Developmental appearance of the Ca2+-binding proteins parvalbumin,calbindin D-28K,S-100 proteins and calmodulin during testicular development in the rat

Summary Calcium and intracellular Ca²⁺-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

In goodeid teleosts, prolonged embryonic development takes place within the ovarian cavity. Apposed maternal and embryonic epithelia interface via a nutritive liquid (embryotrophe) and facilitate aplacental matrotrophy. The role of the internal ovarian epithelium (IOE) in providing proteins for the embryotrophe has been studied using transmission electron-microscopic examinations of both the resting and the active ovarian lining, and isoelectric focusing of embryotrophe and maternal blood serum. The simple IOE is apparently composed of only one, filament-containing cell-type. In the non-gravid ovary these cells are cuboidal to columnar in shape, and are either compact and electron-dense or oedematous and light. During gestation, swelling of the ovarian connective tissue gives rise to dovetailing of the IOE with the subjacent capillary plexus. Part of the IOE overlying the capillaries becomes stretched, resulting in a thin endothelium-like demarcation. The nuclei and the bulk of the cytoplasm are usually recessed between the meshes of the protruding capillary network. The blood-embryotrophe pathway is thus reduced in places, to less than one m. The active form of the IOE contains a well-developed vacuolar apparatus composed of small vesicles, vacuoles, multivesicular bodies, and a few lysosomes. Elements of the RER are sparsely distributed throughout the cytoplasm. Endocytotic activity is observable at the apical and basolateral plasma membrane. Isoelectric focusing of both serum and embryotrophe produces numerous bands each between pI 4–8, which reveal many homologies. The intensity of corresponding bands varies considerably. It is concluded that the cells of the IOE provide a transport pathway for serum-derived macromolecular substances rather than produce proteinaceous secretions.     

Localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats An immunohistochemical study

Summary Immunohistochemical localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats was studied by indirect fluorescent antibody staining technique. A polyclonal antibody for cytochrome P-450_MC purified from hepatic microsomes of 3-methylcholanthrene-pretreated rats was used for this experiment. A strong immunofluorescence was found to be localized in the cytoplasm of the surface epithelium of the mucosa in the colon of 3-methylcholanthrene-pretreated rats. A faint immunofluorescence was also observed in the epithelium of untreated rats. 7-Ethoxycoumarin O-deethylase activity of colonic microsomes was significantly enhanced by 3-methylcholanthrene-pretreatment in parallel with an increase in the intensity of immunostaining for cytochrome P-450_MC in Western blotting analysis. This is the first report on the localization of cytochrome P-450 in the colonic mucosa.     

The efferent innervation of the genital chamber by an identified serotonergic neuron in the female cricket Acheta domestica

Summary The serotonergic innervation of the genital chamber of the female cricket, Acheta domestica, has been investigated applying anti-serotonin (5-HT) immunocyto-chemistry at both light- and electron-microscopic levels as well as using conventional electron microscopy. Whole mount and pre-embedding chopper techniques of immuno-cytochemistry reveal a dense 5-HT-immunoreactive network of varicose fibers in the musculature of the genital chamber. All of these immunoreactive fibers originate from the efferent serotonergic neuron projecting through the nerve 8v to the genital chamber (Hustert and Topel 1986; Elekes et al. 1987). At the electron-microscopic level, 5-HT-immunoreactive nerve terminals, which contain small (50–60 nm) and large ( 100 nm) agranular vesicles as well as granular vesicles (100nm), contact the muscle fibers or the sarcoplasmic processes without establishing specialized neuromuscular connections. In addition to the 5-HT-immunoreactive axons, two types of immunonegative axons can also be found in the musculature. By use of conventional electron microscopy, three ultrastructurally distinct types of axon processes can be observed, one of which resembles 5-HT-immunoreactive axons. While the majority of the varicosities do not synapse on the muscle fibers, terminals containing small (50–60 nm) agranular vesicles occasionally form specialized neuromuscular contacts. It is suggested that the 5-HTergic innervation plays a non-synaptic modulatory role in the regulation circular musculature in the genital chamber of the cricket, while the musculature as a whole may be influenced by both synaptic and modulatory mechanisms.Fellow of the Alexander von Humboldt-Stiftung     

Morphometrical analysis of the gap-junctional area in parenchymal cells of the rat liver after administration of dibutyryl cAMP and aminophylline

Summary In view of the presumed involvement of gap junctions in the coordination of metabolic activities, the influence of cAMP as a regulatory signal of cell metabolism on gap junctions of hepatocytes has been examined. Male rats received two intraperitoneal doses of 10 mg dibutyryl cAMP/100 g body weight with a time interval of 2.5 h and were decapitated 2.5 h later. After this 5-h interval, analysis of freeze-fracture replicas of fixed liver tissue revealed an increase in the mean (± SEM) gap-junctional membrane portion on the lateral hepatocyte membranes from 0.049 + 0.003 (n = 66) in controls to 0.061 ± 0.003 (n = 70) in treated rats, while the configuration of the connexons appeared unaltered. This effect could not be reinforced by prior administration of aminophylline: the relative gapjunctional area is similarly extended from 0.054 ± 0.003 (n = 126) in the control group to 0.065 ± 0.004 (n = 105) in the experimental animals. Probing for the time course of the junctional response, a group of rats was sacrificed 3 h after the onset of treatment. Already within this time, the gapjunctional area is augmented from 0.042 ± 0.004 (n = 63) in the concurrent controls to 0.069 ± 0.006 (n = 42) in the treated rats. These statistically significant increases in area may suggest a stimulating effect of cAMP on gap junctions of hepatocytes in vivo.This investigation was supported by grant No. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium)     

Indications for the presence of two populations of serotonin-containing pinealocytes in the pineal complex of the golden hamster (Mesocricetus auratus)

Summary Serotonin-like immunoreactivity was investigated in the pineal complex of the golden hamster by use of the indirect immunohistochemical technique. The superficial and deep portions of the pineal gland, and also the pineal stalk exhibited an intense cellular immunoreaction for serotonin. In addition, perivascular serotonin-immunoreactive nerve fibers were observed. Some serotonin-immunoreactive processes of the pinealocytes terminated on the surface of the ventricular lumen in the pineal and suprapineal recesses, indicating a receptive or secretory function of these cells. Several serotonin-immunoreactive processes connected the deep pineal with the habenular area. One week after bilateral removal of both superior cervical ganglia the serotonin immunoreaction of the entire pineal complex was greatly decreased. However, some cells in the pineal complex, of which several exhibited a neuron-like morphology, remained intensively stained after ganglionectomy. This indicates that the indoleamine content of some cells in the pineal complex of the golden hamster is independent of the sympathetic innervation.Supported by a Grant from the Italian Society for Veterinary Sciences     

In vitro detachment of bacteria from ruminal digesta by buffered sodium oleate solutions

The effectiveness of a buffered sodium oleate solution was evaluated for detaching bacteria from ruminal digesta samples. A response surface derived from an octagonal design was used to determine the pH and concentration combination for maximum detachment of total and cellulolytic bacteria. The total number of bacteria detached increased up to 81% over control with treatment of a pH 8.8 and 1.5% sodium oleate solution. The recovery of cellulolytic bacteria was decreased to 35% of control with treatment of a pH 9.0 and 0.1% sodium oleate solution. Attempts to improve the recovery of viable bacteria exposed to sodium oleate solutions were unsuccessful. This response surface design identified an optimal pH and concentration that were consistent with existing information regarding detachment of total bacteria, and suggested that sodium oleate, at the concentrations tested, was toxic to the cellulolytic population of the rumen.     

Electron cytochemistry of mycobacteria: Evidence that strongly acidic sulfate groups are present on the surface of H37Rv (virulent) strain ofMycobacterium tuberculosis

Cytochemical characterization of mycobacterial surfaces was carried out on virulent (H37Rv) and avirulent (H37Ra) strains ofMycobacterium tuberculosis. The results were quantified and compared with those obtained with three colony types of the opportunistic pathogenMycobacterium avium. Mycobacterium aurum, a rapidly growing, nonpathogenic species, served as a model for the cytochemical methods. Concanavalin A (ConA) reacted with -d-mannose and -d-glucose residues, whereas negative charged residues were detected with either the ionized ferritin (CF) or the colloidal ferric hydroxide (CIH) method. Strongly acidic sulfate groups were detected by their selective blockage with alcian blue (AB) at pH 1 prior to the CIH labeling at pH 1.8. Weakly acidic groups were demonstrated by AB blockage at pH 2.5 prior to staining with CF stain. Except forM. aurum, all other strains showed a marked heterogeneity in regard to the abundance of their surface labeling. Accessible sulfate groups were present on the cell surface of the virulent H37Rv strain ofM. tuberculosis, but not on the avirulent strain H37Ra. Distribution of ConA receptors, on the other hand, was unrelated to the virulence or pathogenicity of the bacterial strain.     

Construction of transposons encoding genes for β-glucosidase,amylase and polygalacturonatetrans-eliminase fromKlebsiella oxytoca and their expression in a range of gram-negative bacteria

DNA fragments containing theKlebsiella oxytoca genes encoding -glucosidase and amylase were cloned into the kanamycin resistance transposon Tn5. Another DNA fragment containing two genes for polygalacturonatetrans-eliminase was cloned into Tn1721. These newly constructed transposons were then each transposed in vivo onto the broad-host-range plasmid pR751 and conjugally transferred to a variety of Gram-negative bacteria. These were then screened for the newly acquired phenotypes.     

Glucose utilization and growth response to protein hydrolysates byFusobacterium species

Glucose uptake was measured in the supernatants of 18 strains ofFusobacterium species cultured in BM medium. Some species, such asF. nucleatum andF. necrophorum, used between 25% and 48% of the glucose in the medium, but the terminal pH remained near neutral. By contrast, strains ofF. mortiferum andF. necrogenes used on average over 90% of the available glucose in the medium and produced a predictably low acidic pH. Strains ofF. varium used between 86% and 91% of the glucose present but produced a near neutral pH of between 5.8 and 5.9. The metabolic fate of glucose inF. varium was, therefore, examined in more detail. Glucose stimulated the growth of this species, and [¹⁴C]glucose was incorporated into the metabolic end products and various cellular components. Protein hydrolysates, tested for their growth-promoting effects onFusobacterium species, produced two general growth response patterns. Most species grew prolifically on trypticase, proteose peptone, and yeast extract, but poorly in casamino acids and vitamin-free casamino acids. Growth in bactocasitone was poor, but for three species,F. necrophorum, F. varium, andF. nucleatum, there was an approximately linear growth response up to 0.5%. These results suggest a major role for nitrogen metabolism but do not preclude glucose as an energy source in at least some species ofFusobacterium.