Single-stage cell-based cartilage repair in a rabbit model: cell tracking and in vivo chondrogenesis of human umbilical cord blood-derived mesenchymal stem cells and hyaluronic acid hydrogel composite.

OBJECTIVE: Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have gained popularity as a promising cell source for regenerative medicine, but limited in vivo studies have reported cartilage repair. In addition, the roles of MSCs in cartilage repair are not well-understood. The purpose of this study was to investigate the feasibility of transplanting hUCB-MSCs and hyaluronic acid (HA) hydrogel composite to repair articular cartilage defects in a rabbit model and determine whether the transplanted cells persisted or disappeared from the defect site. DESIGN: Osteochondral defects were created in the trochlear grooves of the knees. The hUCB-MSCs and HA composite was transplanted into the defect of experimental knees. Control knees were transplanted by HA or left untreated. Animals were sacrificed at 8 and 16 weeks post-transplantation and additionally at 2 and 4 weeks to evaluate the fate of transplanted cells. The repair tissues were evaluated by gross, histological and immunohistochemical analysis. RESULTS: Transplanting hUCB-MSCs and HA composite resulted in overall superior cartilage repair tissue with better quality than HA alone or no treatment. Cellular architecture and collagen arrangement at 16 weeks were similar to those of surrounding normal articular cartilage tissue. Histological scores also revealed that cartilage repair in experimental knees was better than that in control knees. Immunohistochemical analysis with anti-human nuclear antibody confirmed that the transplanted MSCs disappeared gradually over time. CONCLUSION: Transplanting hUCB-MSCs and HA composite promote cartilage repair and interactions between hUCB-MSCs and host cells initiated by paracrine action may play an important role in cartilage repair.

Inhibition of the multidrug and toxin extrusion (MATE) transporter by pyrimethamine increases the plasma concentration of metformin but does not increase antihyperglycaemic activity in humans.

We hypothesized that the pharmacodynamic (PD) characteristics of metformin would change with inhibition of the multidrug and toxin extrusion (MATE) transporter, which mediates renal elimination of metformin. Twenty healthy male subjects received two doses (750/500 mg) of metformin, with and without 50 mg of pyrimethamine (a potent MATE inhibitor), with 1 week of washout in between each dose. The PD characteristics of metformin were assessed using oral glucose tolerance tests (OGTTs) before and after the metformin dose. Metformin concentrations in plasma and urine were determined using liquid chromatography-electrospray ionization-tandem mass spectrometry. When metformin was co-administered with pyrimethamine, its area under the concentration-time curve from 0 to 12 h was 2.58-fold greater (p < 0.05), whereas the antihyperglycaemic effects of metformin were decreased. The mean differences (90% confidence interval) in mean and maximum serum glucose concentrations and in 2-h-post-OGTT serum glucose concentration were -0.6 (-1, -0.2), -0.9 (-1.6, -0.3) and -0.5 (-1.1, 0.1) mmol/l, respectively. These findings indicate that the response to metformin is not only related to the plasma exposure of metformin but is also related to other factors, such as inhibition of uptake transporters and the gastrointestinal-based pharmacology of metformin.

Nonmyeloablative conditioning generates autoantigen-encoding bone marrow that prevents and cures an experimental autoimmune disease.

Autoimmune diseases result from chronic targeted immune responses that lead to tissue pathology and disease. The potential of autologous hematopoietic stem cells transplantation as a treatment for autoimmunity is currently being trialled but disease relapse is an issue. We have previously shown in a mouse model of experimental autoimmune encephalomyelitis (EAE) that the transplantation of bone marrow (BM) transduced to encode the autoantigen myelin oligodendrocyte glycoprotein (MOG) can prevent disease induction. However these studies were performed using lethal irradiation to generate BM chimeras and a critical factor for translation to humans would be the ability to utilize low toxic preconditioning regimes. In this study, treosulfan was used as a nonmyeloablative agent to generate BM chimeras encoding MOG and assessed in models of EAE induction and reversal. We find that treosulfan conditioning can promote a low degree of chimerism that is sufficient to promote antigen specific tolerance and protect mice from EAE. When incorporated into a curative protocol for treating mice with established EAE, nonmyeloablative conditioning and low chimerism was equally efficient in maintaining disease resistance. These studies further underpin the potential and feasibility of utilizing a gene therapy approach to treat autoimmune disease.

Pharmacodynamic comparison of two formulations of Acarbose 100-mg tablets.

WHAT IS KNOWN AND OBJECTIVE: Acarbose, an α-glycosidase inhibitor, is used to treat diabetic patients. Pharmacokinetic evaluation of acarbose is difficult because <2% is absorbed systemically. The current investigation evaluated the bioequivalence of two formulations of acarbose through pharmacodynamic comparison. METHODS: This investigation consisted of a pilot study and a main study. The pilot study had an open, single-dose, single-sequence design. Subjects received placebo and then two tablets of reference formulation (Glucobay(®) 100 mg tablet; Bayer Healthcare) on two consecutive days with sucrose. The main study was an open, randomized, two-period, two-sequence crossover study. Subjects randomly received placebo and two tablets of either test formulation (generic acarbose 100-mg tablet) or reference formulation with sucrose on two consecutive days in the first period. In the second period, placebo and alternative formulation were administered. Serial blood samples for pharmacodynamic assessment were taken after each administration. The maximum serum glucose concentration (G(max)) and the area under the serum glucose concentration-time profile (AUC(gluc)) were determined and compared. RESULTS AND DISCUSSION: Five subjects completed the pilot study. The AUC(gluc) from dosing until 1 h post-dose (AUC(gluc,1 h)) was significantly different between the placebo and acarbose. A total of 33 subjects completed the main study. The mean differences in G(max) (ΔG(max)) and AUC(gluc,1 h) (ΔAUC(gluc,1 h)) for the reference formulation compared with placebo were 22·0 ± 18·3 mg/dL and 928·2 ± 756·0 mg min/dL, respectively. The corresponding values for the test formulation were 23·3 ± 21·2 mg/dL and 923·0 ± 991·4 0 mg min/dL, respectively. The geometric mean ratios (GMRs) of the test formulation to the reference formulation for ΔG(max) and ΔAUC(gluc, 1 h) were 1·06 and 1·00, respectively, and the 90% confidence intervals (CIs) corresponding values were 0·79-1·39 and 0·64-1·36, respectively. WHAT IS NEW AND CONCLUSION: The 90% CIs of GMRs for the pharmacodynamic parameters chosen for bioequivalence evaluation of two formulations of acarbose did not meet the commonly accepted regulatory criteria for bioequivalence (0·80-1·25).

Efficacy of autologous serum in human adipose-derived stem cells; cell markers, growth factors and differentiation.

Human adipose-derived stem cells (hASCs) are a feasible source of stem cells for use in clinical applications. hASCs are typically cultured in medium containing fetal bovine serum (FBS); however, use of FBS is not recommended due to issues of clinical safety with regard to infections or immune response. Replacement of FBS with autologous human serum (autoHS) can eliminate these problems; however, their maintainability as potent ASCs in autoHS needs to be confirmed. Thus, we conducted an investigation of characterizations and functions of hASCs grown in medium containing autoHS compared to FBS. Cell counting and the WST-8 assay were used in assessment of the proliferation rate. In hASC cultured with culture medium plus autoHS or FBS, cell phenotypes were characterized by flow cytometry (CD13, CD29, CD31, CD34, and CD44) and expression of BDNF, HGF, IGF, LIF, NGF, and VEGF was determined by RT­PCR. Adipogenic differentiation was confirmed by oil red O stain. hASC showed greater expansion in AutoHS than in FBS. Cell surface markers of hASCs grown in autoHS (autoHS-hASCs) were similar to markers of those grown in FBS (FBS-hASCs). AutoHS-hASCs also expressed multiple growth factors as well as FBS-hASCs. In addition, autoHS was effective in growth of hASCs as well as FBS and autoHS-hASCs retained their ability for adipogenic differentiation. In summary, autoHS-hASCs have multiple growth factor expressions with the same cell surface markers as FBS­hASCs in vitro. Our results suggest that autoHS can provide sufficient ex vivo expansion of hASCs.

Calpain cleaved-55kDa N-terminal huntingtin delocalizes from neurons to astrocytes after ischemic injury.

The huntingtin (htt) mutation causes a polyglutamine expansion in the N-terminal region of protein. Mutant N-htt proteolytic fragments aggregate and cause cell death in Huntington's disease (HD). The normal huntingtin also can be cleaved by calpain and produce N-terminal htt fragments following ischemic injury, but the fate of cleaved fragment in dead neurons in the brain are unclear. To determine the localization of huntingtin following proteolysis, we examined htt expression after transient ischemic injury. Huntingtin immunoreactivity in mixed cultures of neuronal and astrocytes-derived clonal cells showed alteration of immunoreactivity from neurons into astrocytes. In the brain, both focal and global ischemia induced reactive astrocytes that were co-immunoreactive for huntingtin with elevated GFAP expression. The immunoreactive huntingtin was 55kDa calpain-cleaved N-terminal fragment, which appeared initially in the process, and extended into the cytoplasm of astrocytes. The results showed, after ischemic injury, huntingtin accumulated in astrocytes indicating that astrocytes may play a role in uptake of cleaved N-htt fragments.

Enhancing DNA vaccine potency by co-administration of xenogenic MHC class-I DNA.

Intramuscular administration of DNA vaccines can lead to the generation of antigen-specific immune responses through cross-priming mechanisms. We propose a strategy that is capable of leading to local inflammation and enhancing cross-priming, thus resulting in improved antigen-specific immune responses. Therefore, in this study, we evaluated the immunological responses elicited through electroporation-mediated intramuscular administration of a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 (CRT-E7) in combination with DNA expressing HLA-A2 as compared with CRT-E7 DNA vaccination alone. We found that the co-administration of a DNA vaccine in conjunction with a DNA encoding a xenogenic major histocompatibility complex (MHC) molecule could significantly enhance the E7-specific CD8+ T-cell immune responses and antitumor effects against an E7-expressing tumor, TC-1, in C57BL/6 tumor-bearing mice. Furthermore, a similar enhancement in E7-specific immune responses was observed by the co-administration of CRT-E7 DNA with DNA encoding other types of xenogenic MHC class-I molecules. This strategy was also applicable to another antigenic system, ovalbumin. Further characterization of the injection site revealed that the co-administration of HLA-A2 DNA led to a significant increase in the number of infiltrating CD8+ T lymphocytes and CD11b/c+ antigen-presenting cells. Furthermore, the E7-specific immune responses generated by intramuscular co-administration of CRT-E7 with HLA-A2 DNA were reduced in HLA-A2 transgenic mice. Thus, our data suggest that intramuscular co-administration of DNA encoding xenogenic MHC class-I can further improve the antigen-specific immune responses, as well as antitumor effects generated by DNA vaccines through enhancement of cross-priming mechanisms.

Wrinkling and strain softening in single-wall carbon nanotube membranes.

The nonlinear elasticity of thin supported membranes assembled from length purified single-wall carbon nanotubes is analyzed through the wrinkling instability that develops under uniaxial compression. In contrast with thin polymer films, pristine nanotube membranes exhibit strong softening under finite strain associated with bond slip and network fracture. We model the response as a shift in percolation threshold generated by strain-induced nanotube alignment in accordance with theoretical predictions.

Effects of SLCO1B1 and ABCB1 genotypes on the pharmacokinetics of atorvastatin and 2-hydroxyatorvastatin in healthy Korean subjects.

OBJECTIVE: This study aimed to evaluate the effect of genetic polymorphisms of SLCO1B1 and ABCB1 on the pharmacokinetics of atorvastatin and its metabolites. METHODS: 290 Koreans were genotyped for SLCO1B1, ABCB1 and CYP3A5, and 28 subjects were selected for the pharmacokinetic study. Each subject received a single oral dose of 20 mg atorvastatin and blood samples were collected up to 48 hr after dosing. The relationship between the genotypes and atorvastatin pharmacokinetics was examined. RESULTS: For SLCO1B1 genotypes, the mean area under the concentration-time curve from time 0 to infinity (AUC0-infinity) of atorvastatin was 148.2 ng x hr/ml for *15/*15 subjects (n = 3), which was significantly larger than for 1a/*15 and *1b/*15 (n = 8) (80.7 ng x hr/ml, p = 0.0121) and also larger than for *1a/*1a, *1a/*1b and *1b/*1b (n = 17) (66.3 ng x hr/ml, p = 0.0018). The mean AUC0-infinity of 2-hydroxyatorvastatin for *15/*15 was also larger than in *1a/*1a, *1a/*1b and *1b/*1b (p = 0.012). In lactone forms, no significant pharmacokinetic difference was found among the genotypes. For ABCB1 genotypes, the half-lives of atorvastatin, atorvastatin lactone, 2-hydroxyatorvastatin and 2-hydroxyatorvastatin lactone were significantly longer in c.2677TT-c.3435TT (n = 3) vs. c.2677GG-c.3435CC and c.2677GT-c.3435CT (n = 10), yielding p = 0.049, 0.007, 0.007 and 0.007, respectively. CONCLUSION: This study shows that the SLCO1B1*15 allele may be associated with the individual difference in the AUC0-infinity of atorvastatin whereas the ABCB1 TT-TT diplotype may affect the elimination half-life of the drug in the Korean population.

Isolated sarcoid granulomatous interstitial nephritis in pediatrics: a case report and review of literature.

Sarcoidosis is a multisystem disease of unknown etiology primarily affecting the lungs, skin, and lymph nodes. The disease usually manifests in young adults and is uncommon in childhood. Renal involvement, including granulomatous interstitial nephritis (GIN), is rare, and few cases of isolated sarcoid GIN have been reported in pediatrics. We report a case and review the literature.

Preferential activation and expansion of human peripheral blood gamma delta T cells in response to Toxoplasma gondii in vitro and their cytokine production and cytotoxic activity against T. gondii-infected cells.

Studies were conducted to determine if gamma delta T cells participate in the immune response to Toxoplasma gondii. Preferential expansion of human gamma delta T cells occurred when peripheral blood T cells from either T. gondii-seronegative or seropositive individuals were incubated with autologous PBMC infected with the parasite. That gamma delta T cells proliferated after incubation with infected cells was confirmed using purified of gamma delta T cells. These T. gondii-induced gamma delta T cell responses did not require prior exposure to the parasite since T cells obtained from umbilical cord blood from seronegative newborns also exhibited preferential expansion of gamma delta T cells. Cytofluorometric analysis of T cells obtained from either umbilical cord blood or peripheral blood from adults revealed that V gamma 9+ and V delta 2+ gamma delta T cells responded to stimulation with infected cells. Preferential expansion of gamma delta T cells was not restricted by polymorphic determinants of MHC molecules. PBMC that had internalized killed parasites but not PBMC incubated with T. gondii lysate antigens also stimulated preferential expansion and activation of gamma delta T cells as assessed by expression of CD25 and HLA-DR molecules. V gamma 9+V delta 2+ gamma delta T cells were cytotoxic for T. gondii-infected cells in an MHC-unrestricted manner, and produced IFN-gamma, IL-2, TNF-alpha, but not IL-4 when incubated with cells infected with the parasite. These results suggest that rapid induction of a remarkable primary gamma delta T cell response may be important in the early protective immune response to T. gondii.

Exploration of Biomarkers for Amoxicillin/Clavulanate-Induced Liver Injury: Multi-Omics Approaches.

To explore potential biomarkers for amoxicillin/clavulanate-induced liver injury (AC-DILI), we conducted a clinical trial in 32 healthy subjects based on multi-omics approaches. Every subject was administered amoxicillin/clavulanate for 14 days. The liver-specific microRNA-122 (miR-122) level increased prior to and correlated well with the observed alanine aminotransferase (ALT) level increase. This result indicates its potential as a sensitive early marker for AC-DILI. We also identified urinary metabolites, such as azelaic acid and 7-methylxanthine, with levels that significantly differed among the groups classified by ALT elevation level on day 8 after drug administration (P < 0.05). Lymphocyte proliferation in response to the drug was also observed. These findings demonstrate sequential changes in the process of AC-DILI, including metabolic changes, increased miR-122 level, increased liver enzyme activity, and enhanced lymphocyte proliferation after drug administration. In conclusion, this study provides potential biomarkers for AC-DILI based on currently known mechanisms using comprehensive multi-omics approaches.

API5 confers cancer stem cell-like properties through the FGF2-NANOG axis.

Immune selection drives the evolution of tumor cells toward an immune-resistant and cancer stem cell (CSC)-like phenotype. We reported that apoptosis inhibitor-5 (API5) acts as an immune escape factor, which has a significant role in controlling immune resistance to antigen-specific T cells, but its functional association with CSC-like properties remains largely unknown. In this study, we demonstrated for the first time that API5 confers CSC-like properties, including NANOG expression, the frequency of CD44-positive cells and sphere-forming capacity. Critically, these CSC-like properties mediated by API5 are dependent on FGFR1 signaling, which is triggered by E2F1-dependent FGF2 expression. Furthermore, we uncovered the FGF2-NANOG molecular axis as a downstream component of API5 signaling that is conserved in cervical cancer patients. Finally, we found that the blockade of FGFR signaling is an effective strategy to control API5high human cancer. Thus, our findings reveal a crucial role of API5 in linking immune resistance and CSC-like properties, and provide the rationale for its therapeutic application for the treatment of API5+ refractory tumors.

Inhibition of activator protein 1 activity and cell growth by purified green tea and black tea polyphenols in H-ras-transformed cells: structure-activity relationship and mechanisms involved.

ras gene mutation, which perpetually turns on the growth signal transduction pathway, occurs frequently in many cancer types. The mouse epidermal JB6 cell line has been transfected with a mutant H-ras gene to mimic carcinogenesis in vitro. These transformed cells (30.7b Ras 12) are able to grow in soft agar, exhibiting anchorage independence and high endogenous activator protein 1 (AP-1) activity, which can be detected by a stable AP-1 luciferase reporter. The present study investigated the ability of different pure green and black tea polyphenols to inhibit this ras signaling pathway. The major green tea polyphenols (catechins), (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin-3-gallate, (-)-epicatechin, and their epimers, and black tea polyphenols, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate (TFdiG), were compared with respect to their ability to inhibit the growth of 30.7b Ras 12 cells and AP-1 activity. All of the tea polyphenols except (-)-epicatechin showed strong inhibition of cell growth and AP-1 activity. Among the catechins, both the galloyl structure on the B ring and the gallate moiety contributed to the growth inhibition and AP-1 activity; the galloyl structure appeared to have a stronger effect on the inhibitory action than the gallate moiety. The epimers of the catechins showed similar inhibitory effects on AP-1 activity. The addition of catalase to the incubation of the cells with EGCG or TFdiG did not prevent the inhibitory effect on AP-1 activity, suggesting that H2O2 does not play a significant role in the inhibition by tea polyphenols. Both EGCG and TFdiG inhibited the phosphorylation of p44/42 (extracellular signal-regulated kinase 1 and 2) and c-jun without affecting the levels of phosphorylated-c-jun-NH2-terminal kinase. TFdiG inhibited the phosphorylation of p38, but EGCG did not. EGCG lowered the level of c-jun, whereas TFdiG decreased the level of fra-1. These results suggest that tea polyphenols inhibited AP-1 activity and the mitogen-activated protein kinase pathway, which contributed to the growth inhibition; however, different mechanisms may be involved in the inhibition by catechins and theaflavins.

Feasibility of baculovirus-expressed recombinant 10-kDa antigen in the serodiagnosis of Taenia solium neurocysticercosis.

Bacterially expressed recombinant 10-kDa protein of Taenia solium metacestode (TsM) was previously found to be reliable in the diagnosis of active stage neurocysticercosis (NCC) by immunoblotting but not by ELISA. In this study, we evaluated the diagnostic feasibility of detecting eukaryote-expressed recombinant 10-kDa protein of TsM by ELISA (rTsM10-ELISA) in the serum and cerebrospinal fluid (CSF) from NCC patients. In 45 cases of active NCC, 91.1 and 97.8% cases showed positive reactions for serum and CSF by rTsM10-ELISA. ELISA employing the crude cyst fluid antigen (CF-ELISA) also revealed a similar result. Negligible cross-reactions were observed in serum samples from control subjects and from subjects with other helminthic diseases by rTsM10-ELISA (5/139 cases, 3.6%). By contrast, CF-ELISA demonstrated a high degree of cross-reactivity (24/139, 17.3%) especially from those patients with alveolar and cystic echinococcoses. The overall sensitivity and specificity of rTsM10-ELISA were 94.3 and 96.4%; and those of CF-ELISA were 95.7 and 84.5%, for serum and CSF, respectively. Antibody responses to rTsM10 were detected as early as 3 months after experimental infection of T. solium eggs in pigs. Our results show that ELISA with rTsM10 could be highly applicable in the serodiagnosis of NCC from early stage of infection.

Intraocular pharmacokinetics of intravitreal vascular endothelial growth factor-Trap in a rabbit model.

PURPOSE: To determine intraocular pharmacokinetic properties of intravitreally injected vascular endothelial growth factor (VEGF)-Trap in a rabbit model. METHODS: VEGF-Trap was intravitreally injected in 18 rabbit eyes. Eyes were enucleated 1 h and 1, 2, 5, 14, and 30 days after injections and immediately frozen at -80 °C. Concentration of VEGF-Trap in vitreous, aqueous humor, and retina/choroid was determined using an indirect enzyme-linked immunosorbent assay and analyzed to obtain pharmacokinetic properties. RESULTS: Maximum concentration of VEGF-Trap was achieved at 1 h in all three tissues. A one-compartment model of distribution was selected as the final model for all tissues studied. Estimated half-life of VEGF-Trap in vitreous, aqueous humor, and retinal/choroid was 87.1, 36.8, and 35.0 h, respectively, and estimated mean residence time was 125.7, 53.1, and 50.5 h, respectively. Area under the curve from time 0 to the end point was 10009.8, 3945.1, and 1189.3, respectively. Total exposure of the aqueous humor and retina/choroid to VEGF-Trap was 39.4% and 11.9% of vitreous exposure, respectively. CONCLUSION: The vitreous half-life of VEGF-Trap is 3.63 days. This is shorter than that of bevacizumab (6.99 days) and longer than that of ranibizumab (2.51 days), as shown in studies using the same experimental settings. The concentration of VEGF-Trap peaked at 1 h after injections in all eye tissues studied.

Synthesis and in vitro characterization of novel amino terminally modified oxotremorine derivatives for brain muscarinic receptors.

A series of novel 2-substituted acetylenic pyrrolidines and piperidines related to oxotremorine (1) were prepared and evaluated in vitro as muscarinic cholinergic agents at brain M1 and M2 receptors. One analogue, 3-(2-oxo-1-pyrrolidinyl)-1-[2(R)-pyrrolidinyl]-1-propyne hydrogen oxalate (6a), was found to be a partial agonist producing a PI hydrolysis response at cortical M1 receptors approximately 3-fold larger than that produced by 1. The intrinsic activity profile of 6a at brain muscarinic receptors is similar to those of azetidine oxo analogue 2 and dimethylamino oxo analogue. All three compounds are partial M1 agonists and full M2 agonists; however, the profile of 6a in binding studies is significantly different. While 2 and 3 exhibit large M2 selectivities ranging between 8-fold to several hundred-fold, the binding profile of 6a shows almost no subtype selectivity.

Morphological selection of parental Chinese hamster ovary cell clones exhibiting high-level expression of recombinant protein.

To facilitate the establishment of recombinant Chinese hamster ovary (rCHO) cell lines with dihydrofolate reductase (dhfr)-mediated gene amplification, a primary selection method based on morphology of parental CHO clones has been developed. Morphology of parental clones that were made by transfecting various plasmids encoding thrombopoietin (TPO) and its analogs and humanized antibodies into dhfr-deficient (dhfr-) CHO cells was not uniform. Morphology of many parental clones exhibiting high-level expression of the introduced gene was similar to that of nontransfected dhfr- CHO cells. On the other hand, most parental clones with low-level expression experienced noticeable morphological changes such as bipolar fibroblast-like morphology. In case of selection of parental clones with TPO expression level higher than 200 ng/mL, morphological selection improved selection efficiency by 3.5-fold compared with random selection. Furthermore, when subjected to methotrexate for gene amplification, parental clones that were selected based on morphology elevated the expression level as much as those that were selected randomly. Taken together, morphological selection of parental clones can facilitate the establishment of rCHO cell lines expressing recombinant proteins.

Synthesis of an anti-methicillin-resistant Staphylococcus aureus (MRSA) carbapenem via stannatrane-mediated Stille coupling.

[formula: see text] A short synthesis of carbapenem 1 is described. They key step involves the cross-coupling of an enol triflate with an amino-substituted sp3 carbon. This cross-couping, which allows the introduction of the complete side chain in one step, utilizes a stannatrane as the heteroalkyl transfer reagent.

The capsule biosynthetic locus of Pasteurella multocida A:1.

Pasteurella multocida is the aetiological agent of fowl cholera, bovine haemorrhagic septicaemia and atrophic rhinitis in pigs. Many strains of P. multocida express a capsule on their surface. However, nothing is known about the capsule biosynthetic locus in P. multocida although the capsule has been implicated as a virulence factor. The entire capsule locus of P. multocida A:1 was cloned and sequenced. The locus is divided into three regions. Region 1 comprises four ORFs which are involved in the transport of the capsule polysaccharide to the surface. Region 2 comprises five ORFs whose postulated protein products are involved in the biosynthesis of the polysaccharide capsule. Region 3 comprises two ORFs whose postulated products show similarity to proteins that are involved in the phospholipid substitution of the polysaccharide capsule.