Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes

Abstract Three phyletic groups of Borrelia associated with Lyme disease, B. burgdorferi, B. garinii and group VS461 can be distinguished from each other and other species of Borrelia by Bfa I restriction site polymorphisms in PCR amplified 16S rRNA genes. One strain isolated from an Ixodes pacificus tick in California that was previously unclassifiable was distinguishable from B. burgdorferi by an Mnl I restriction site polymorphism.     

Altered differentiation and clustering of Sertoli cells in transgenic mice showing a Sertoli cell specific knockout of the connexin 43 gene

Histological analysis revealed that Sertoli cell specific knockout of the predominant testicular gap junction protein connexin 43 results in a spermatogenic arrest at the level of spermatogonia or Sertoli cell-only syndrome, intratubular cell clusters and still proliferating adult Sertoli cells, implying an important role for connexin 43 in the Sertoli and germ cell development. This study aimed to determine the (1) Sertoli cell maturation state, (2) time of occurrence and (3) composition, differentiation and fate of clustered cells in knockout mice. Using immunohistochemistry connexin 43 deficient Sertoli cells showed an accurate start of the mature markers androgen receptor and GATA-1 during puberty and a vimentin expression from neonatal to adult. Expression of anti-Muellerian hormone, as a marker of Sertoli cell immaturity, was finally down-regulated during puberty, but its disappearance was delayed. This observed extended anti-Müllerian hormone synthesis during puberty was confirmed by western blot and Real-Time PCR and suggests a partial alteration in the Sertoli cell differentiation program. Additionally, Sertoli cells of adult knockouts showed a permanent and uniform expression of GATA-1 at protein and mRNA level, maybe caused by the lack of maturing germ cells and missing negative feedback signals. At ultrastructural level, basally located adult Sertoli cells obtained their mature appearance, demonstrated by the tripartite nucleolus as a typical feature of differentiated Sertoli cells. Intratubular clustered cells were mainly formed by abnormal Sertoli cells and single attached apoptotic germ cells, verified by immunohistochemistry, TUNEL staining and transmission electron microscopy. Clusters first appeared during puberty and became more numerous in adulthood with increasing cell numbers per cluster suggesting an age-related process. In conclusion, adult connexin 43 deficient Sertoli cells seem to proliferate while maintaining expression of mature markers and their adult morphology, indicating a unique and abnormal intermediate phenotype with characteristics common to both undifferentiated and differentiated Sertoli cells.     

The phosphate clamp: a small and independent motif for nucleic acid backbone recognition

The 1.7 Å X-ray crystal structure of the B-DNA dodecamer, [d(CGCGAATTCGCG)]₂ (DDD)-bound non-covalently to a platinum(II) complex, [{Pt(NH₃)₃}₂-µ-{trans-Pt(NH₃)₂(NH₂(CH₂)₆NH₂)₂}](NO₃)₆ (1, TriplatinNC-A,) shows the trinuclear cation extended along the phosphate backbone and bridging the minor groove. The square planar tetra-am(m)ine Pt(II) units form bidentate N-O-N complexes with OP atoms, in a Phosphate Clamp motif. The geometry is conserved and the interaction prefers O2P over O1P atoms (frequency of interaction is O2P > O1P, base and sugar oxygens > N). The binding mode is very similar to that reported for the DDD and [{trans-Pt(NH₃)₂(NH₂(CH₂)₆(NH₃⁺)}₂-µ-{trans-Pt(NH₃)₂(NH₂(CH₂)₆NH₂)₂}](NO₃)₈ (3, TriplatinNC), which exhibits in vivo anti-tumour activity. In the present case, only three sets of Phosphate Clamps were found because one of the three Pt(II) coordination spheres was not clearly observed and was characterized as a bare Pt²⁺ ion. Based on the electron density, the relative occupancy of DDD and the sum of three Pt(II) atoms in the DDD-1 complex was 1:1.69, whereas the ratio for DDD-2 was 1:2.85, almost the mixing ratio in the crystallization drop. The high repetition and geometric regularity of the motif suggests that it can be developed as a modular nucleic acid binding device with general utility.     

A New Single Chamber Implantable Defibrillator with Atrial Sensing: A Practical Demonstration of Sensing and Ease of Implantation

Implantable cardioverter-defibrillators (ICDs) terminate ventricular tachycardia (VT) and ventricular fibrillation (VF) with high efficacy and can protect patients from sudden cardiac death (SCD). However, inappropriate shocks may occur if tachycardias are misdiagnosed. Inappropriate shocks are harmful and impair patient quality of life. The risk of inappropriate therapy increases with lower detection rates programmed in the ICD. Single-chamber detection poses greater risks for misdiagnosis when compared with dual-chamber devices that have the benefit of additional atrial information. However, using a dual-chamber device merely for the sake of detection is generally not accepted, since the risks associated with the second electrode may outweigh the benefits of detection. Therefore, BIOTRONIK developed a ventricular lead called the Linox^SMART S DX, which allows for the detection of atrial signals from two electrodes positioned at the atrial part of the ventricular electrode. This device contains two ring electrodes; one that contacts the atrial wall at the junction of the superior vena cava (SVC) and one positioned at the free floating part of the electrode in the atrium. The excellent signal quality can only be achieved by a special filter setting in the ICD (Lumax 540 and 740 VR-T DX, BIOTRONIK). Here, the ease of implantation of the system will be demonstrated.     

Biochemical properties of alligator (Alligator mississippiensis) bone collagen

Acid-soluble collagen (ASC) and pepsin solubilized collagen (PSC) isolated and purified from alligator (Alligator mississippiensis) bone were studied for molecular size, amino acid profile, secondary structure, and denaturation temperature by SDS-PAGE, HPLC, circular dichroism, and viscometry. Two collagen subunits, alpha1 and alpha2 were identified by SDS-PAGE. The molecular masses for alpha1 and alpha2 chains of ASC were 124 kDa and 111 kDa, respectively. The molecular masses were 123 kDa for alpha1 and 110 kDa for alpha2 chains of the PSC preparation. The molecular masses for ([alpha1](2) alpha2) of ASC and PSC were 359 kDa and 356 kDa, respectively. The major composition of alligator bone ASC and PSC was found to be typical type I collagen. The amino acid profiles of alligator ASC and PSC were similar to amino acid profile of subtropical fish black drum (Pogonias cromis, Sciaenidae) bone. Comparison of amino acid profiles with shark cartilage PSC, showed differences in alanine, hydroxylysine, lysine, and histidine contents. The denaturation temperatures (T(d)) of alligator ASC and PSC collagen measured by viscometry were 38.1 and 38.2 degrees C, respectively. Thermal denaturation temperatures, measured by melting point using circular dichroism, were 37.6 and 37.9 degrees C, respectively. Taken together, these results suggest that alligator bone collagen may find a wide range of applications in biological research, functional foods and nutraceuticals, and biomedical and pharmaceutical research.     

The mechanism of nitrogen monoxide (NO)-mediated iron mobilization from cells. NO intercepts iron before incorporation into ferritin and indirectly mobilizes iron from ferritin in a glutathione-dependent manner.

Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO-mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem. 276, 4724-4732]. We hypothesized that GSH completes the coordination shell of an NO[bond]Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO-mediated Fe mobilization. Native PAGE 59Fe-autoradiography shows that NO decreased ferritin-59Fe levels in cells prelabelled with [59Fe]transferrin. In prelabelled cells, ferritin-59Fe levels increased 3.5-fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin-59Fe levels decreased 10-fold compared with control cells after a 240-min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59Fe on route to ferritin, and indirectly facilitates removal of 59Fe from the protein. Studies using the GSH-depleting agent, L-buthionine-(S,R)-sulphoximine, indicated that the reduction in ferritin-59Fe levels via NO was GSH-dependent. Competition experiments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentration gradient. Based on our results, we propose a model of glucose-dependent NO-mediated Fe mobilization.     

Weak functional coupling of the melanocortin-1 receptor expressed in human adipocytes

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40-49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-alpha), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-alpha upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R-effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.