Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Predator-induced behavioral and morphological plasticity in the tropical marine Gastropod Strombus gigas

Florida queen conch stocks once supported a significant fishery, but overfishing prompted the state of Florida to institute a harvest moratorium in 1985. Despite the closure of the fishery, the queen conch population has been slow to recover. One method used in the efforts to restore the Florida conch population has been to release hatchery-reared juvenile conch into the wild; however, suboptimal predator avoidance responses and lighter shell weights relative to their wild counterparts have been implicated in the high mortality rates of released hatchery juveniles. We conducted a series of experiments in which hatchery-reared juvenile conch were exposed to a predator, the spiny lobster (Panulirus argus), to determine whether they could develop behavioral and morphological characteristics that would improve survival. Experiments were conducted in tanks with a calcareous sand substrate to simulate a natural environment. Conditioned conch were exposed to caged lobsters while conch in the control tanks were exposed to empty cages. Conditioned conch moved significantly less and buried themselves more frequently than the naive control conch. Morphometric data indicated that the conditioned conch grew at a significantly slower rate than the naive conch, but the shell weights of the two groups were not significantly different. This implies that the conditioned conch had thicker or denser shells than the control group. As a result, the conditioned conch had significantly higher survival than naive conch in a subsequent predation experiment in which a lobster was allowed to roam free in each tank for 24 hours. In the future, the conditioning protocols documented in this study will be used to increase the survival of hatchery-reared conch in the wild.     

Functional analysis of mouse hepatocytes differing in DNA content: volume,receptor expression,and effect of IFNgamma

Polyploidy and binuclearity are characteristics of the mammalian liver. Increasing polyploidisation occurs with age and after administration of various drugs and chemicals. This study was designed to examine the function of ploidy by addressing several questions: (1) Does the increase in size of polyploid hepatocytes have any physiological function by altering surface receptor expression such as intercellular adhesion molecule‐1 (ICAM‐1, CD54) or IFNγR? and (2) Do polyploid cells respond differently to inflammatory cytokines such as interferon gamma (IFNγ)? We have developed a method to accurately measure the volume of live isolated hepatocytes using confocal microscopy and image analysis. Using flow cytometry, we have shown that the expression of ICAM‐1 increases with increasing DNA content and IFNγR is not detectable on isolated mouse hepatocytes. Diploid (2n), tetraploid (4n) and octoploid (8n) hepatocytes were found to be equally susceptible to IFNγ‐induced apoptosis in vitro. Although the function of polyploidy remains unanswered, we have described some of the characteristics of polyploidy in isolated hepatocytes and in vitro. J. Cell. Physiol. 191: 138–144, 2002. © 2002 Wiley‐Liss, Inc.     

Evidence for male XO sex-chromosome system in Pentodon bidens punctatum (Coleoptera Scarabaeoidea: Scarabaeidae) with X-linked 18S-28S rDNA clusters

In scarab beetle species of the genus Pentodon, the lack of analysis of sex chromosomes in females along with the poor characterization of sex chromosomes in the males, prevented all previous investigations from conclusively stating sex determination system. In this study, somatic chromosomes from females and spermatogonial chromosomes from males of Pentodon bidens punctatum (Coleoptera: Scarabaeoidea: Scarabaeidae) from Sicily have been analyzed using non-differential Giemsa staining. Two modal numbers of chromosomes were obtained: 2n = 20 and 19 in females and males, respectively. This finding along with other karyological characteristics such as the occurrence of one unpaired, heterotypic chromosome at metaphase-I and two types of metaphase-II spreads in spermatocytes demonstrate that a XO male/XX female sex determining mechanism - quite unusual among Scarabaeoidea - operates in the species investigated here. Spermatocyte chromosomes have also been examined after a number of banding techniques and fluorescent in situ hybridization with ribosomal sequences as a probe (rDNA FISH). The results obtained showed that silver and CMA(3) staining were inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) due to the over-all stainability of both constitutive heterochromatin and heterochromatin associated to the NORs. This suggests that heterochromatic DNA of P. b. punctatum is peculiar as compared with other types of heterochromatin studied so far in other invertebrate taxa. By rDNA FISH major ribosomal genes were mapped on the X chromosome.     

Helicobacter pylori strains expressing the vacuolating cytotoxin interrupt phagosome maturation in macrophages by recruiting and retaining TACO (coronin 1) protein

Recent evidence suggests that persistence of Helicobacter pylori can be explained, at least in part, by the failure of macrophages to kill bacteria. The fate of type 1 H. pylori strain LC11, which expresses the cag pathogenicity island (PAI) and the vacuolating cytotoxin, and type 2 strain LC20, which lacks both these virulence factors, was determined following infection of the murine macrophage cell line RAW 264.7 or the human macrophage-like cell line THP-1. Helicobacter pylori strain LC11 displayed enhanced survival in macrophages in comparison with strain LC20 (4.0 +/- 0.2 versus 2.1 +/- 0.6 log CFU ml-1, P < 0.01) at 24 h. Phagosomes containing strain LC11 showed reduced co-localization with LysoTracker Red, higher levels of expression of the early endosome marker EEA1 expression and lower expression of the late endosome/lysosome marker LAMP1 relative to internalized strain LC20, both at 2 h and 24 h. These findings indicate that, in contrast to strain LC20, strain LC11 resides in a compartment with early endosome properties and does not fuse with lysosomes. In addition, phagosomes containing LC11 recruited and retained a higher percentage of TACO (coronin 1) protein in comparison with phagosomes containing strain LC20. Furthermore, IFN-gamma stimulation facilitated maturation of phagosomes containing strain LC11 in association with the release of TACO and a reduction in bacterial survival. We have demonstrated through the use of isogenic cagA-, cagE-/picB- and vacA- mutant strains, that VacA plays a significant role in the interruption of the phagosome maturation. Taken together, these results indicate that, following phagocytosis, H. pylori strains expressing the vacuolating cytotoxin arrest phagosome maturation in association with the retention of TACO.     

Mother's mitochondria and optimal offspring sex ratio

In certain cases, predicted by evolutionary theory of sex-allocation and confirmed by empirical evidence, animals adaptively change their progeny sex-ratio according to individual circumstances. Here we argue that a similar response of offspring sex-ratio must exist in relation to genetic variation of mothers' mitochondria, as a consequence of maternal inheritance of these organelles and of their influence on fitness resulting from their crucial role in metabolism. In fact, a mathematical analysis of evolutionary dynamics of sex-allocation mutants demonstrates that natural selection promotes an evolutionarily stable allocation policy where mothers with defective mitochondria generate only sons, while those with optimal mitochondria have female biased progenies.     

Innervation of vas deferens and accessory male genital glands in the water buffalo (Bubalus bubalis). Neurochemical characteristics and relationships to the reproductive activity

Autonomic nerves supplying mammalian male internal genital organs have an important role in the regulation of reproductive function. To find out the relationships between the neurochemical content of these nerves and the reproductive activity, we performed a histochemical and immunohistochemical study in a species, the water buffalo, exhibiting a seasonal sexual behaviour. The distribution of noradrenergic and nitric oxide synthase (NOS)- and peptide-containing nerves was evaluated during the mating and non-mating periods. Fresh segments of vas deferens and accessory genital glands were collected immediately after slaughter and immersed in 4% paraformaldehyde. Frozen sections were obtained and processed according to single and double labelling immunofluorescent procedures or NADPH-diaphorase histochemistry. During the mating period, a dense noradrenergic innervation was observed to supply the vas deferens as well as the accessory genital glands. NOS- and peptide-containing nerves were also observed but with a lower density. During the non-mating period noradrenergic nerves dramatically reduced. In addition, neuropeptide Y (NPY)- and vasoactive intestinal peptide (VIP)-containing nerves were also reduced. These findings suggest the presence of complex interactions between androgen hormones and the autonomic nerve supply in the regulation of male water buffalo reproductive functions.     

Nontyphoidal salmonellae in United Kingdom badgers: prevalence and spatial distribution

Eighteen (72%) of 25 badger social groups were found to excrete Salmonella enterica serovar Ried, S. enterica serovar Binza, S. enterica serovar Agama, or S. enterica serovar Lomita. Each serovar was susceptible to a panel of antimicrobials. Based on results of pulsed-field gel electrophoresis, the S. enterica serovar Agama and S. enterica serovar Binza isolates were very similar, but two clones each of S. enterica serovar Lomita and S. enterica serovar Ried were found. Badgers excreting S. enterica serovar Agama were spatially clustered.     

Matrilineal Cohesion and Social Networks in Macaca fuscata

In a provisioned troop of Japanese macaques (Macaca fuscata) in Arashiyama, Japan, greater adherence to Kawamura's rules of matrilineal rank inheritance and youngest ascendancy occurred among high-ranking females versus low-ranking females. Accordingly, high-ranking females formed more clustered hierarchies and low-ranking females had more dispersed hierarchies. A proximate explanation for this finding may be related to differences in how females maintain their social networks. To determine whether the clustering in the hierarchy was reflected in patterns of social cohesiveness, I compared network sizes of coalition and grooming partners for females in each third of the hierarchy. I calculated the proportion of available partners that were coalition and grooming partners within each category of relatedness (0.5 r 0.004 and r = 0). High-ranking females formed coalitions with a large proportion of their close relatives and a small proportion of their distant relatives; middle-ranking females supported an intermediate proportion of their close relatives and a small proportion of their distant relatives; and, low-ranking females formed coalitions with very few available close and distant relatives. High-ranking females groomed nearly all available close relatives and an intermediate proportion of distant relatives, whilst middle- and low-ranking females groomed a large proportion of available close relatives and a very small proportion of distant relatives. Thus, levels of clustering within the hierarchy appeared to reflect levels of social cohesion, in terms of grooming and coalition formation.