Screening of phage libraries expressing random peptides for binding to prostate cancer cells primarily yielded peptides that had a C-terminal arginine (or rarely lysine) residue, usually in a consensus context R/KXXR/K. Phage expressing these sequences and synthetic nanoparticles coated with them bound to and were internalized into cells. The C-terminal arginine (or lysine) was essential to the activity; adding another amino acid, or even blocking the free carboxyl group of this arginine residue by amidation, eliminated the binding and internalizing activity. An internal R/KXXR/K can be exposed and switched on by a cleavage by a protease. The strict requirement for C-terminal exposure of the motif prompted us to term the phenomenon the C-end rule (CendR). Affinity chromatography showed that the CendR peptides bind to neuropilin-1 (NRP-1) on the target cells. NRP-1 is a cell-surface receptor that plays an essential role in angiogenesis, regulation of vascular permeability, and the development of the nervous system. VEGF-A165 and other ligands of NRP-1 possess a C-terminal CendR sequence that interacts with the b1 domain of NRP-1 and causes cellular internalization and vascular leakage. Our CendR peptides have similar effects, particularly when made multivalent through coupling to a particle. We also noted a unique and important activity of these peptides: penetration and transportation through tissues. The peptides were able to take payloads up to the nanoparticle size scale deep into extravascular tissue. Our observations have implications in drug delivery and penetration of tissue barriers and tumors.

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes.

Antimicrobial peptides (AMPs) are an important component of the natural defences of most living organisms against invading pathogens. These are relatively small (< 10kDa), cationic and amphipathic peptides of variable length, sequence and structure. During the past two decades several AMPs have been isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. Most of these peptides are obtained from different sources like macrophages, neutrophils, epithelial cells, haemocytes, fat body, reproductive tract, etc. These peptides exhibit broad-spectrum activity against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, protozoa, yeast, fungi and viruses. A few peptides have also been found to be cytotoxic to sperm and tumour cells. AMPs are classified based on the three dimensional structural studies carried out with the help of NMR. The peptides are broadly classified into five major groups namely (a) peptides that form alpha-helical structures, (b) peptides rich in cysteine residues, (c) peptides that form beta-sheet, (d) peptides rich in regular amino acids namely histatin, arginine and proline and (e) peptides composed of rare and modified amino acids. Most of these peptides are believed to act by disrupting the plasma membrane leading to the lysis of the cell. AMPs have been found to be excellent candidates for developing novel antimicrobial agents and a few of these peptides show antimicrobial activity against pathogens causing sexually transmitted infection (STI), including HIV/HSV. Peptides, namely magainin and nisin have been shown to demonstrate contraceptive properties in vitro and in vivo. A few peptides have already entered clinical trials for the treatment of impetigo, diabetic foot ulcers and gastric helicobacter infections. In this review, we discuss the source, structures and mode of action with special reference to therapeutic considerations of various AMPs.

An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design. We report here the 2.5 angstrom resolution X-ray crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator peptide. The protease has a chymotrypsin-like fold and features a tetrahedrally coordinated metal ion distal to the active site. The NS4A peptide intercalates within a beta sheet of the enzyme core.

Membrane phosphoinositides control a variety of cellular processes through the recruitment and/or regulation of cytosolic proteins. One mechanism ensuring spatial specificity in phosphoinositide signalling is the targeting of enzymes that mediate their metabolism to specific subcellular sites. Phosphatidylinositol phosphate kinase type 1 gamma (PtdInsPKI gamma) is a phosphatidylinositol-4-phosphate 5-kinase that is expressed at high levels in brain, and is concentrated at synapses. Here we show that the predominant brain splice variant of PtdInsPKI gamma (PtdInsPKI gamma-90) binds, by means of a short carboxy-terminal peptide, to the FERM domain of talin, and is strongly activated by this interaction. Talin, a principal component of focal adhesion plaques, is also present at synapses. PtdInsPKI gamma-90 is expressed in non-neuronal cells, albeit at much lower levels than in neurons, and is concentrated at focal adhesion plaques, where phosphatidylinositol-4,5-bisphosphate has an important regulatory role. Overexpression of PtdInsPKI gamma-90, or expression of its C-terminal domain, disrupts focal adhesion plaques, probably by local disruption of normal phosphoinositide balance. These findings define an interaction that has a regulatory role in cell adhesion and suggest new similarities between molecular interactions underlying synaptic junctions and general mechanisms of cell adhesion.

BACKGROUND

The 65-kD isoform of glutamic acid decarboxylase (GAD) is a major autoantigen in patients with type 1 diabetes mellitus. This trial assessed the ability of alum-formulated GAD (GAD-alum) to reverse recent-onset type 1 diabetes in patients 10 to 18 years of age.

METHODS

We randomly assigned 70 patients with type 1 diabetes who had fasting C-peptide levels above 0.1 nmol per liter (0.3 ng per milliliter) and GAD autoantibodies, recruited within 18 months after receiving the diagnosis of diabetes, to receive subcutaneous injections of 20 microg of GAD-alum (35 patients) or placebo (alum alone, 35 patients) on study days 1 and 30. At day 1 and months 3, 9, 15, 21, and 30, patients underwent a mixed-meal tolerance test to stimulate residual insulin secretion (measured as the C-peptide level). The effect of GAD-alum on the immune system was also studied.

RESULTS

Insulin secretion gradually decreased in both study groups. The study treatment had no significant effect on change in fasting C-peptide level after 15 months (the primary end point). Fasting C-peptide levels declined from baseline levels significantly less over 30 months in the GAD-alum group than in the placebo group (-0.21 vs. -0.27 nmol per liter [-0.62 vs. -0.81 ng per milliliter], P=0.045), as did stimulated secretion measured as the area under the curve (-0.72 vs. -1.02 nmol per liter per 2 hours [-2.20 vs. -3.08 ng per milliliter per 2 hours], P=0.04). No protective effect was seen in patients treated 6 months or more after receiving the diagnosis. Adverse events appeared to be mild and similar in frequency between the two groups. The GAD-alum treatment induced a GAD-specific immune response.

CONCLUSIONS

GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent-onset type 1 diabetes, although it did not change the insulin requirement. (ClinicalTrials.gov number, NCT00435981.)

Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.

The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.

The major structural elements of retroviruses are contained in a single polyprotein, Gag, which in human immunodeficiency virus type 1 (HIV-1) comprises the MA, CA, spacer peptide 1 (SP1), NC, SP2, and p6 polypeptides. In the immature HIV-1 virion, the domains of Gag are arranged radially with the N-terminal MA domain at the membrane and C-terminal NC-SP2-p6 region nearest to the center. Here, we report the three-dimensional structures of individual immature HIV-1 virions, as obtained by electron cryotomography. The concentric shells of the Gag polyprotein are clearly visible, and radial projections of the different Gag layers reveal patches of hexagonal order within the CA and SP1 shells. Averaging well-ordered unit cells leads to a model in which each CA hexamer is stabilized by a bundle of six SP1 helices. This model suggests why the SP1 spacer is essential for assembly of the Gag lattice and how cleavage between SP1 and CA acts as a structural switch controlling maturation.

Cilia play an essential role in protecting the respiratory tract by providing the force necessary for mucociliary clearance. Although the major structural components of human cilia have been described, a complete understanding of cilia function and regulation will require identification and characterization of all ciliary components. Estimates from studies of Chlamydomonas flagella predict that an axoneme contains>> or = 250 proteins. To identify all the components of human cilia, we have begun a comprehensive proteomic analysis of isolated ciliary axonemes. Analysis by two-dimensional (2-D) PAGE resulted in a highly reproducible 2-D map consisting of over 240 well resolved components. Individual protein spots were digested with trypsin and sequenced using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Peptide matches were obtained to 38 potential ciliary proteins by this approach. To identify ciliary components not resolved by 2-D PAGE, axonemal proteins were separated on a one-dimensional gel. The gel lane was divided into 45 individual slices, each of which was analyzed by LC/MS/MS. This experiment resulted in peptide matches to an additional 110 proteins. In a third approach, preparations of isolated axonemes were digested with Lys-C, and the resulting peptides were analyzed directly by LC/MS/MS or by multidimensional LC/MS/MS, leading to the identification of a further 66 proteins. Each of the four approaches resulted in the identification of a subset of the proteins present. In total, sequence data were obtained on over 1400 peptides, and over 200 potential axonemal proteins were identified. Peptide matches were also obtained to over 200 human expressed sequence tags. As an approach to validate the mass spectrometry results, additional studies examined the expression of several identified proteins (annexin I, sperm protein Sp17, retinitis pigmentosa protein RP1) in cilia or ciliated cells. These studies represent the first proteomic analysis of the human ciliary axoneme and have identified many potentially novel components of this complex organelle.

BACKGROUND

Islet allografts from 2 to 4 donors can reverse type 1 diabetes. However, for islet transplants to become a widespread clinical reality, diabetes reversal must be achieved with a single donor to reduce risks and costs and increase the availability of transplantation.

OBJECTIVE

To assess the safety of a single-donor, marginal-dose islet transplant protocol using potent induction immunotherapy and less diabetogenic maintenance immunosuppression in recipients with type 1 diabetes. A secondary objective was to assess the proportion of islet transplant recipients who achieve insulin independence in the first year after single-donor islet transplantation.

METHODS

Prospective, 1-year follow-up trial conducted July 2001 to August 2003 at a single US center and enrolling 8 women with type 1 diabetes accompanied by recurrent hypoglycemia unawareness or advanced secondary complications.

METHODS

Study participants underwent a primary islet allotransplant with 7271 (SD, 1035) islet equivalents/kg prepared from a single cadaver donor pancreas. Induction immunosuppression was with antithymocyte globulin, daclizumab, and etanercept. Maintenance immunosuppression consisted of mycophenolate mofetil, sirolimus, and no or low-dose tacrolimus.

METHODS

Safety (assessed by monitoring the severity and duration of adverse events) and efficacy (assessed by studying the recipients' insulin requirements, C-peptide levels, oral and intravenous glucose tolerance results, intravenous arginine stimulation responses, glycosylated hemoglobin levels, and hypoglycemic episodes) associated with the study transplant protocol.

RESULTS

There were no serious, unexpected, or procedure- or immunosuppression-related adverse events. All 8 recipients achieved insulin independence and freedom from hypoglycemia. Five remained insulin-independent for longer than 1 year. Graft failure in 3 recipients was preceded by subtherapeutic sirolimus exposure in the absence of measurable tacrolimus trough levels.

CONCLUSIONS

The tested transplant protocol restored insulin independence and protected against hypoglycemia after single-donor, marginal-dose islet transplantation in 8 of 8 recipients. These results may be related to improved islet engraftment secondary to peritransplant administration of antithymocyte globulin and etanercept. These findings may have implications for the ongoing transition of islet transplantation from clinical investigation to routine clinical care.

All plus-strand RNA viruses replicate in association with cytoplasmic membranes of infected cells. The RNA replication complex of many virus families is associated with the endoplasmic reticulum membranes, for example, picorna-, flavi-, arteri-, and bromoviruses. However, endosomes and lysosomes (togaviruses), peroxisomes and chloroplasts (tombusviruses), and mitochondria (nodaviruses) are also used as sites for RNA replication. Studies of individual nonstructural proteins, the virus-specific components of the RNA replicase, have revealed that the replication complexes are associated with the membranes and targeted to the respective organelle by the ns proteins rather than RNA. Many ns proteins have hydrophobic sequences and may transverse the membrane like polytopic integral membrane proteins, whereas others interact with membranes monotopically. Hepatitis C virus ns proteins offer examples of polytopic transmembrane proteins (NS2, NS4B), a "tip-anchored" protein attached to the membrane by an amphipathic alpha-helix (NS5A) and a "tail-anchored" posttranslationally inserted protein (NS5B). Semliki Forest virus nsP1 is attached to the plasma membrane by a specific binding peptide in the middle of the protein, which forms an amphipathic alpha-helix. Interaction of nsP1 with membrane lipids is essential for its capping enzyme activities. The other soluble replicase proteins are directed to the endo-lysosomal membranes only as part of the initial polyprotein. Poliovirus ns proteins utilize endoplasmic reticulum membranes from which vesicles are released in COPII coats. However, these vesicles are not directed to the normal secretory pathway, but accumulate in the cytoplasm. In many cases the replicase proteins induce membrane invaginations or vesicles, which function as protective environments for RNA replication.

Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear-encoded mitochondrial chaperone genes in C. elegans (UPR(mt)). Here, we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPR(mt) and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP dependent and required HAF-1 and the protease ClpP. Defective UPR(mt) signaling in the haf-1-deleted worms was associated with failure of the bZIP protein, ZCClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPR(mt) signaling across the mitochondrial inner membrane.

Studies conducted in many laboratories over the past several years have resulted in the identification and initial characterization of a large superfamily of structurally and functionally related inflammatory cytokines. This superfamily currently includes 14 distinct members: platelet factor 4, beta-thromboglobulin, neutrophil activating peptide-1/interleukin-8, gro, IP-10, mig, ENA-78, macrophage inflammatory proteins-1 alpha and -1 beta, monocyte chemoattractant protein-1/JE, RANTES, HC-14, C-10, and I-309. Although numerous biological activities have been assigned to these molecules, a common theme is their ability to stimulate the chemotactic migration of distinct sets of cells, including neutrophils, monocytes, lymphocytes, and fibroblasts. Accumulating evidence indicates that these molecules play important roles in mediating cell recruitment and activation necessary for inflammation and the repair of tissue damage.

Changes of bone turnover with aging are responsible for bone loss and play a major role in osteoporosis. Although an increase of bone turnover has been documented at the time of menopause, the subsequent abnormalities of bone resorption and formation and their potential role in determining bone mass in the elderly have not been investigated. To address this issue, we have measured a battery of new sensitive and specific markers of bone turnover in a population-based study of 653 healthy women analyzed cross-sectionally, including 432 women postmenopausal from 1 to 40 years, and the data were correlated with bone mineral density (BMD) measured by dual-energy X-ray absorptiometry (DXA) at different skeletal sites. Bone formation was assessed by serum osteocalcin (OC), serum bone-specific alkaline phosphatase (B-ALP), serum C-propeptide of type I collagen (PICP), and bone resorption by the urinary excretion of two pyridinoline cross-linked peptides (NTX and CTX). Bone turnover increased in perimenopausal women with both irregular menses and elevated serum follicle stimulating hormone (FSH). Menopause induced a 37-52% and 79-97% increase in the bone formation and bone resorption marker levels, respectively (p < 0.0001 except for PICP). In postmenopausal women, bone formation markers did not decrease with age. When resorption markers were corrected by whole body bone mineral content (BMC), the fraction of bone resorbed per day was not correlated with age in postmenopausal women and remained elevated for up to 40 years after menopause. In premenopausal women, the bone turnover rate accounted for only 0-10% of the variation in whole body BMC, total hip, distal radius, and lumbar spine BMD. With increasing time after menopause, the importance of the bone turnover rate as a determinant of bone mass increased at all sites and accounted for up to 52% of the BMD variance in elderly women. Thus, in women 20 years or more postmenopause, bone turnover was higher in those in the lowest quartile than in those in the highest quartile of BMD. In elderly women, 20 years since menopause and over, but not in younger ones, serum PTH was negatively correlated with serum 25-hydroxyvitamin D (r = -0.22, p < 0.05) and explained only 5-8% of the bone turnover variance (p < 0.01-0.001). These data indicate that the overall rates of both bone formation and bone resorption remain high in elderly women. The rate of bone turnover appears to play an increasing role as a determinant of bone mass with increasing time since menopause with a high bone turnover rate being associated with a low bone mass. Thus assessing bone marker levels may be useful in the evaluation of osteoporosis risk. In elderly women, secondary hyperparathyroidism caused in part by reduced serum 25-hydroxyvitamin D appears to be a marginal determinant of an increased bone turnover rate.

OBJECTIVE

To determine associations of gestational diabetes mellitus (GDM) and obesity with adverse pregnancy outcomes in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study.

METHODS

Participants underwent a 75-g oral glucose tolerance test (OGTT) between 24 and 32 weeks. GDM was diagnosed post hoc using International Association of Diabetes and Pregnancy Study Groups criteria. Neonatal anthropometrics and cord serum C-peptide were measured. Adverse pregnancy outcomes included birth weight, newborn percent body fat, and cord C-peptide >90th percentiles, primary cesarean delivery, preeclampsia, and shoulder dystocia/birth injury. BMI was determined at the OGTT. Multiple logistic regression was used to examine associations of GDM and obesity with outcomes.

RESULTS

Mean maternal BMI was 27.7, 13.7% were obese (BMI ≥33.0 kg/m(2)), and GDM was diagnosed in 16.1%. Relative to non-GDM and nonobese women, odds ratio for birth weight >90th percentile for GDM alone was 2.19 (1.93-2.47), for obesity alone 1.73 (1.50-2.00), and for both GDM and obesity 3.62 (3.04-4.32). Results for primary cesarean delivery and preeclampsia and for cord C-peptide and newborn percent body fat >90th percentiles were similar. Odds for birth weight >90th percentile were progressively greater with both higher OGTT glucose and higher maternal BMI. There was a 339-g difference in birth weight for babies of obese GDM women, compared with babies of normal/underweight women (64.2% of all women) with normal glucose based on a composite OGTT measure of fasting plasma glucose and 1- and 2-h plasma glucose values (61.8% of all women).

CONCLUSIONS

Both maternal GDM and obesity are independently associated with adverse pregnancy outcomes. Their combination has a greater impact than either one alone.

We report the isolation of three full-length cDNAs corresponding to the mRNAs of closely related glutathione S-transferase (GST) Pi genes, designated hGSTP1*A, hGSTP1*B, and hGSTP1*C, expressed in normal cells and malignant gliomas. The variant cDNAs result from A ->> G and C ->> T transitions at nucleotides +313 and +341, respectively. The transitions changed codon 104 from ATC (Ile) in hGSTP1*A to GTC (Val) in hGSTP1*B and hGSTP1*C and changed codon 113 from GCG (Ala) to GTG (Val) in hGSTP1*C. Both amino changes are in the electrophile-binding active site of the GST Pi peptide. Computer modeling of the deduced crystal structures of the encoded peptides showed significant deviations in the interatomic distances of critical electrophile-binding active site amino acids as a consequence of the amino acid changes. The encoded proteins expressed in Escherichia coli and purified by GSH affinity chromatography showed a 3-fold lower Km (CDNB) and a 3-4-fold higher Kcat/Km for the hGSTP1*A encoded protein than the proteins encoded by hGSTP1*B and hGSTP1*C. Analysis of 75 cases showed the relative frequency of hGSTP1*C to be 4-fold higher in malignant gliomas than in normal tissues. These data provide conclusive molecular evidence of allelopolymorphism of the human GST Pi gene locus, resulting in active, functionally different GST Pi proteins, and should facilitate studies of the role of this gene in xenobiotic metabolism, cancer, and other human diseases.

We have isolated about 30 to 40 different BALB/c mouse sperm DNA genomic clones that hybridize to cDNA clones encoding proteins homologous to transplantation antigens. One of these clones (27.1) was selected for sequence because it was polymorphic in Southern blot analysis of the DNAs from BALB/c and CBA mice. A fragment of 5.7 kilobases of this clone was completely sequenced and found to contain a pseudogene whose sequence is highly homologous to the sequences of known transplantation antigens. Pseudogene 27.1 is split into eight exons that correlate with the structurally defined protein domains of transplantation antigens. Using Southern blot hybridization on the DNAs of different inbred mouse strains, we mapped the pseudogene to the Qa-2,3 region, a part of the Tla complex on chromosome 17 that is adjacent to the major histocompatibility complex. The Qa2,3 region encodes lymphoid differentiation antigens homologous to the transplantation antigens in size, in peptide map profiles and in their association with beta2-microglobulin. These mapping studies suggest that gene 27.1 may be a pseudogene for eigher a Qa antigen or an as yet undefined transplantation antigen. Accordingly, we may have isolate genes encoding lymphoid differentiation antigens of the Tla complex as well as those encoding transplantation antigens among the 30 to 40 different genomic clones isolated from our sperm library.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

Many innate immune mechanisms are conserved throughout the animal kingdom. Manduca sexta, a widely used model for insect biochemical research, employs these mechanisms to defend against invading pathogens and parasites. We have isolated from M. sexta hemolymph a group of proteins (hemolin, peptidoglycan recognition proteins, beta-1,3-glucan recognition proteins, and C-type lectins), which serve as a surveillance mechanism by binding to microbial surface molecules (e.g. peptidoglycan, lipopolysaccharide, lipoteichoic acid, and beta-1,3-glucan). The binding triggers diverse responses such as phagocytosis, nodule formation, encapsulation, melanization, and synthesis of anti-microbial peptides/proteins. Some of these responses are mediated and coordinated by serine proteinase cascades, analogous to the complement system in mammals. Our current research is focused on the proteolytic activation of prophenoloxidase (proPO)--a reaction implicated in melanotic encapsulation, wound healing, and protein cross-linking. We have isolated three proPO-activating proteinases, each of which requires serine proteinase homologs as a cofactor for generating active phenoloxidase. The proteinases and proteinase-like molecules, containing one to two clip domains at their amino-terminus, are acute-phase proteins induced upon an immune challenge. Inhibitory regulation of the proteinases by serpins and association of the proteinase homologs with a bacteria-binding lectin are important for ensuring a localized defense response. Additional serine proteinases expressed in M. sexta hemocytes and fat body have been discovered. Future research efforts will be aimed at elucidating the proteinase cascade for proPO activation and investigating the roles of proteinases in other immune responses such as processing of plasmatocyte-spreading peptide.

Inability to form new memories is an early clinical sign of Alzheimer's disease (AD). There is ample evidence that the amyloid-beta (Abeta) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of Abeta are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of Abeta-mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic Abeta(1-42) oligomers impaired consolidation of the long-term recognition memory, whereas mature Abeta(1-42) fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-Abeta antibody. It has been suggested that the cellular prion protein (PrP(C)) mediates the impairment of synaptic plasticity induced by Abeta. We confirmed that Abeta(1-42) oligomers interact with PrP(C), with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that Abeta(1-42) oligomers are responsible for cognitive impairment in AD and that PrP(C) is not required.

Structures of prM-containing dengue and yellow fever virus particles were determined to 16 and 25 A resolution, respectively, by cryoelectron microscopy and image reconstruction techniques. The closely similar structures show 60 icosahedrally organized trimeric spikes on the particle surface. Each spike consists of three prM:E heterodimers, where E is an envelope glycoprotein and prM is the precursor to the membrane protein M. The pre-peptide components of the prM proteins in each spike cover the fusion peptides at the distal ends of the E glycoproteins in a manner similar to the organization of the glycoproteins in the alphavirus spikes. Each heterodimer is associated with an E and a prM transmembrane density. These transmembrane densities represent either an EE or prMprM antiparallel coiled coil by which each protein spans the membrane twice, leaving the C-terminus of each protein on the exterior of the viral membrane, consistent with the predicted membrane-spanning domains of the unprocessed polyprotein.

Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to beta cells. Generation of glucose-responsive, insulin-producing beta cells from self-renewing, pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol, hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Ptf1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development, and the final population contained representatives of the ductal, exocrine, and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells, as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/mug of DNA was measured in the ILCs, representing levels higher than that of human fetal islets. In addition, the hESC-derived ILCs contained numerous secretory granules, as determined by electron microscopy, and secreted human C-peptide in a glucose-dependent manner. Disclosure of potential conflicts of interest is found at the end of this article.