GH3 cell secretion of growth hormone and prolactin increaseases spontaneously during perfifusion

Summary GH₃ cell secretory activity was studied in long-term perifusion to define previously reported spontaneous increases in growth hormone (GH) and prolactin production (PRL). Mechanically harvested cells (1×10⁷/column) were perifused at 4 ml/h for 72 h. A basal period of variable duration (8 to 12 h), during which hormone secretion was stable, was followed by steadily increasing secretion rates. Changes in cell number were not sufficient to acount for increased jormone secretion rates: a) there was no significant change in cell count after 72 h (0.97±0.03×10⁷;n=18); b) mean cell column DNA content increased 25.5% above the base value, whereas GH secretion rose 385% and PRL rose 178% (n=5). Observed differences in the duration of the basal secretion period, the basal secretory rate, and the magnitude of secretory rate increase were associated with several variables: a) variablility within a subline was a function of passage number: GH secretion decreased and PRL secretion increased with subculture number; b) cells with identical lot and freeze numbers, but received at different times, behaved differently; c) the presence of an antifungal agent (nystatin) altered hormone secretion reproducibly. Conclusions: a) rates of GH and PRL secretion rise spontaneously in perifusion without a proportional increase in GH₃ cell number; b) fluctuations in the rate of GH₃ cell secretion of GH and PRL are not entirely random but are determined by several definable variables. Supported by a grant to MES from the National Institutes of Health (AM33388) and in part by the Medical Research Service of the Veterans Administration.     

Acute effect of cigarette smoke on laryngeal receptors

Upper airway exposure to cigarette smoke elicits reflex changes in breathing pattern. To examine whether laryngeal afferents are affected by cigarette smoke, neural activity was recorded from the peripheral cut end of superior laryngeal nerve in anesthetized dogs. A box-balloon system, connected to the breathing circuit, allowed smoke to be inhaled spontaneously through the isolated upper airway while preserving its normal respiratory flow and pressure. Our results showed the following. Inhalation of cigarette smoke (25-50% concentration, 300-400 ml) caused a marked increase in activity of laryngeal irritant receptors which were either silent or randomly discharging during control breathing [their activity increased from a control value of 1.67 +/- 0.50 (mean +/- SE; n = 21) to a peak of 5.03 +/- 0.85 impulses/s in 11-15 s]. The activity of laryngeal cold receptors was reduced to 77.3 and 63.8% of control (n = 9) during the two breaths of smoke inhalation, respectively. After returning toward the base-line activity, a more pronounced inhibition (26.3% of control) occurred at three to nine breaths after the smoke inhalation. A small but significant decrease (88.5% of control) in the inspiratory discharge of laryngeal mechanoreceptors was observed during the first test breath. These effects were independent of the CO2 content of the smoke. Furthermore, there was no difference between the responses of these laryngeal afferents to high- and low-nicotine cigarette smoke.     

Iodoazomycin riboside (1-(5'-iodo-5'-deoxyribofuranosyl)-2-nitroimidazole), a hypoxic cell marker. I. Synthesis and in vitro characterization

Misonidazole (MISO), a selective radiosensitizer of hypoxic cells, forms adducts with cellular biomolecules with rates which are 30-50 X higher under hypoxic as compared to aerobic conditions of incubation. This technique of sensitizer adduct formation was proposed as a possible means of measuring the hypoxic fraction of solid tumors by noninvasive procedures. Iodoazomycin riboside (5'-IAZR) and 5'-[125I]AZR were synthesized and chemically characterized. Measurements of in vitro cytotoxicity and radiosensitizing ability with EMT-6 tumor cells in vitro indicated that 5'-IAZR is approximately 3 X more toxic and effective than is azomycin riboside (AZR) and approximately 10 X more toxic and effective than is MISO. 5'-[125I]AZR was shown to selectively bind to hypoxic EMT-6 cells at rates which were 2.5-3 X faster than those of MISO. The absolute rates of binding of 5'-IAZR to hypoxic cells at concentrations of 10-100 microM are the highest observed in this laboratory for any hypoxic cell radiosensitizer tested to date. These data suggest that 5'-IAZR, when labeled with an appropriate radioisotope (e.g., 131I), might be a useful marker for hypoxic cells in solid tumors amenable to noninvasive detection. Additional studies with animal tumor models appear to be warranted.     

High-speed preparative-scale separation and purification of ribosomal 5S and 5.8S RNA's via Sephacryl S-300 gel filtration chromatography

In this work we present a rapid and economical alternative to Sephadex and high performance size exclusion chromatography (HPSEC) for preparative-scale separation and purification of low molecular weight RNA's: 5.8S RNA, 5S RNA, and tRNA's. These three RNA species can be well resolved from each other and from higher molecular weight RNA species via Sephacryl S-300 gel filtration chromatography under mild eluting conditions: 10 mM Tris-HCl buffer, pH 7.5, containing 1.0 M NaCl. For a sample load of about 250 mg, the resolving power of a Sephacryl S-300 column (78 X 3.2 cm) is comparable to that of a 4.5 times larger Sephadex G-75 column (144 X 5 cm). Moreover, the total separation period is 2.5 times shorter for the Sephacryl method. Up to 500 mg or more of crude ribosomal RNA mixtures could be separated via two Sephacryl S-300 columns operated in tandem.     

Mutants of Pachysolen tannophilus with Improved Production of Ethanol from d-Xylose

The conversion of d-xylose to ethanol by the yeast Pachysolen tannophilus is relatively inefficient in batch culture. The inefficiency has been attributed in part to concurrent utilization of ethanol in the presence of appreciable concentrations of d-xylose and to the formation of xylitol and other by-products. To increase the concentration of ethanol accumulated in batch cultures, UV-induced mutants of P. tannophilus were selected on the basis of diminished growth on ethanol. Eleven independent mutant loci that conferred the ethanol-defective phenotype were identified. Three led to a greater yield and volumetric rate of production of ethanol than the wild type. One also produced less xylitol and was characterized by a deficiency in activity for malate dehydrogenase.     

In vitro regeneration of plantlets in Fagraea fragrans Roxb. — a tropical tree

Different vegetative parts of Fagraea fragrans Roxb., a valuable timber tree of South East Asia, were used as explants in in vitro studies.Nodal segments showed the best growth response as numerous adventitious shoots were regenerated in MS medium supplemented with Benzyl adenine (BA, 8.8 um) and 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 um). Shoot buds also developed from the leaf and root segments that were subcultured. The detailed process of callus growth and differentiation leading to the formation of whole plantlets is described. Uniform plantlets obtained could be transplanted successfully in soil.     

Urine dopamine in starving obese subjects

In seven obese female subjects undergoing a period of therapeutic starvation, the excretion of sodium, potassium and dopamine and plasma levels of renin and aldosterone were measured. Sodium excretion increased during starvation and was maximal on the 2nd day. The urinary excretion of dopamine was significantly higher on day 4 and it remained elevated till the end of the study. Plasma renin activity and plasma aldosterone levels were also higher on the 4th-6th days of starvation. These findings suggest that dopamine may not play a significant role in the natriuresis of starvation.     

Hydroxyl-radical production and ethanol oxidation by liver microsomes isolated from ethanol-treated rats.

In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an 'uncoupling' of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO.     

A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties.

We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers.