Reduction of tetrazolium salts by sulfate-reducing bacteria

Abstract The reduction of tetrazolium salts by the sulfate-reducing bacteria, Desulfovibrio desulfuricans and Desulfotomaculum orientis , was examined. D. desulfuricans and D. orientis reduced triphenyltetrazolium chloride (TTC) and 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyltetrazolium chloride (INT) forming intracellular formazan deposits. The reduction rate of INT was higher than that of TTC. INT reduction was not inhibited by the addition of sulfate or molybdate, and sulfate uptake was inhibited by the addition of both INT and molybdate. The ratio of intracellular formazan forming cells to acridine orange direct counts in both strains decreased with culture age and starvation time.     

Patterns of paternal care in primates

An interspecific comparison was carried out to understand better the relationships among paternal care, paternal certainty, and reproductive burden in primates. Although monogamy is generally rare among mammals, a number of primate species are monogamous. Extensive paternal care is a related issue but is one that is not necessarily associated with monogamy or with paternal certainty. For example, despite paternal certainty, primate mothers in monogamous species with body weights over 2 kg still remain the primary infant caretakers, while males in the communally breeding tamarins carry infants more frequently than mothers do, even in the absence of paternal certainty. Several different tactics are used by small-bodied primates to cope with the energetic burden of raising proportionately large infants in an arboreal environment: (1) infant carrying by subadult and/or related nulliparous females (Saimiri, Lemur monogoz); (2) infant carrying by fathers and offspring (Aotus, Callicebus, Saguinus, Cebuella, Leontopithecus); (3) parking infants while family members forage (Tarsius, Galago, Microcebus, Cheirogaleus, Varecia); or (4) some combination of the above (Callithrix, Hapalemur, Loris). Lactation length and infant growth patterns appear to influence which of these tactics is employed by a given species. Moreover, although most small-bodied, mated, monogamous female primates spend no more than 9 months annually in gestation and lactation,Aotus andCallicebus mated females are either pregnant or lactating on a year-round basis. It is this heavy female reproductive burden that may be an important factor in selection for extensive paternal care in these monogamous cebids.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Detection of Glycogen-Debranching System in Trophozoites of Entamoeba histolytica

ABSTRACT. Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4- α -glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl- α -glucoside yielding successively 4-nitrophenyl- α -maltoside and 4-nitrophenyl- α -maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of M_r= 180,000 ± 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

Two Types of Apoptotic Cell Death of Rat Central Nervous System-Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 m M DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.     

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

Effects of Chronic Ethanol Consumption and Aging on Dopamine, Serotonin, and Metabolites

Abstract: This study examined the hypothesis that chronic ethanol consumption results in significant abnormalities in both the dopaminergic and the serotonergic system of aged rats. Levels of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindole-3-acetic acid (5-HIAA) were determined in brain areas of both the nigrostriatal and mesocorticolimbic DA systems in 5-, 14-, and 24-month-old male Fischer 344 rats. Aging was associated with a reduced concentration of DA in the striatum (ST), ventral tegmental area (VTA), and ventral pallidum (VP) and an increased concentration of 5-HIAA in the ST, globus pallidus, nucleus accumbens, frontal cortex, and VP. In addition, there was an increase in the 5-HIAA/5-HT ratio in all brain areas analyzed. Six weeks of ethanol consumption was accompanied by significant changes in mesocorticolimbic brain areas. In the VTA of 5-month-old ethanol-fed rats DA content was decreased to the levels found in aged rats, e.g., 24 months of age. Ethanol also significantly lowered 5-HT and 5-HIAA contents in the VTA and reduced DOPAC and 5-HIAA levels in the VP. In addition, ethanol blunted the normal age-related increase in 5-HIAA/5-HT ratio in the VTA, VP, and substantia nigra. It is interesting that although the age-related changes were found in both nigrostriatal and mesocorticolimbic brain areas, the ethanol-associated effects were found only in brain areas of the mesocorticolimbic system. The changes in DA and 5-HT function that accompany aging and ethanol consumption may contribute to the problems in motor function and ethanol abuse found in the aged.     

FIXATION PROBABILITIES OF SELFING RATE MODIFIERS IN SIMULATIONS WITH SEVERAL DELETERIOUS ALLELES WITH LINKAGE

The fixation rates of selfing rate modifiers were found by stochastic simulation in an infinite site model, including effects of several deleterious alleles with variable effects, which were randomly distributed in the genome without assuming any pollen discounting. Previous results on the evolution of selfing obtained by more precise methods were in this study further validated, and it was concluded that the effect of genetic associations on the evolution of mating systems is small except in the case of full pollen discounting. Furthermore, attention was given to the uneven distribution of the genetic load in the population, and the accompanying large among-genome variation in fixation rates. This among-genome variation will be of significance for the evolution of mating systems.     

CORRELATED EVOLUTION OF SELF-FERTILIZATION AND INBREEDING DEPRESSION: AN EXPERIMENTAL STUDY OF NINE POPULATIONS OF AMSINCKIA (BORAGINACEAE)

The relation between inbreeding depression and rate of self-fertilization was studied in nine natural populations of the annual genus Amsinckia. The study included two clades (phylogenetic lineages) in which small-flowered, homostylous populations or species are believed to have evolved from large-flowered, heterostylous, self-compatible ones. In one lineage the small-flowered species is tetraploid with disomic inheritance. Rates of self-fertilization were 25% to 55% in the four large-flowered, heterostylous populations; 72% in a large-flowered but homostylous population; and greater than 99.5% in the four small-flowered, homostylous populations, which produce seed autonomously. When present, inbreeding depression occurred in the fertility but not the survival components of fitness. Using a cumulative fitness measure incorporating both survival and fertility (flower number), we found inbreeding depression to be lower in the four very highly self-fertilizing populations than in the five intermediate ones. The Spearman rank correlation between inbreeding depression and selfing rate for the nine populations was –0.50, but was not statistically significant (P = 0.12). Inbreeding depression was greater in the two tetraploid populations than in the very highly self-fertilizing, diploid ones. Phenotypic stability of progeny from self-fertilization tended to be higher in populations with lower inbreeding depression. We conclude that levels of self-fertilization and inbreeding depression in Amsinckia are determined more by other factors than by each other. Estimates of mutation rates and dominance coefficients of deleterious alleles, obtained from a companion study of the four highly self-fertilizing populations, suggest that a strong relationship may not be expected. We discuss the relationship of the present results to current theory of the coevolution of self-fertilization and inbreeding depression.