohistochemical approach. Thirty-four bladder tumors (1 Pis, 6 Pa, 5 P1, 3 P3a, 18 P3b, 1 P4; 8 low grade and 26 high grade tumors) have been analyzed. Restriction fragment length polymorphism analysis directed at 5 suspected or established tumor suppressor gene regions (3p21-25, 11p15, 13q14, 17p11-13, and 18q21) was combined with immunohistochemical using Rb-PMG3-245 monoclonal antibody directed at the retinoblastoma (Rb) gene product. Tumor grade correlated with deletions of 3p (P = 0.004) and 17p (P = 0.063). Tumor stage correlated with deletions of 3p (P = 0.010), 17p (P = 0.015) and altered Rb expression (P = 0.054). Vascular invasion correlated only with deletions of 17p (P = 0.038). No marker correlated with positive lymph nodes. Our results suggest that altered Rb expression occurs in all grades and stages of bladder cancer but is more commonly associated with invasive tumors. Genetic alterations of 3p, 11p, 17p, and 18q are rare events in low grade, superficial tumors, whereas they are more common in high grade and invasive bladder cancer. The role of these genetic alterations in the prognosis of bladder cancer will require additional follow-up and further studies.

1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5'-[beta gamma-imido]triphosphate, adenosine 5'-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5'-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.

Type IV P-type ATPases (P4-ATPases) are a large family of putative phospholipid translocases (flippases) implicated in the generation of phospholipid asymmetry in biological membranes. P4-ATPases are typically the largest P-type ATPase subgroup found in eukaryotic cells, with five members in Saccharomyces cerevisiae, six members in Caenorhabditis elegans, 12 members in Arabidopsis thaliana and 14 members in humans. In addition, many of the P4-ATPases require interaction with a noncatalytic subunit from the CDC50 gene family for their transport out of the endoplasmic reticulum (ER). Deficiency of a P4-ATPase (Atp8b1) causes liver disease in humans, and studies in a variety of model systems indicate that P4-ATPases play diverse and essential roles in membrane biogenesis. In addition to their proposed role in establishing and maintaining plasma membrane asymmetry, P4-ATPases are linked to vesicle-mediated protein transport in the exocytic and endocytic pathways. Recent studies have also suggested a role for P4-ATPases in the nonvesicular intracellular trafficking of sterols. Here, we discuss the physiological requirements for yeast P4-ATPases in phospholipid translocase activity, transport vesicle budding and ergosterol metabolism, with an emphasis on Drs2p and its noncatalytic subunit, Cdc50p.

Synthetic peptides, 14-16 residues in length, were used as substrates for purified recombinant poliovirus proteinase 3C. The sequences of the substrates correspond to the sequences of authentic cleavage sites in the poliovirus polyprotein, all of which contain Gln-Gly at the scissile bond. Specificity of cleavages was demonstrated by analysis of 3C digests of synthetic peptides. Relative rate constants for the cleavages were derived by competition experiments. The rate constants roughly correlated with the estimated half-life of the homologous precursor proteins detected in poliovirus-infected cells. The peptide most resistant to cleavage corresponded to the 3C/3D junction, a site known to be cleaved very slowly by 3C in vivo. Substitution of threonine for alanine in P4 position of this peptide, however, resulted in significant cleavage. This observation supports the hypothesis that the residue in P4 position, in addition to the Gln-Gly in P1 and P1', respectively, contributes to substrate recognition. Ac-Gln-Gly-NH2 was not a substrate for 3C.

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Delta cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.

To isolate new plant genes involved in the defense against oxidative stress, an Arabidopsis cDNA library in a yeast expression vector was transformed into a yeast strain deficient in the YAP1 gene, which encodes a b-Zip transcription factor and regulates general stress response in yeasts. Cells from approximately 10(5) primary transformants were subjected to a tolerance screen toward the thiol-oxidizing drug diamide, which depletes the reduced glutathione in the cell. Four types of Arabidopsis cDNAs were isolated. Three of these cDNAs (P1, P2, and P4) belong to a plant zeta-crystallin family and P3 is an Arabidopsis homolog of isoflavonoid reductases. As such, all four isolated cDNAs are homologous to NADPH oxidoreductases. P1, P2, and P3 steady-state mRNAs accumulated rapidly in Arabidopsis plants under various oxidative stress conditions, such as treatment with paraquat, t-butylhydroperoxide, diamide, and menadione. The data suggested that proteins encoded by the isolated cDNAs play a distinct role in plant antioxidant defense and are possibly involved in NAD(P)/NAD(P)H homeostasis.

Ferritins concentrate and store iron as a mineral in all bacterial, plant, and animal cells. The two ferritin subunit types, H or M (fast) and L (slow), differ in rates of iron uptake and mineralization and assemble in vivo to form heteropolymeric protein shells made up of 24 subunits; H/L subunit ratios reflect cell specificity of H and L subunit gene expression. A diferric peroxo species that is the initial reaction product of Fe(II) in H-type ferritins, as well as in ribonucleotide reductase (R2) and methane monooxygenase hydroxylase (MMOH), has recently been characterized, exploiting the relatively high accumulation of the peroxo intermediate in frog H-subunit type recombinant ferritin with the M sequence. The stability of the diferric reaction centers in R2 and MMOH contrasts with the instability of diferric centers in ferritin, which are precursors of the ferric mineral. We have determined the crystal structure of the homopolymer of recombinant frog M ferritin in two crystal forms: P4(1)2(1)2, a = b = 170.0 A and c = 481.5 A; and P3(1)21, a = b = 210.8 A and c = 328.1 A. The structural model for the trigonal form was refined to a crystallographic R value of 19.0% (Rfree = 19.4%); the two structures have an r.m.s.d. of approximately 0.22 A for all C alpha atoms. Comparison with the previously determined crystal structure of frog L ferritin indicates that the subunit interface at the molecular twofold axes is most variable, which may relate to the presence of the ferroxidase site in H-type ferritin subunits. Two metal ions (Mg) from the crystallization buffer were found in the ferroxidase site of the M ferritin crystals and interact with Glu23, Glu58, His61, Glu103, Gln137 and, unique to the M subunit, Asp140. The data suggest that Gln137 and Asp140 are a vestige of the second GluxxHis site, resulting from single nucleotide mutations of Glu and His codons and giving rise to Ala140 or Ser140 present in other eukaryotic H-type ferritins, by additional single nucleotide mutations. The observation of the Gln137xxAsp140 site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared to MMOH and R2 and the observed kinetic variability of the diferric peroxo species in different H-type ferritin sequences.

The MHC class II molecule DQ0602 confers strong susceptibility to narcolepsy but dominant protection against type 1 diabetes. The crystal structure of DQ0602 reveals the molecular features underlying these contrasting genetic properties. Structural comparisons to homologous DQ molecules with differential disease associations highlight a previously unrecognized interplay between the volume of the P6 pocket and the specificity of the P9 pocket, which implies that presentation of an expanded peptide repertoire is critical for dominant protection against type 1 diabetes. In narcolepsy, the volume of the P4 pocket appears central to the susceptibility, suggesting that the presentation of a specific peptide population plays a major role.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) may be formed in the back reaction of the amino acid-activation reaction [Zamecnik, Stephenson, Janeway & Randerath (1966) Biochem. Biophys. Res. Commun. 24, 91-98]. On the basis of a number of observations of the properties of Ap4A it has been suggested that it may have a signal function for the initiation of DNA replication in eukaryotic cells] Grummt (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 371-375]. In the present paper human platelets have been shown to contain relatively large amounts of Ap4A. The compound is apparently metabolic inactive in platelets, but it is almost quantitatively released when platelets are activated to aggregate by treatment with thrombin. The results are discussed in connection with the known growth-stimulating activity of platelets.

Genomic imprinting is a mechanism whereby only one of the two parental alleles is expressed. Loss or relaxation of genomic imprinting has been proposed as an epigenetic mechanism for oncogenesis in a variety of human tumours. Although the mechanism of imprinting is unknown, differential CpG methylation of the parental alleles has been implicated. The human insulin-like growth factor-II (IGF2) gene, which is transcribed from four promoters, P1-P4 (ref. 13), is imprinted in fetal liver but biallelic expression occurs in adult liver. Like most tissues, fetal liver uses primarily promoters P3 and P4 (ref. 17). Adult liver, however, transcribes IGF2 from promoter P1, and it has been suggested that the recruitment of P1 may be responsible for the absence of imprinting in human liver, and in choroid plexus and leptomeninges. We report here that in liver and chondrocytes, IGF2 transcripts from promoter P1 are always derived from both parental alleles, whereas transcripts from promoters P2, P3 and P4 are always from one parental allele. These findings demonstrate that imprinting and a lack of imprinting can both occur within a single gene in a single tissue, suggesting that regional imprinting factors may be important.

In the injured nervous system, myelin-associated glycoprotein (MAG) on residual myelin binds to receptors on axons, inhibits axon outgrowth, and limits functional recovery. Conflicting reports identify gangliosides (GD1a and GT1b) and glycosylphosphatidylinositol-anchored Nogo receptors (NgRs) as exclusive axonal receptors for MAG. We used enzymes and pharmacological agents to distinguish the relative roles of gangliosides and NgRs in MAG-mediated inhibition of neurite outgrowth from three nerve cell types, dorsal root ganglion neurons (DRGNs), cerebellar granule neurons (CGNs), and hippocampal neurons. Primary rat neurons were cultured on control substrata and substrata adsorbed with full-length native MAG extracted from purified myelin. The receptors responsible for MAG inhibition of neurite outgrowth varied with nerve cell type. In DRGNs, most of the MAG inhibition was via NgRs, evidenced by reversal of inhibition by phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves glycosylphosphatidylinositol anchors, or by NEP1-40, a peptide inhibitor of NgR. A smaller percentage of MAG inhibition of DRGN outgrowth was via gangliosides, evidenced by partial reversal by addition of sialidase to cleave GD1a and GT1b or by P4, an inhibitor of ganglioside biosynthesis. Combining either PI-PLC and sialidase or NEP1-40 and P4 was additive. In contrast to DRGNs, in CGNs MAG inhibition was exclusively via gangliosides, whereas inhibition of hippocampal neuron outgrowth was mostly reversed by sialidase or P4 and only modestly reversed by PI-PLC or NEP1-40 in a non-additive fashion. A soluble proteolytic fragment of native MAG, dMAG, also inhibited neurite outgrowth. In DRGNs, dMAG inhibition was exclusively NgR-dependent, whereas in CGNs it was exclusively ganglioside-dependent. An inhibitor of Rho kinase reversed MAG-mediated inhibition in all nerve cells, whereas a peptide inhibitor of the transducer p75(NTR) had cell-specific effects quantitatively similar to NgR blockers. Our data indicate that MAG inhibits axon outgrowth via two independent receptors, gangliosides and NgRs.

The purpose of this study was to examine how gonadal steroid hormones modulate basal nociception and morphine antinociception relative to regulating reproduction in the adult rat. Male and female Sprague-Dawley rats were either gonadectomized (GDX) or sham-gonadectomized (sham); GDX males were implanted subcutaneously with capsules containing testosterone (T), estradiol (E2), dihydrotestosterone (DHT), E2 and DHT, or nothing (0). GDX females received E2, T, or empty (0) capsules immediately after surgery, and vehicle or progesterone (P4) injections at 4-day intervals. Basal nociception and morphine antinociception were tested 28 days after surgery on 50 degrees C and 54 degrees C hotplate tests, and reproductive behavior and physiology were assessed shortly thereafter. There were no significant differences in baseline hotplate latencies among the male treatment groups, but morphine was significantly more potent in sham and GDX+T males than in GDX+0 males. The ability of T to increase morphine's potency was approximated by its major metabolites E2 and DHT, given together but not alone. Baseline hotplate latencies were higher in sham females tested during diestrus than in those tested during estrus. Morphine was significantly more potent in sham females tested during proestrus and diestrus than in those tested during estrus. Baseline hotplate latencies were significantly higher, and morphine was significantly less potent in GDX+E2, GDX+E2/P4 and GDX+T females than in GDX+0 females. All group differences in basal nociception and morphine antinociception observed on the 50 degrees C hotplate test were smaller and generally non-significant on the 54 degrees C hotplate test. Steroid manipulations produced the expected changes in reproductive behaviors and steroid-sensitive organs. These results demonstrate that in adult rats, gonadal steroid manipulations, that are physiologically relevant, modulate (1) basal nociception in females but not males, and (2) morphine's antinociceptive potency in both males and females.

The solute carrier (SLC) family 10 (SLC10) comprises influx transporters of bile acids, steroidal hormones, various drugs, and several other substrates. Because the seminal transporters of this family, namely, sodium/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT; SLC10A2), were primarily bile acid transporters, the term "sodium bile salt cotransporting family" was used for the SLC10 family. However, this notion became obsolete with the finding of other SLC10 members that do not transport bile acids. For example, the sodium-dependent organic anion transporter (SOAT; SLC10A6) transports primarily sulfated steroids. Moreover, NTCP was shown to also transport steroids and xenobiotics, including HMG-CoA inhibitors (statins). The SLC10 family contains four additional members, namely, P3 (SLC10A3; SLC10A3), P4 (SLC10A4; SLC10A4), P5 (SLC10A5; SLC10A5) and SLC10A7 (SLC10A7), several of which were unknown or considered hypothetical until approximately a decade ago. While their substrate specificity remains undetermined, great progress has been made towards their characterization in recent years. Explicitly, SLC10A4 may participate in vesicular storage or exocytosis of neurotransmitters or mastocyte mediators, whereas SLC10A5 and SLC10A7 may be involved in solute transport and SLC10A3 may have a role as a housekeeping protein. Finally, the newly found role of bile acids in glucose and energy homeostasis, via the TGR5 receptor, sheds new light on the clinical relevance of ASBT and NTCP. The present mini-review provides a brief summary of recent progress on members of the SLC10 family.

The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA (dsRNA). We prepared cDNA copies of the viral genome and cloned this material in plasmids that replicate in Escherichia coli and Pseudomonas phaseolicola, the natural host of phi 6. These plasmids direct the formation of viral proteins and the assembly of structures similar to viral procapsids containing proteins P1, P2, P4, and P7. We found that these particles are capable of taking up viral single-stranded RNA and synthesizing the minus strands to produce dsRNA structures. Once the dsRNA is formed, it is then used as a template for the production of viral plus strands in a reaction that resembles normal transcription. The particles were also capable of directly transcribing exogenous dsRNA. The replicase reactions were specific for phi 6 RNA, were specific for procapsids, and resulted in substantial incorporation of product dsRNA into particles. These results offer strong support to a model in which genomic packaging is done by preformed procapsids.

DNA methylation is an important component of the epigenetic control of genome functions. Understanding the regulation of the DNA Methyltransferase (dnmt1) gene expression is critical for comprehending how DNA methylation is coordinated with other critical biological processes. In this paper, we investigate the transcriptional regulatory region of the human dnmt1 gene using a combination of RACE, RNase protection analysis and CAT assays. We identified one major and three minor transcription initiation sites in vivo (P1-P4), which are regulated by independent enhancers and promoter sequences. The minimal promoter elements of P1, P2 and P4 are mapped within 256 bp upstream of their respective transcription initiation sites. P1 is nested within a CG-rich area, similar to other housekeeping genes, whereas P2-P4 are found in CG-poor areas. Three c-Jun-dependent enhancers are located downstream to P1 and upstream to P2-P4, thus providing a molecular explanation for the responsiveness of dnmt1 to oncogenic signals that are mediated by the Ras-c-Jun oncogenic signaling pathway.

BACKGROUND

The proteasome is a large multicatalytic protease complex (700 kDa) involved in a number of highly regulated processes. It has three major catalytic activities: a chymotrypsin-like activity, a trypsin-like activity and a post-glutamyl peptide hydrolyzing (PGPH) activity. To be useful as molecular probes, which could help dissect the cellular functions of the proteasome, inhibitors should be specific for the proteasome, active in vivo and selectively block only one of the three catalytic activities. To date, few inhibitors fulfill these requirements so we set out to make novel proteasome inhibitors that incorporate these characteristics.

RESULTS

A panel of amino-terminally acetylated peptide alpha',beta'-epoxyketones with leucine in P1 and various aliphatic or aromatic amino acids in P2-P4 were prepared and evaluated. Most compounds selectively inhibited the chymotrypsin-like activity, while only weakly inhibiting the trypsin-like and PGPH activities. After optimization, one inhibitor, Ac-hFLFL-epoxide, was found to be more potent and selective for the inhibition of the chymotrypsin-like activity than several previously described inhibitors. This inhibitor also exhibited strong in vivo anti-inflammatory activity.

CONCLUSIONS

Optimization of amino-terminally acetylated peptide alpha',beta'-epoxyketones furnished a potent proteasome inhibitor, Ac-hFLFL-epoxide, that has an excellent selectivity for the chymotrypsin-like activity. The inhibitor also proved to be a potent antiproliferative and anti-inflammatory agent. The strong in vivo and in vitro activities suggest that this class of proteasome inhibitors could be both molecular probes and therapeutic agents.

We have augmented our previous studies [Storey, Shears, Kirk & Michell (1984) Nature (London) 312, 374-376] on the subcellular location and properties of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) phosphatases in rat liver and human erythrocytes. We also investigate Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) metabolism by rat liver. Membrane-bound and cytosolic Ins(1,4,5)P3 phosphatases both attack the 5-phosphate. The membrane-bound enzyme is located on the inner face of the plasma membrane, and there is little or no activity associated with Golgi apparatus. Cytosolic Ins(1,4,5)P3 5-phosphatase (Mr 77,000) was separated by gel filtration from Ins(1,4)P2 (inositol 1,4-bisphosphate) and inositol 1-phosphate phosphatases (Mr 54,000). Ins(1,4,5)P3 5-phosphatase activity in hepatocytes was unaffected by treatment of the cells with insulin, vasopressin, glucagon or dibutyryl cyclic AMP. Ins(1,4,5)P3 5-phosphatase activity in cell homogenates was unaffected by changes in [Ca2+] from 0.1 to 2 microM. After centrifugation of a liver homogenate at 100,000 g, Ins(1,3,4)P3 phosphatase activity was largely confined to the supernatant. The sum of the activities in the supernatant and the pellet exceeded that in the original homogenate. When these fractions were recombined, Ins(1,3,4)P3 phosphatase activity was restored to that observed in unfractionated homogenate. Ins(1,3,4)P3 was produced from Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) and was metabolized to a novel InsP2 that was the 3,4-isomer. Ins(1,3,4)P3 phosphatase activity was not changed by 50 mM-Li+ or 0.07 mM-Ins(1,4)P2 alone, but when added together these agents inhibited Ins(1,3,4)P3 metabolism. In Li+-treated and vasopressin-stimulated hepatocytes, Ins(1,4)P2 may reach concentrations sufficient to inhibit Ins(1,3,4)P3 metabolism, with little effect on Ins(1,4,5)P3 hydrolysis.

Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNA(val), and yeast tRNA(phe) that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.

Hydroxyl radicals (.OH) can cleave the phosphodiester backbone of nucleic acids and are valuable reagents in the study of nucleic acid structure and protein-nucleic acid interactions. Irradiation of solutions by high flux "white light" X-ray beams based on bending magnet beamlines at the National Synchrotron Light Source (NSLS) yields sufficient concentrations of .OH so that quantitative nuclease protection ("footprinting") studies of DNA and RNA can be conducted with a duration of exposure in the range of 50 to 100 ms. The sensitivity of DNA and RNA to X-ray mediated .OH cleavage is equivalent. Both nucleic acids are completely protected from synchrotron X-ray induced cleavage by the presence of thiourea in the sample solution, demonstrating that cleavage is suppressed by a free radical scavenger. The utility of this time-dependent approach to footprinting is demonstrated with a synchrotron X-ray footprint of a protein-DNA complex and by a time-resolved footprinting analysis of the Mg(2+)-dependent folding of the Tetrahymena thermophilia L-21 ScaI ribozyme RNA. Equilibrium titrations reveal differences among the ribozyme domains in the cooperativity of Mg(2+)-dependent .OH protection. RNA .OH protection progress curves were obtained for several regions of the ribozyme over timescales of 30 seconds to several minutes. Progress curves ranging from>> or = 3.5 to 0.4 min-1 were obtained for the P4-P6 and P5 sub-domains and the P3-P7 domain, respectively. The .OH protection progress curves have been correlated with the available biochemical, structural and modeling data to generate a model of the ribozyme folding pathway. Rate differences observed for specific regions within domains provide evidence for steps in the folding pathway not previously observed. Synchrotron X-ray footprinting is a new approach of general applicability for the study of time-resolved structural changes of nucleic acid conformation and protein-nucleic acid complexes.