The CD4 (T4) antigen is a cell-surface glycoprotein that is expressed predominantly on the surface of helper T cells and has been implicated in the regulation of T-cell activation and in the associative recognition of class II antigens of the major histocompatibility complex. In addition, the CD4 antigen appears to serve as a receptor for the human immunodeficiency virus (HIV). An important question has been whether the CD4 receptor is linked to an intracellular mediator that could regulate the activation of the CD4+ subset. In this paper, we provide preliminary evidence that the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (PTK) of 55-60 kDa, which is expressed specifically in T cells. The PTK is the human analogue of the murine pp56LSTRA (pp56lck) and has significant homology with c-src, c-yes, and other members of the src family. The identification of the PTK associated with CD4 receptor was made by use of an antiserum to a synthetic peptide that was deduced from the DNA sequence of PTK. Two-dimensional nonequilibrium pH gradient gel electrophoresis/NaDodSO4/PAGE revealed the kinase to focus as a heterogeneous collection of spots in the pH range of 4.0-5.0. Furthermore, in vitro phosphorylation revealed the phosphorylation of two additional polypeptides at 40 and 80 kDa, in addition to the autophosphorylation of the PTK at 55-60 kDa. The potential importance of the association between the CD4 receptor and the PTK of T cells is discussed in relation to T-cell activation and HIV infectivity.

The base sequence of the ribosome binding site region of the Gram-positive Staphylococcus aureus beta-lactamase gene has been determined. The leader peptide sequence of 24 amino acids which precedes the NH2 terminus of extracellular S. aureus beta-lactamase has also been established. This initiation site possesses two unique features not observed for most initiation sites recognized by Escherichia coli ribosomes. A novel initiation codon, UUG, initiates protein synthesis with methionine; and a very strong Shine-Dalgarno complementarity containing five G-C base pairs precedes the UUG initiation codon. The strong Shine-Dalgarno complementarity may explain the reduced translational dependence on initiation factor IF-3 function that has been observed for the beta-lactamase mRNA and other mRNAs from Gram-positive bacteria. We suggest that this extent of complementarity between the mRNA and this extent of complementarity between the mRNA and the ribosome may be a requirement for efficient initiation by Bacillus subtilis and other Gram-positive ribosomes, and may provide the basis for the observed inability of the Gram-positive systems to translate most of the mRNAs from Gram-negative bacteria.

The complete amino acid sequence of human insulin-like growth factor I (IGF-I), a polypeptide isolated from serum, has been determined. IGF-I is a single chain polypeptide of 70 amino acid residues cross-linked by three disulfide bridges. The calculated molecular weight is 7649. IGF-I displays obvious homology to proinsulin: positions 1 to 29 are homologous to insulin B chain and positions 42 to 62 to insulin A chain. A shortened "connecting" peptide with 12 residues (positions 30 to 41) compared to 30 to 35 in proinsulins shows no homology to proinsulin C peptide. An octapeptide sequence at the COOH-terminal end is also a feature not found in proinsulins. The number of differences in amino acid positions between IGF-I and insulins suggests that duplication of the gene of the common ancestor of proinsulin and IGF occurred before the time of appearance of the vertebrates. Of the 19 residues known to be invariant in all insulins so far sequenced, only glutamine A5 and asparagine A21 are replaced in IGF-I by glutamic acid and alanine, respectively. The fact that all half-cystine and glycine residues and most nonpolar core residues of the insulin monomer are conserved is compatible with a three-dimensional structure of IGF-I similar to that of insulin.

PDZ domains are protein-protein recognition modules that play a central role in organizing diverse cell signaling assemblies. These domains specifically recognize short C-terminal peptide motifs, but can also recognize internal sequences that structurally mimic a terminus. PDZ domains can therefore be used in combination to bind an array of target proteins or to oligomerize into branched networks. Several PDZ-domain-containing proteins play an important role in the transport, localization and assembly of supramolecular signaling complexes. Examples of such PDZ-mediated assemblies exist in Drosophila photoreceptor cells and at mammalian synapses. The predominance of PDZ domains in metazoans indicates that this highly specialized scaffolding module probably evolved in response to the increased signaling needs of multicellular organisms.

Cathelicidins comprise a family of mammalian proteins containing a C-terminal cationic antimicrobial domain that becomes active after being freed from the N-terminal cathelin portion of the holoprotein. Many other members of this family have been identified since the first cathelicidin sequences were reported 10 years ago. The mature peptides generally show a wide spectrum of antimicrobial activity and, more recently, some of them have also been found to exert other biological activities. The human cathelicidin peptide LL-37 is chemotactic for neutrophils, monocytes, mast cells, and T cells; induces degranulation of mast cells; alters transcriptional responses in macrophages; stimulates wound vascularization and re-epithelialization of healing skin. The porcine PR-39 has also been involved in a variety of processes, including promotion of wound repair, induction of angiogenesis, neutrophils chemotaxis, and inhibition of the phagocyte NADPH oxidase activity, whereas the bovine BMAP-28 induces apoptosis in transformed cell lines and activated lymphocytes and may thus help with clearance of unwanted cells at inflammation sites. These multiple actions provide evidence for active participation of cathelicidin peptides in the regulation of the antimicrobial host defenses.

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61-101) and to a lesser extent the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.

To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D alpha 1 subunits were produced. Anti-CNCC immunoprecipitated 75% of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type alpha 1 subunit, LCCCNCCND1 antibodies revealed that the alpha 1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNCCA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calcium-dependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.

We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection, gas phase ion/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites. From a 30-microg (approximately 600 pmol) sample of total yeast protein, we identify 1,252 phosphorylation sites on 629 proteins. Identified phosphoproteins have expression levels that range from <50 to 1,200,000 copies per cell and are encoded by genes involved in a wide variety of cellular processes. We identify a consensus site that likely represents a motif for one or more uncharacterized kinases and show that yeast kinases, themselves, contain a disproportionately large number of phosphorylation sites. Detection of a pHis containing peptide from the yeast protein, Cdc10, suggests an unexpected role for histidine phosphorylation in septin biology. From diverse functional genomics data, we show that phosphoproteins have a higher number of interactions than an average protein and interact with each other more than with a random protein. They are also likely to be conserved across large evolutionary distances.

The neurodegenerative diseases, Alzheimer's disease (AD) and Parkinson's disease (PD), are age-related disorders characterized by the deposition of abnormal forms of specific proteins in the brain. AD is characterized by the presence of extracellular amyloid plaques and intraneuronal neurofibrillary tangles in the brain. Biochemical analysis of amyloid plaques revealed that the main constituent is fibrillar aggregates of a 39-42 residue peptide referred to as the amyloid-β protein (Aβ). PD is associated with the degeneration of dopaminergic neurons in the substantia nigra pars compacta. One of the pathological hallmarks of PD is the presence of intracellular inclusions called Lewy bodies that consist of aggregates of the presynaptic soluble protein called α-synuclein. There are various factors influencing the pathological depositions, and in general, the cause of neuronal death in neurological disorders appears to be multifactorial. However, it is clear, that the underlying factor in the neurological disorders is increased oxidative stress substantiated by the findings that the protein side-chains are modified either directly by reactive oxygen species (ROS) or reactive nitrogen species (RNS), or indirectly, by the products of lipid peroxidation. The increased level of oxidative stress in AD brain is reflected by the increased brain content of iron (Fe) and copper (Cu) both capable of stimulating free radical formation (e.g. hydroxyl radicals via Fenton reaction), increased protein and DNA oxidation in the AD brain, enhanced lipid peroxidation, decreased level of cytochrome c oxidase and advanced glycation end products (AGEs), carbonyls, malondialdehyde (MDA), peroxynitrite, and heme oxygenase-1 (HO-1). AGEs, mainly through their interaction with receptors for advanced glycation end products (RAGEs), further activate signaling pathways, inducing formation of proinflammatory cytokines such as interleukin-6 (IL-6). The conjugated aromatic ring of tyrosine residues is a target for free-radical attack, and accumulation of dityrosine and 3-nitrotyrosine has also been reported in AD brain. The oxidative stress linked with PD is supported by both postmortem studies and by studies showing the increased level of oxidative stress in the substantia nigra pars compacta, demonstrating thus the capacity of oxidative stress to induce nigral cell degeneration. Markers of lipid peroxidation include 4-hydroxy-trans-2-nonenal (HNE), 4-oxo-trans-2-nonenal (4-ONE), acrolein, and 4-oxo-trans-2-hexenal, all of which are well recognized neurotoxic agents. In addition, other important factors, involving inflammation, toxic action of nitric oxide (NO·), defects in protein clearance, and mitochondrial dysfunction all contribute to the etiology of PD. It has been suggested that several individual antioxidants or their combinations can be neuroprotective and decrease the risk of AD or slow its progression. The aim of this review is to discuss the role of redox metals Fe and Cu and non-redox metal zinc (Zn) in oxidative stress-related etiology of AD and PD. Attention is focused on the metal-induced formation of free radicals and the protective role of antioxidants [glutathione (GSH), vitamin C (ascorbic acid)], vitamin E (α-Tocopherol), lipoic acid, flavonoids [catechins, epigallocatechin gallate (EGCG)], and curcumin. An alternate hypothesis topic in AD is also discussed.

SELEX stands for systematic evolution of ligands by exponential enrichment. This method, described primarily in 1990 [Ellington, A.D., Szostak, J.W., 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822; Tuerk, C., Gold, L., 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510] aims at the development of aptamers, which are oligonucleotides (RNA or ssDNA) binding to their target with high selectivity and sensitivity because of their three-dimensional shape. Aptamers are all new ligands with a high affinity for considerably differing molecules ranging from large targets as proteins over peptides, complex molecules to drugs and organic small molecules or even metal ions. Aptamers are widely used, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools bearing aptamers as molecular recognition and binding elements are another big field of application. Moreover, aptamers are used for the investigation of binding phenomena in proteomics. The SELEX method was modified over the years in different ways to become more efficient and less time consuming, to reach higher affinities of the aptamers selected and for automation of the process. This review is focused on the development of aptamers by use of SELEX and gives an overview about technologies, advantages, limitations, and applications of aptamers.

Insulin secretion rates can be accurately estimated from plasma C-peptide levels with a two-compartment model for C-peptide distribution and degradation. In previous studies, the kinetic parameters of C-peptide clearance were derived in each subject from the decay curve observed after bolus intravenous injection of biosynthetic human C-peptide. To determine whether standard parameters for C-peptide clearance could be defined and used to calculate insulin secretion without obtaining a decay curve in each subject, we analyzed 200 decay curves of biosynthetic human C-peptide obtained in normal, obese, and non-insulin-dependent diabetes mellitus subjects studied in our laboratory. This analysis showed that the volume of distribution and kinetic parameters of C-peptide distribution and metabolism vary by less than 30% in a population highly heterogeneous in terms of age, sex, degree of obesity, and degree of glucose tolerance. The volume of distribution correlated with the degree of obesity as quantified by body surface area (BSA). This dependence of C-peptide distribution volume on BSA was more marked in men than in women. The long half-life was slightly longer in elderly subjects than in younger adults. When effects of BSA, sex, and age were taken into account, the parameters of C-peptide kinetics were very similar in normal, obese, and diabetic subjects. Based on these findings, a simple procedure to derive standard parameters for C-peptide clearance taking into account degree of obesity, sex, and age was defined. These standard parameters resulted in estimations of mean insulin secretion rates, which differed in each subject by only 10-12% from those obtained with individual parameters. The approach of using standard rather than individual parameters did not systematically underestimate or overestimate insulin secretion so that group values for the fasting secretion rate, the mean 24-h secretion rate, and the number and the amplitude of secretory pulses obtained with standard parameters differed by only 1-2% from the values obtained with individual parameters. Furthermore, the accuracy of measurements based on standard parameters was not different from that associated with replicate determinations of the parameters of C-peptide clearance in the same subject. We conclude that it is possible to estimate insulin secretion rates from plasma C-peptide levels with standard parameters for C-peptide clearance rather than individually derived parameters without significant loss of accuracy.

Defensins are antimicrobial and cytotoxic peptides that contain 29-35 amino acid residues, including six invariant cysteines whose intramolecular disulfide bonds cyclize and stabilize them in a complexly folded, triple-stranded beta-sheet configuration. Generated by the proteolytic processing of 93-95 amino acid precursor peptides, the constitute>> 5% of the total cellular protein in human and rabbit neutrophils (polymorphonucleated neutrophils--PMN) and are also produced by rabbit lung macrophages and by mouse and rabbit small intestinal Paneth cells. Despite their prominence in rat PMN, defensins are not found in murine PMN. The antimicrobial spectrum of defensins includes gram positive and gram negative bacteria, mycobacteria, T. pallidum, many fungi, and some enveloped viruses. Defensins exert nonspecific cytotoxic activity against a wide range of normal and malignant targets, including cells resistant to TNF-alpha and NK-cytolytic factor. They appear to kill mammalian target cells and microorganisms by a common mechanism, which involves initial electrostatic interactions with negatively charged target cell surface molecules (likely the head groups of polar membrane lipids), followed by insertion into the cell membranes which they permeabilize, forming voltage-regulated channels. In addition to their antimicrobial and cytotoxic properties, some defensins act as opsonins, while others inhibit protein kinase C, bind specifically to the ACTH receptor and block steroidogenesis or act as selective chemoattractants for monocytes. Defensins are a newly delineated family of effector molecules whose contribution to host defense, inflammation, and cytotoxicity may be considerable for humans, even though it is unlikely to be revealed by experimentation with mice.

Extracellular signals activate mitogen-activated protein kinase (MAPK) cascades to execute complex cellular programs, like proliferation, differentiation and apoptosis. In mammalian cells, three MAPK families have been characterized: extracellular signal-regulated kinase (ERK), which is activated by growth factors, peptide hormones and neurotransmitters, and Jun kinase (JNK) and p38 MAPK, which are activated by cellular stress stimulus as well as growth factors. This review describes the family of 90 kDa ribosomal S6 kinases (RSK; also known as p90rsk or MAPK-activated protein kinase-1, MAPKAP-K1), which were among the first substrates of ERK to be discovered and which has proven to be a ubiquitous and versatile mediator of ERK signal transduction. RSK is composed of two functional kinase domains that are activated in a sequential manner by a series of phosphorylations. Recently, a family of RSK-related kinases that are activated by ERK as well as p38 MAPK were discovered and named mitogen- and stress-activated protein kinases (MSK). A number of cellular functions of RSK have been proposed. (1) Regulation of gene expression via association and phosphorylation of transcriptional regulators including c-Fos, estrogen receptor, NFkappaB/IkappaB alpha, cAMP-response element-binding protein (CREB) and CREB-binding protein; (2) RSK is implicated in cell cycle regulation in Xenopus laevis oocytes by inactivation of the Myt1 protein kinase leading to activation of the cyclin-dependent kinase p34cdccogen synthase kinase-3; and (4) RSK phosphorylates the Ras GTP/GDP-exchange factor, Sos leading to feedback inhibition of the Ras-ERK pathway.

Previous studies have demonstrated that stress may increase prodynorphin gene expression, and kappa opioid agonists suppress drug reward. Therefore, we tested the hypothesis that stress-induced release of endogenous dynorphin may mediate behavioral responses to stress and oppose the rewarding effects of cocaine. <em>C</em>57Bl/6 mice subjected to repeated forced swim testing (FST) using a modified Porsolt procedure at 30 degrees <em>C</em> showed a characteristic stress-induced immobility response and a stress-induced analgesia observed with a tail withdrawal latency assay. Pretreatment with the kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI; 10 mg/kg, i.p.) blocked the stress-induced analgesia and significantly reduced the stress-induced immobility. The nor-BNI sensitivity of the behavioral responses suggests an activation of the kappa opioid receptor by a stress-induced release of dynorphin <em>peptides</em>. Supporting this hypothesis, transgenic mice possessing a disrupted prodynorphin gene showed no increase in immobility or stress-induced analgesia after exposure to repeated FST. Because both stress and the kappa opioid system can modulate the response to drugs of abuse, we tested the effects of forced swim stress on cocaine-conditioned place preference (<em>C</em>PP). FST-exposed mice conditioned with cocaine (15 mg/kg, s.c.) showed significant potentiation of place preference for the drug-paired chamber over the responses of unstressed mice. Surprisingly, nor-BNI pretreatment blocked stress-induced potentiation of cocaine <em>C</em>PP. <em>C</em>onsistent with this result, mice lacking the prodynorphin gene did not show a stress-induced potentiation of cocaine <em>C</em>PP, whereas wild-type littermates did. The findings suggest that chronic swim stress may activate the kappa opioid system to produce analgesia, immobility, and potentiation of the acute rewarding properties of cocaine in <em>C</em>57Bl/6 mice.

Neuronal death in cerebral ischemia is largely due to excitotoxic mechanisms, which are known to activate the c-Jun N-terminal kinase (JNK) pathway. We have evaluated the neuroprotective power of a cell-penetrating, protease-resistant peptide that blocks the access of JNK to many of its targets. We obtained strong protection in two models of middle cerebral artery occlusion (MCAO): transient occlusion in adult mice and permanent occlusion in 14-d-old rat pups. In the first model, intraventricular administration as late as 6 h after occlusion reduced the lesion volume by more than 90% for at least 14 d and prevented behavioral consequences. In the second model, systemic delivery reduced the lesion by 78% and 49% at 6 and 12 h after ischemia, respectively. Protection correlated with prevention of an increase in c-Jun activation and c-Fos transcription. In view of its potency and long therapeutic window, this protease-resistant peptide is a promising neuroprotective agent for stroke.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity.

Homopolymers or peptides containing a high percentage of cationic amino acids have been shown to have a unique ability to cross the plasma membrane of cells, and consequently have been used to facilitate the uptake of a variety of biopolymers and small molecules. To investigate whether the polycationic character of these molecules, or some other structural feature, was the molecular basis for the effect, the ability of a variety of homopolymers to enter cells was assayed by confocal microscopy and flow cytometry. Polymers of L- or D-arginine containing six or more amino acids entered cells far more effectively than polymers of equal length composed of lysine, ornithine and histidine. Peptides of fewer than six amino acids were ineffective. The length of the arginine side-chain could be varied without significant loss of activity. These data combined with the inability of polymers of citrulline to enter cells demonstrated that the guanidine headgroup of arginine was the critical structural component responsible for the biological activity. Cellular uptake could be inhibited by preincubation of the cells with sodium azide, but not by low temperature (3 degrees C), indicating that the process was energy dependent, but did not involve endocytosis.

The peptide binding specificities of HLA-DRB1*0401, DRB1*0101, and DRB1*0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1*0101, DRB1*0401, and DRB1*0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1*0101, DRB1*0401, DRB1*0701, DRB5*0101, DRB1*1501, DRB1*0901, and DRB1*1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.

OBJECTIVE

AKT signaling is frequently deregulated in human cancers. MK-2206 is a potent, oral allosteric inhibitor of all AKT isoforms with antitumor activity in preclinical models. A phase I study of MK-2206 was conducted to investigate its safety, maximum-tolerated dose (MTD), pharmacokinetics (PKs), pharmacodynamics (PDs), and preliminary antitumor efficacy.

METHODS

Patients with advanced solid tumors received MK-2206 on alternate days. Paired tumor biopsies were mandated at the MTD for biomarker studies. PD studies incorporated tumor and hair follicle analyses, and putative predictive biomarker studies included tumor somatic mutation analyses and immunohistochemistry for phosphatase and tensin homolog (PTEN) loss.

RESULTS

Thirty-three patients received MK-2206 at 30, 60, 75, or 90 mg on alternate days. Dose-limiting toxicities included skin rash and stomatitis, establishing the MTD at 60 mg. Drug-related toxicities included skin rash (51.5%), nausea (36.4%), pruritus (24.2%), hyperglycemia (21.2%), and diarrhea (21.2%). PKs (area under the concentration-time curve from 0 to 48 hours and maximum measured plasma concentration) were dose proportional. Phosphorylated serine 473 AKT declined in all tumor biopsies assessed (P = .015), and phosphorylated threonine 246 proline-rich AKT substrate 40 was suppressed in hair follicles at 6 hours (P = .008), on days 7 (P = .028) and 15 (P = .025) with MK-2206; reversible hyperglycemia and increases in insulin c-peptide were also observed, confirming target modulation. A patient with pancreatic adenocarcinoma (PTEN loss; KRAS G12D mutation) treated at 60 mg on alternate days experienced a decrease of approximately 60% in cancer antigen 19-9 levels and 23% shrinkage in tumor measurements. Two patients with pancreatic neuroendocrine tumors had minor tumor responses.

CONCLUSIONS

MK-2206 was well tolerated, with evidence of AKT signaling blockade. Rational combination trials are ongoing to maximize clinical benefit with this therapeutic strategy.

Glucagon-like peptide 1 (GLP-1) (7-36 amide) is a physiological incretin hormone that is released after nutrient intake from the lower gut and stimulates insulin secretion at elevated plasma glucose concentrations. Previous work has shown that even in Type 2 (non-insulin-dependent) diabetic patients GLP-1 (7-36 amide) retains much of its insulinotropic action. However, it is not known whether the magnitude of this response is sufficient to normalize plasma glucose in Type 2 diabetic patients with poor metabolic control. Therefore, in 10 Type 2 diabetic patients with unsatisfactory metabolic control (HbA1c 11.6 +/- 1.7%) on diet and sulphonylurea therapy (in some patients supplemented by metformin or acarbose), 1.2 pmol x kg-1 x min-1 GLP-1 (7-36 amide) or placebo was infused intravenously in the fasting state (plasma glucose 13.1 +/- 0.6 mmol/l). In all patients, insulin (by 17.4 +/- 4.7 nmol x 1-1 x min; p = 0.0157) and C-peptide (by 228.0 +/- 39.1 nmol x 1-1 x min; p = 0.0019) increased significantly over basal levels, glucagon was reduced (by -1418 +/- 308 pmol x 1-1 x min) and plasma glucose reached normal fasting concentrations (4.9 +/- 0.3 mmol/l) within 4 h of GLP-1 (7-36 amide) administration, but not with placebo. When normal fasting plasma glucose concentrations were reached insulin returned towards basal levels and plasma glucose concentrations remained stable despite the ongoing infusion of GLP-1 (7-36 amide). Therefore, exogenous GLP-1 (7-36 amide) is an effective means of normalizing fasting plasma glucose concentrations in poorly-controlled Type 2 diabetic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

During differentiation, B lymphocytes undergo a shift from expression of membrane-bound IgM to IgM secretion. The mu chains of membrane and secreted IgM, mum and mus, respectively, differ in the amino acid sequence of their carboxy terminal regions. In this paper, we demonstrate that mum and mus heavy chains are encoded by separate mRNAs of 2.7 and 2.4 kb, respectively. Restriction mapping and sequence analysis of mu cDNA clones from a myeloma tumor that produces both types of mu chain indicate that the mum and mus mRNAs are identical throughout the coding region up to the 3' end of the fourth constant region (Cmu 4) domain, but differ in their C terminal coding and 3' untranslated segments. From the nucleotide sequence of the mum cDNA clone, we predict the amino acid sequence of the 41-residue mum C terminal segment or "M" (membrane) segment. This sequence has characteristics consistent with its being a transmembrane peptide. Thus the mus chain has a 20-residue hydrophilic C terminal segment after the Cmu 4 domain, and the mum chain has a 41-residue C terminal segment containing a hydrophobic sequence. We propose that comparable C terminal segments also will be found in other membrane-bound immunoglobulin heavy chains.

pE194 is a small plasmid (isolated originally in Staphylococcus aureus) which confers erythromycin-inducible resistance to macrolide, lincosamide, and streptogramin type B (MLS) antibiotics. The nucleotide sequence of pE194 contains 3,728 base pairs (bp), corresponding to a molecular mass of 2.4 million daltons. By means of site-specific cleavage with restriction endonucleases and cloning resultant fragments, determinants of the two major biological functions of p E194, i.e., inducible MLS resistance and replication, could be localized and assigned to specific sequences in the plasmid. Restriction endonuclease TaqI cut pE194 at three sites. TaqI fragment A (1,443 bp) contained the determinant for inducible MLS resistance, whereas TaqI fragment B (1,354 bp) contained a determinant necessary for plasmid replication. Regulatory mutations resulting in constitutive expression of MLS resistance mapped in TaqI fragment A, whereas a mutation associated with elevated plasmid copy number was mapped in TaqI fragment B. Also mapping in TaqI fragment B was a plasmid replication determinant comprising two sets of inverted complementary repeat sequences, one of which spanned 124 bp and was adjacent to a second smaller set which was rich in guanine and cytosine residues. pE194 contained six open reading frames which were theoretically capable of coding for proteins with maximum molecular masses as follows (in daltons): A, 48,300; B, 29,200; C, 14,000; D, 13,900; E, 12,600; and F, 2,700. Insertion of plasmid pBR322 into the single PstI site located in frame A of pE194 resulted in a composite plasmid which could replicate in both Bacillus subtilis and Escherichia coli, suggesting that an intact polypeptide A is dispensable for both replication of pE194 and for MLS resistance. Frame B specified inducible MLS resistance, whereas frame F specified the putative peptide associated with the proposed B determinant translational attenuator. The extent to which frames C, D, and E, all contained in TaqI fragment B, were translated into polypeptide products is not known; however, a base change in frame E was found in a comparison between the high-copy-number mutant, cop-6, and the wild-type strains.

New parameter sets of the GROMOS biomolecular force field, 54A7 and 54B7, are introduced. These parameter sets summarise some previously published force field modifications: The 53A6 helical propensities are corrected through new φ/ψ torsional angle terms and a modification of the N-H, C=O repulsion, a new atom type for a charged -CH(3) in the choline moiety is added, the Na(+) and Cl(-) ions are modified to reproduce the free energy of hydration, and additional improper torsional angle types for free energy calculations involving a chirality change are introduced. The new helical propensity modification is tested using the benchmark proteins hen egg-white lysozyme, fox1 RNA binding domain, chorismate mutase and the GCN4-p1 peptide. The stability of the proteins is improved in comparison with the 53A6 force field, and good agreement with a range of primary experimental data is obtained.

HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anti-coagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay-based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.