Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA.

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.     

Histidine:pyruvate transamination and phyenylalanine:pyruvate transamination may be catalyzed by the same enzyme.

Some properties of histidine:pyruvate transaminase (HPT) and phenylalanine:pyruvate transaminase (PPT) in the cytosol of rat liver were studied. HPT and PPT activity could not be separated by DEAE-Sephadex A-50 or hydroxylapatite column chromatography, and the ratio of $\text{HPT}\text{PPT}$ activity remained constant during these purification procedures. The two enzyme activities also showed similar heat stability and responses to glucagon injection. Based on these findings, we suggest that a single enzyme may specifically catalyze histidine:pyruvate and phenylalanine:pyruvate transamination.     

Nuclear phosphoprotein kinase activities in normal and neoplastic tissues.

Nuclear phosphoprotein kinases from normal rat liver and transplantable neoplasms were fractionated and compared. A phosphoprotein kinase fraction activated by Mn2+ was found to be present only in the neoplasms. This nuclear protein kinase phosphorylated nuclear proteins represented by one major and several minor bands as determined by polyacrylamide gel electrophoresis (M approximately 50,000).     

In vivo expression of a nonselected gene transferred into murine hematopoietic stem cells by electroporation

Mouse bone marrow cells were subjected to electroporation in the presence of RSVCAT and SV2NEO plasmids. CAT activity was detected in the G-418 resistant granulocyte-macrophage colonies. RSVCAT electroporated into primary bone marrow cells, repopulated lethally irradiated mice as demonstrated by the persistence of CAT activity in the hematopoietic tissues showing that electroporation can offer a powerful mode of gene transfer into bone marrow cells.     

Alignment and merging of electron microscope images of frozen hydrated crystals of the T4 DNA helix destabilizing protein gp32*I.

Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.     

The responses of atrial natriuretic factor concentrations to acute volume changes in conscious rats

A rapid and sensitive radioimmunoassay has been developed for measurements of atrial natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181 +/- 24 pg/ml (N = 24) in the unstressed conscious rats (Charles River CD, male). The ANF immunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major immunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

Temperature-dependent development and population growth of Tetraneura nigriabdominalis (Homoptera: Pemphigidae) on three host plants

The root aphid Tetraneura nigriabdominalis (Sasaki) (Homoptera: Pemphigidae) is a pest of many Gramineae species; however, little is known about its biology and relationships with host plants. The objectives of this study were to quantify the effects of temperature on development, longevity, fecundity, and population growth of T. nigriabdominalis and to assess the effects of host plant on development of T. nigriabdominalis. The effects of temperature on performance of this root aphid were determined at 10, 15, 20, 25, 30, and 35 +/- 1 degrees C on rice roots, Oryza sativa L. Nymphal stages from birth to adult decreased from 46.3 d at 10 degrees C to 8.5 d at 30 degrees C. Aphid survival and development were lowest at 35 degrees C, and no aphid produced progeny at this temperature. Average adult longevity decreased from 23.3 d at 15 degrees C to 8.2 d at temperatures up to 35 degrees C. Average number of nymphs produced per female was highest at 25 degrees C; averaging near 30 nymphs per female, but it dropped to near zero at both 10 and 35 degrees C. The highest intrinsic rate of increase (r(m)) was 0.241 at 30 degrees C. Net reproductive rate (R(0)) ranged from 29.8 at 25 degrees C to 0.2 at 10 degrees C. The generation time (GT) decreased with increasing temperatures from 60.3 d at 10 degrees C to 13.8 d at 30 degrees C. In addition, root aphids reared at 30 degrees C on three host plants [O. sativa, Zea mays L. and Sorghum bicolor (L.) Moench] revealed that the developmental time of the nymphal stages averaged 6.9 d when reared on O. sativa and 10.7 d when reared on Z. mays. Comparison of the nitrogen content of the three host plants indicated that the root nitrogen content was highest in O. sativa. The effect of nitrogen content on aphid performance, however, is still not clear. Other factors, such as plant secondary chemistry, may play a role in affecting aphid performance.