Walid G. Yasmineh

Satellite DNA in constitutive heterochromatin of the guinea pig.

Total DNA and DNA from the heterochromatin and euchromatin fractions of male guinea pig liver nuclei were analyzed by cesium sulfate-silver density-gradient centrifugation. Total DNA is composed of three components: a heavy satellite DNA, a main DNA of intermediate density, and a light satellite DNA. Heterochromatin DNA shows a fourfold enrichment in the satellite components while euchromatin DNA is relatively devoid of them. The strands of both satellite DNAs are separable by centrifugation in alkaline cesium chloride. Base analyses on the separate strands demonstrate that the two satellite DNAs represent different species.

Satellite DNA in mouse autosomal hetero chromatin

Abstract Within the last several years numerous investigations have been concerned with the existence of a minor DNA component, termed satellite DNA, in the genome of a number of higher organisms ( Kit, 1961 ; Chun and Littlefield, 1963 ; Corneo et al., 1968 ; Britten and Kohne, 1968 ). In the present communication, we wish to report that mouse autosomal heterochromatin, isolated by sonication of purified nuclei, is composed primarily of satellite DNA.

Effect of tumor necrosis factor on enzymes of gluconeogenesis in the rat

Abstract The effect of human recombinant tumor necrosis factor (TNF)-α on enzymes of gluconeogenesis in the rat was investigated by determining the activity of glucose 6-phosphatase, fructose 1,6-diphosphatase (FDP), and phosphoenolpyruvate carboxykinase in the liver and kidney of fed and fasted rats. The activity of transaldolase in the pentose phosphate pathway was also measured. Starvation of rats for 24 hr resulted in a 1.6- to 3.1-fold increase in liver and kidney glucose 6-phosphatase and phosphoenolpyruvate carboxykinase (P ≤ 0.05), a decrease in liver and kidney FDP (P < 0.002), and an increase in liver and kidney transaldolase (P = 0.0001). Injection of 50 and 100 μg/kg/day of TNF for 5 days resulted in a significant (P < 0.03) decrease in kidney FDP only. Injection of 100 μg/kg/day of TNF for 5 days with a 24-hr fast on Day 5 resulted in a significant (P = 0.04) increase in liver transaldolase, and a significant decrease in kidney FDP and phosphoenolpyruvate carboxykinase. Comparison of the enzyme activities of rats injected with 100 μg/kg/day of TNF for 5 days with those of their pair-fed control partners revealed additionally a significant decrease in glucose 6-phosphatase in the liver (P < 0.001). It is concluded that TNF administration in the rat has different effects on the enzymes of gluconeogenesis in the liver and kidney, and these effects differ from those seen in starved or tumor-bearing rats.

Repetitive DNA of microtus agrestis

Abstract DNA from the European field vole Microtus agrestis was sheared to fragments approximately 500 nucleotides in length in a French pressure cell and fractionated according to repetitiveness on hydroxyapatite. Four fractions, amounting to 26% of the total DNA, were obtained at Cot values between 2×10−4 and 8×10−1. Analysis of the fractions by density gradient centrifugation in CsCl and determination of base composition and melting profiles, revealed the presence of 3 main components: two minor highly repetitive components, representing about 5 and 3% of the total DNA and rich in GC and AT, respectively, and a major component of intermediate repetitiveness with a base composition similar to that of total DNA.

Determination of serum catalase activity on a centrifugal analyzer by an NADP/NADPH coupled enzyme reaction system

We developed a simple, kinetic method for the determination of catalase activity in which i) the enzyme catalyzes the peroxidation of ethanol by hydrogen peroxide to acetaldehyde and water, and ii) the acetaldehyde so formed is rapidly oxidized to acetic acid and NADPH by the addition of an excess of NADP+ and aldehyde dehydrogenase. The rate of NADPH production was monitored at 340 nm in a COBAS centrifugal analyzer. The reaction was linear to 800 U/L or a delta A of 0.020/min. Using human serum pools containing 80 and 460 U/L of peroxidase activity, the within-run coefficients of variation (CV) were 1.9 and 1.3%, respectively. Between-run CV values were 5% for both pools. The reference range for sera from 72 males and 52 females was 23 to 158 U/L (mean + 2 SD) by log normal transformation. The activity in red cells was 600 U/g hemoglobin but did not change the reference range appreciably provided that serum without visible hemolysis was used. Preliminary observations on sera from nine patients with various pancreatic disorders showed a poor correlation between the activities of catalase (peroxidase) and amylase in serum. The reasons for this discrepancy are under investigation.

Do immature T cells accumulate in advanced age

The hypothesis that decreased T cell function in the elderly involves an increased number of less differentiated T cells was examined. Three markers known to change during thymocyte development were analyzed; ratio of adenosine deaminase (ADA) to purine nucleoside phosphorylase (PNP), lactate dehydrogenase (LD) H/M subunit ratios and the T cell associated antigens, T3, T4, T8 and T10. Cells tested were from 10 old (greater than 75 years) and 10 young (less than 35 years) persons with equal numbers of males and females in each group. Before analysis, cells were purified into three groups; unfractionated, and monocyte depleted T cell and B cell enriched populations. Results for ADA/PNP ratios showed no significant differences between old and young in any of the fractions analyzed. H/M ratios however, were significantly reduced in all three fractions from old donors when compared with young. Surface marker distribution pattern as illustrated by the T3 - (T4 + T8) difference was lower in samples from old donors but not significantly so. There was a very significant reduction in percent cells positive for T3 in all three fractions from old persons. Although some of the changes seen in these markers could be due to a failure of normal differentiation, they could also be caused by the general phenomenon of altered gene expression known to occur with advanced age in a variety of non-lymphoid cells. The absence of any difference in the ADA/PNP ratio suggests that T cell dysfunction in the elderly may not be due to increased numbers of less differentiated cells as a result of thymic involution.

Chapter 11 Isolation of Mammalian Heterochromatin and Euchromatin1

Publisher Summary This chapter discusses the isolation of mammalian heterochromatin and euchromatin. One of the essential requirements for the isolation of relatively pure heterochromatin and euchromatin fractions from mammalian cells is the isolation of clean nuclei in which the architecture of the chromatin has not been significantly altered. The techniques used in the isolation of nuclei from mammals, as well as other species, are divided into those utilizing aqueous or nonaqueous media, depending upon the nuclear component under investigation. Techniques involving nonaqueous media are usually time consuming, but are desirable when nuclear components, such as the soluble proteins, are to be retained in the nucleus. Sucrose solutions are usually most efficient for the isolation of nuclei from normal tissue cells, where the cytoplasmic constituents can be easily stripped off. In this case, numerous recipes exist that differ primarily in the concentration of sucrose and divalent cations. Nearly 100% recovery of nuclei may be obtained by homogenization of the tissue in isotonic sucrose solutions containing divalent cations.

Chromatographie behavior of immunoglobulin-bound creatine kinase on DEAE-Sephadex A-50

We investigated the chromatographic behavior of CK isoenzymes and immunoglobulin-bound macro-CK by discontinuous gradient elution from DEAE-Sephadex A-50 at pH 7 and 8. In four of five patients with macro-CK, the macro-CK was eluted with the MB buffer at pH 8, but a significant portion of the complex was eluted with the MM buffer at pH 7, in a region between CK-MM and CK-MB. In the fifth patient, the macro-CK was eluted with the MB buffer at both pH values. The immunoglobulin patterns of all five patients and of normal serum showed that IgG and IgM are eluted mainly with the MM and MB buffers, respectively, at both pH values, whereas IgA is eluted mainly with the MB buffer at pH 8, but at pH 7, shows a significant shift similar to that of macro-CK. These characteristic patterns allowed the identification of the immunoglobulin in macro-CK as IgA in the first four patients, and as IgM in the fifth patient.

Homogeneous trinder-coupled assay for the determination of glucose-6-phosphatase activity in tissue extracts

We describe an automated, homogeneous, glucose oxidase-coupled method for the determination of glucose-6-phosphatase activity in tissue extracts. The method is based on measurement of the rate of glucose formation by the Trinder reaction, in which the end product is a quinoneimine dye which absorbs maximally at 505 nm and has a molar extinction coefficient of 5700. The incubation mixture contains 20 microL of tissue extract, 25 microL of 0.5 M phosphate buffer, pH 7.0, 175 microL of Trinder/glucose-6-phosphate reagent, and 30 microL of distilled water. After a delay period of 15 min, to exhaust any glucose endogenously present in the extract, glucose production from glucose-6-phosphate is monitored at 505 nm for 5 min in a centrifugal analyzer. The Km was 13 mM over a 10-fold range in glucose-6-phosphate concentration and the reaction was linear up to about 250 U/L. Within-run CV of the assay at activities of 48 and 190 U/L ranged between 2.5-5.0%. The between-run CV at 190 U/L was 5.1%.

Hepatic mitochondrial enzyme activity and serum amino acid composition in rats treated with tumor necrosis factor

The biochemical integrity of hepatocellular mitochondria was investigated in rats treated with small doses of human recombinant tumor necrosis factor-alpha (Hur-TNF;50-100 micrograms/kg/d injected intraperitoneally for 5 d) by measuring the activities of three mitochondrial enzymes, glutamate dehydrogenase, succinate dehydrogenase and malate dehydrogenase. The activity of glutamate dehydrogenase (a mitochondrial matrix enzyme) was 20% to 34% lower than that of control rats (P = 0.02 to 0.0003). The activities of succinate dehydrogenase (an inner mitochondrial membrane enzyme) and malate dehydrogenase (a mitochondrial matrix and cytosolic enzyme) showed no significant difference. The effect of TNF on serum amino acid composition was studied using pair-fed, weight-matched partners to eliminate any effect of the reduction of food intake due to TNF treatment. The results for the TNF-treated rats showed a significant (P < 0.05) increase in the concentration of 12 of the 21 amino acids measured (range = 33% to 140%). Of these, major increases were observed in the urea cycle intermediates, ornithine (140%) and arginine (59%), as well as proline (94%), alanine (41%), valine (61%), leucine (64%), isoleucine (63%), and aspargine (71%). Since previous studies have shown that the treatment of rats with the same low doses of TNF did not cause any change in mitochondrial ultrastructure detectable by electron microscopy, these results suggest that significant biochemical changes in amino acid metabolism occur as a result of a decrease in mitochondrial glutamate dehydrogenase activity.