In an effort to determine the localization of fibroblast growth factor (FGF) receptors (FGFR) that could mediate the intracellular action of FGF-2, we discovered the presence of high-affinity FGF-2 binding sites in the nuclei of bovine adrenal medullary cells (BAMC). Western blot analysis demonstrated the presence of 103-, 118-, and 145-kDa forms of FGFR1 in nuclei isolated from BAMC. 125I-FGF-2 cross-linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 can account for the nuclear FGF-2 binding sites. Nuclear FGFR1 has kinase activity and undergoes autophosphorylation. Immunocytochemistry with the use of confocal and electron microscopes demonstrated the presence of FGFR1 within the nuclear interior. Nuclear subfractionation followed by Western blot or immunoelectron microscopic analysis showed that the nuclear FGFR1 is contained in the nuclear matrix and the nucleoplasm. Agents that induce translocation of endogenous FGF-2 to the nucleus (forskolin, carbachol, or angiotensin II) increased the intranuclear accumulation of FGFR1. This accumulation was accompanied by an overall increase in FGF-2-inducible tyrosine kinase activity. Our findings suggest a novel mode for growth factor action whereby growth factor receptors translocate to the nucleus in parallel with their ligand and act as direct mediators of nuclear responses to cell stimulation.

Gardnerella vaginalis is an important component of the human vaginal microflora. It is proposed to play a key role in the pathogenesis of bacterial vaginosis (BV), the most common vaginal condition. Here we describe the development, validation and comparative analysis of a novel molecular approach capable of G. vaginalis identification, quantification and subtyping in noncultured vaginal specimens. Using two quantitative PCR (qPCR) assays, we analysed G. vaginalis bacterial loads and clade distribution in 60 clinical vaginal-swab samples. A very high pathogen prevalence was revealed by species-specific qPCR not only among BV patients (100 %), but also in healthy women (97 %), although the G. vaginalis concentration was significantly lower in non-BV samples. G. vaginalis clades identified in vaginal specimens by subtyping multiplex qPCR, which targets four clade-specific genetic markers, had frequencies of 53 % for

In this study we describe the presence of high anity FGF-2 binding sites in the nuclei of U251MG glioma cells (K d =7 pM). Immunoprecipitation of total cell extracts with FGF receptor (FGFR) 1-4 antibodies showed that U251MG glioma cells express only FGFR1. [ 125 I]FGF-2 cross linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 may account for the nuclear FGF-2 binding sites. Western blot analysis demonstrated the presence of 103, 118 kDa and small amounts of 145 kDa FGFR1 isoforms in the nuclei of glioma cells. All isoforms contain both the Cand N-terminal domains. Nuclear FGFR1 retains kinase activity. Immunocytochemistry using confocal microscopy showed speci®c FGFR1 immunoreactivity within the nuclear interior. In continuously proliferating glioma cells, nuclear FGFR1 is constitutively expressed, independent of cell density. In contrast, in nontransformed human astrocytes, nuclear FGFR1 levels¯uc-tuate with the proliferative state of the cell. In quiescent, con¯uent astrocytes nuclear FGFR1 protein was depleted. An accumulation of nuclear FGFR1 was observed following the transition to a subcon¯uent, proliferating state. Transfection of a pcDNA3.1-FGFR1 expression vector into glioma cells that do not express FGFR1 resulted in the nuclear accumulation of FGFR1, increased cell proliferation, and stimulated transition from the G 0 /G 1 to the S-phase of the cell cycle. The increased proliferative rate was resistant to inhibition by the cell-impermeable FGF binding antagonist, myoinositol hexakis [dihydrogen phosphate]. Our results suggest that the constitutive nuclear presence of FGFR1 contributes to the increased proliferation of glioma cells while the transient nuclear accumulation of FGFR1 in normal astrocytes may play a role in the transition to a reactive state.

FGF-2 has been implicated in the neoplastic transformation of glioma cells and in the transition of normal quiescent astrocytes to a proliferating, reactive state. In the present study we have observed that in human glial cells, levels and subcellular localization of FGF-2 are di erent in quiescent and proliferating cells. FGF-2 was detected in the cytoplasm of non-reactive astrocytes in human brain sections. In contrast FGF-2 was located within the cytoplasm and nuclei of reactive astrocytes in gliotic brain tissue and in neoplastic cells of glioma tumors. In vitro, FGF-2 was found predominantly in the nucleus of subcon¯uent proliferating astrocytes, but was detected only in the cytoplasm of density arrested quiescent astrocytes. Our results suggest that reduced cell contact stimulates nuclear accumulation of FGF-2, accompanying mitotic activation of reactive human astrocytes. FGF-2 was constitutively localized to the nucleus of continuously proliferating glioma cells independent of cell density. A role for intracellular FGF-2 was further suggested by the observation that glioma cells that are not stimulated to proliferate by extracellular FGF-2 proliferated faster when transfected with FGF-2 expressing vectors. This increased proliferation correlated with nuclear accumulation of FGF-2. Cell proliferation was attenuated by 5'-deoxy-5'-methylthioadenosine, a FGF-2 receptor tyrosine kinase inhibitor that acts within the cell, but was una ected by myo-inositol hexakis [dihydrogen phosphate] that disrupts FGF-2 binding to plasma membrane receptors. Our results indicate that FGF-2 serves as a nuclear regulator of proliferation in astrocytic cells. In glioma cells, the constitutive presence of FGF-2 in the nucleus may promote proliferation that is insensitive to cell contact inhibition.

No abstract

Our data implicate B henselae as a potential human tick-borne pathogen. Patients with a history of neuroborreliosis who have incomplete resolution of symptoms should be evaluated for B henselae infection.

PCR analysis of Ixodes scapularis ticks collected in New Jersey identified infections with Borrelia burgdorferi (33.6%), Babesia microti (8.4%), Anaplasma phagocytophila (1.9%), and Bartonella spp. (34.5%). The I. scapularis tick is a potential pathogen vector that can cause coinfection and contribute to the variety of clinical responses noted in some tick-borne disease patients

Erythromycin (EM) and clindamycin (CM) susceptibility testing was performed on 222 clinical isolates of group B Streptococcus. A multiplex PCR assay was used to detect the ermB, ermTR, and mefA/E antibiotic resistance genes. These results were compared to the phenotypes as determined by the standard EM/CM double disk diffusion assay.Group B Streptococcus (GBS) is one of the leading causes of neonatal bacterial infection. This type of infection commonly leads to pneumonia, septicemia, or meningitis. Because of the serious nature of neonatal GBS infections, the suggested standard protocol for the obstetrician/gynecologist is that pregnant women should be tested for the presence of GBS at 35 to 37 weeks of gestation (7,15). Once GBS colonization is diagnosed, the typical treatment for these patients is penicillin, to which there is no known resistance. However, there is a significant population of penicillin-allergic patients, a reported 12% of pregnant women (12), for whom the macrolide (erythromycin [EM]) or lincosamide (clindamycin [CM]) class of drugs needs to be administered, in particular, for those patients who are at high risk for anaphylactic shock. Previous reports have cited resistance of GBS to EM and CM of up to 37% and 17%, respectively (7). The resistance is commonly caused by three genes: ermB, ermTR, and mefA/E (1, 9, 10). The ermB and ermTR genes encode 23S rRNA methylases, which alter the binding of the antibiotic target site. The expression of these genes leads to the constitutively expressed and the erythromycin-induced macrolide, lincosamide, and streptogramin B (cMLS and iMLS, respectively) resistance phenotypes (9). The mefA and mefE genes, which are 90% identical, encode 14-and 15-member macrolide efflux pumps and lead to the macrolide only (M) resistance phenotype (1). Because of the presence of ermB, ermTR, mefA/E, and other antibiotic resistance genes on plasmids and/or transposons, these genes can pass from organism to organism, and the monitoring of the antibiotic resistance of GBS should occur regularly (13). We used a multiplex PCR assay to screen for the prevalence of the ermB, ermTR, and mefA/E genes in GBS clinical isolates from 222 patients for whom physicians ordered GBS testing. The samples, representing 20 states in the United States and 60% of which were from Florida, New Jersey, and Texas, were chosen at random. Patient ages ranged from 15 to 82 years, with an average of 31.3 Ϯ 11.8 years. These results were compared to the antibiotic resistance phenotypes as determined by the standard EM/CM double disk diffusion assay (3,11,15)