Effect of adenosine receptor blockade on pial arteriolar dilation during sciatic nerve stimulation

In the present study, we report the effects of adenosine receptor antagonists on pial vasodilatation during contralateral sciatic nerve stimulation (SNS). The pial circulation was observed through a closed cranial window in alpha-chloralose-anesthetized rats. In artificial cerebrospinal fluid (CSF), SNS resulted in a 30.5 +/- 13.2% increase in pial arteriolar diameter in the hindlimb somatosensory cortex. Systemic administration of the selective adenosine A2A receptor antagonist, 4-(2-[7-amino-2-[2-furyl][3,2,4]triazolol[2,3-a][1,3,5]triazin-5-yl-amino] ethyl)phenol (ZM-241385), significantly (P < 0.05, n = 6) attenuated the SNS-induced vasodilatation. Systemic administration of 8-(p-sulfophenyl)theophylline (8SPT), a nonselective antagonist that is blood-brain barrier (BBB) impermeable, had no effect on vasodilatation to SNS. In contrast, systemic theophylline, which readily penetrates the BBB, nearly abolished the SNS-induced vasodilatation (P < 0.01; n = 7). Topical superfusion of 8SPT significantly (P < 0.01; n = 6) attenuated vasodilatation during SNS. Topical superfusion of 8- cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist, significantly potentiated SNS-induced vasodilatation (P < 0.01; n > or = 5). Hypercarbic vasodilatation and somatosensory-evoked potentials were not affected by any of the compounds tested. Our findings suggest that luminal endothelial adenosine receptors are not involved in the arteriolar response to SNS, as demonstrated by a lack of effect with systemic 8SPT. Furthermore, the adenosine A2A receptor subtype appears to be involved in the dilator response to SNS. Finally, the neuromodulatory action of adenosine, via the A1 receptor subtype, significantly influences SNS-induced vasodilatation. Thus the present study provides further evidence for a role of adenosine in the regulation of CBF during somatosensory stimulation.     

CD43 gene expression is mediated by a nuclear factor which binds pyrimidine-rich single-stranded DNA

CD43 is a leukocyte-specific surface molecule which plays an important role both in adhesion and signal transduction. We have identified a site spanning nucleotides +18 to +39 within the human CD43 gene promoter which in vitro is hypersensitive to cleavage by nuclease S1. Repeats of this region are sufficient to activate expression of a heterologous promoter in CD43-positive cell lines. Two nuclear factors, PyRo1 and PyRo2, interact with the hypersensitive site. PyRo1 is a single-stranded DNA-binding protein which binds the pyrimidine-rich sense strand. Mutation analysis demonstrates that the motif TCCCCT is critical for PyRo1 interaction. Replacement of this motif with the sequence CATATA abolishes PyRo1 binding and reduces expression of the CD43 promoter by 35% in Jurkat T lymphocytic cells and by 52% in the pre-erythroid/pre-megakaryocytic cell line K562. However, this same replacement failed to affect expression in U937 monocytic cells or in CEM T lymphocytic cells. PyRo1, therefore, exhibits cell-specific differences in its functional activity. Further analysis demonstrated that PyRo1 not only interacts with the CD43 gene promoter but also motifs present within the promoters of the CD11a, CD11b, CD11c and CD11d genes. These genes encode the α subunits of the β2 integrin family of leukocyte adhesion receptors. Deletion of the PyRo1 binding site within the CD11c gene reduced promoter activity in T lymphocytic cells by 47%. However, consistent with our analysis of the CD43 gene, the effect of this same deletion within U937 monocytic cells was less severe. That PyRo1 binds preferentially to single-stranded DNA and sequences within the CD43 and CD11 gene promoters suggests that expression of these genes is influenced by DNA secondary structure.     

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli . We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA . These shuA -positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA , so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae . The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA . The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA -positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae , indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae , and it is located in the same relative position on the chromosome.     

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set ( tonB1, exbB1, exbD1 ) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system ( tonB2, exbB2, exbD2 ). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes ( hutBCD ), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1 . A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae , the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae .     

The Anti-(+)-Methamphetamine Monoclonal Antibody mAb7F9 Attenuates Acute (+)-Methamphetamine Effects on Intracranial Self-Stimulation in Rats

Passive immunization with monoclonal antibodies (mAbs) against (+)-methamphetamine (METH) is being evaluated for the treatment of METH addiction. A human/mouse chimeric form of the murine anti-METH mAb7F9 has entered clinical trials. This study examined the effects of murine mAb7F9 on certain addiction-related behavioral effects of METH in rats as measured using intracranial self-stimulation (ICSS). Initial studies indicated that acute METH (0.1-0.56 mg/kg, s.c.) lowered the minimal (threshold) stimulation intensity that maintained ICSS. METH (0.3 mg/kg, s.c.) also blocked elevations in ICSS thresholds (anhedonia-like behavior) during spontaneous withdrawal from a chronic METH infusion (10 mg/kg/day x 7 days). In studies examining effects of i.v. pretreatment with mAb7F9 (at 30, 100, or 200 mg/kg), 200 mg/kg blocked the ability of an initial injection of METH (0.3 mg/kg, s.c.) to reduce baseline ICSS thresholds, but was less capable of attenuating the effect of subsequent daily injections of METH. MAb7F9 (200 mg/kg) also produced a small but significant reduction in the ability of METH (0.3 mg/kg, s.c.) to reverse METH withdrawal-induced elevations in ICSS thresholds. These studies demonstrate that mAb7F9 can partially attenuate some addiction-related effects of acute METH in an ICSS model, and provide some support for the therapeutic potential of mAb7F9 for the treatment of METH addiction.     

Effects of High Fat Feeding and Diabetes on Regression of Atherosclerosis Induced by Low-Density Lipoprotein Receptor Gene Therapy in LDL Receptor-Deficient Mice

We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr^-/-) mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR). After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr^-/- mice in different metabolic states.     

The very low birth weight infant microbiome and childhood health

This review describes current understandings about the nature of the very low birth weight infant (VLBW) gut microbiome. VLBW infants often experience disruptive pregnancies and births, and prenatal factors can influence the maturity of the gut and immune system, and disturb microbial balance and succession. Many VLBWs experience rapid vaginal or Caesarean births. After birth these infants often have delays in enteral feeding, and many receive little or no mother's own milk. Furthermore the stressors of neonatal life in the hospital environment, common use of antibiotics, invasive procedures and maternal separation can contribute to dysbiosis. These infants experience gastrointestinal dysfunction, sepsis, transfusions, necrotizing enterocolitis, oxygen toxicity, and other pathophysiological consditions that affect the normal microbiota. The skin is susceptible to dysbiosis, due to its fragility and contact with NICU organisms. Dysbiosis in early life may resolve but little is known about the timing of the development of the signature gut microbiome in VLBWs. Dysbiosis has been associated with a number of physical and behavioral problems, including autism spectrum disorders, allergy and asthma, gastrointestinal disease, obesity, depression, and anxiety. Dysbiosis may be prevented or ameliorated in part by prenatal care, breast milk feeding, skin to skin contact, use of antibiotics only when necessary, and vigilance during infancy and early childhood. Birth Defects Research (Part C) 105:252–264, 2015. © 2015 Wiley Periodicals, Inc.     

Contribution of the Two Genes Encoding Histone Variant H3.3 to Viability and Fertility in Mice

Histones package DNA and regulate epigenetic states. For the latter, probably the most important histone is H3. Mammals have three near-identical H3 isoforms: canonical H3.1 and H3.2, and the replication-independent variant H3.3. This variant can accumulate in slowly dividing somatic cells, replacing canonical H3. Some replication-independent histones, through their ability to incorporate outside S-phase, are functionally important in the very slowly dividing mammalian germ line. Much remains to be learned of H3.3 functions in germ cell development.Histone H3.3 presents a unique genetic paradigm in that two conventional intron-containing genes encode the identical protein. Here, we present a comprehensive analysis of the developmental effects of null mutations in each of these genes. H3f3a mutants were viable to adulthood. Females were fertile, while males were subfertile with dysmorphic spermatozoa. H3f3b mutants were growth-deficient, dying at birth. H3f3b heterozygotes were also growth-deficient, with males being sterile because of arrest of round spermatids. This sterility was not accompanied by abnormalities in sex chromosome inactivation in meiosis I. Conditional ablation of H3f3b at the beginning of folliculogenesis resulted in zygote cleavage failure, establishing H3f3b as a maternal-effect gene, and revealing a requirement for H3.3 in the first mitosis. Simultaneous ablation of H3f3a and H3f3b in folliculogenesis resulted in early primary oocyte death, demonstrating a crucial role for H3.3 in oogenesis.These findings reveal a heavy reliance on H3.3 for growth, gametogenesis, and fertilization, identifying developmental processes that are particularly susceptible to H3.3 deficiency. They also reveal partial redundancy in function of H3f3a and H3f3b, with the latter gene being generally the most important.