A serine/threonine protein kinase that binds phorbol esters and diacylglycerol (named protein kinase D, PKD) has been identified. PKD contains membrane localization signals and a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC). A bacterially expressed N-terminal domain of PKD exhibited high-affinity phorbol ester binding activity (Kd = 35 nM). The diacylglycerol analog 1-oleoyl-2-acetylglycerol inhibited phorbol ester binding in a dose-dependent manner. The catalytic domain of PKD contains all characteristic sequence motifs of serine protein kinases but shows only a low degree of sequence similarity to PKCs. The highest identity is with the catalytic domain of myosin light-chain kinase from Dictyostelium (41%). The bacterially expressed catalytic domain of PKD efficiently phosphorylated the exogenous peptide substrate syntide 2 in serine but did not catalyze significant phosphorylation of a variety of other substrates used by PKCs and other major second messenger regulated kinases. PKD may be an unusual component in the transduction of diacylglycerol and phorbol ester signals.

With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found to influence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation by screening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885 mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolished swarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also had a wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold more exopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Whole transcriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for the synthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein (which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of the PA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMP enhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein has phosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensing positively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

OBJECTIVE

Hyperinsulinemic hypoglycemia is newly recognized as a rare but important complication after Roux-en-Y gastric bypass (GB). The etiology of the syndrome and metabolic characteristics remain incompletely understood. Recent studies suggest that levels of incretin hormones are increased after GB and may promote excessive beta-cell function and/or growth.

METHODS

We performed a cross-sectional analysis of metabolic variables, in both the fasting state and after a liquid mixed-meal challenge, in four subject groups: 1) with clinically significant hypoglycemia [neuroglycopenia (NG)] after GB surgery, 2) with no symptoms of hypoglycemia at similar duration after GB surgery, 3) without GB similar to preoperative body mass index of the surgical cohorts, and 4) without GB similar to current body mass index of the surgical cohorts.

RESULTS

Insulin and C-peptide after the liquid mixed meal were both higher relative to the glucose level achieved in persons after GB with NG compared with asymptomatic individuals. Glucagon, glucagon-like peptide 1, and glucose-dependent insulinotropic peptide levels were higher in both post-GB surgical groups compared with both overweight and morbidly obese persons, and glucagon-like peptide 1 was markedly higher in the group with NG. Insulin resistance, assessed by homeostasis model assessment of insulin resistance, the composite insulin sensitivity index, or adiponectin, was similar in both post-GB groups. Dumping score was also higher in both GB groups but did not discriminate between asymptomatic and symptomatic patients. Notably, the frequency of asymptomatic hypoglycemia after a liquid mixed meal was high in post-GB patients.

CONCLUSIONS

A robust insulin secretory response was associated with postprandial hypoglycemia in patients after GB presenting with NG. Increased incretin levels may contribute to the increased insulin secretory response.

Flagellins from three strains of Campylobacter jejuni and one strain of Campylobacter coli were shown to be extensively modified by glycosyl residues, imparting an approximate 6000-Da shift from the molecular mass of the protein predicted from the DNA sequence. Tryptic peptides from C. jejuni 81-176 flagellin were subjected to capillary liquid chromatography-electrospray mass spectrometry with a high/low orifice stepping to identify peptide segments of aberrant masses together with their corresponding glycosyl appendages. These modified peptides were further characterized by tandem mass spectrometry and preparative high performance liquid chromatography followed by nano-NMR spectroscopy to identify the nature and precise site of glycosylation. These analyses have shown that there are 19 modified Ser/Thr residues in C. jejuni 81-176 flagellin. The predominant modification found on C. jejuni flagellin was O-linked 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) with additional heterogeneity conferred by substitution of the acetamido groups with acetamidino and hydroxyproprionyl groups. In C. jejuni 81-176, the gene Cj1316c, encoding a protein of unknown function, was shown to be involved in the biosynthesis and/or the addition of the acetamidino group on Pse5Ac7Ac. Glycosylation is not random, since 19 of the total 107 Ser/Thr residues are modified, and all but one of these are restricted to the central, surface-exposed domain of flagellin when folded in the filament. The mechanism of attachment appears unrelated to a consensus peptide sequence but is rather based on surface accessibility of Ser/Thr residues in the folded protein.

Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

A neuropathological hallmark of Alzheimer's disease is the deposition of amyloid-beta (Abeta) peptides in senile plaques in the hippocampus and cerebral cortex. Abeta is derived from larger integral membrane proteins termed amyloid precursor proteins (APP). We demonstrated previously that APP, synthesized by neurons in the entorhinal cortex, is transported via the perforant pathway to presynaptic terminals in the dentate gyrus. We reported that, although full-length APP and membrane-tethered, C-terminal APP derivatives (APP-CTFs) accumulate at terminal fields, the production of Abeta peptides at these sites was indeterminate. To test the hypothesis that APP-CTFs, generated from axonally transported APP, are further metabolized to Abeta peptides that are subsequently released and deposited proximal to nerve terminals, we created unilateral knife lesions of the perforant pathway of transgenic mice that exhibit hippocampal amyloid deposits. We observed pronounced reductions in amyloid burden in the ipsilateral dentate gyrus, findings that lead us to conclude that axonally transported APP gives rise to Abeta peptides that are released from presynaptic sites in the dentate gyrus and deposited in extracellular plaques. Moreover, our findings are consistent with the view that Abeta deposits are dynamic structures and that the perforant path lesion alters the equilibrium between Abeta production-deposition toward clearance as a consequence of blocked axonal transport of APP from the entorhinal cortex to terminal fields in the hippocampus.

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.

Adiponectin, the gene product of the adipose most abundant gene transcript 1, is a novel adipocyte-derived peptide that has been considered to have antiinflammatory and antiatherogenic effects. To characterize the relationship between adiponectin and lipids metabolism, we measured fasting plasma adiponectin concentration by ELISA, serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), and apolipoprotein (apo) levels in 352 nondiabetic women, 16-86 yr old, with a wide range of body weight [body mass index (BMI), 14.8-36.3 kg/m(2)]. Plasma adiponectin concentrations in women with the highest tertile of TG (1.69 mM < or approximately) were decreased, compared with the middle (1.13 < or = approximately < 1.69) or lowest tertile of TG (approximately < 1.13) (5.9 +/- 0.5 vs. 7.5 +/- 0.3, 9.2 +/- 0.2 microg/ml; P < 0.005, 0.001). Plasma adiponectin with the lowest tertile of HDL-C (approximately < 1.16 mM) was decreased, compared with the middle (1.16 < or = approximately < 1.81) or highest tertile of HDL-C (1.81 < or approximately ) (5.7 +/- 0.5 vs. 7.8 +/- 0.2, 10.1 +/- 0.4 microg/ml; both P < 0.001). These relationships had similar tendencies after adjustment for BMI, body fat mass, age, or diastolic blood pressure. Adiponectin was negatively correlated with serum TG (r = -0.33, P < 0.0001), atherogenic index [(total cholesterol - HDL-C)/HDL-C] (r = -0.34, P < 0.0001), apo B (r = -0.45, P < 0.0001), or apo E (r = -0.29, P < 0.05), and positively correlated with serum HDL-C (r = 0.39, P < 0.0001) or apo A-I levels (r = 0.42, P < 0.002). Those negative relationships became stronger after adjusting for BMI or body fat mass. The slightly positive correlation between adiponectin and age, blood urea nitrogen, or creatinine levels was also observed (all P < 0.001). These results indicate that high-TGnemia and low-HDL-Cnemia are associated with low plasma adiponectin concentrations in nondiabetic women. Further efforts must now be targeted to determine whether adiponectin causes these lipid abnormalities and thus whether it is partly responsible for the atherogenic risk seen in the metabolic syndrome.

The name surfactin refers to a bacterial cyclic lipopeptide, primarily renowned for its exceptional surfactant power since it lowers the surface tension of water from 72 mN m-1 to 27 mN m-1 at a concentration as low as 20 microM. Although surfactin was discovered about 30 years ago, there has been a revival of interest in this compound over the past decade, triggered by an increasing demand for effective biosurfactants for difficult contemporary ecological problems. This simple molecule also looks very promising as an antitumoral, antiviral and anti-Mycoplasma agent. Structural characteristics show the presence of a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a beta-hydroxy fatty acid with 13-15 C atoms. In solution, the molecule exhibits a characteristic "horse saddle" conformation that accounts for its large spectrum of biological activity, making it very attractive for both industrial applications and academic studies. Surfactin biosynthesis is catalysed non-ribosomally by the action of a large multienzyme complex consisting of four modular building blocks, called the surfactin synthetase. The biosynthetic activity involves the multicarrier thiotemplate mechanism and the enzyme is organized in structural domains that place it in the family of peptide synthetases, a class of enzymes involved in peptidic secondary-metabolite synthesis. The srfA operon, the sfp gene encoding a 4'-phosphopantetheinyltransferase and the comA regulatory gene work together for surfactin biosynthesis, while the gene encoding the acyltransferase remains to be isolated. Concerning surfactin production, there is no indication whether the genetic regulation, involving a quorum-sensing mechanism, overrides other regulation factors promoted by the fermentation conditions. Knowledge of the modular arrangement of the peptide synthetases is of the utmost relevance to combinatorial biosynthetic approaches and has been successfully used at the gene level to modify the surfactin template. Biosynthetic and genetic rationales have been described for building variants. A fine study of the structure/function relationships associated with the three-dimensional structure has led to the recognition of the specific residues required for activity. These studies will assist researchers in the selection of molecules with improved and/or refined properties useful in oil and biomedical industries.

NMDA receptors play key roles in synaptic plasticity and neuronal development, and may be involved in learning, memory, and compensation following injury. A polyclonal antibody that recognizes four of seven splice variants of NMDAR1 was made using a C-terminus peptide (30 amino acid residues). NMDAR1 is the major NMDA receptor subunit, found in most or all NMDA receptor complexes. On immunoblots, this antibody labeled a single major band migrating at M(r) = 120,000. The antibody did not cross-react with extracts from transfected cells expressing other glutamate receptor subunits, nor did it label non-neuronal tissues. Immunostained vibratome sections of rat tissue showed labeling in many neurons in most structures in the brain, as well as in the cervical spinal cord, dorsal root and vestibular ganglia, and in pineal and pituitary glands. Staining was moderate to dense in the olfactory bulb, neocortex, striatum, some thalamic and hypothalamic nuclei, the colliculi, and many reticular, sensory, and motor neurons of the brainstem and spinal cord. The densest stained cells included the pyramidal and hilar neurons of the CA3 region of the hippocampus, Purkinje cells of the cerebellum, supraoptic and magnocellular paraventricular neurons of the hypothalamus, inferior olive, red nucleus, lateral reticular nucleus, peripheral dorsal cochlear nucleus, and motor nuclei of the lower brainstem and spinal cord. Ultrastructural localization of immunostaining was examined in the hippocampus, cerebral cortex, and cerebellar cortex. The major staining was in postsynaptic densities apposed by unstained presynaptic terminals with round or mainly round vesicles, and in associated dendrites. The pattern of staining matched that of previous in situ hybridization but differed somewhat from that of binding studies, implying that multiple types of NMDA receptors exist. Comparison with previous studies of localization of other glutamate receptor types revealed that NMDAR1 may colocalize with these other types in many neurons throughout the nervous system.

All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys-1His array (CCHC = C-X2-C-X4-H-X4-C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc-binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc-edge extended X-ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus-bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840-847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys-1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV-1 NC protein and have determined its three-dimensional solution-state structure. The CCHC arrays of HIV-1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV-1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid-binding results provide a basis for the rational design of new NC-targeted antiviral agents and vaccines for the control of AIDS.

Diversity in 39 HLA-A, -B, and -C molecules is derived from 20 amino acid positions of high variability and 71 positions of low variability. Variation in the structurally homologous alpha 1 and alpha 2 domains is distinct and may correlate with partial segregation of peptide and T-cell receptor binding functions. Comparison of 15 HLA-A with 20 HLA-B molecules reveals considerable locus-specific character, due primarily to differences at polymorphic residues. The results indicate that genetic exchange between alleles of the same locus has been a more important mechanism in the generation of HLA-A, -B, and -C diversity than genetic exchange events between alleles of different loci.

The P2X7 receptor is a bifunctional molecule. The binding of ATP induces within milliseconds the opening of a channel selective for small cations, and within seconds a larger pore opens which allows permeation by molecules as large as propidium dyes (629 Da). In situ hybridization using a digoxigenin-labelled riboprobe, and immunohistochemistry using an antibody raised against a C-terminal peptide sequence, were used to determine the distribution of the P2X7 receptor mRNA and protein in rat and mouse tissues and cell lines. The brain of newborn rats showed a 6 kb RNA by Northern blotting, but this was not detectable in adult brain. By in situ hybridization and immunohistochemistry, there was heavy labelling of ependymal cells in both newborn and adult brain, but the brain parenchyma showed no labelling. However, P2X7 receptor-immunoreactive cells appeared in the penumbral region around an area of necrosis evoked by prior occlusion of the middle cerebral artery, suggesting expression of the receptor by activated microglia. NTW8 cells, a mouse microglial cell line, strongly expressed the P2X7 receptor mRNA and protein. The P2X7 receptor mRNA and protein were also observed in the majority of bone marrow cells, including those separately identified by their expression of other antigens as granulocytes, monocyte/macrophages and B lymphocytes. The expression of P2X7 receptor by brain macrophages rather than neurons would be consistent with a role in brain repair following inflammation, infarction or immune insult.

Despite its importance in Parkinson's disease, a detailed understanding of the structure and mechanism of alpha-synuclein fibril formation remains elusive. In this study, we used site-directed spin labeling and electron paramagnetic resonance spectroscopy to study the structural features of monomeric and fibrillar alpha-synuclein. Our results indicate that monomeric alpha-synuclein, in solution, has a highly dynamic structure, in agreement with the notion that alpha-synuclein is a natively unfolded protein. In contrast, fibrillar aggregates of alpha-synuclein exhibit a distinct domain organization. Our data identify a highly ordered and specifically folded central core region of approximately 70 amino acids, whereas the N terminus is structurally more heterogeneous and the C terminus ( approximately 40 amino acids) is completely unfolded. Interestingly, the central core region of alpha-synuclein exhibits several features reminiscent of those observed in the core region of fibrillar Alzheimer's amyloid beta peptide, including an in-register parallel structure. Although the lengths of the respective core regions differ, fibrils from different amyloid proteins nevertheless appear to be able to take up highly similar, and possibly conserved, structures.

Differential expression of ion channels contributes functional diversity to sensory neuron signaling. We find nerve injury induced by the Chung model of neuropathic pain leads to striking reductions in voltage-gated K(+) (Kv) channel subunit expression in dorsal root ganglia (DRG) neurons, suggesting a potential molecular mechanism for hyperexcitability of injured nerves. Moreover, specific classes of DRG neurons express distinct Kv channel subunit combinations. Importantly, Kv1.4 is the sole Kv1 alpha subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in A delta and C fibers arising from these cells. These neurons are presumably nociceptors, because they also express the VR-1 capsaicin receptor, calcitonin gene-related peptide, and/or Na(+) channel SNS/PN3/Nav1.8. In contrast, larger diameter neurons associated with mechanoreception and proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 alpha subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells.

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids Cpeptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.

beta-Secretase (BACE, Asp-2) is a transmembrane aspartic proteinase responsible for cleaving the amyloid precursor protein (APP) to generate the soluble ectodomain sAPPbeta and its C-terminal fragment CTFbeta. CTFbeta is subsequently cleaved by gamma-secretase to produce the neurotoxic/synaptotoxic amyloid-beta peptide (Abeta) that accumulates in Alzheimer's disease. Indirect evidence has suggested that amyloidogenic APP processing may preferentially occur in lipid rafts. Here, we show that relatively little wild-type BACE is found in rafts prepared from a human neuroblastoma cell line (SH-SY5Y) by using Triton X-100 as detergent. To investigate further the significance of lipid rafts in APP processing, a glycosylphosphatidylinositol (GPI) anchor has been added to BACE, replacing the transmembrane and C-terminal domains. The GPI anchor targets the enzyme exclusively to lipid raft domains. Expression of GPIBACE substantially up-regulates the secretion of both sAPPbeta and amyloid-beta peptide over levels observed from cells overexpressing wild-type BACE. This effect was reversed when the lipid rafts were disrupted by depleting cellular cholesterol levels. These results suggest that processing of APP to the amyloid-beta peptide occurs predominantly in lipid rafts and that BACE is the rate-limiting enzyme in this process. The processing of the APP695 isoform by GPI-BACE was up-regulated 20-fold compared with wild-type BACE, whereas only a 2-fold increase in the processing of APP751/770 was seen, implying a differential compartmentation of the APP isoforms. Changes in the local membrane environment during aging may facilitate the cosegregation of APP and BACE leading to increased beta-amyloid production.

Transportan is a 27 amino acid-long peptide containing 12 functional amino acids from the amino terminus of the neuropeptide galanin and mastoparan in the carboxyl terminus, connected via a lysine. Transportan is a cell-penetrating peptide as judged by indirect immunofluorescence using N epsilon13-biotinyl-transportan. The internalization of biotinyl-transportan is energy independent and takes place efficiently at 37 degrees, 4 degrees, and 0 degrees C. Cellular uptake of transportan is probably not mediated by endocytosis, since it cannot be blocked by treating the cells with phenylarsine oxide or hyperosmolar sucrose solution and is nonsaturable. The kinetics of internalization was studied with the aid of the 125I-labeled peptide. At 37 degrees C, the maximal intracellular concentration is reached in about 20 min. The internalized transportan is protected from trypsin. The cell-penetrating ability of transportan is not restricted by cell type, but seems to be a general feature of this peptide. In Bowes' melanoma cells, transportan first localizes in the outer membrane and cytoplasmatic membrane structures. This is followed by a redistribution into the nuclear membrane and uptake into the nuclei where transportan concentrates in distinct substructures, probably the nucleoli.

FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides (FaRPs) have been found throughout the animal kingdom, where they are involved in many behaviors. We previously identified 22 genes comprising the flp gene family that encodes FaRPs in Caenorhabditis elegans; in this paper we report the identification of another flp gene, flp-23. As a first step toward determining their functional roles in C. elegans, we examined the cell-specific expression pattern of the flp gene family. Of the 19 flp genes examined, each gene is expressed in a distinct set of cells; these cells include interneurons, motor neurons, and sensory neurons that are involved in multiple behaviors, as well as supporting cells, muscle cells, and epidermal cells. Several flp genes show sex-specific expression patterns. Furthermore, we find that expression of two flp genes changes in response to the developmental state of the animal. Many neurons express multiple flp genes. To investigate how flp genes are regulated in different neuronal subtypes, we examined flp expression in a small, well-defined subset of neurons, the mechanosensory neurons. Mutations in the unc-86 and mec-3 genes, which are necessary for the production and differentiation of the mechanosensory neurons, result in the complete loss of flp-4, flp-8, and flp-20 expression in mechanosensory neurons. Collectively, these data indicate that members of the flp gene family are likely to influence multiple behaviors and that their regulation can be dependent on the developmental state of the organism.

Store-operated Ca(2+) entry (SOCE) is a universal mechanism to increase intracellular Ca(2+) concentrations in non-excitable cells. It is initiated by the depletion of ER Ca(2+) stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca(2+) release activated Ca(2+) (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca(2+) influx and CRAC currents (I(CRAC)) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 39 [corrected] amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I(CRAC) consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca(2+) stores.

Compressed and stretched polyacrylamide hydrogels previously have been shown to offer a robust method for aligning proteins. A simple, funnel-like apparatus is described for generating uniformly stretched hydrogels. For prolate-shaped proteins, gels stretched in the direction of the magnetic field yield two-fold larger alignment than gels compressed to the same aspect ratio in this direction. Empirically, protein alignment is found to be proportional to (c-2.3)2 [(d(o/dN)3-1], where do and dN are the diameters of the cylindrical gels before and after stretching, respectively, and c is the polyacrylamide weight fraction in percent. Low gel densities, in the 4-7% range, are found to have minimal effects on macromolecular rotational correlation times, tauc, and no effect of the compression ratio on tauc could be discerned over the range studied (do/dN < or = 1.4). Application is demonstrated for a sample containing the first Ig-binding domain of protein G, and for a detergent-solubilized peptide.

Pheochromocytomas, intra-adrenal paraganglioma, and extra-adrenal sympathetic and parasympathetic paragangliomas are neuroendocrine tumors derived from adrenal chromaffin cells or similar cells in extra-adrenal sympathetic and parasympathetic paraganglia, respectively. Serious morbidity and mortality rates associated with these tumors are related to the potent effects of catecholamines on various organs, especially those of the cardiovascular system. Before any surgical procedure is done, preoperative blockade is necessary to protect the patient against significant release of catecholamines due to anesthesia and surgical manipulation of the tumor. Treatment options vary with the extent of the disease, with laparoscopic surgery being the preferred treatment for removal of primary tumors. Medullary thyroid cancer (MTC) is a malignancy of the thyroid C cells or parafollicular cells. Thyroid C cells elaborate a number of peptides and hormones, such as calcitonin, carcinoembryonic antigen, and chromogranin A. Some or all of these markers are elevated in patients with MTC and can be used to confirm the diagnosis as well as to follow patients longitudinally for recurrence. Medullary thyroid cancer consists of a spectrum of diseases that ranges from extremely indolent tumors that are stable for many years to aggressive types associated with a high mortality rate. Genetic testing for RET mutations has allowed identification of familial cases and prophylactic thyroidectomy for cure. The only curative treatment is complete surgical resection.

Vascular smooth muscle (VSM) cells, endothelial cells (EC), and pericytes that form the walls of vessels in the microcirculation express a diverse array of ion channels that play an important role in the function of these cells and the microcirculation in both health and disease. This brief review focuses on the K+ channels expressed in smooth muscle and endothelial cells in arterioles. Microvascular VSM cells express at least four different classes of K+ channels, including inward-rectifier K+ channels (Kin), ATP-sensitive K+ channels (KATP), voltage-gated K+ channels (Kv), and large conductance Ca2+-activated K+ channels (BKCa). VSM KIR participate in dilation induced by elevated extracellular K+ and may also be activated by C-type natriuretic peptide, a putative endothelium-derived hyperpolarizing factor (EDHF). Vasodilators acting through cAMP or cGMP signaling pathways in VSM may open KATP, Kv, and BKCa, causing membrane hyperpolarization and vasodilation. VSMBKc. may also be activated by epoxides of arachidonic acid (EETs) identified as EDHF in some systems. Conversely, vasoconstrictors may close KATP, Kv, and BKCa through protein kinase C, Rho-kinase, or c-Src pathways and contribute to VSM depolarization and vasoconstriction. At the same time Kv and BKCa act in a negative feedback manner to limit depolarization and prevent vasospasm. Microvascular EC express at least 5 classes of K+ channels, including small (sKCa) and intermediate(IKCa) conductance Ca2+-activated K+ channels, Kin, KATP, and Kv. Both sK and IK are opened by endothelium-dependent vasodilators that increase EC intracellular Ca2+ to cause membrane hyper-polarization that may be conducted through myoendothelial gap junctions to hyperpolarize and relax arteriolar VSM. KIR may serve to amplify sKCa- and IKCa-induced hyperpolarization and allow active transmission of hyperpolarization along EC through gap junctions. EC KIR channels may also be opened by elevated extracellular K+ and participate in K+-induced vasodilation. EC KATP channels may be activated by vasodilators as in VSM. Kv channels may provide a negative feedback mechanism to limit depolarization in some endothelial cells.

OBJECTIVE

To evaluate the efficacy and safety of four doses of pioglitazone monotherapy in the treatment of patients with type 2 diabetes.

METHODS

There were 408 patients randomized in this multicenter double-blind placebo-controlled clinical trial. Patients who had HbA1c>> or = 7.0%, fasting plasma glucose (FPG)>> or = 140 mg/dl, and C-peptide>> 1 ng/ml were randomized to receive placebo or 7.5, 15, 30, or 45 mg pioglitazone administered once a day for 26 weeks.

RESULTS

Patients treated with 15, 30, or 45 mg pioglitazone had significant mean decreases in HbA1c (range -1.00 to -1.60% difference from placebo) and FPG (-39.1 to -65.3 mg/dl difference from placebo). The decreases in FPG were observed as early as the second week of therapy; maximal decreases occurred after 10-14 weeks and were maintained until the end of therapy (week 26). In the 15-, 30-, or 45-mg pioglitazone groups, there were significant mean percent decreases in triglycerides, significant mean percent increases in HDL cholesterol, and only small percent changes in total cholesterol and LDL. The subset of patients naive to therapy had greater improvements in HbA1c and FPG (difference from placebo of -2.55% and -79.9 mg/dl for the 45-mg group) compared with previously treated patients. The overall adverse event profile of pioglitazone was similar to that of placebo. There was no evidence of drug-induced hepatotoxicity or drug-induced elevations of alanine aminotransferase levels in this study

CONCLUSIONS

Pioglitazone monotherapy significantly improves HbA1c and FPG while producing beneficial effects on serum lipids in patients with type 2 diabetes with no evidence of drug-induced hepatotoxicity.