Immunolocalization of α1A-adrenoceptors in rat and human epididymis

Immunohistochemistry was conducted to analyze the cellular localization of alpha(1A)-adrenoceptors along rat and human epididymis. ADR-A, a polyclonal antibody that recognizes the specific C-terminal region of alpha(1A)-adrenoceptors, immunostained this adrenoceptor subtype in smooth muscle cells surrounding the epididymal tubules and interstitial blood vessels and in subpopulations of epithelial cells from adult rat and human caput and cauda epididymidis. The same cell types from rat epididymidis were immunostained by ADR-1, a polyclonal antibody that recognizes a common region of the three alpha(1)-adrenoceptor subtypes, alpha(1A), alpha(1B), and alpha(1D). Immunostaining with both antibodies was also conducted in adult rat and human vas deferens and seminal vesicle used as positive controls because of the abundance of alpha(1A)-adrenoceptors in these tissues. ADR-A- and ADR-1-positive immunostaining was differentially distributed depending on the antibody, method of tissue fixation (Bouin-fixed and fresh frozen tissues), species (rat and human), tissue (caput and cauda epididymidis), and age (immature and adult rats) analyzed. This is the first report immunolocalizing alpha(1A)-adrenoceptor along rat and human epididymis. The presence of this adrenoceptor subtype in epididymal smooth muscle and epithelial cells indicates their contribution to smooth muscle contractile responses and a possible role in the absorptive and/or secretory activities of the epithelium lining the epididymal duct. Taken together, our results should contribute to a better understanding of the physiological role of alpha(1)-adrenoceptors in the epididymidis and the importance of the sympathetic nervous system for male (in)fertility.     

Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex--a system capable of producing nitric oxide and singlet oxygen

The synthesis, structural aspects, pharmacological assays, and in vitro photoinduced cytotoxic properties of [Ru(NO)(ONO)(pc)] (pc = phthalocyanine) are described. Its biological effect on the B16F10 cell line was studied in the presence and absence of visible light irradiation. At comparable irradiation levels, [Ru(NO)(ONO)(pc)] was more effective than [Ru(pc)] at inhibiting cell growth, suggesting that occurrence of nitric oxide release following singlet oxygen production upon light irradiation may be an important mechanism by which the nitrosyl ruthenium complex exhibits enhanced biological activity in cells. Following visible light activation, the [Ru(NO)(ONO)(pc)] complex displayed increased potency in B16F10 cells upon modifications to the photoinduced dose; indeed, enhanced potency was detected when the nitrosyl ruthenium complex was encapsulated in a drug delivery system. The liposome containing the [Ru(NO)(ONO)(pc)] complex was over 25% more active than the corresponding ruthenium complex in phosphate buffer solution. The activity of the complex was directly proportional to the ruthenium amount present inside the cell, as determined by inductively coupled plasma mass spectroscopy. Flow cytometry analysis revealed that the photocytotoxic activity was mainly due to apoptosis. Furthermore, the vasorelaxation induced by [Ru(NO)(ONO)(pc)], proposed as NO carrier, was studied in rat isolated aorta. The observed vasodilation was concentration-dependent. Taken together, the present findings demonstrate that the [Ru(NO)(ONO)(pc)] complex induces vascular relaxation and could be a potent anti-tumor agent. Nitric oxide release following singlet oxygen production upon visible light irradiation on a nitrosyl ruthenium complex produces two radicals and may elicit phototoxic responses that may find useful applications in photodynamic therapy.     

Nutritional development of feeding strategies in arctic ruminants: digestive morphometry of reindeer, Rangifer tarandus, and muskoxen, Ovibos moschatus

Reindeer have been classified as intermediate feeders and muskoxen as grazers based on differences in digestive morphology and consumption of fibrous plants. We hypothesized that the digestive morphology of young (<2 months) reindeer and muskoxen anticipates transitions in diet and determines the feeding strategy of each species at adulthood. We compared structural morphology and rates of cell division in the rumen, abomasum, duodenum and liver of reindeer and muskoxen as neonates (1 day old), during the transition from milk to forage (30–60  days old) and in adults (>7 yr). Development in utero provided the neonate with a functioning mucosa of the gastric abomasum and duodenal mucosa with high surface enlargement for digestion and absorption of concentrated milks. Transition to forage was preceded by changes in ruminal papillae structure that increased surface area and likely contributed to active fermentation by 60 days of age. The abomasum also increased in acid-secreting parietal cells during the transition to forage, which may enhance digestion of plant and microbial proteins. Rates of cell division also indicated a sustained differentiation of tissue structure during the transitional period. Young arctic ruminants expressed digestive structures that preceded full function, which indicated the ultimate feeding strategy of each species. For example, the rumen of young muskoxen had thick cornified epithelia and muscle layers that would provide ruminal mucosa with better protection from fibrous abrasion and enhance motility of bulky diets. Conversely, young reindeer had more complex papillary shapes in the rumen and more foliate villi in the duodenum, indicating a greater absorptive capacity of these structures than in muskoxen. Ontogenetic programs, therefore, play the primary role for digestive development of reindeer and muskoxen and determine the nutritional strategies of adults.     

Copper status of muskoxen: a comparison of wild and captive populations

We compared wild muskoxen (Ovibos moschatus) on Banks Island (Northwest Territories, Canada) with captive animals maintained on grass (Bromus sp.) hay and supplemental minerals. We measured copper (Cu) in liver, whole serum, and deproteinated serum (unbound Cu) as well as serum activity of the Cu-enzyme ceruloplasmin. Unbound serum Cu concentrations did not change with season in captive animals (n = 53). Ceruloplasmin activity was similar between seasons in females but elevated in males during breeding in autumn. Increasing concentrations of Cu in whole sera were mainly associated with protein whereas unbound Cu predominated at low concentrations of whole serum Cu. Ceruloplasmin activity and serum Cu concentration were linearly related to liver Cu in female muskoxen. Measures of copper status in females were lower in the wild (n = 19) than in captivity (n = 16): 8 vs. 160 ug Cu.g-1 of whole liver; 0.67 vs. 1.15 microgram unbound Cu.ml-1 whole serum and; 22 vs. 33 IU.l-1 ceruloplasmin activity. Bioavailability of Cu may limit the population on Banks Island especially when density of animals is high. The wide range of hepatic Cu concentrations in muskoxen indicated accumulation of Cu without apparent ill effect in captive animals. Hepatic storage of Cu may allow wild muskoxen to contend with low and fluctuating availability of Cu in small foraging areas at high latitudes.     

Trypanosoma cruzi: partial characterization of minor cruzipain isoforms non-adsorbed to Concanavalin A-Sepharose

The present paper reports the partial characterization of a subset of atypical cruzipain molecules which do not bind to Concanavalin A-Sepharose column. They are present in different strains of epimastigote forms of Trypanosoma cruzi and represent a 2-4% of total cruzipain. They were purified by affinity chromatography on Cystatin-Sepharose, recognized by the polyclonal anti-cruzipain serum, and their activity in gelatin-containing gels was completely abolished by E-64, TLCK, leupeptin, and aprotinin but not by PMSF, pepstatin A, EDTA or 1,10-phenantroline. These cysteine proteinases, as well as cruzipain showed to be endoproteinases able to hydrolize azocasein, hemoglobin, and bovine serum albumin at acidic pHs. However, evidences are presented indicating that this subset of cruzipain isoforms were also able to use the same blocked chromogenic peptidyl substrates than cruzipain at similar optimal alkaline pH values although with a different order of preference. Moreover, they showed a different oligosaccharide pattern after enzymatic treatment by high pH anion exchange chromatography, suggesting that this structural difference may account for the atypical behaviour in the lectin column.     

Myosin light-chain expression during avian muscle development

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.     

Vasorelaxant activity of phthalazinones and related compounds

Several series of dihydrostilbenamide, imidazo[2,1-a]isoindole, pyrimido[2,1-a]isoindole and phthalazinone derivatives were obtained and their vasorelaxant activity was measured on isolated rat aorta rings pre-contracted with phenylephrine (10(-5)M). Some phthalazinones attained, practically, the total relaxation of the organ at micromolar concentrations. For the most potent compound 9h (EC(50)=0.43microM) the affinities for alpha(1A), alpha(1B) and alpha(1D) adrenergic sub-receptors were determined.     

Body protein stores and isotopic indicators of N balance in female reindeer (Rangifer tarandus) during winter

We studied bred and unbred female reindeer (Rangifer tarandus tarandus) during 12 wk of winter when ambient temperatures were low and nitrogen (N) demand for fetal growth is highest in pregnant females. Animals were fed a complete pelleted diet ad lib. that contained 2.54% N in dry matter that was 80% +/- 2% (X +/- SD) digestible. Female reindeer lost 64% +/- 14% of body fat but gained 34% +/- 11% of lean mass from 10 wk prepartum to parturition. These changes were equivalent to average balances of -14.14 +/- 2.35 MJ d(-1) and 10 +/- 3 g N d(-1). Blood cells, serum, and urine declined in (15)N/(14)N in late winter as body protein was gained from the diet. Blood cells of newborn calves were more enriched in (15)N and (13)C than that of their mothers, indicating the deposition of fetal protein from maternal stores. To quantify pathways of N flow in reindeer, N balance was measured by confining animals to cages for 10 d at 4 wk from parturition. N balance was inversely related to (15)N/(14)N in urea-N but not related to (15)N/(14)N of blood cells, creatinine, and feces. The proportion of urea-N derived from body protein increased above 0.46 as N balance fell below -200 mg N kg(-0.75) d(-1). Proportions of urea-N from body protein were -0.01 +/- 0.21 in pregnant females before and after caging and were consistent with average body protein gain in winter. Storage of protein allows reindeer and caribou to tolerate diets that are low in N without impairing fetal development.