EcoRI-sensitive mutation of T7 phage

Summary It was found that yeast cells contain an endonuclease specific for apurinic sites in DNA which has no effect on DNA with normal strands or on strands with alkylated sites. The enzyme activity was studied in the RAD strain and in rad6, rad18-2 and rad21 mutants, all very sensitive to MMS, as compared to the wild type. The level of endonuclease activity does not differ much between the tested strains, regardless of their differences in susceptibility to MMS. The enzyme activity is not induced by pretreatment of the cells with this mutagen.     

The ultrahistochemical picture of the so-called reversed ATPase in the gastrocnemius muscle of the rat

Summary The ultrahistochemical localization of the reversed ATPase activity was investigated. Red muscle fibres showed permanent sarcomere contraction, enzymatic activity in the inner membrane and matrix of mitochondria, and large, osmiophilic, probably calcium-containing structures within mitochondria and on their outside. White muscle fibre sarcomeres were relaxed, and activity within their sarcoplasmic reticulum was marked, but slight in the mitochondria. The relaxed state of the sarcomere in the white muscle fibres is supposed to be connected with inactivation of myofibrillar ATPase by acid preincubation, whereas red muscle contraction indicates that acid preincubation does not inactivate their myofibrillar ATPase. That the product of its activity failed to become visible in the sarcomeres is probably due to imperfection of the method.Two sub-types of red muscle fibres were distinguished: those showing only enzymatic activity in mitochondria, and those containing large intra-and extramitochondrial osmiophilic structures. The origin and composition of these structures is difficult to explain. A relation seems to exist between their presence within mitochondria and outside.This work was supported by a Fellowship from the Muscular Dystrophy Association of America and under Research Project No. 05-002-1 of the National Institutes of Health, IND, Bethesda, USA. Preliminary experiments were made at the Medical Neurology Branch, INDS, NIH, Bethesda, USA     

Sorption of Cadmium by Microorganisms in Competition with Other Soil Constituents

The fate of cadmium in soil is influenced to a great extent by microbial activity. Microorganisms were compared with abiotic soil components for their ability to sorb Cd from a liquid medium. When the same amount (on a dry weight basis) of bacterial cells (Serratia marcescens and Paracoccus sp.), clay (montmorillonite), or sand was separately incubated in 0.05 M phosphate buffer, pH 7.2, containing 10 ppm of Cd (10 μg/ml), bacterial cells removed the largest quantity of Cd. Dead cells sorbed much more Cd from the medium than live cells. A comparative study of Cd removal from the medium by seven soil bacteria and four fungi did not indicate appreciable differences. With increasing microbial biomass, the relative efficiency of 0.1 M NaOH as an extractant of sorbed Cd increased, whereas the extraction efficiency of 0.005 M DTPA (diethylenetriaminepentaacetic acid) decreased. It appeared that NaOH and DTPA extracted different chemical forms of Cd. This assumption was supported by vastly different correlation coefficients in the relative amount of Cd extracted by the two solvents.     

The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts

The levels of cdc2 mRNA increase when quiescent cells are stimulated by growth factors. In BALB/c 3T3, both platelet-derived growth factor and insulin-like growth factor 1 (IGF-1) are required to increase cdc2 mRNA levels. In p6 cells, which constitutively overexpress the IGF-1 receptor, IGF-1 is sufficient. The importance of the IGF-1/IGF-1 receptor interaction in regulating the levels of cdc2 mRNA was further confirmed by showing that an antisense oligodeoxynucleotide to the IGF-1 receptor RNA inhibited the IGF-1-mediated increase.     

Effect of Oxotremorine on Ornithine Decarboxylase Activity of the Adrenal Gland in Rat

Abstract: In this work we have studied the mechanism for the increase of adrenal ODC (ornithine decarboxylase, EC 4.1.1.17) activity provoked by oxotremorine, a muscarinic agonist. 1. Oxotremorine increased medullary ODC activity maximally at 2 h. Cortical enzyme responded much more slowly. 2. Blockade of peripheral muscarinic receptors with methylatropine partially reduced the response to oxotremorine in the medulla, but not cortex. 3. Hy-pophysectomy abolished the cortical, but not the medullary, responses to oxotremorine. Methylatropine reduced the effect of oxotremorine on medullary ODC in hypophysectomized rats. 4. In unilaterally splanchnicotomized rats oxotremorine caused an increase of ODC activity of the denervated adrenal gland relative to control value; activities in both medulla and cortex were significantly lower than those observed in the innervated gland. Evidence was obtained for a compensatory increase of ODC activity of the adrenal cortex (but not medulla) on the intact side of unilaterally operated rats. 5. Surgical intervention, in the form of a sham operation for transection of the spinal cord, leads to an increase of ODC activity in both parts of the adrenal gland. Transection of the cord attenuates these increases. 6. The additional increase of medullary ODC activity owing to the administration of oxotremorine to sham-operated rats is partially reduced in the adrenal medulla by muscarinic blockade, and completely in the cortex. This effect of methylatropine in regard to cortical ODC activity was not apparent in the other experiments with intact or unilaterally splanchnicotomized (unoperated side) rats. The results with unilaterally splanchnicotomized rats and those with transected spinal cord suggest that oxotremorine-induced modifications of adrenal ODC activity are centrally mediated, above the level of origin of the splanchnic nerves in the spinal cord (T_8–10). Experiments with hypophysectomized rats show that the response of the adrenal cortex to oxotremorine is entirely mediated by the hypophysis.     

Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4 degrees C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ;maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The k(cat.) and K(m) values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a k(cat.)/K(m) value near 10(7)s(-1).m(-1), which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.     

The acetyl-CoA pathway of autotrophic growth

Abstract The most direct conceivable route for synthesis of multicarbon compounds from CO₂ is to join two molecules of CO₂ together to make a 2-carbon compound and then polymerize the 2-carbon compound or add CO₂ successively to the 2-carbon compound to make multicarbon compounds. Recently, it has been demonstrated that the bacterium, Clostridium thermoaceticum , grows autotrophically by such a process. The mechanism involves the reduction of one molecule of CO₂ to a methyl group and then its combination with a second molecule of CO₂ and CoA to form acetyl-CoA. We have designated this autotrophic pathway the acetyl-CoA pathway [1]. Evidence is accumulating that this pathway is utilized by other bacteria that grow with CO₂ and H₂ as the source of carbon and energy. This group includes bacteria which, like C. thermoaceticum , produce acetate as a major end product and are called acetogens or acetogenic bacteria. It also includes the methane-producing bacteria and sulfate-reducing bacteria.
The purpose of this review is to examine critically the evidence that the acetyl-CoA pathway occurs in other bacteria by a mechanism that is the same or similar to that found in C. thermoaceticum . For this purpose, the mechanism of the acetyl-CoA pathway, as found in C. thermoaceticum , is described and hypothetical mechanisms for other organisms are presented based on the acetyl-CoA pathway of C. thermoaceticum . The available data have been reviewed to determine if the hypothetical schemes are in accord with presently known facts. We conclude that the formation of acetyl-CoA by other acetogens, the methanogens and sulphate-reducing bacteria occurs by a mechanism very similar to that of C. thermoaceticum .