Novel and unique diagnostic biomarkers for Bacillus anthracis infection.

A search for bacterium-specific biomarkers in peripheral blood following infection with Bacillus anthracis was carried out with rabbits, using a battery of specific antibodies generated by DNA vaccination against 10 preselected highly immunogenic bacterial antigens which were identified previously by a genomic/proteomic/serologic screen of the B. anthracis secretome. Detection of infection biomarkers in the circulation of infected rabbits could be achieved only after removal of highly abundant serum proteins by chromatography using a random-ligand affinity column. Besides the toxin component protective antigen, the following three secreted proteins were detected in the circulation of infected animals: the chaperone and protease HtrA (BA3660), an NlpC/P60 endopeptidase (BA1952), and a protein of unknown function harboring two SH3 (Src homology 3) domains (BA0796). The three proteins could be detected in plasma samples from infected animals exhibiting 10(3) to 10(5) CFU/ml blood and also in standard blood cultures at 3 to 6 h post-bacterial inoculation at a bacteremic level as low as 10(3) CFU/ml. Furthermore, the three biomarkers appear to be present only in the secretome of B. anthracis, not in those of the related pathogens B. thuringiensis and B. cereus. To the best of our knowledge, this is the first report of direct detection of B. anthracis-specific proteins, other than the toxin components, in the circulation of infected animals.

Inhibition of rat oocyte maturation and ovulation by nitric oxide: mechanism of action.

Established gap junctional communication (GJC) in the ovarian follicle is essential for maintaining the oocytes in meiotic arrest. Alternatively, LH-induced reinitiation of meiosis is subsequent to breakdown of GJC. It was recently reported that nitric oxide (NO) inhibits maturation in rat follicle-enclosed oocytes and elevates GJC in cultured mesangial cells. Taking these observations into account, we hypothesized that NO prevents reinitiation of meiosis by antagonizing the effect of LH on GJC in the ovarian follicle. Indeed, we found that NO interferes with LH-induced disruption of GJC as well as with the decrease of the expression of the gap junction protein GJA1 (previously known as CONNEXIN43). We also demonstrated that NO prevents activation of LH-induced mitogen-activated protein kinases (MAPKs) 1 and 2 and inhibits cumulus expansion. Along this line, incubation of ovarian follicles with an inhibitor of soluble guanylate cyclase, which is a downstream NO effector, induced on its own oocyte maturation as well as cumulus expansion. Unlike previous studies, we show here that elevation of NO resulted in inhibition of ovulation. We conclude that the mechanism by which NO inhibits LH-induced oocyte maturation possibly involves a negative effect on MAPK activation and, in turn, interference with interruption of GJC. This action of NO in the ovarian follicle is apparently mediated by cGMP. In addition, the negative effect of NO on ovulation may be subsequent to its inhibitory effect on cumulus expansion. Together, this study suggests that the preovulatory decrease in NO concentrations is a prerequisite for the ovarian response to LH.

Oocyte-directed depletion of connexin43 using the Cre-LoxP system leads to subfertility in female mice.

Gap junctions, predominantly comprising connexin43 (Cx43), mediate cell-to-cell communication within the ovarian follicle. However, the partaking of Cx43 in the formation of the gap junction channels, between the oocyte and the somatic cells, is controversial. We addressed this dispute by crossing females that carry a Cx43 coding region, flanked by loxP recognition sites, with males expressing the Cre recombinase under the control of Zp3 promoter. Oocytes of the resultant Zp3Cre;Gja1(lox/lox) mice did not express Cx43 and were referred to as Cx43(del/del). Unexpectedly, a decrease in Cx43 was observed in cumulus/granulosa cells of some follicles as well. Nevertheless, no histological abnormalities were detected in the ovaries of the Zp3Cre;Gja1(lox/lox) mice. Furthermore, these mice ovulated normally and developed fully functional corpora lutea. Additionally, the ovarian Cx43(del/del) oocytes were meiotically arrested and transferred Lucifer yellow to the surrounding cumulus cells. However, mating Zp3Cre;Gja1(lox/lox) females with wild-type males resulted in a reduced rate of parturition and a substantial decrease in litter size. Further examination revealed that although preimplantation development of Zp3Cre;Gja1(lox/+) embryos was normal, the blactocysts exhibited impaired implantation. Our data suggest that total ablation of Cx43 in the oocyte, combined with its decrease in the surrounding somatic cells, allows normal oogenesis and folliculogenesis, ovulation and early embryonic development but severely impairs the implantation capacity of the resulting blactocysts.

Meiotic arrest of oocytes depends on cell-to-cell communication in the ovarian follicle.

The source of the inhibitory levels of cAMP that maintain oocytes meiotically arrested is under controversy. A model for regulation of the meiotic division that suggests the transfer of a somatic follicular cells-derived cAMP into the oocyte via gap junctions was first proposed by us in 1978. Later studies provide strong evidence that established gap-junctional communication within the ovarian follicle is indispensable for maintenance of meiotic arrest. On the other hand, other recent reports suggest that oocytes generate their own inhibitory cAMP by a G protein-coupled receptor-activated Gs. These studies as well as other recent reports related to this topic are thoroughly discussed in this chapter.

Disruption of gap junctional communication within the ovarian follicle induces oocyte maturation.

Meiotically arrested mammalian oocytes are stimulated to resume meiosis by LH. This response, which can be reversed by elevation of intraoocyte cAMP levels, is associated with interruption of gap junctional communication (GJC) within the ovarian follicle. In the present study, we examined the hypothesis that disruption of GJC within the ovarian follicle is sufficient for induction of oocyte maturation. For this purpose, we incubated rat follicle-enclosed oocytes with carbenoxolone (CBX), a known blocker of gap junctions. We found that this selective disruptor of GJC promoted maturation of almost all the follicle-enclosed oocytes after 5 h of incubation; this response was also obtained by a transient (2 h) exposure to this agent. CBX-induced oocyte maturation was accompanied by a substantial decrease in intraoocyte concentrations of cAMP that was not associated with elevated activity of type 3A phosphodiesterase (PDE3A). The effect of CBX on reinitiation of meiosis was blocked by isobutylmethylxanthine, a phosphodiesterase inhibitor. Unlike LH, CBX did not activate MAPK in the follicular cells, and inhibition of the MAPK signaling pathway by means of UO126 did not prevent the resumption of meiosis. Injection of CBX into the ovarian bursa of intact animals stimulated maturation in 30% of the oocytes, whereas no maturation was observed in the contralateral ovary injected with PBS. We conclude that, because experimentally induced breakdown of communication within the ovarian follicle is associated with a drop in intraoocyte cAMP concentrations and results in resumption of meiosis, this could be the physiological mechanism employed by LH to stimulate oocyte maturation.

Mitogen-activated protein kinase mediates luteinizing hormone-induced breakdown of communication and oocyte maturation in rat ovarian follicles.

Resumption of meiosis, induced by LH, is preceded by the breakdown of gap junctional communication, which terminates the supply of cAMP from the somatic cells of the ovarian follicle to the oocyte. It has recently been shown that LH-induced reinitiation of meiosis is mediated by MAPK; however, the underlying molecular mechanism involved in the action of this enzyme remains unknown. We hypothesized that activation of MAPK interrupts junctional communication within the ovarian follicle, leading, in turn, to oocyte maturation. To test this hypothesis, we blocked the activation of MAPK by UO126, which specifically inhibits the MAPK signaling pathway. We analyzed junctional communication using three complementary methods: 1) patch-clamp analysis, which determined changes in the electrical coupling between two adjacent granulosa cells; 2) the scrape-loading technique, which monitored the spread of dyes through a granulosa cell layer; and 3) a metabolic coupling assay, which evaluated the transfer of radiolabeled uridine from the cumulus cells to the oocyte. We show, herein, that the somatic follicle cells, rather than the oocyte, activate MAPK immediately after their exposure to LH. Moreover, inhibition of LH-induced MAPK activation not only prevents oocyte maturation but also blocks the reduction in junctional communication. In addition, the appearance of the two phosphorylated forms of the gap junction protein, connexin 43, in response to LH, was avoided by UO126. We concluded that MAPK mediates LH-induced oocyte maturation by interrupting cell-to-cell communication within the ovarian follicle, possibly through phosphorylation of connexin 43.