The covalent anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria occurs by a universal mechanism requiring sortases, extracellular transpeptidases that are positioned in the plasma membrane. Surface protein precursors are first initiated into the secretory pathway of Gram-positive bacteria via N-terminal signal peptides. C-terminal sorting signals of surface proteins, bearing an LPXTG motif or other recognition sequences, provide for sortase-mediated cleavage and acyl enzyme formation, a thioester linkage between the active site cysteine residue of sortase and the C-terminal carboxyl group of cleaved surface proteins. During cell wall anchoring, sortase acyl enzymes are resolved by the nucleophilic attack of peptidoglycan substrates, resulting in amide bond formation between the C-terminal end of surface proteins and peptidoglycan cross-bridges within the bacterial cell wall envelope. The genomes of Gram-positive bacteria encode multiple sortase genes. Recent evidence suggests that sortase enzymes catalyze protein anchoring reactions of multiple different substrate classes with different sorting signal motif sequences, protein linkage to unique cell wall anchor structures as well as protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria.

Group I metabotropic glutamate receptors (mGluRs) increase cellular levels of inositol-1,4,5-triphosphate (IP3) and thereby trigger intracellular Ca2+ release. Also, group I mGluRs are organized with members of Homer scaffold proteins into multiprotein complexes involved in postreceptor signaling. In this study, we investigated the relative importance of the IP3/Ca2+ signaling and novel Homer proteins in group I mGluR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in cultured rat striatal neurons. We found that selective activation of mGluR5, but not mGluR1, increased ERK1/2 phosphorylation. Whereas the IP3/Ca2+ cascade transmits a small portion of signals from mGluR5 to ERK1/2, the member of Homer family Homer1b/c forms a central signaling pathway linking mGluR5 to ERK1/2 in a Ca2+-independent manner. This was demonstrated by the findings that the mGluR5-mediated ERK1/2 phosphorylation was mostly reduced by a cell-permeable Tat-fusion peptide that selectively disrupted the interaction of mGluR5 with the Homer1b/c and by small interfering RNAs that selectively knocked down cellular levels of Homer1b/c proteins. Furthermore, ERK1/2, when only coactivated by both IP3/Ca2+- and Homer1b/c-dependent pathways, showed the ability to phosphorylate two transcription factors, Elk-1 and cAMP response element-binding protein, and thereby facilitated c-Fos expression. Together, we have identified two coordinated signaling pathways (a conventional IP3/Ca2+ vs a novel Homer pathway) that differentially mediate the mGluR5-ERK coupling in neurons. Both the Ca2+-dependent and -independent pathways are corequired to activate ERK1/2 to a level sufficient to achieve the mGluR5-dependent synapse-to-nucleus communication imperative for the transcriptional regulation.

The simultaneous assessment of quantitative indexes of insulin secretion and action in a single individual is important when quantifying their relative role in the evolution of glucose tolerance in different physiopathological states. Available methods quantify these indexes in relatively nonphysiological conditions, e.g., during glucose clamps or intravenous glucose tolerance tests. Here, we present a method based on a physiological test applicable to large-scale genetic and epidemiologic studies-the oral glucose tolerance test (OGTT). Plasma C-peptide, insulin, and glucose data from a frequently sampled OGTT with 22 samples throughout 300 min (FSOGTT300-22) were analyzed in 11 subjects with various degrees of glucose tolerance. In each individual, two indexes of pancreatic sensitivity to glucose (phis [10(9) min(-1)] and phid [10(9)]) and the insulin sensitivity index (SI) (10(5) dl/kg per min per pmol/l) were estimated by using the minimal model of C-peptide secretion and kinetics originally proposed for intravenous graded glucose infusion and the minimal model approach recently proposed for meal/OGTTs. The indexes obtained from FSOGTT300-22 were used as a reference for internal validation of OGTT protocols with reduced sampling schedules. Our results show that 11 samples in a 300-min period (OGTT300-11) is the test of choice because the indexes it provides (phis = 36 +/- 3 [means +/- SE]; phid = 710 +/- 111; SI = 10.2 +/- 2.4) show excellent correlation and are not statistically different from those of FSOGTT300-22 (phis = 33 +/- 3; phid = 715 +/- 120; SI = 10.1 +/- 2.3). In conclusion, OGTT300-11, interpreted with C-peptide and glucose minimal models, provides a quantitative description of beta-cell function and insulin sensitivity in a single individual while preserving the important clinical classification of glucose tolerance provided by the standard 120-min OGTT.

The initiation and progression of adult-onset periodontitis has been associated with infection of the gingival sulcus by Porphyromonas gingivalis. This organism utilizes a multitude of virulence factors to evade host defenses as it establishes itself as one of the predominant pathogens in periodontal pockets. A feature common to many other oral pathogens is the production of ammonia due to its protective effect during acidic cleansing cycles in the mouth. Additionally, ammonia production by P. gingivalis has been proposed as a virulence factor due to its negative effects on neutrophil function. In this study, we describe the first purification of a peptidylarginine deiminase (PAD) from a prokaryote. PAD exhibits biochemical characteristics and properties that suggest that it may be a virulence agent. PAD deiminates the guanidino group of carboxyl-terminal arginine residues on a variety of peptides, including the vasoregulatory peptide-hormone bradykinin, to yield ammonia and a citrulline residue. The soluble protein has an apparent mass of 46 kDa, while the DNA sequence predicts a full-length protein of 61.7 kDa. PAD is optimally active at 55 degrees C, stable at low pH, and shows the greatest activity above pH 9.0. Interestingly, in the presence of stabilizing factors, PAD is resistant to limited proteolysis and retains significant activity after short-term boiling. We propose that PAD, acting in concert with arginine-specific proteinases from P. gingivalis, promotes the growth of the pathogen in the periodontal pocket, initially by enhancing its survivability and then by assisting the organism in its circumvention of host humoral defenses.

The candidate division Korarchaeota comprises a group of uncultivated microorganisms that, by their small subunit rRNA phylogeny, may have diverged early from the major archaeal phyla Crenarchaeota and Euryarchaeota. Here, we report the initial characterization of a member of the Korarchaeota with the proposed name, "Candidatus Korarchaeum cryptofilum," which exhibits an ultrathin filamentous morphology. To investigate possible ancestral relationships between deep-branching Korarchaeota and other phyla, we used whole-genome shotgun sequencing to construct a complete composite korarchaeal genome from enriched cells. The genome was assembled into a single contig 1.59 Mb in length with a G + C content of 49%. Of the 1,617 predicted protein-coding genes, 1,382 (85%) could be assigned to a revised set of archaeal Clusters of Orthologous Groups (COGs). The predicted gene functions suggest that the organism relies on a simple mode of peptide fermentation for carbon and energy and lacks the ability to synthesize de novo purines, CoA, and several other cofactors. Phylogenetic analyses based on conserved single genes and concatenated protein sequences positioned the korarchaeote as a deep archaeal lineage with an apparent affinity to the Crenarchaeota. However, the predicted gene content revealed that several conserved cellular systems, such as cell division, DNA replication, and tRNA maturation, resemble the counterparts in the Euryarchaeota. In light of the known composition of archaeal genomes, the Korarchaeota might have retained a set of cellular features that represents the ancestral archaeal form.

The development of vaccines and other immunotherapies has been complicated by heterogeneous antigen display and the use of incompletely defined immune adjuvants with complex mechanisms of action. We have observed strong antibody responses in mice without the coadministration of any additional adjuvant by noncovalently assembling a T and B cell epitope peptide into nanofibers using a short C-terminal peptide extension. Self-assembling peptides have been explored recently as scaffolds for tissue engineering and regenerative medicine, but our results indicate that these materials may also be useful as chemically defined adjuvants. In physiological conditions, these peptides self-assembled into long, unbranched fibrils that displayed the epitope on their surfaces. IgG1, IgG2a, and IgG3 were raised against epitope-bearing fibrils in levels similar to the epitope peptide delivered in complete Freund's adjuvant (CFA), and IgM production was even greater for the self-assembled epitope. This response was dependent on self-assembly, and the self-assembling sequence was not immunogenic by itself, even when delivered in CFA. Undetectable levels of interferon-gamma, IL-2, and IL-4 in cultures of peptide-challenged splenocytes from immunized mice suggested that the antibody responses did not involve significant T cell help.

BACKGROUND

Sibutramine is a tertiary amine that has been shown to induce dose-dependent weight loss and to enhance the effects of a low-calorie diet for up to a year. We did a randomised, double-blind trial to assess the usefulness of sibutramine in maintaining substantial weight loss over 2 years.

METHODS

Eight European centres recruited 605 obese patients (body-mass index 30-45 kg/m2) for a 6-month period of weight loss with sibutramine (10 mg/day) and an individualised 600 kcal/day deficit programme based on measured resting metabolic rates. 467 (77%) patients with more than 5% weight loss were then randomly assigned 10 mg/day sibutramine (n=352) or placebo (n=115) for a further 18 months. Sibutramine was increased up to 20 mg/day if weight regain occurred. The primary outcome measure was the number of patients at year 2 maintaining at least 80% of the weight lost between baseline and month 6. Secondary outcomes included changes in uric acid concentrations and glycaemic and lipid variables. Analysis was by intention to treat.

RESULTS

148 (42%) individuals in the sibutramine group and 58 (50%) in the placebo group dropped out. Of the 204 sibutramine-treated individuals who completed the trial, 89 (43%) maintained 80% or more of their original weight loss, compared with nine (16%) of the 57 individuals in the placebo group (odds ratio 4.64, p<0.001). Patients had substantial decreases over the first 6 months with respect to triglycerides, VLDL cholesterol, insulin, C peptide, and uric acid; these changes were sustained in the sibutramine group but not the placebo group. HDL cholesterol concentrations rose substantially in the second year: overall increases were 20.7% (sibutramine) and 11.7% (placebo, p<0.001). 20 (3%) patients were withdrawn because of increases in blood pressure; in the sibutramine group, systolic blood pressure rose from baseline to 2 years by 0.1 mm Hg (SD 12.9), diastolic blood pressure by 2.3 mm Hg (9.4), and pulse rate by 4.1 beats/min (11.9).

CONCLUSIONS

This individualised management programme achieved weight loss in 77% of obese patients and sustained weight loss in most patients continuing therapy for 2 years. Changes in concentrations of HDL cholesterol, VLDL cholesterol, and triglyceride, but not LDL cholesterol, exceed those expected either from weight loss alone or when induced by other selective therapies for low concentrations of HDL cholesterol relating to coronary heart disease.

BACKGROUND

Impaired activity of natural killer (NK) cells has been proposed as a mechanism contributing to viral persistence in hepatitis C virus (HCV) infection. As the function of NK cells is primarily regulated by NK cell receptors (NKR), we analysed whether decreased NK cell function in hepatitis C may be related to dysregulated NKR expression.

METHODS

Expression of NK cell was analysed by flow cytometry on lymphocytes from HCV(+) subjects (n = 30), patients who became HCV(-) after antiviral therapy (n = 10), healthy individuals (n = 10), and hepatitis B virus (HBV) infected patients (n = 9). Cytolytic function of lymphocytes was studied in a redirected lysis assay and in a standard 51chromium release cytotoxicity assay, respectively.

RESULTS

In patients with chronic hepatitis C, we found a significantly reduced proportion of NKp46 and NKp30 expressing NK cells compared with healthy and HBV infected subjects. Low expression of natural cytotoxicity receptor (NCR) was also confirmed in in vitro activated NK cell populations derived from HCV patients compared with uninfected donors. In contrast, patients who cleared HCV under antiviral therapy showed normal expression of NKp44, NKp30, and NKp46. Reduced NCR expression in chronic hepatitis C was associated with a parallel decrease in NCR mediated target cell killing. Furthermore, we found a significantly increased proportion of NKG2A expressing NK cells and CD8+ T cells in HCV positive patients, resulting in a reduced cytolytic activity against cells incubated with the HLA-E stabilising peptide HCV core35-44.

CONCLUSIONS

The present study indicates that defective expression of NKR represents a novel mechanism contributing to impaired function of NK cells and CD8+ T cells in chronic hepatitis C.

We have shown previously that the betagamma subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gbetagamma is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gbetagamma directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gbetagamma-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gbetagamma with PKD regulates the dynamics of Golgi membranes and protein secretion.

The cathelicidin peptides comprise one of several families of antimicrobial peptides that are found in neutrophils and epithelia as components of the early host defenses of mammals against infection. All cathelicidin family members are synthesized and stored in cells as two-domain proteins. These are split on demand to produce a cathelin protein and an antimicrobial peptide. Accumulating evidence indicates that both the cathelin portion and the C-terminal peptide exert biological activities connected with host protection. This review presents an overview of the structure and biology of cathelicidins and discusses recent progress in cathelicidin research with emphasis on the functional properties and role in host defense of the human cathelicidin hCAP18/LL-37. Although investigators initially concentrated their attention on antibiotic activity, it is becoming clear now that LL-37 is a multifunctional molecule that may mediate various host responses, and thus represents an essential component of the innate immune system in humans.

NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes.

Integrin alpha(v)beta(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive imaging of alpha(v)beta(3) expression and targeted radionuclide therapy. In this study, we developed (64)Cu-labeled multimeric RGD peptides, E{E[c(RGDyK)](2)}(2) (RGD tetramer) and E(E{E[c(RGDyK)](2)}(2))(2) (RGD octamer), for PET imaging of tumor integrin alpha(v)beta(3) expression.

METHODS

Both RGD tetramer and RGD octamer were synthesized with glutamate as the linker. After conjugation with 1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), the peptides were labeled with (64)Cu for biodistribution and small-animal PET imaging studies (U87MG human glioblastoma xenograft model and c-neu oncomouse model). A cell adhesion assay, a cell-binding assay, receptor blocking experiments, and immunohistochemistry were also performed to evaluate the alpha(v)beta(3)-binding affinity/specificity of the RGD peptide-based conjugates in vitro and in vivo.

RESULTS

RGD octamer had significantly higher integrin alpha(v)beta(3)-binding affinity and specificity than RGD tetramer analog (inhibitory concentration of 50% was 10 nM for octamer vs. 35 nM for tetramer). (64)Cu-DOTA-RGD octamer had higher tumor uptake and longer tumor retention than (64)Cu-DOTA-RGD tetramer in both tumor models tested. The integrin alpha(v)beta(3) specificity of both tracers was confirmed by successful receptor-blocking experiments. The high uptake and slow clearance of (64)Cu-DOTA-RGD octamer in the kidneys was attributed mainly to the integrin positivity of the kidneys, significantly higher integrin alpha(v)beta(3)-binding affinity, and the larger molecular size of the octamer, as compared with the other RGD analogs.

CONCLUSIONS

Polyvalency has a profound effect on the receptor-binding affinity and in vivo kinetics of radiolabeled RGD multimers. The information obtained here may guide the future development of RGD peptide-based imaging and internal radiotherapeutic agents targeting integrin alpha(v)beta(3).

The classification of adults with diabetes mellitus can be invalidated by patients who initially present as NIDDM but who later become frankly insulin dependent. In some of these, the pathogenesis could be similar to that in IDDM, namely autoimmune destruction of the pancreatic beta-cells. We studied 102 patients>> 35 yr of age at diabetes onset who had initially been nonketotic and non-insulin-dependent for>> or = 6 mo. They were classified according to glucagon-stimulated C-peptide levels into an insulin-deficient group (n = 33) and a non-insulin-deficient group (n = 69). We measured antibodies to GAD, islet cell cytoplasm, thyroid antigens, and gastric parietal cells in both groups. Anti-GAD was significantly higher in the insulin deficient group, 76% (25 of 33), than in the non-insulin deficient group, 12% (8 of 69), and this difference was substantially greater than that shown for ICAs. Thus, in a proportion of adults who present with NIDDM, a slowly evolving autoimmune insulitis can be revealed by testing for anti-GAD. This could have important connotations not only for early intervention, but also for the correct classification of diabetes.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.

PCCCpeptide and contains a 292-residue domain closely related to the catalytic modules of PCCCCCCCCCCC-terminal amphipathic helical segment similar to the putative membrane anchor of carboxypeptidase H. These and other differences suggest that these proteins carry out compartmentalized proteolysis within cells, such as processing within regulated versus constitutive secretory pathways.

The turnover and processing of the Alzheimer beta/A4 amyloid precursor protein (beta APP) has been studied in PCC, decreased the levels of mature beta APP and increased the levels of 15- and 19-kDa peptides. These peptides appeared to be COOH-terminal fragments of beta APP, which arose when phorbol 12,13-dibutyrate increased the rate of proteolytic processing of mature forms of beta APP. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, also led to decreased levels of mature beta APP and increased levels of the 15- and 19-kDa peptides. H-7, an inhibitor of protein kinase C and of several other protein kinases, apparently decreased the rate of proteolytic processing of mature beta APP. The sizes of the putative COOH-terminal fragments observed after treatment with either phorbol 12,13-dibutyrate or okadaic acid suggest that one or both may contain the entire beta/A4 region of beta APP and thus be amyloidogenic. Our results support the hypothesis that abnormal protein phosphorylation may play a role in the development of the cerebral amyloidosis that accompanies Alzheimer disease.

Naturally occurring peptides with biological actions have in most cases been detected by observing their biological activities in crude extracts and their isolation has been followed using bioassays. As a complement to the classical biological detection systems, we have proposed a chemical detection system based on fragmentation of peptides in tissue extracts followed by identification of certain of these peptide fragments having distinct chemical features. One such chemical feature is the C-terminal amide structure which is characteristic of many biologically active peptides. We have devised a chemical assay method for peptides having such a structure and have found several previously unknown peptide amides in procine upper small intestinal tissues. We report here the isolation and characterization of two of them, designated PHI and PYY. PHI is related to secretin, vasoactive intestinal polypeptide (VIP, glucagon and gastric inhibitory polypeptide (GIP); PYY is related to the pancreatic polypeptide and to neurotensin. Both peptides exhibit biological activities and appear to be present not only in the intestine but also in brain.

Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.

PlcR is a pleiotropic regulator that activates the expression of genes encoding various virulence factors, such as phospholipases C, proteases and hemolysins, in Bacillus thuringiensis and Bacillus cereus. Here we show that the activation mechanism is under the control of a small peptide: PapR. The papR gene belongs to the PlcR regulon and is located 70 bp downstream from plcR. It encodes a 48-amino-acid peptide. Disruption of the papR gene abolished expression of the PlcR regulon, resulting in a large decrease in hemolysis and virulence in insect larvae. We demonstrated that the PapR polypeptide was secreted, then reimported via the oligopeptide permease Opp. Once inside the cell, a processed form of PapR, presumably a pentapeptide, activated the PlcR regulon by allowing PlcR to bind to its DNA target. This activating mechanism was found to be strain specific, with this specificity determined by the first residue of the penta peptide.

Protein kinase C (PKC) isozymes translocate to unique subcellular sites following activation. We previously suggested that translocation of activated isozymes is required for their function and that in addition to binding to lipids, translocation involves binding of the activated isozymes to specific anchoring proteins (receptors for activated protein kinase C. Using cultured cardiomyocytes we identified inhibitors, the V1 fragment of epsilonPKC (epsilonV1), and an 8-amino acid peptide derived from it that selectively inhibited the translocation of epsilonPKC. Inhibition of epsilonPKC translocation but not inhibition of delta or betaPKC translocation specifically blocked phorbol ester- or norepinephrine-mediated regulation of contraction. These isozyme-selective translocation inhibitors provide novel tools to determine the function of individual PKC isozymes in intact cells.

There are nine known expanded CAG repeat neurological diseases, including Huntington's disease (HD), each involving the repeat expansion of polyglutamine (polyGln) in a different protein. Similar conditions can be induced in animal models by expression of the polyGln sequence alone or in other protein contexts. Besides the polyGln sequence, the cellular context of the disease protein, and the sequence context of the polyGln within the disease protein, are both likely to contribute to polyGln physical behavior and to pathology. In HD, the N-terminal, exon-1 segment of the protein huntingtin contains the polyGln sequence immediately followed by an oligoproline region. We show here that introduction of a P10 sequence C-terminal to polyGln in synthetic peptides decreases both the rate of formation and the apparent stability of the amyloid-like aggregates associated with this family of diseases. The sequence can be trimmed to P6 without altering the suppression, but a P3 sequence is ineffective. Spacers up to at least three amino acid residues in length can be inserted between polyGln and P10 without altering this effect. There is no suppression, however, when the P10 sequence is either placed on the N-terminal side of polyGln or attached to polyGln via a side-chain tether. The nucleation mechanism of a Q40 sequence is unchanged upon addition of a P10 C-terminal extension, yielding a critical nucleus of one. The effects of oligoPro length and structural context on polyGln aggregation are correlated strongly with alterations in the circular dichroism spectra of the monomeric peptides. For example, the P10 sequence eliminates the small amount of alpha helical content otherwise exhibited by the Q40 sequence. The P10 sequence may suppress aggregation by stabilizing an aggregation-incompetent conformation of the monomer. The effect is transportable: a P10 sequence fixed to the C terminus of the sequence Abeta similarly modulates amyloid fibril formation.

Nociceptive dorsal root ganglion (DRG) neurones have fibres that conduct in the C, Adelta and Aalpha/beta conduction velocity range. The properties of nociceptive compared with non-nociceptive somatic afferent dorsal root ganglion neurones appear to fall into two patterns, A and B. Pattern A properties of nociceptive neurones, the more common type, include longer action potential duration and slower maximum rate of fibre firing, as well as a greater expression of substance P and calcitonin gene-related peptide immunoreactivity. The values of pattern A properties appear to be graded according to the conduction velocity group (C, Adelta or Aalpha/beta) of the fibres. The most pronounced forms of A-type properties are expressed by nociceptive neurones with C-fibres, and these become less pronounced in nociceptive neurones with Adelta-fibres and least pronounced in those with Aalpha/beta fibres (C>> Adelta>> Aalpha/beta). Some of these properties are also expressed in a less extreme but similarly graded manner through C, Adelta and Aalpha/beta groups of non-nociceptive low threshold mechanoreceptive (LTM) neurone. The less common pattern B properties of nociceptive neurones have similar values in C-, Adelta- and Aalpha/beta-fibre nociceptive neurones but these clearly differ from LTM units with C-, Adelta- and Aalpha/beta-fibre conduction velocities. These features of nociceptive neurones include consistently larger action potential overshoots and longer after-hyperpolarisation durations in nociceptive than in LTM neurones.

The beta gamma subunits of heterotrimeric G proteins play important roles in regulating receptor-stimulated signal transduction processes. Recently appreciated among these is their role in the signaling events that lead to the phosphorylation and subsequent desensitization of muscarinic cholinergic (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227) and beta-adrenergic (Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267) receptors. Beta gamma mediates the membrane targeting of the beta-adrenergic receptor kinase (beta ARK), in response to receptor activation, through a specific beta ARK-beta gamma interaction. This process utilizes the membrane-anchoring properties of the isoprenylated gamma subunit of beta gamma. In the present study, we have employed three distinct approaches to identify the region within the carboxyl terminus of beta ARK which binds beta gamma and thereby results in membrane translocation. We studied the ability of beta gamma to enhance the enzymatic activity of a series of truncated mutants of bovine beta ARK1, the ability of glutathione S-transferase fusion proteins containing various lengths of the carboxyl terminus of beta ARK to bind beta gamma subunits, and the ability of synthetic peptides comprised of beta ARK sequences to inhibit beta gamma activation of beta ARK1. We find that the minimal beta gamma binding domain of beta ARK is localized to a 125-amino acid residue stretch, the distal end of which is located 19 residues from the carboxyl terminus. A single 28-mer peptide (Trp643 to Ser670) derived from this sequence effectively inhibited beta gamma activation of beta ARK1, with an ICpeptide inhibitors provide important tools for the study of G protein-coupled receptor desensitization, as well as for the investigation of beta gamma activation of other G protein-effector systems.

Obesity has become an epidemic problem in western societies, contributing to metabolic diseases, hypertension, and cardiovascular disease. Overweight and obesity are frequently associated with increased plasma levels of aldosterone. Recent evidence suggests that human fat is a highly active endocrine tissue. Therefore, we tested the hypothesis that adipocyte secretory products directly stimulate adrenocortical aldosterone secretion. Secretory products from isolated human adipocytes strongly stimulated steroidogenesis in human adrenocortical cells (NCI-H295R) with a predominant effect on mineralocorticoid secretion. Aldosterone secretion increased 7-fold during 24 h of incubation. This stimulation was comparable to maximal stimulation of these cells with forskolin (2 x 10(-5) M). On the molecular level, there was a 10-fold increase in the expression of steroid acute regulatory peptide mRNA. This effect was independent of adipose angiotensin II as revealed by the stimulatory effect of fat cell-conditioned medium even in the presence of the angiotensin type 1 receptor antagonist, valsartan. None of the recently defined adipocytokines accounted for the effect. Mineralocorticoid-stimulating activity was heat sensitive and could be blunted by heating fat cell-conditioned medium to 99 degrees C. Centrifugal filtration based on molecular mass revealed at least two releasing factors: a heat sensitive fraction (molecular mass >50 kDa) representing 60% of total activity, and an inactive fraction (molecular mass <50 kDa). However, the recovery rate increased to 92% when combining these two fractions, indicating the interaction of at least two factors. In conclusion, human adipocytes secrete potent mineralocorticoid-releasing factors, suggesting a direct link between obesity and hypertension.