Transformation of NIH 3T3 cells by overexpression of the normal coding sequence of the rat neu gene.

While the normal human erbB-2 gene is potently transforming when overexpressed in NIH 3T3 cells, its rat homolog, the neu gene, seems to acquire transforming properties only upon alteration of its coding sequence. In this study, we compared the effects of different levels of expression of normal erbB-2 and neu in NIH 3T3 cells. Our results revealed that the normal rat neu gene acts as a potent oncogene when sufficiently overexpressed in NIH 3T3 cells.     

Transposon-generated Tn10 insertion mutations at thearo genes ofEscherichia coli K-12

We have obtained a set ofEscherichia coli K-12 derivatives with transposon-generated Tn10 insertion mutations at thearo genes of their aromatic biosynthetic pathway. Bacteriophage NK561 (Tn10) has been used for transposon mutagenesis ofE. coli, strain BW545. Tetracycline (Tc)-resistant derivatives were screened by their Aro^– phenotype by growth on a minimal medium with adequate requirements. Sixaro mutant types were mapped; two strains werearoA, twoaroD, onearoB oraroE, and onearoC. A selective medium and ad-cycloserine enrichment in the presence of tetracycline were used to select for Aro^–, Tc-sensitive derivatives. The reversion index to aromatic-independent colonies of some derivatives was less than 2 × 10^–11 per bacterium per generation. P1 transduction experiments transferred an aroA::Tn10 insertion fromE. coli BW545 to an enterotoxigenicE. coli strain from porcine origin. Derivatives of this strain beingaro, Tc-sensitive and not reverting toaro ⁺ at a detectable frequency, and many others transduced at will, may prove their usefulness as live vaccines.     

Trophic level assessment of profundal sediments of the artificial Lake Campotosto (Central Italy), using midge larval community (Diptera: Chironomidae)

A study on the profundal chironomids of the artificial Lake Campotosto (Central Italy) was carried out during the summer/early autumn of 1983 and 1984, in order to analyse their composition and community structure in relation to the lake trophic level assessed by water chemical analysis.A total of about 2000 specimens belonging to 15 taxa were collected during the study.Chironomus plumosus group andTanytarsus spp. dominated in 1983 and 1984, respectively, showing a competitive relationship probably due to the larval size. The functional feeding organization was mostly composed of collectors (percentages greater than 90%), revealing the presence of abundant fine organic deposits (FPOM). Diversity and evenness appeared to be negatively affected by the monotony of food, which seems to constitute the key factor in governing both the taxonomic and the trophic structure of chironomid fauna.A clear discrepancy between water chemical data and profundal chironomid analyses was observed in the assessment of the lake trophic level. Sediments exhibited eutrophicated conditions, whereas overlying waters indicated an oligotrophic status. The relevance of profundal macrobenthic investigations in detecting eutrophication is stressed.     

Collagen increases the synthesis of membrane-associated proteoglycans produced by Sertoli cells.

Sertoli cells in culture produce two isoforms of proteoglycans which are found in the culture medium and associated with the cell membrane. The amount of both types of proteoglycans increased when Sertoli cells were plated on type I collagen-coated dishes as compared to uncoated dishes. The effect is due to an increase in the synthesis of proteoglycans rather than a diminished rate of degradation of these molecules. The collagen substrate also affects the distribution of these macromolecules; an increase in the amount of membrane-associated proteoglycans occurs at the expense of the proteoglycans released to the culture medium.     

Effect of potassium humate on apple cv. ‘Golden Delicious’ cultured in vitro

The effects of humic substances on in vitro culture of Golden Delicious apple are reported. Potassium humate (KH) when used in proliferation showed a negative interaction with BA while it enhanced rooting when IBA was not present in the culture medium. In the presence of IBA, KH increased root number and reduced root growth. The highest concentration tested, 500 mg l^-1, caused a drastic reduction in root system development. 50 mg l^-1 KH hastened rooting and plants grew more rapidly when transferred to soil.     

Effects of prolonged sodium restriction on the morphology and function of rat adrenocortical autotransplants

Summary Regenerated adrenocortical nodules were obtained by implanting fragments of the capsular tissue of excised adrenal glands into the musculus gracilis of rats (Belloni et al. 1990). Five months after the operation, operated rats showed a normal basal blood level of corticosterone, but a very low concentration of circulating aldosterone associated with a slightly increased plasma renin activity (PRA). Regenerated nodules were well encapsulated and some septa extended into the parenchyma from the connective-tissue capsule. The majority of parenchymal cells were similar to those of the zonae fasciculata and reticularis of the normal adrenal gland, while zona glomerulosa-like cells were exclusively located around septa (juxta-septal zone; JZ). In vitro studies demonstrated that nodules were functioning as far as glucocorticoid production was concerned, while mineralocorticoid yield was very low. Prolonged sodium restriction significantly increased PRA and plasma aldosterone concentration, and provoked a marked hypertrophy of JZ, which was due to increases in both the number and average volume of JZ cells. Accordingly, the in vitro basal production of aldosterone and other 18-hydroxylated steroids was notably enhanced. The plasma level of corticosterone, as well as zona fasciculata/reticularis-like cells and in vitro production of glucocorticoids by regenerated nodules were not affected. These findings, indicating that autotransplanted adrenocortical nodules respond to a prolonged sodium restriction similar to the normal adrenal glands, suggest that the relative deficit in mineralocorticoid production is not due to an intrinsic defect of the zona glomerulosa-like JZ, but is probably caused by the impairment of its adequate stimulation under basal conditions. The hypothesis is advanced that the lack of splanchnic nerve supply and chromaffin medullary tissue in regenerated nodules may be the cause of such an impairment.     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Photo-CIDNP nuclear magnetic resonance as a probe for conformational changes in epidermal growth factor

Photo-chemically induced dynamic nuclear polarization (CIDNP)-NMR spectroscopy at 360 MHz has been used to investigate pH-induced conformational transitions in mouse epidermal growth factor. At about pH 9, all five tyrosine residues and both tryptophan residues are, to various extents, solvent-exposed, while the His-22 residue is buried in the protein matrix. Tyr-13 is the least exposed of the tyrosine residues and also the most immobilized. As the pH is decreased to 5.9, the tryptophan residues gradually become less exposed, while the Tyr-13 residue becomes internalized in the protein. These data suggest that the C-terminus and part of the N-terminal structural domain are affected by a conformational transition in mouse epidermal growth factor occurring between pH 6 and 8 via breakage of the His-22 inter-residue linkage. Above pH 9, a decreased photo-CIDNP effect is evident for both tryptophans and for Tyr-10 and Try-13; this information suggests that a second conformational change takes place at basic pH, which may simply be incipient denaturation.     

Role of mevalonate-5-pyrophosphate decarboxylase in the regulation of chick intestinal cholesterogenesis

The response to different dietary conditions of the enzymes responsible for the transformation of mevalonic acid to isopentenyl pyrophosphate has been studied for the first time in the small bowel of the chick to elucidate the role of these enzymes in the regulation of intestinal cholesterogenesis. Feeding a 2% cholesterol diet from hatching resulted in a small but significant inhibition of mevalonate-5-pyrophosphate decarboxylase, while mevalonate kinase and mevalonate-5-phosphate kinase remained unaltered. Similar results were obtained for the three enzymes when 13-day-old chicks fed a standard fat-free diet were switched to a 5% cholesterol diet. Starved chicks exhibited lower intestinal decarboxylase activity than chicks fed a standard diet, while refeeding resulted in levels of activity similar or slightly greater than controls. None of the enzymes effecting the conversion of mevalonate to isopentenyl pyrophosphate in the small intestine presented diurnal variations. Results obtained suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in the regulation of cholesterol synthesis in the small intestine.