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Tissue ischemia results in an accumulation of lactate and local or systemic lactic acidosis. In nociceptive sensory neurons, lactate was reported to sensitize or activate the transient receptor potential ion channel TRPA1 and acid-sensing ion channels (ASICs). However, it is unclear how lactate modulates the TRPV1 regarded as the main sensor for acidosis in sensory neurons. In this study we investigated the effects of lactate (LA) on recombinant and native TRPV1 channels and on TRPV1-mediated release of neuropeptides from mouse nerves. TRPV1-mediated membrane currents evoked by protons, capsaicin or heat are inhibited by LA at concentrations ranging from 3 μM to 100 mM. LA inhibits TRPV1-mediated proton-induced Ca2+-influx in dorsal root ganglion neurons as well as proton-evoked neuropeptide release from mouse nerves. Inhibition of TRPV1 by LA is significantly stronger on inward currents as compared to outward currents since LA affects channel gating, shifting the activation curve towards more positive potentials. The mutation I680A in the pore lower gate displays no LA inhibition. Cell-attached as well as excised inside- and outside-out patches suggest an interaction through an extracellular binding site. In conclusion, our data demonstrate that lactate at physiologically relevant concentrations is a potent endogenous inhibitor of TRPV1.

We identified three isolates of Streptococcus agalactiae (group B streptococcus [GBS]), of human origin, which failed to react with antisera against any of the nine known GBS serotypes. Polyclonal rabbit antisera raised against these isolates and standard GBS typing sera were used in capillary precipitation and Ouchterlony tests to compare the strains with known GBS serotype reference strains. All three previously nontypeable isolates reacted with all three new antisera, producing lines of identity in the Ouchterlony test. Weak cross-reactions with antisera against several GBS serotypes were observed but were removed by absorption with corresponding antigens. The new antisera were used to test 227 GBS isolates that had been nontypeable or difficult to type using standard antisera. Of these, five reacted with the new antisera. These results suggested that all eight isolates belong to the previously unrecognized GBS serotype. They were tested by Western blotting for the Cα and Cβ proteins and by PCR to identify molecular serotypes and surface protein antigen genes. Two segments of the cps gene cluster (3′ end of cpsE - cpsF and 5′ end of cpsG , approximately 700 bp; 3′ end of cpsH and 5′ end of cpsM , approximately 560 bp) were sequenced. All eight isolates expressed Cα, and seven expressing the Cβ protein and the corresponding genes, bca and bac , respectively, were identified. They all share the same, unique partial cps sequence. These results indicate that these eight isolates represent a new S. agalactiae serotype, which we propose should be designated serotype IX.

Objective: Molecular pathogenesis of digestive neuroendocrine tumors (dNETs) is largely unknown. Recently, the serine-threonine kinase B-Raf was identified as an oncogene in endocrine cancer such as thyroid carcinoma. In endocrine cells, the small G-protein Rap1 stimulates mitogen-activated protein kinase (MAPK) signaling by activating B-Raf. We examined the expression of Rap1 and B-Raf in dNETs and their contribution to MAPK signaling in neuroendocrine cell lines. In addition, we explored the effect of suppressing B-Raf kinase by the recently developed inhibitor BAY43-9006 (Sorafinib) on growth, apoptosis and MAPK activation neuroendocrine cell lines. Methods and Results: Expression of Rap1 and B-Raf in dNETs (19 insulinomas, 15 carcinoid tumors and 10 gastrinomas) was examined by immunohistochemistry, which revealed that Rap1 and B-Raf were highly prevalent in the majority of dNETs. Overexpression of Rap1 and B-Raf activated MAPK extracellular dependent kinase (ERK) ERK-2 and ERK-dependent transcription factor Elk-1 in neuroendocrine cell lines Bon and INS-1. Suppression of B-Raf by BAY43-9006 inhibited growth and induced apoptosis in Bon and INS-1 cells. In addition, BAY43-9006 suppressed phosphorylation of MAPK ERK1/2 and its upstream kinase MEK1/2 in Bon and INS-1 cells. Conclusion: These results indicate that Rap1-B-Raf signaling may contribute to pathogenesis of dNETs and provides a molecular target for treatment of dNETs.

The taxonomic characteristics of β-hemolytic streptococcal strains that reacted with Lancefield group M antisera were investigated. Group M streptococci have not been proposed as a species to date. Four strains of the group M streptococci isolated from dog were located within the pyogenic group of the genus Streptococcus on 16S rRNA gene-based phylogenetic analysis; the group M strains were located a short distance away from all other members of the group. The homology values of 16S rRNA gene sequences between group M strains and all other streptococci were95.6%. Group M strains exhibited low levels of DNA-DNA homology to other streptococcal species. Some biochemical traits, such as β-galactosidase activity and acid production from glycogen, could distinguish these group M strains from other closely related species. Thus, these strains are proposed to constitute a new species -Streptococcus fryi sp. nov. The type strain is PAGU 653(T) (=NCTC 10235(T)=JCM 16387(T)).

Group B streptococci (GBS) are generally known to be pathogenic for humans, particularly for newborn infants, but they are also recognized as a cause of bacteremia in parturient women, elderly people, and immunocompromised patients (2). At present, GBS strains are typed in reference laboratories around the world by use of typing schemes based on serotyping, followed, if necessary, by phage typing (3). The primary serologic test used for classification in the laboratories is the capillary precipitation test introduced by Lancefield in 1934 (5). This test is based on the presence of capsular antigens extracted with hot hydrochloric acid (HCl) and allows classification of GBS strains into nine serotypes (Ia, Ib, II, III, IV, V, VI, VII, and VIII) (2, 3, 7). Most GBS strains can be classified as belonging to one of these serotypes, but always a certain percentage of strains remains nontypeable (NT) (2, 4). We have observed that the normality of the HCl used in the extract antigen can affect the number of strains that are determined to be NT in surveillance studies. Routinely, the microbiology departments of various institutions in Denmark submit GBS strains (confirmed to be such by pure culture) for typing in our facility (the Streptococcus Unit of the Statens Serum Institut). However, before we carry out the serotyping for a given strain, we always verify that it is a GBS strain by evaluating the strain visually on a blood-agar plate and by testing it with GBS antiserum by the Lancefield method. Of the 383 human isolates of GBS received at the Streptococcus Unit during the years 1997 to 2000, 154 were classified as serotype III. Of these, 103 isolates reacted with the specific antiserum when extracted with both 0.1 and 0.2 N HCl, and 51 isolates reacted only when extracted with 0.2 N HCl. A total of 26 of the isolates did not react when extracted with either HCl concentration. Based on this observation, we decided that, beginning in the year 2001, we would test all GBS isolates with both 0.1 and 0.2 N HCl to determine if we could reduce the number of NT GBS isolates. We had previously used only one extract concentration (0.2 N HCl) for typing GBS isolates. The antisera used for typing the GBS isolates as belonging generally to group B and more specifically to one of the serotypes (Ia to VIII) were produced at the Streptococcus Unit. The Lancefield extracts used as controls were based on reference strains of GBS serotypes Ia to VIII, as well as of group B, that were obtained within the Streptococcus Unit, which serves as a national reference center for streptococci. A modified version of the Lancefield method (which modification is used as the standard serotyping method in the Streptococcus Unit) was performed as follows. A freeze-dried culture of GBS was suspended into serum broth. From this suspension, a control for purity was made by plating a droplet onto a 5% horse blood-agar plate. The agar plate was incubated at 36°C overnight. The next day, a few colonies from the agar plate were added to 5 ml of glucose broth, and the bacterial suspension was incubated at 36°C overnight. After incubation, the suspension was centrifuged and the supernatant was removed. A 0.1-ml volume of either 0.1 or 0.2 N hydrochloric acid was added to the bacteria pellet. The suspension with the acid added was held at 100°C (i.e., placed in boiling water) for 15 min and thereafter cooled under tap water. After cooling, 0.2 N NaOH was added to adjust the pH to 7.0 (it was also recommended that a droplet of phenol red be added to the acid suspension as an indicator). The suspension was then centrifuged for 10 min to obtain a crystal-clear supernatant, and serotyping was performed as described by Lancefield (3, 5). We tested all the GBS isolates that we received from January to mid-November 2001. During this period, a total of 94 isolates were identified as GBS and typed. Of these, 82 (87.2%) could be identified with both normalities. Of the remaining isolates, 10 (10.6%) could be identified only with the 0.2 N extract and 2 (2.1%) could be identified only with the 0.1 N extract. Serotyping of GBS is essential for surveillance studies as part of the development and formulation of protective human GBS vaccines. There may be many reasons why some isolates are NT, and one of the explanations could be that some of the NT isolates represent new serotypes that still remain to be described. However, many studies (1, 2, 3, 6) of the distribution of GBS serotypes among human isolates are mainly based on serotyping by use of the capillary precipitation method with only one standard extract antigen normality (5). Given the results described above, if different extract normalities had been used, it is possible that fewer NT isolates would have been found. A suggestion for better use of the precipitation test in the future is to retest all the strains defined as NT, using the standard 0.2 N HCl extract and other, different extract normalities (such as 0.1 N HCl) in order to reduce the number of false-negative responses.