Sinorhizobium meliloti is an alpha-proteobacterium that forms agronomically important N(2)-fixing root nodules in legumes. We report here the complete sequence of the largest constituent of its genome, a 62.7% GC-rich 3,654,135-bp circular chromosome. Annotation allowed assignment of a function to 59% of the 3,341 predicted protein-coding ORFs, the rest exhibiting partial, weak, or no similarity with any known sequence. Unexpectedly, the level of reiteration within this replicon is low, with only two genes duplicated with more than 90% nucleotide sequence identity, transposon elements accounting for 2.2% of the sequence, and a few hundred short repeated palindromic motifs (RIME1, RIME2, and C) widespread over the chromosome. Three regions with a significantly lower GC content are most likely of external origin. Detailed annotation revealed that this replicon contains all housekeeping genes except two essential genes that are located on pSymB. Amino acid/peptide transport and degradation and sugar metabolism appear as two major features of the S. meliloti chromosome. The presence in this replicon of a large number of nucleotide cyclases with a peculiar structure, as well as of genes homologous to virulence determinants of animal and plant pathogens, opens perspectives in the study of this bacterium both as a free-living soil microorganism and as a plant symbiont.

The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central role in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an "opening" of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221; Spyracopoulos et al. (1997) Biochemistry 36, 12138-12146], which is in contrast to the regulatory N-domain of skeletal TnC (sTnC). This implies that the mode of interaction between cTnC and cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115-131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol. Chem. 272, 28494-28500]. The present study demonstrates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six times weaker than that between the skeletal counterparts. Thus, while Ca2+ does not cause opening, it is required for muscle regulation. The solution structure of the cNTnC.Ca2+.cTnI147-163 complex has been determined by multinuclear multidimensional NMR spectroscopy. The structure reveals an open conformation for cNTnC, similar to that of Ca2+-saturated sNTnC. The bound peptide adopts a alpha-helical conformation spanning residues 150-157. The C-terminus of the peptide is unstructured. The open conformation for Ca2+-saturated cNTnC in the presence of cTnI (residues 147-163) accommodates hydrophobic interactions between side chains of the peptide and side chains at the interface of A and B helices of cNTnC. Thus the mechanistic differences between the regulation of cardiac and skeletal muscle contraction can be understood in terms of different thermodynamics and kinetics equilibria between essentially the same structure states.

We have studied the effects of 3 wk of continuous subcutaneous insulin infusion (CSII) on endogenous insulin secretion and action in a group of 14 type II diabetic subjects with a mean (+/-SEM) fasting glucose level of 286 +/- 17 mg/dl. Normal basal and postprandial glucose levels were achieved during insulin therapy at the expense of marked peripheral hyperinsulinemia. During the week of posttreatment evaluation, the subjects maintained a mean fasting glucose level of 155 +/- 11 mg/dl off insulin therapy, indicating a persistent improvement in carbohydrate homeostasis. Adipocyte insulin binding and in vivo insulin dose-response curves for glucose disposal using the euglycemic clamp technique were measured before and after therapy to assess the effect on receptor and postreceptor insulin action. Adipocyte insulin binding did not change. The insulin dose-response curve for overall glucose disposal remained right-shifted compared with age-matched controls, but the mean maximal glucose disposal rate increased by 74% from 160 +/- 14 to 278 +/- 18 mg/m2/min (P less than 0.0005). The effect of insulin treatment on basal hepatic glucose output was also assessed; the mean rate was initially elevated at 159 +/- 8 mg/m2/min but fell to 90 +/- 5 mg/m2/min in the posttreatment period (P less than 0.001), a value similar to that in control subjects. Endogenous insulin secretion was assessed in detail and found to be improved after exogenous insulin therapy. Mean 24-h integrated serum insulin and C-peptide concentrations were increased from 21,377 +/- 2766 to 35,584 +/- 4549 microU/ml/min (P less than 0.01) and from 1653 +/- 215 to 2112 +/- 188 pmol/ml/min (P less than 0.05), respectively, despite lower glycemia. Second-phase insulin response to an intravenous (i.v.) glucose challenge was enhanced from 170 +/- 53 to 1022 +/- 376 microU/ml/min (P less than 0.025), although first-phase response remained minimal. Finally, the mean insulin and C-peptide responses to an i.v. glucagon pulse were unchanged in the posttreatment period, but when glucose levels were increased by exogenous glucose infusion to approximate the levels observed before therapy and the glucagon pulse repeated, responses were markedly enhanced. Simple and multivariate correlation analysis showed that only measures of basal hepatic glucose output and the magnitude of the postbinding defect in the untreated state could be related to the respective fasting glucose levels in individual subjects.(ABSTRACT TRUNCATED AT 400 WORDS)

The capsid (CA) and nucleocapsid domains of the human immunodeficiency virus type 1 Gag polyprotein are separated by the p2 spacer peptide, which is essential for virus replication. Previous studies have revealed that p2 has an important role in virus morphogenesis. In this paper, we show that a crucial assembly determinant maps to the highly conserved N terminus of p2, which is predicted to form part of an alpha-helix that begins in CA. A mutational analysis indicates that the ability of the N terminus of p2 to adopt an alpha-helical structure is essential for its function during virus assembly. To prevent CA-p2 processing, it was necessary to mutate both the CA-p2 cleavage site and an internal cleavage site within p2. Virions produced by the double mutant lacked a conical core shell and instead contained a thin electron-dense shell about 10 nm underneath the virion membrane. These results suggest that p2 is transiently required for proper assembly, but needs to be removed from the C terminus of CA to weaken CA-CA interactions and allow the rearrangement of the virion core shell during virus maturation.

A synthetic peptide (Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr), homologous to the amino terminus of a protein purified from cerebrovascular amyloid (beta protein), induced antibodies in BALB/c mice that were used immunohistochemically to stain not only amyloid-laden cerebral vessels but neuritic plaques as well. These findings suggest that the amyloid in neuritic plaques shares antigenic determinants with beta protein of cerebral vessels. Since the amino acid compositions of plaque amyloid and cerebrovascular amyloid are similar, it is likely that plaque amyloid also consists of beta protein. This possibility suggests a model for the pathogenesis of Alzheimer disease involving beta protein.

Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Kinetochore assembly depends on specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). We have defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A and H2B. We further found that the more broadly conserved CENP-C motif uses the same mechanism for CENP-A nucleosome recognition. Our findings reveal a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through hydrophobic interactions facilitated by nucleosome docking.

The dnaX gene (previously called dnaZX) of Escherichia coli has only one open reading frame for a 71-kDa polypeptide from which two distinct DNA polymerase III holoenzyme subunits, tau (71 kDa) and gamma (47 kDa), are produced. To determine how the gamma subunit is generated, we examined the influence of mutations in the dnaX gene on the pattern of tau and gamma production in overproducing cells. Important structural elements in dnaX mRNA include a stretch of six adenines (nucleotides 1425-1430), a stable hairpin structure (nucleotides 1437-1466), and a UGA stop codon in a -1 frame (nucleotides 1434-1436) between the stretch of adenines and the hairpin structure. Disruption of this stop codon generates a slightly larger gamma subunit, indicative of the use of a -1 stop codon farther downstream (nucleotides 1470-1472). These results suggest that a -1 frameshift during translation allows the use of this UGA codon to terminate translation of the gamma polypeptide. The amino acid composition, sequence, and mass spectra of a C-terminal peptide from mild digestion of the purified gamma protein with endoproteinase Lys-C confirms that this frameshift occurs at either of the two lysine codons in the region of the adenine stretch. Remarkable features of this frameshifting are its high frequency (i.e., about 80% in an overproducing cell) and the striking structural similarity to the frameshifting signal responsible for expression of the pol and pro genes in many retroviruses.

Rapamycin/interleukin-2 (IL-2) combination treatment of NOD mice effectively treats autoimmune diabetes. We performed a phase 1 clinical trial to test the safety and immunologic effects of rapamycin/IL-2 combination therapy in type 1 diabetic (T1D) patients. Nine T1D subjects were treated with 2-4 mg/day rapamycin orally for 3 months and 4.5 × 10(6) IU IL-2 s.c. three times per week for 1 month. β-Cell function was monitored by measuring C-peptide. Immunologic changes were monitored using flow cytometry and serum analyses. Regulatory T cells (Tregs) increased within the first month of therapy, yet clinical and metabolic data demonstrated a transient worsening in all subjects. The increase in Tregs was transient, paralleling IL-2 treatment, whereas the response of Tregs to IL-2, as measured by STAT5 phosphorylation, increased and persisted after treatment. No differences were observed in effector T-cell subset frequencies, but an increase in natural killer cells and eosinophils occurred with IL-2 therapy. Rapamycin/IL-2 therapy, as given in this phase 1 study, resulted in transient β-cell dysfunction despite an increase in Tregs. Such results highlight the difficulties in translating therapies to the clinic and emphasize the importance of broadly interrogating the immune system to evaluate the effects of therapy.

A generic statistical mechanical model is presented for the self-assembly of chiral rod-like units, such as beta-sheet-forming peptides, into helical tapes, which with increasing concentration associate into twisted ribbons (double tapes), fibrils (twisted stacks of ribbons), and fibers (entwined fibrils). The finite fibril width and helicity is shown to stem from a competition between the free energy gain from attraction between ribbons and the penalty because of elastic distortion of the intrinsically twisted ribbons on incorporation into a growing fibril. Fibers are stabilized similarly. The behavior of two rationally designed 11-aa residue peptides, P(11)-I and P(11)-II, is illustrative of the proposed scheme. P(11)-I and P(11)-II are designed to adopt the beta-strand conformation and to self-assemble in one dimension to form antiparallel beta-sheet tapes, ribbons, fibrils, and fibers in well-defined solution conditions. The energetic parameters governing self-assembly have been estimated from the experimental data using the model. The 8-nm-wide fibrils consist of eight tapes, are extremely robust (scission energy approximately 200 k(B)T), and sufficiently rigid (persistence length l(fibril) approximately 20-70 microm) to form nematic solutions at peptide concentration c approximately 0.9 mM (volume fraction approximately 0.0009 vol/vol), which convert to self-supporting nematic gels at c>> 4 mM. More generally, these observations provide a new insight into the generic self-assembling properties of beta-sheet-forming peptides and shed new light on the factors governing the structures and stability of pathological amyloid fibrils in vivo. The model also provides a prescription of routes to novel macromolecules based on a variety of self-assembling chiral units, and protocols for extraction of the associated energy changes.

BACKGROUND

Diatoms are unicellular algae responsible for approximately 20% of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids.

RESULTS

The whole genome sequence of the diatom Phaeodactylum tricornutum has recently been completed. We identified and annotated genes for enzymes involved in carbohydrate pathways based on extensive EST support and comparison to the whole genome sequence of a second diatom, Thalassiosira pseudonana. Protein localization to mitochondria was predicted based on identified similarities to mitochondrial localization motifs in other eukaryotes, whereas protein localization to plastids was based on the presence of signal peptide motifs in combination with plastid localization motifs previously shown to be required in diatoms. We identified genes potentially involved in a CCO(2) fixation between the two diatoms. We also identified genes encoding enzymes involved in photorespiration with one interesting exception: glycerate kinase was not found in either P. tricornutum or T. pseudonana. Various Calvin cycle enzymes were found in up to five different isoforms, distributed between plastids, mitochondria and the cytosol. Diatoms store energy either as lipids or as chrysolaminaran (a beta-1,3-glucan) outside of the plastids. We identified various beta-glucanases and large membrane-bound glucan synthases. Interestingly most of the glucanases appear to contain C-terminal anchor domains that may attach the enzymes to membranes.

CONCLUSIONS

Here we present a detailed synthesis of carbohydrate metabolism in diatoms based on the genome sequences of Thalassiosira pseudonana and Phaeodactylum tricornutum. This model provides novel insights into acquisition of dissolved inorganic carbon and primary metabolic pathways of carbon in two different diatoms, which is of significance for an improved understanding of global carbon cycles.

We have isolated the full-length cDNA of a novel human serine/threonine protein kinase gene. The deduced protein sequence shows strong homology to conserved domains of members of the protein kinase C (PKC) subfamily. Homologies reside in the duplex zinc-finger-like cysteine-rich motif and in the protein kinase domain. The lack of the CCa(2+)-dependent PKCs and the presence of a unique NH2-terminal sequence with a potential signal peptide and a transmembrane domain suggest that PKC mu is a novel member of the subgroup of atypical PKCs. An open reading frame coding for 912 amino acids directs an in vitro translation product with an apparent M(r) of 115,000. In vitro phorbol ester binding studies and kinase assays with lysates of cells overexpressing PKC mu showed phorbol ester-independent kinase activity, autophosphorylation, and, in normal rat kidney (NRK) cells, predominant phosphorylation of a 30-kDa protein at serine residues. Southern analysis revealed that PKC mu is a single copy gene located on human chromosome 21. There is constitutive low level expression of the human PKC mu gene in normal tissues with a single transcript of 3.8 kilobases and elevated expression levels in selected tumor cell lines. These data suggest a role of PKC mu in signal transduction pathways related to growth control.

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

The amino acid sequences of major outer membrane proteins (MOMPs) from Chlamydia trachomatis serovars A, B, C, L1, and L2 are predominantly conserved but have four variable domains (VDs) in which major neutralizing and serotyping antigenic determinants are located. Because these MOMP VDs are primarily responsible for antigenic differences between serovars and are associated with important immunological and biological properties, we undertook studies focused on defining these sequences within the MOMPs of all 15 C. trachomatis serovars. We used oligonucleotide primer extension sequencing of MOMP mRNA to determine the nucleotide and deduced amino acid sequences of the four MOMP VDs of the 15 C. trachomatis serovars. Comparative amino acid sequence homologies of all four domains separated the serovars into three groups: group 1, serovars B, Ba, D, E, L1, and L2; group 2, serovars G and F; and group 3, serovars A, C, H, I, J, K, and L3. Hydrophilicity and charge values for each domain were determined. The MOMP VDs of given serovars with the greatest total hydrophilicity and charge values were found to be the location of antigenic determinants recognized by MOMP-specific monoclonal antibodies. These findings should be useful for predicting MOMP antigenic determinants and testing the antigenic properties of these VDs by using synthetic peptides corresponding to each MOMP VD. The potential usefulness of the VD sequence information is discussed in relation to the development of defined synthetic peptides and oligonucleotides that may be used to develop new serological and diagnostic assays for C. trachomatis infections.

OBJECTIVE

Glucagon-like-peptide-1 (GLP-1) is strongly insulinotropic in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas glucose-dependent insulinotropic polypeptide (GIP) is less effective. Our investigation evaluated "early" (protocol 1) - and "late phase" (protocol 2) insulin and C-peptide responses to GLP-1 and GIP stimulation in patients with Type II diabetes.

METHODS

Protocol 1: eight Type II diabetic patients and eight matched healthy subjects received i.v. bolus injections of GLP-1(2.5 nmol) or GIP(7.5 nmol) concomitant with an increase of plasma glucose to 15 mmol/l. Protocol 2: eight Type II diabetic patients underwent a hyperglycaemic clamp (15 mmol/l) with infusion (per kg body weight/min) of either: 1 pmol GLP-1 (7-36) amide (n=8), 4 pmol GIP (n=8), 16 pmol GIP (n=4) or no incretin hormone (n=5). For comparison, six matched healthy subjects were examined.

RESULTS

Protocol 1: Type II diabetic patients were characterised by a decreased "early phase" response to both stimuli, but their relative response to GIP versus GLP-1 stimulation was exactly the same as in healthy subjects [insulin (C-peptide): patients 59+/-9% (74+/-6%) and healthy subjects 62+/-5% (71+/-9%)]. Protocol 2, "Early phase" (0-20 min) insulin response to glucose was delayed and reduced in the patients, but enhanced slightly and similarly by GIP and GLP-1. GLP-1 augmented the "late phase" (20-120 min) insulin secretion to levels similar to those observed in healthy subjects. In contrast, the "late phase" responses to both doses of GIP were not different from those obtained with glucose alone. Accordingly, glucose infusion rates required to maintain the hyperglycaemic clamp in the "late phase" period (20-120 min) were similar with glucose alone and glucose plus GIP, whereas a doubling of the infusion rate was required during GLP-1 stimulation.

CONCLUSIONS

Lack of GIP amplification of the late phase insulin response to glucose, which contrasts markedly to the normalising effect of GLP-1, could be a key defect in insulin secretion in Type II diabetic patients.

Besides their microbicidal functions, human beta-defensins (hBD) and LL-37 activate different immune and inflammatory cells, and their expression is enhanced in inflamed skin and cutaneous wound sites. To protect against pathogens, the skin produces antimicrobial peptides including hBDs and LL-37. Therefore, the aim of our study was to investigate whether hBDs participate in cutaneous inflammation and wound healing by inducing keratinocyte migration, proliferation, and production of proinflammatory cytokines/chemokines. We found that hBD-2, -3, and -4 but not hBD-1 stimulated human keratinocytes to increase their gene expression and protein production of IL-6, IL-10, IP-10, monocyte chemoattractant protein-1, macrophage inflammatory protein-3alpha, and RANTES. This stimulatory effect was markedly suppressed by pertussis toxin and U-73122, inhibitors for G protein and phospholipase C, respectively. We also demonstrated that hBDs elicited intracellular Ca2+ mobilization, and increased keratinocyte migration, and proliferation. In addition, these peptides induced phosphorylation of EGFR, signal transducer and activator of transcription (STAT)1, and STAT3, which are intracellular signaling molecules involved in keratinocyte migration and proliferation. In our study, inhibition of these molecules significantly reduced hBD-mediated keratinocyte migration and proliferation. In conclusion, this study provides evidence that human antimicrobial peptides may be involved in skin immunity through stimulating cytokine/chemokine production, and participate in wound healing by promoting keratinocyte migration and proliferation.

The collagen prolyl 4-hydroxylases (P4Hs), enzymes residing within the endoplasmic reticulum, have a central role in the biosynthesis of collagens. In addition, cytoplasmic P4Hs play a critical role in the regulation of the hypoxia-inducible transcription factor HIFalpha. Collagen and HIF P4Hs constitute enzyme families as several isoenzymes have been identified. Two catalytic alpha subunit isoforms have been cloned and characterized for collagen P4Hs from vertebrates, both of them assembling into alpha(2)beta(2) P4H tetramers in which protein disulfide isomerase (PDI) acts as the beta subunit. The catalytic properties of the two isoenzymes are very similar, but distinct differences are found in the binding properties of peptide substrates and inhibitors, and major differences are seen in the expression patterns of the isoenzymes. The nematode Caenorhabditis elegans has five P4H alpha subunit isoforms, PHY1-PHY5. The C. elegans PHY1 and PHY2, together with PDI, are expressed in the collagen synthesizing hypodermal cells and three P4H forms are assembled from them, a PHY-1/PHY-2/PDI(2) mixed tetramer and PHY-1/PDI and PHY-2/PDI dimers. The mixed tetramer is the main P4H form in wild-type C. elegans. PHY-3 is much shorter than PHY-1 and PHY-2, has a unique expression pattern, and is most likely involved in the synthesis of collagens in early embryos. The genome of Drosophila melanogaster contains approximately 20 P4H alpha subunit-related genes, and that of Arabidopsis thaliana six. One A. thaliana P4H has been cloned and shown to be a soluble monomer with several unexpected properties. It effectively hydroxylates poly(L-proline), (Pro-Pro-Gly)(10) and many other proline-containing peptides.

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development.

Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup alpha-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. Alpha-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.

Stress induces the release of the peptide corticotropin-releasing factor (CRF) into the ventral tegmental area (VTA), and also increases dopamine levels in brain regions receiving dense VTA input. Therefore, stress may activate the mesolimbic dopamine system in part through the actions of CRF in the VTA. Here, we explored the mechanism by which CRF affects VTA dopamine neuron firing. Using patch-clamp recordings from brain slices we first determined that the presence of I(h) is an excellent predictor of dopamine content in mice. We next showed that CRF dose-dependently increased VTA dopamine neuron firing, which was prevented by antagonism of the CRF receptor-1 (CRF-R1), and was mimicked by CRF-R1 agonists. Inhibition of the phospholipase C (PLC)-protein kinase C (PKC) signalling pathway, but not the cAMP-protein kinase A (PKA) signalling pathway, prevented the increase in dopamine neuron firing by CRF. Furthermore, the effect of CRF on VTA dopamine neurons was not attenuated by blockade of I(A), I(K(Ca)) or I(Kir), but was completely eliminated by inhibition of I(h). Although cAMP-dependent modulation of I(h) through changes in the voltage dependence of activation is well established, we surprisingly found that CRF, through a PKC-dependent mechanism, enhanced I(h) independent of changes in the voltage dependence of activation. Thus, our results demonstrated that CRF acted on the CRF-R1 to stimulate the PLC-PKC signalling pathway, which in turn enhanced I(h) to increase VTA dopamine neuron firing. These findings provide a cellular mechanism of the interaction between CRF and dopamine, which can be involved in promoting the avoidance of threatening stimuli, the pursuit of appetitive behaviours, as well as various psychiatric conditions.

Natural killer (NK) cells preferentially lyse targets that express reduced levels of major histocompatibility complex (MHC) class I proteins. To date, the only known mouse NK receptors for MHC class I belong to the Ly49 family of C-type lectin homodimers. Here, we report the cloning of mouse NKG2A, and demonstrate it forms an additional and distinct class I receptor, a CD94/NKG2A heterodimer. Using soluble tetramers of the nonclassical class I molecule Qa-1(b), we provide direct evidence that CD94/NKG2A recognizes Qa-1(b). We further demonstrate that NK recognition of Qa-1(b) results in the inhibition of target cell lysis. Inhibition appears to depend on the presence of Qdm, a Qa-1(b)-binding peptide derived from the signal sequences of some classical class I molecules. Mouse NKG2A maps adjacent to CD94 in the heart of the NK complex on mouse chromosome six, one of a small cluster of NKG2-like genes. Our findings suggest that mouse NK cells, like their human counterparts, use multiple mechanisms to survey class I expression on target cells.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.

c-Jun, a major component of the inducible transcription factor AP-1, is a phosphoprotein. In nonstimulated fibroblasts and epithelial cells, c-Jun is phosphorylated on a cluster of two to three sites abutting its DNA-binding domain. Phosphorylation of these sites inhibits DNA binding, and their dephosphorylation correlates with increased AP-1 activity. We show that two of these sites, Thr-231 and Ser-249, are phosphorylated by casein kinase II (CKII). Substitution of the third site, Ser-243, by Phe interferes with phosphorylation of the inhibitory sites in vivo and by purified CKII in vitro. Microinjection into living cells of synthetic peptides that are specific competitive substrates or inhibitors of CKII results in induction of AP-1 activity and c-Jun expression. Microinjection of CKII suppresses induction of AP-1 by either phorbol ester or an inhibitory peptide. These results suggest that one of the roles of CKII, a major nuclear protein kinase with no known functions, is to attenuate AP-1 activity through phosphorylation of c-Jun.

OBJECTIVE

This 24-week trial assessed the efficacy and safety of saxagliptin as add-on therapy in patients with type 2 diabetes with inadequate glycemic control with metformin alone.

METHODS

This was a randomized, double-blind, placebo-controlled study of saxagliptin (2.5, 5, or 10 mg once daily) or placebo plus a stable dose of metformin (1,500-2,500 mg) in 743 patients (A1C>> or =7.0 and < or =10.0%). Efficacy analyses were performed using an ANCOVA model using last observation carried forward methodology on primary (A1C) and secondary (fasting plasma glucose [FPG] and postprandial glucose [PPG] area under the curve [AUC]) end points.

RESULTS

Saxagliptin (2.5, 5, and 10 mg) plus metformin demonstrated statistically significant adjusted mean decreases from baseline to week 24 versus placebo in A1C (-0.59, -0.69, and -0.58 vs. +0.13%; all P < 0.0001), FPG (-14.31, -22.03, and -20.50 vs. +1.24 mg/dl; all P < 0.0001), and PPG AUC (-8,891, -9,586, and -8,137 vs. -3,291 mg . min/dl; all P < 0.0001). More than twice as many patients achieved A1C <7.0% with 2.5, 5, and 10 mg saxagliptin versus placebo (37, 44, and 44 vs. 17%; all P < 0.0001). beta-Cell function and postprandial C-peptide, insulin, and glucagon AUCs improved in all saxagliptin treatment groups at week 24. Incidence of hypoglycemic adverse events and weight reductions were similar to those with placebo.

CONCLUSIONS

Saxagliptin once daily added to metformin therapy was generally well tolerated and led to statistically significant improvements in glycemic indexes versus placebo added to metformin in patients with type 2 diabetes inadequately controlled with metformin alone.

The loss of T helper cell (TH) function in asymptomatic HIV type 1-infected individuals occurs before the decline in CD4+ T cells. At least part of the loss in TH function results from changes in immunoregulatory cytokine profiles. To investigate the role of IL-10 in such dysregulation, we tested whether: (a) expression of IL-10-specific mRNA would be upregulated in PBMC from asymptomatic, HIV-infected (HIV+) individuals; (b) PBMC from these same individuals would produce increased levels of IL-10 when stimulated in vitro with phytohemagglutinin; and (c) defective antigen-specific TH function could be restored by anti-IL-10 antibody. We observed that IL-10-specific mRNA was marginally upregulated, and increased levels of IL-10 were produced by PBMC from HIV+ individuals compared with PBMC from uninfected individuals. Those individuals whose TH function was more severely compromised produced higher levels of IL-10. Additionally, defective antigen-specific TH function in vitro could be reversed by anti-IL-10 antibody, including the response to HIV envelope synthetic peptides. Furthermore, the antigen-specific TH responses of HIV-uninfected PBMC could be reduced with IL-10, a process reversed by anti-IL-10. These results confirm that the early loss of TH function in HIV+ individuals is due at least in part to cytokine-induced immune dysregulation, and support the hypothesis of a switch from a predominant type 1 state to a predominant type 2 condition in HIV infection.