Asymmetric induction in the [4+2]cycloaddition of cyclopentadiene and furan to chiral derivatives of fumaric acid.

[4+2]Cycloaddition reactions of cyclopentadiene (1a) and furan (1b) to N,N'-fumaroyldi[(2R)-bornane-10,2-sultam] (2) and to N,N'-fumaroyldi[(2R)-bornane-10,2-(2'-phenyl-pyrazol-3'-one)] (3) are presented. A correlation between the solvent polarity and the logarithm of the diastereoisomer ratio (dr) was found for the uncatalyzed [4+2]cycloaddition of 1a to 3.     

Delayed pharmacological effects of proctolin and CCAP on heartbeat in pupae of the tobacco hornworm, Manduca sexta

Abstract. The effects in vivo of cardioactive peptides proctolin, CCAP and leucomyosuppressin (LMS) are investigated by means of noninvasive optocardiographic or thermographic techniques in postdiapause pupae of Manduca sexta. A constant pattern of heartbeat reversal in these pupae is manifested by regular alternations of the forward orientated (anterograde) and the backward orientated (retrograde) cardiac pulsations, with a periodicity of some 5–10 min. The heartbeat pattern is monitored continuously for several hours before and 24 h after injection of the investigated peptides. Injections of Ringer solution alone cause a slight, almost immediate increase of the rate of the pupal heartbeat (0–10%), which lasts only for 20–30 min. Injection of proctolin, CCAP or LMS does not show any immediate cardiostimulating effects (beyond those of Ringer) at concentrations up to 2 × 10⁻⁶ M (calculated from µg of the injected peptide and 70% pupal water content; 5–7 g pupal body mass). By contrast, injections of proctolin and CCAP in the range of 10^-9 − 10^-6 M concentrations cause delayed effects on the heartbeat, which are manifested only several hours after the injections. The delayed effects involve prolonged, or even continuous periods of unidirectional, more efficient and faster anterograde pulsations. Consequently, the flow of haemolymph through the head and thoracic parts of the pupal body increases. In the case of proctolin, the prolonged anterograde cardiac activity usually starts 5 h after the injections and the effect persists for 7–12 h. Using CCAP, the stimulation of anterograde activity starts 2.5–3 h after injections and lasts usually 7–8 h. Very small doses of peptides (10^-8 − 10^-9 M) do not change the latency period significantly, but they decrease the duration of the response. The frequency of the systolic contractions of the heart does not increase during the prolonged anterograde phase. Injections of LMS to produce a final concentration of 10⁻⁶ M in the pupa induce pathophysiological disturbances of heartbeat reversal and peristalsis. The effects start with a delay of some 1.5–2.5 h after the injections. By contrast to the effects of proctolin and CCAP, LMS does not produce delayed anterograde cardiac pulsations. These results show that the most intensively investigated cardiostimulating peptides in vitro, proctolin and CCAP, have no direct cardiostimulating activity under physiological conditions in vivo. It is concluded therefore that the delayed pharmacological effects of these peptides observed in the pupae of M. sexta, represent a secondary effect, resulting from stimulation of nonspecific, extracardiac myotropic or other physiological functions.     

Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats

The aim of the study was to determine whether the selection of immature oocytes by a combination of cumulus-oocyte-complexes (COCs) morphology and staining with brilliant cresyl blue (BCB) would be helpful in selecting developmentally competent oocytes, and thereby increase the efficiency of blastocyst production from ovarian oocytes of FSH-primed, adult goats. In a second experiment the interaction between oocyte quality and semen donor was assessed. In a third experiment the usefulness of Vero cells for co-culture with goat embryos was investigated. In the pool of morphologically normal COCs recovered from ovaries following slicing (21.9+/-11.0), the mean rate of COCs classified as BCB+ was 85.6%, and the BCB- was approximately 11%. Oocytes classified as grade 1 and BCB+ exhibited the highest developmental competence (P<0.001) after in vitro maturation and fertilization compared with oocytes of grade 1 BCB- and grade 2 BCB+ or BCB-. There were no significant differences in developmental competence in grade 2 oocytes, regardless of BCB coloration. No significant differences in embryo cleavage and blastocyst formation rates among three bucks were observed when morphologically normal, BCB+ oocytes were used. For all tested bucks, differences in embryo production efficiency were related only to the oocyte quality. Similar blastocyst rates were developed from embryos co-cultured with goat oviduct epithelial cells (34.3%) and with Vero cells (33.3%). These results show that the most important criterion for selection of COCs before maturation is the visual assessment of morphological features. Staining with BCB of COCs recovered from adult goats does not enhance efficiency of selection of developmentally competent oocytes for IVF.     

Testicular morphology and expression of aromatase in testes of mice with the mosaic mutation (Atp7a mo-ms)

The aim of this study was to determine whether testicular cells of mice with the mosaic mutation, associated with abnormal copper metabolism, are able to aromatize androgens to estrogens, and what is the putative role of estrogens in the gonad of the mutant male. Mosaic is a lethal mutation; affected males usually die on about day 16. Those, which survive to reach sexual maturity, are valuable research subjects. In testes of young and adult mutants, histological analysis revealed the presence of many degenerating seminiferous tubules besides normal-looking ones. Additionally, high numbers of apoptotic germ cells were observed, especially in young mutants when compared with the controls. Positive immunostaining for aromatase was found in cultured Leydig cells and testicular sections of both control and mutant males. The intensity of immunostaining was always stronger in the mosaic mice. In both groups, Western-blot analysis revealed the presence of aromatase protein as a single band of approximately 55 kDa. In the mosaic males, levels of testosterone in cultured Leydig cells, whole testes, and in blood plasma were lower than in those of the respective controls. On the contrary, estradiol concentrations were always higher in the mutants. Both in vivo and in vitro studies indicate that morphological and functional changes in the testes of the mosaic mice mainly result from defective copper metabolism. The higher level of endogenous estrogens can additionally enhance morphological alterations within the testes. It seems also likely that excess estrogens may affect the survival rate of the mosaic males.     

The role of oxidative stress induced by growth regulators in the regeneration process of wheat

As part of work to optimize the regeneration processes of winter wheat callus culture the effects of two auxins (2,4-D, IAA), two cytokinins (kinetin, zeatin), and the fungal mycotoxin zearalenone, were tested individually in vitro using embryo-, and inflorescence-derived callus. To determine the role of oxidative stress in cell regeneration, changes in the basic antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and peroxidases (PODs) were investigated. In general, zearalenone (ZEN) was found to be more effective than cytokinin treatments for inducing shoot production, whereas auxins suppressed the regeneration process. Regenerating callus showed higher induction of these antioxidant enzymes in comparison with non-regenerating callus. SOD, CAT and POD activities were higher in callus derived from inflorescence than in callus derived from immature embryo. Activities of SOD, CAT and POD in culture derived from immature embryos were depending on type of growth regulator in medium. The highest enzyme activities were observed in non-regenerating tissues after auxins treatment and in regenerating tissues after cytokinins treatment. The effect of ZEN was similar to that of cytokinins. One MnSOD band and two Cu/ZnSOD bands were detected in all cultures. Changes in SOD izoform patterns occurred in callus culture on media with auxins and ZEN, but not on media with cytokinins. Our results suggest that callus regeneration is associated with reactive oxygen species production induced by specific growth regulators. Reactive oxygen species under the control of cellular antioxidant machinery can mediate signalling pathways between exogenously applied growth regulators and the induction and/or creation of the direction of morphogenesis.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> strain MP-1: a new source of mutanase

A mutan-degrading bacterium, closely related to Paenibacillus curdlanolyticus, was isolated from soil. It produced 0.4 U mutanase ml⁻¹ in 2 days in shake-flask cultures when bacterial mutan was the sole carbon source. Mutanase activity was optimal at pH 6.2 and 45°C over 1 h and was stable between pH 5.8 and 12 at 4°C for 24 h and up to 40°C for 1 h. Mutan produced by Streptococcus mutans was rapidly hydrolyzed by this enzyme. The hydrolysis of mutan (1 g l⁻¹) resulted in 17% saccharification over 2 h and, at the same time, glucan was entirely solubilized.     

Pigment epithelium-derived factor in ulcerative colitis: possible relationship with disease activity

OBJECTIVES: Pigment epithelium-derived factor (PEDF) is an endogenous most potential angiogenic inhibitor and increased expression of PEDF in intestinal mucosa specimens was shown in the course of ulcerative colitis (UC). The aim of the present study was to evaluate serum concentration of pigment epithelium-derived growth factor, a potent anti-angiogenic factor and its possible association with vascular endothelial growth factor (VEGF) levels and disease activity. METHODS: Concentrations of PEDF and VEGF were measured in sera of 33 patients (13 females and 20 males) with active UC. RESULTS: There was significant increase of serum PEDF (32.3+/-2.9 vs. 20.6+/-4.7 ng/mL, P<0.05) as well as VEGF (326.4+/-58.1 vs. 110.9+/-15.7 pg/mL, P<0.05) in UC patients compared to healthy controls. Serum PEDF showed strong, positive correlation with endoscopic score (r=0.622, P<0.001), while such association was absent in respect to VEGF (r=0.05, P=0.77). In contrast serum VEGF decreased in severe UC comparing to patients with a mild course of disease, however the difference was not significant (274.9+/-64.9 vs. 360.4+/-103.4 pg/mL, P=0.53). CONCLUSIONS: Increase in serum PEDF during UC, especially in severe forms of disease suggests its involvement in UC pathogenesis.     

Interaction of AT1 receptors and V1a receptors-mediated effects in the central cardiovascular control during the post-infarct state

Experimental objectives. Because myocardial infarct is associated with overactivation of brain angiotensin II (ANG II) and vasopressin (AVP) V1a receptors we decided to determine whether AT1 and V1a receptors-mediated effects of ANG II and AVP interact in central cardiovascular control during the post-infarct state. Four groups of infarcted and four groups of sham-operated conscious rats entered the study. Results. In the infarcted rats cerebroventricular infusion of AT1 (AT1ANT, losartan) and V1a antagonist {V1aANT,d(CH(2))(5)[Tyr(Me)(2)Ala-NH(2)(9)]VP} and combined infusion of both these compounds performed 4 weeks after induction of the infarct significantly and comparably reduced mean arterial blood pressure (MABP) in comparison to control experiments (artificial cerebrospinal fluid infusion). In the sham rats MABP was not affected by any of the infusions. In control experiments MABP and HR responses to an alarming air jet stress were significantly higher in the infarcted than in the sham rats. Both responses were normalized with the same effectiveness by administration of AT1ANT, V1aANT and AT1ANT+V1aANT. In the sham rats administration of these compounds did not affect MABP and HR responses to stress. Conclusion: The results provide evidence for interaction of AT1 and V1a receptors-mediated effects of ANG II and AVP in the central cardiovascular control during the post-infarct state.     

Survey of susceptibility of clinical Clostridium diffiicile strains isolated from patients hospitalised in different departments of paediatric hospital to antimicrobial agents

This study was performed to determine the susceptibility of 50 C. difficile strains isolated from faecal samples of children suspected to antibiotic associated diarrhea (AAD) to antimicrobial agents: metronidazole, vancomycin, erythromycin, clindamycin, ciprofloxacin, moxifloksacin, gatifloksacin and imipenem. The all C. difficile strains were sensitived to metronidazole and vancomycin. Twenty six per cent of strains were resistant to erythromycin and clindamycin (MLS(B) type resistance). Resitance to ciprofloxacin, moxifloxacin, gatifloxacin and imipenem was detected in 98%, 8%, 8% and 30% of C. difficile strains, respectively.