Nerve Growth Factor Effect on Adenosine Transport in Cultured Chromaffin Cells

Chromaffin cells both recently isolated or in culture present a high-affinity adenosine transporter with a Km value of 1 microM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3 microM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (approximately 200-260%) in NGF-cultured cells throughout the period studied.     

Autolysis of the cell wall. Its possible role in endogenous and IAA-induced growth in epicotyls of Cicer arietinum

Indoleacetic acid (IAA), a factor that induces growth in epicotyls of cicer arietinum L. cv. Castellana, increases the autolytic capacity of the cell walls by 50%, suggesting that autolysis is related to the processes of cell wall loosening that accompany growth. IAA promotes an increase in the specific activities of the enzymes involved in autolysis, mainly α-galactosidase (EC 3.2.1.22). This relationship autolysis-growth. was also observed in a study of the autolytic capacity of cell walls from regions of the epicotyl with different growth capacity. The sugars released and the level of enzymatic protein were higher in the subapical region that towards the base.     

The zooplankton community in the vicinity of the ice edge,western Weddell Sea,March 1986

Summary The zooplankton community in the vicinity of the ice edge in the west central Weddell Sea was investigated in the late austral summer (March 1986). Sampling was done with two ships operating concurrently, one in the pack ice and the other in the adjcent open sea. Metazoan microzooplankton (<1 mm) was most abundant in the epipelagic zone. It consisted mostly of copepod nauplii and copepods of the genera Oithona, Oncaea, Ctenocalanus and Microcalanus. While species composition was similar in both areas, vertical patterns differed in that the microzooplankton had sparse populations in the upper 50 m under the ice. This may have been related to water temperature which in the upper 50 m under the ice was more than 1°C cooler than in the open sea. Zooplankton in the 1–20 mm size range was dominated by the calanoid copepods Metridia gerlachei, Calanus propinquus and Calanoides acutus which constituted half the biomass in the upper 1000 m. Their populations had highest densities in the upper 150 m, though much of the C. acutus population resided below 300 m. Metridia gerlachei and C. propinquus underwent diel vertical migrations in both areas whereas C. acutus did not migrate. Species diversity in the epipelagic zone was moderate and the fauna was characterized by species typical of the oceanic east wind drift. Diversity increased with depth and was due primarily to the appearance of circumpolar mesopelagic copepods in Weddell Warm Deep Water. Biomass of 1–20 mm zooplankton in the 0–1000 m zone was low (1.1–1.3 gDWm^-2) compared to other Southern Ocean areas investigated with comparable methods. It is suggested that this is related to Weddell circulation patterns and the resulting low annual primary production in the central Weddell Sea.     

Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.

A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.     

Human adenovirus type 9-induced rat mammary tumors.

Following subcutaneous inoculation of newborn Wistar-Furth rats with human adenovirus type 9 (Ad9), 16 of 16 female and 0 of 11 male rats developed mammary tumors. Tumor-positive animals usually developed tumors in multiple glands. Histopathological analyses indicated that three general categories of tumor could be identified. Mammary fibroadenomas were the most common tumor type encountered, but phyllodeslike tumors and solid sarcomas were also frequently found. In situ hybridization and immunohistochemical techniques established that benign fibroadenomas were derived from mammary fibroblasts (collagen type I- and vimentin-positive cells) and that malignant tumors were derived from myoepithelial cells (collagen type IV-, vimentin-, and muscle-specific actin-positive cells). The fact that mammary tumors were limited to female rats suggested that female hormones are essential for tumor growth and development. In this regard, ovariectomy of Ad9-infected female rats prevented tumor development, while subsequent diethylstilbestrol (DES) treatment elicited tumor formation. In addition, Ad9-infected and castrated male rats which received DES also developed mammary tumors. Established male mammary tumors regressed when DES treatment was stopped and reappeared after DES treatment was resumed. Together, these results indicate that estrogen is required for both initiation and maintenance of Ad9-induced mammary tumors. Southern blot analysis of high-molecular-weight tumor DNA showed that mammary tumor cells contained single or multiple integrated copies of the entire Ad9 genome. RNase protection experiments established that estrogen receptor as well as Ad9 E1a and E4 mRNAs were expressed in mammary tumors, but Ad9 E3 and, surprisingly, E1b mRNAs were not expressed at detectable levels.     

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.     

Physical and chemical characterization of a horse serum carboxylesterase

The serine carboxylesterase from horse serum was characterized by amino acid composition, peptide mapping, molecular and subunit weights, and sequencing of the amino acids around the essential serine residue at the active site. A protocol was developed for using reversed-phase high-performance liquid chromatography as the final step to obtain homogeneous preparations of horse serum carboxylesterase. Amounts sufficient for determining the amino acid composition and for peptide maps were obtained from a partially purified starting material which contained approximately 55% carboxylesterase. The amino acid composition, like the subunit weight (70,800 +/- 1400), was similar to the corresponding values reported for other serine carboxylesterases. However, the amino acid sequence of the tryptic digest fragment containing the essential nucleophilic seryl residue differed significantly from the corresponding sequences of other mammalian serine carboxylesterases.