Microtechnique for isolating fecal coliforms from soil.

A most-probable-number microtitration technique for isolating fecal coliforms from soil was developed. A correlation coefficient of 0.86, with a 95% confidence interval of 0.76 less than zeta less than 0.92, was obtained when this technique was compared with the standard elevated-temperature fecal coliform most-probable-number procedure.     

The effect of fish and aquatic habitat complexity on amphibians

Fish introductions are considered one of the most widespread anthropogenic threats to aquatic ecosystems. Their negative impact on native amphibian communities has received increasing attention in recent years. We investigated the relationship between the introduced fish, emergent vegetation cover and native amphibians in man-made ponds generated by regulation and dam building along the Tarnava Mare Valley (Romania) during the last 40 years. We inventoried amphibians and fish inhabiting 85 permanent ponds and estimated habitat complexity focusing on emergent vegetation cover. Four amphibian species were found to be negatively associated with the presence of predatory fish. Species richness of ponds without fish and ponds without predatory fish did not differ significantly, whereas ponds containing only predatory fish had significantly lower amphibian richness. A significant positive relationship was found between the emergent vegetation cover and pond occupancy of six amphibian species and amphibian species richness. As a management recommendation, we suggest the restriction of fish introductions to non predatory fish and the maintenance of high emergent vegetation cover in the ponds. Handling editor: S. Declerck     

Cell surface glycoproteins of hepatocytes and hepatoma cells identified by monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MABs) directed to cell surface components of rat hepatocytes were isolated. The antigens of seven MABs were identified as glycosylated plasma membrane proteins. The presence of these glycoproteins on normal hepatocytes and hepatocellular carcinoma cells was analyzed. A semi-quantitative enzyme-linked immunosorbent assay revealed that only two MABs (Be 8.7, Ne 11.3) recognized proteins which were expressed not only in normal liver but also in chemically induced transplantable Morris hepatomas and hepatoma-derived cell lines. The expression of six antigens was found to be sensitive to transformation. The domain specificity of the MABs was determined by indirect immunofluorescence on sections of liver tissue containing neoplastic nodules. Three MABs (Be 8.4, Ne 11.1, Ne 11.3) specifically bound to the sinusoidal domain and two MABs (Be 9.2, De 13.4) to the bile canalicular domain. These five antigens were transformation-sensitive except for the glycoprotein recognized by the MAB Ne 11.3. Three MABs (Be 8.7, Be 9.1, De 13.2) also showed intracellular immunofluorescence. Two of the antigens (Be 9.1, De 13.2) were not present in hepatomas. The relative molar masses (Mr) of the glycoproteins were determined after protein immunoblotting and immunoprecipitation. Four MABs (Be 8.7, Be 9.1, Be 9.2, De 13.4) recognized antigens with a Mr of 110 000 but did not mutually cross-react. The antigen recognized by MAB De 13.4 was identified as the ectoenzyme dipeptidyl peptidase IV (EC 3.4.14.-).     

Kinetic and mechanistic basis of the nonprocessive Kinesin-3 motor NcKin3

Kinesin-3 motors have been shown to transport cellular cargo along microtubules and to function according to mechanisms that differ from the conventional hand-over-hand mechanism. To find out whether the mechanisms described for Kif1A and CeUnc104 cover the full spectrum of Kinesin-3 motors, we characterize here NcKin3, a novel member of the Kinesin-3 family that localizes to mitochondria of ascomycetes. We show that NcKin3 does not move in a K-loop-dependent way as Kif1A or in a cluster-dependent way as CeUnc104. Its in vitro gliding velocity ranges between 0.30 and 0.64 mum/s and correlates positively with motor density. The processivity index (k(bi,ratio)) of approximately 3 reveals that not more than three ATP molecules are hydrolyzed per productive microtubule encounter. The NcKin3 duty ratio of 0.03 indicates that the motor spends only a minute fraction of the ATPase cycle attached to the filament. Unlike other Kinesin-3 family members, NcKin3 forms stable dimers, but only one subunit releases ADP in a microtubule-dependent fashion. Together, these data exclude a processive hand-over-hand mechanism of movement and suggest a power-stroke mechanism where nucleotide-dependent structural changes in a single motor domain lead to displacement of the motor along the filament. Thus, NcKin3 is the first plus end-directed kinesin motor that is dimeric but moves in a nonprocessive fashion to its destination.     

Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere

The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere.     

Characterisation of gene expression patterns in 22RV1 cells for determination of environmental androgenic/antiandrogenic compounds

Alteration of androgen receptor function due to hormonally active compounds in the environment, may be responsible for impaired reproductive function in aquatic wildlife. Based on human prostate carcinoma 22RV1 cells, a cell culture expression system was established to test effects of putative androgenic/antiandrogenic compounds on endogenous gene expression. 22RV1 cells were shown to express human androgen receptor, but not human progestin (hPR) or human oestrogen receptor (hER) alpha and beta. Six androgen-regulated genes (ARGs) were chosen to determine androgenic/antiandrogenic action using highly sensitive real-time RT-PCR. Results showed that gene expression is altered in a time-dependent manner. After stimulation of cells by DHT (10nM), synthetic androgen R1881 (1 nM), or organic pesticides (difenoconazole, fentinacetate, tetramethrin) TMPRSS2 mRNA expression was down-regulated by the factor 0.6 after 24h of DHT treatment. Similar results were obtained when cells were assayed for mRNA expression of PSA after fentinacetate and R1881 stimulation. In contrast, TMPRSS2 expression was up-regulated by the factor 0.9 when cells were stimulated by tetramethrin. Final goal of the work is a sensitive determination of differential gene expression by different compounds under study, achievement of substance-specific expression patterns and function related analysis of potential androgens/antiandrogens.     

Direct negative density‐dependence in a pond‐breeding frog population

Understanding population dynamics is critical for the management of animal populations. Comparatively little is known about the relative importance of endogenous (i.e. density‐dependent) and exogenous (i.e. density‐independent) factors on the population dynamics of amphibians with complex life cycles. We examined the potential effects of density‐dependent and ‐independent (i.e. climatic) factors on population dynamics by analyzing a 15‐yr time series data of the agile frog Rana dalmatina population from Târnava Mare Valley, Romania. We used two statistical models: 1) the partial rate correlation function to identify the feedback structure and the potential time lags in the time series data and 2) a Gompertz state‐space model to simultaneously investigate direct and delayed density dependence as well as climatic effects on population growth rate. We found evidence for direct negative density dependence, whereas delayed density dependence and climate did not show a strong influence on population growth rate. Here we demonstrated that direct density dependence rather than delayed density dependence or climate determined the dynamics of our study population. Our results confirm the findings of many experimental studies and suggest that density dependence may buffer amphibian populations against environmental stress. Consequently, it may not be easy to scale up from individual‐level effects to population‐level effects.     

Social communities and spatiotemporal dynamics of association patterns in estuarine bottlenose dolphins

Network analysis has recently been used to delve into the dynamics of cetacean sociality. Few studies, however, have addressed how habitat shape influences sociality, specifically how linear water bodies constrain the space where individuals might interact. We utilized network and spatiotemporal analyses to investigate association patterns and community structure in a bottlenose dolphin population in a linear estuarine system, the Indian River Lagoon (IRL), Florida. Using sighting histories from a multiyear photo‐identification study we examined association patterns for 185 individuals collected over a 6.5 yr period (2002–2008). The population was highly differentiated (S = 0.723) and organized into six distinct social communities (Q = 0.544), spread in an overlapping pattern along the linear system. Social organization differed between communities, with some displaying highly interconnected networks and others comprising loosely affiliated individuals with more ephemeral associations. Temporal patterns indicated short‐term associations were a significant feature of the fission‐fusion dynamics of this population. Spatial analyses revealed that social structure was shaped by an individual's ranging patterns and by social processes including preference and avoidance behavior. Finally, we found that habitat “narrowness” may be a major driving force behind the sociality observed.     

Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry

Immunohistochemistry and activity histochemistry were used to study the localization of dipeptidyl peptidase (DPP) IV in rats. For immunohistochemistry, polyclonal as well as monoclonal anti-DPP IV antibodies were employed. The pattern of DPP IV immunoreactivity, determined with polyclonal anti-DPP IV antibody, corresponds to the histochemical pattern found for the enzymic activity of DPP IV. Immunoreactivity was present, in addition, in nerve cells, lateral membranes of certain surface epithelia, e.g., Fallopian tube, uterus and vesicular gland, in the luminal cytoplasm of e.g., vesicular gland epithelium, and in mucous cells of Brunner's gland. The monoclonal antibodies against DPP IV recognized four different epitopes (A-D) of the DPP IV molecule, and revealed that certain epitopes were not detectable by immunohistochemistry in some organs. Generally, the staining intensities for epitopes A, B, C and D decreased in that order. Usually, the monoclonal antibodies against epitopes A and B showed similar reaction patterns to those as obtained with the polyclonal antibody. Epitope D was recognized in the lumen of the duct system of exocrine glands and the intestine. Furthermore, high reactivity of this epitope was detected in goblet cells of the intestine, where no DPP IV activity was present.