Effects of kinins on mammalian spermatozoa and the impact of peptidolytic enzymes.

Effects of kinins, mainly bradykinin (Bk), and other components of the kallikrein-kinin system on sperm motility and further fertility-related functions have been described repeatedly. However, reported data are in part controversial and the mechanism of kinin effects on sperm motility is not yet understood. In the present report we describe a significant promoting effect of Bk on sperm motility at subnanomolar concentrations. This effect was stabilized and even increased by suppression of Bk hydrolysis in semen samples. As sperm membrane-bound angiotensin-converting enzyme and neutral metalloendopeptidase are mainly involved in Bk hydrolysis, an effective cocktail of enzyme inhibitors promoting the sperm motility consists of phosphoramidon and lisinopril (both at 10-7 m). The effects of Bk on sperm cells are not mediated by the B2 Bk receptor. Using several biochemical, molecular and genetic methods we could not detect any Bk receptor on spermatozoa.

Neutral endopeptidase and alcohol consumption, experiments in neutral endopeptidase-deficient mice.

Alcohol consumption was investigated in mice which were rendered deficient in the peptide-degrading enzyme neutral endopeptidase (EC 3.4.24.11) (NEP-/-) by gene targeting and compared to alcohol consumption in corresponding wild type mice (NEP+/+). Mice were offered a free choice to drink tap water or 10% alcohol. The NEP-/- mice consumed significantly more alcohol ( approximately 42%) than the NEP+/+ mice, whereas no significant differences were observed in the total fluid consumption. The daily food consumption of alcohol naive NEP-/- animals was elevated ( approximately 29%). Furthermore, the activities of peptidases closely related to neutral endopeptidase were analysed ex vivo in several brain regions from NEP-/- and NEP+/+ mice not treated with alcohol. There was no obvious compensation for the total loss of neutral endopeptidase by the functionally related peptidases angiotensin-converting enzyme and aminopeptidase N. In vitro, the degradation of exogenously applied [Leu(5)]enkephalin was not reduced in membrane preparations of those brain regions assayed in NEP-/- mice. A small reduction in [Leu(5)]enkephalin degradation was detected in striatal membrane preparations of NEP-/- mice, if aminopeptidase N was additionally blocked by bestatin or amastatin.

mRNA quantification of C-type natriuretic peptide in brain areas of rodents.

C-type natriuretic peptide (CNP) is regarded as an endothelium-derived vasodilator and might therefore have an important role in controlling vascular tone and remodeling. Because CNP also is expressed in the brain, it is considered to be a neurotransmitter. The present study compares expression levels of CNP mRNA in distinct areas of the mouse brain with the expression pattern in the rat brain. A distinct expression of CNP was found in all investigated areas with the exception of the mouse striatum. In both rodents, high CNP expression was detected in the tegmentum.

Expression of the sigmaB-dependent general stress regulon confers multiple stress resistance in Bacillus subtilis.

The alternative sigma factor sigmaB of Bacillus subtilis is required for the induction of approximately 100 genes after the imposition of a whole range of stresses and energy limitation. In this study, we investigated the impact of a null mutation in sigB on the stress and starvation survival of B. subtilis. sigB mutants which failed to induce the regulon following stress displayed an at least 50- to 100-fold decrease in survival of severe heat (54 degrees C) or ethanol (9%) shock, salt (10%) stress, and acid (pH 4.3) stress, as well as freezing and desiccation, compared to the wild type. Preloading cells with sigmaB-dependent general stress proteins prior to growth-inhibiting stress conferred considerable protection against heat and salt. Exhaustion of glucose or phosphate induced the sigmaB response, but surprisingly, sigmaB did not seem to be required for starvation survival. Starved wild-type cells exhibited about 10-fold greater resistance to salt stress than exponentially growing cells. The data argue that the expression of sigmaB-dependent genes provides nonsporulated B. subtilis cells with a nonspecific multiple stress resistance that may be relevant for stress survival in the natural ecosystem.

Separate mechanisms activate sigma B of Bacillus subtilis in response to environmental and metabolic stresses.

sigma B is a secondary sigma factor that controls the general stress response of Bacillus subtilis. sigma B-dependent transcription is induced by the activation of sigma B itself, a process that involves release of sigma B from an inhibitory complex with its primary regulator, RsbW. sigma B becomes available to RNA polymerase when RsbW forms a complex with an additional regulatory protein (RsbV) and, because of this, fails to bind sigma B. Using Western blot (immunoblot) analyses, reporter gene fusion assays, and measurements of nucleotide pool sizes, we provide evidence for two independent processes that promote the binding of RsbW to RsbV. The first occurs during carbon limitation or entry into stationary phase. Activation of sigma B under these circumstances correlates with a drop in the intracellular levels of ATP and may be a direct consequence of ATP levels on RsbW's binding preference. The second activation process relies on the product of a third regulatory gene, rsbU. RsbU is dispensable for sigma B activation during carbon limitation or stationary phase but is needed for activation of sigma B in response to any of a number of different environmental insults (ethanol treatment, salt or acid shock, etc.). RsbU, or a process dependent on it, alters RsbW binding without regard for intracellular levels of ATP. In at least some instances, the effects of multiple inducing stimuli are additive. The data are consistent with RsbW being a regulator at which distinct signals from separate effectors can be integrated to modulate sigma B activity.

sigma B-dependent regulation of gsiB in response to multiple stimuli in Bacillus subtilis.

The expression of the gsiB gene of Bacillus subtilis in response to a wide variety of stress conditions was analysed, and the results provide evidence that gsiB is subject to a sigma B-dependent regulation. Primer extension experiments established identical start points for gsiB transcription during growth and after the induction by heat shock, salt or ethanol stress, and glucose limitation. The sequence upstream of the transcription start point shows great similarity to the sequences of sigma B-dependent promoters of B. subtilis. sigma B was absolutely required for the increase in gsiB mRNA level and in the synthesis rate of GsiB in response to various stimuli. Measurements of the ATP pool indicated that changes in the level of ATP might be one of the signals that regulate the activity of sigma B in B. subtilis.

Analysis of the induction of general stress proteins of Bacillus subtilis.

In Bacillus subtilis stress proteins are induced in response to different environmental conditions such as heat shock, salt stress, glucose and oxygen limitation or oxidative stress. These stress proteins have been previously grouped into general stress proteins (Gsps) and heat-specific stress proteins (Hsps). In this investigation the N-terminal sequences of 13 stress proteins of B. subtilis were determined. The quantification of the mRNA and the analysis of the protein synthesis pattern support the initial hypothesis that the chaperones DnaK and GroEL are Hsps in B. subtilis. In contrast, the recently described proteins GsiB, Ctc and RsbW belong to a class of Gsps that are induced by various stresses including heat shock. The main part of the Gsps described in this study failed to be induced in the sigB deletion mutant ML6 in response to heat shock. However, all the five Hsps were induced in this mutant in response to heat shock. These data indicate that SigB plays a crucial role in the induction of general stress genes, but is dispensable for the induction of Hsps.