Nitric oxide mediates sexual behavior in female rats

Nitric oxide (NO), an active free radical formed during the conversion of arginine to citrulline by the enzyme NO synthase (NOS), mediates vasorelaxation, cytotoxicity, and neurotransmission. Neurons containing NOS (NOergic) are located in the hypothalamus. These NOergic neurons control the release of several hypothalamic peptides. Release of NO from these NOergic neurons stimulates pulsatile release of luteinizing hormone-releasing hormone (LHRH) in vivo and LHRH release in vitro. LHRH not only induces LH release, which induces ovulation, but also facilitates female sexual behavior. Sexual behavior can be induced reliably in estrogen-primed ovariectomized female rats by progesterone (P). This behavior consists of proceptive behavior to attract the male and the assumption of a clear characteristic posture, lordosis, when mounted by the male. To ascertain the role of NO in the control of sexual behavior in female rats, an inhibitor of NOS, NG-monomethyl-L-arginine was microinjected into the third cerebral ventricle (3V) of conscious, ovariectomized, estrogen-primed rats with indwelling cannulae. NG-Monomethyl-L-arginine (10-1000 micrograms) prevented P-facilitated lordosis when administered intracerebroventricularly into the 3V, 20 min prior to the 3V injection of P. NG-Monomethyl-D-arginine, which does not inhibit NOS, did not inhibit lordosis under the same experimental conditions. Microinjection into the 3V of sodium nitroprusside (SNP), which spontaneously releases NO, facilitated lordosis in estrogen-primed rats in the absence of P. The facilitation of lordosis induced by either P or SNP was prevented by intracerebroventricular injection of hemoglobin, which binds NO. Lordosis facilitated by P or SNP was blocked by injection of LHRH antiserum into the 3V. The results are interpreted to mean that the P-facilitated lordosis response is mediated by LHRH release. Furthermore, since NO release from SNP also facilitates lordosis in the absence of P and this response could be blocked by LHRH antiserum, we conclude that P brings about the release of NO, which stimulates LHRH release that facilitates lordosis. Thus, the results indicate that NO induces LHRH release and that LHRH then plays a crucial role in mediation of sexual behavior in the female rats.     

A new tool for studying the molecular architecture of the fungal cell wall: one-step purification of recombinant trichoderma beta-(1-6)-glucanase expressed in Pichia pastoris

We assessed the anesthetic properties of helium and neon at hyperbaric pressures by testing their capacity to decrease anesthetic requirement for desflurane using electrical stimulation of the tail as the anesthetic endpoint (i.e., the minimum alveolar anesthetic concentration [MAC]) in rats. Partial pressures of helium or neon near those predicted to produce anesthesia by the Meyer-Overton hypothesis (approximately 80-90 atm), tended to increase desflurane MAC, and these partial pressures of helium and neon produced convulsions when administered alone. In contrast, the noble gases argon, krypton, and xenon were anesthetic with mean MAC values of (+/- SD) of 27.0 +/- 2.6, 7.31 +/- 0.54, and 1.61 +/- 0.17 atm, respectively. Because the lethal partial pressures of nitrogen and sulfur hexafluoride overlapped their anesthetic partial pressures, MAC values were determined for these gases by additivity studies with desflurane. Nitrogen and sulfur hexafluoride MAC values were estimated to be 110 and 14.6 atm, respectively. Of the gases with anesthetic properties, nitrogen deviated the most from the Meyer-Overton hypothesis. Implications: It has been thought that the high pressures of helium and neon that might be needed to produce anesthesia antagonize their anesthetic properties (pressure reversal of anesthesia). We propose an alternative explanation: like other compounds with a low affinity to water, helium and neon are intrinsically without anesthetic effect.     

5-[125I]iodo-2'-deoxyuridine in the radiotherapy of brain tumors in rats

Glial neoplasms of the human central nervous system have defied treatment, in part because of the limited selectivity of available cytotoxic agents. The thymidine analog 5-iodo-2'-deoxyuridine radiolabeled with the Auger electron emitter 125I (125IUdR) is highly toxic to dividing cells when it is deoxyribonucleic acid incorporated, but it is relatively innocuous when located outside the nucleus. Previous studies have shown that 125IUdR has significant antineoplastic potential against mammalian cells in vitro and direct administration of 125IUdR is effective therapy for ovarian ascites tumors in mice and neoplastic meningitis in rats. Studies using external gamma imaging and autoradiography have also shown that direct intratumoral administration of 123IUdR/125IUdR into intracerebral 9L gliosarcomas in rats results in selective uptake of the radionuclide into tumor cells. Based on these encouraging results, we have evaluated the therapeutic potential of 125IUdR in rats bearing intracerebral 9L gliosarcomas. METHODS: Iodine-125-IUdR was infused intracerebrally over a 2-day period into rats bearing 1-day-old 9L tumors and over a 6-day period into animals with 9-day-old 9L tumors; equimolar concentrations of 127IUdR were infused into control animals. Tumor growth was monitored by contrast-enhanced 1H MRI and animal survival was followed over time. RESULTS: Intracerebral tumors (3-7 mm) were readily detected by MRI. Tumor-bearing rats treated with 127IUdR succumbed within 17-24 days, whereas tumor-bearing animals treated with 125IUdR survived significantly longer, and 10%-20% of the animals were cured of tumors. CONCLUSION: These data substantiate the antineoplastic potential of 5-[125I]iodo-2'-deoxyuridine and indicate that it may be a useful agent for the therapy of solid tumors that are accessible to direct radiopharmaceutical administration.     

Development and utility of anti-PepT1 anti-peptide polyclonal antibodies

We have considered the access resistance (AR) of a single conducting channel placed in a membrane bathed by an electrolyte. The classical expression for AR is due to Hall, who modeled the electrolyte as an ohmic conducting homogeneous medium. This approach is discussed in the present paper and it is shown that it is not valid in all cases, but depends on the ion concentration in solution and the ratio between solution and channel resistivities. To get a new expression for AR, we have combined the use of one-dimensional Nernst-Planck and Poisson (NPP) equations for the mouth of the channel and three-dimensional NPP equations for the outside solution. The influence of ion gradients and the channel itself on AR tums out to be considerable in diluted solutions (and also in the case of small channels in any solution). This influence is weaker in concentrated solutions, for which AR is well described by Hall's expression.     

Monocrotaline pyrrole induces apoptosis in pulmonary artery endothelial cells

Eosinophilic myocarditis followed by fibrosis of the cardiac muscle was observed in addition to peripheral blood eosinophilia in CBA/J mice infected with Toxocara canis. The infected mice were used as an experimental model of eosinophilic endomyocarditis associated with hypereosinophilic syndrome. Effects of in vivo treatment with MoAbs to adhesion molecules on eosinophilic myocarditis were examined using this experimental model. Expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells of capillaries in myocardium were increased 1 and 2 weeks after infection. Infiltration of very late antigen (VLA)-4+ and/or CD11a+ cells into the cardiac muscles was also observed 1 and 2 weeks after infection. Infiltration of eosinophils into the heart was significantly suppressed by anti-CD18 MoAb and anti-VLA-4 MoAb, and focal fibrosis of the cardiac muscle was also significantly suppressed by combined administration of anti-CD18 and anti-ICAM-1 MoAbs. These results indicate that adhesion molecules may play important roles in eosinophilic myocarditis, and that blockade of interaction between adhesion molecules and their ligands may help to control it.     

The impact of genetic markers on selection

Genetic marker technologies, such as marker-assisted selection, parentage identification, and gene introgression can be applied to livestock selection programs. Highly saturated genetic maps are now available for cattle, swine, and sheep to provide the genetic framework for developing MAS programs. These programs rely on three phases for commercialization of the technology: the detection phase, in which quantitative trait loci are located and their effects on the phenotype measured; the evaluation phase, in which the markers are evaluated in commercial populations; and the implementation phase, in which markers are combined with phenotypic and pedigree information in genetic evaluation for predicting the genetic merit of individuals within the population. Predicting the economic impact of genetic technologies is a complex process that requires quantitative prediction and economic analysis. Evaluating the impact of these benefits across an industry can be achieved through a process in which gains from implementation of a genetic technology are assessed at the individual, enterprise, and industry levels. A pattern of annual benefits and costs can be predicted using gene flows that can be evaluated by conventional economic analysis.     

Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage

Through a differential screening technique, we have identified a cDNA clone with differential expression in normal versus tumor cells. This clone, designated rit42 (reduced in tumor, 42 kDa), was previously isolated as a homocysteine-inducible gene in human endothelial cells (RTP), and the same or a highly related androgen-responsive gene in mouse has also been identified. Both Northern blot analysis and in situ hybridization demonstrated a significantly diminished expression in tumor cells, including those derived from breast and prostate when compared with normal cells. It was shown that RTP/rit42 mRNA cycles with cell division, peaking at G1 and G2-M, with lower expression in S phase. The biphasic expression of RTP/rit42 mRNA was absent in tumor cells. Introduction of rit42 cDNA into human cancer cells reduced cell growth both in vitro and in nude mice. Moreover, analysis of a tetracycline-regulated p53-inducible system in null-p53 cell lines showed that RTP/rit42 mRNA expression increased concomitantly with p53 expression and followed a similar time course. In addition, DNA-damaging agents induced RTP/rit42 expression in a p53-dependent manner but independent of a p53-mediated G1 arrest. Immunofluorescence analysis of a FLAG epitope-tagged RTP/rit42 protein revealed a cytoplasmic localization pattern with redistribution to the nucleus upon DNA damage. We have localized RTP/rit42 to human chromosome 8q24.3. Taken together, these results are consistent with a growth inhibitory role for RTP/rit42, and its down-regulation may contribute to the tumor malignant phenotype.     

Biochemical and immunochemical characterization of Brassica juncea glyoxalase I

A homogenous preparation of glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1.5) was obtained from Brassica juncea seedlings. The enzyme is a heterodimer with 27,000 and 29,000 M(r) subunits and native M(r) of 56,000. The circular dichroic spectra of the protein showed characteristics of a distinctly helical protein, and magnesium affected the secondary structure. It is a zinc metalloenzyme. Amino acid modification studies suggested the involvement of histidine residues in catalysis. Apo-glyoxalase I was reactivated by divalent cations Mn2+ (0.5 Mm) > Mg2+ (5 Mm) > Zn2+ (0.05 Mm) and Ca2+ (0.01 Mm). Monospecific, polyclonal anti-glyoxalase I antibodies were raised, which showed its presence in seeds, roots, hypocotyl, cotyledon and different flower parts. They showed varied degree of cross reactivity with the extracts from various plants, yeast, bacteria and animal system.     

The effects of reverse micelles on the reaction mechanism of alpha-chymotrypsin

Reverse micelles were employed to test the accuracy of the widely accepted mechanism for alpha-chymotrypsin in a highly structured aqueous system similar to intracellular conditions. Results yielded from spectrophotometrical assays of the alpha-chymotrypsin catalyzed hydrolysis of both p-nitrophenyl acetate (p-NPA) and p-nitrophenyl trimethylacetate (p-NPTA) were kinetically analyzed to determine constants typical of the proposed mechanistic model. This was accomplished through the establishment of a control, i.e. the well studied buffer system, for comparison between the reverse micellular environment and a bulk aqueous solution. Control group results yielded kinetic constants in favor of the proposed mechanism (Km = 1.55 x 10(-5) +/- 1.40 x 10(-6) M for p-NPA and a Km = 4.97 x 10(-6) +/- 2.29 x 10(-7) M, Km(app) = 4.92 x 10(-6) +/- 2.33 x 10(-8) M, k2 = 4.34 x 10(-3) +/- 1.31 x 10(-3), k(cat) = 1.96 x 10(-3) +/- 2.47 x 10(-4), and Ks = 1.60 x 10(-5) +/- 4.61 x 10(-6) M for p-NPTA). In contrast, similar reactions of the enzyme in a reverse micellular system produced kinetic constants atypical to that representative of the textbook mechanism. (Km = 1.59 x 10(-4) +/- 2.70 x 10(-5) M, Ks = -8.67 x 10(-5) +/- 4.46 x 10(-5) M and Km(app) = -4.80 x 10(-5) +/- 7.05 x 10(-5) M for p-NPA and Km = 1.95 x 10(-4) +/- 9.28 x 10(-5) M, Km(app) = -1.79 x 10(-4) +/- 2.36 x 10(-5) M, and Ks = -3.95 x 10(-4) +/- 1.18 x 10(-4) M for p-NPTA). In addition to negative kinetic constants, alpha-chymotrypsin seemed to display characteristics indicative of super-activity and a hysteretic response. Overall, the widely accepted mechanism for alpha-chymotrypsin appeared to fail within the confines of reverse micelles, due to the direct influence of the system's highly structured form.