Mutagenic consequences of the incorporation of 6-thioguanine into DNA

The stimulatory effect of phytin added to skim milk on acid production of Lactobacillus casei was examined. Phytin stimulated acid production of L. casei fairly well. The stimulatory effect of phytin on acid production was not shown when phytin was treated with Dowex 50 (H+) and neutralized by NaOH solution. The incinerated product of phytin maintained almost equal stimulatory effect on acid production as that before processing. The addition of Mn2+ in the amount contained in a reagent phytin augmented the stimulatory effect on acid production markedly. The further addition of Fe3+, Ca2+, Mg2+ and PO4(3-) in amounts corresponding to their contents in the preparation of phytin as well as Mn2+ increased the effect slightly. The four preparations of phytin contained 0.045-0.20% of Mn, and the greater the Mn content was, the greater the potentiation of acid production.     

Production of the 19-kDa antigen of Mycobacterium tuberculosis in Escherichia coli and its purification

The 19-kDa antigen (19Ag) of Mycobacterium tuberculosis (Mt) is a lipoprotein which is released from the organism during growth. In order to study the possible involvement of this antigen in the host protective response against Mt infection, it would be helpful to obtain high-level production of 19Ag from a recombinant organism. We have found that overexpression of the native 19Ag gene in Escherichia coli or yeast leads to products which are aggregated and insoluble. By site-directed mutagenesis of the 19Ag lipoprotein leader sequence, we have generated a mutant gene which directs the production of 19Ag into the periplasmic space of E. coli, from where it can be easily purified in high yield. 19Ag obtained from this mutant construct lacks the lipid-modified N-terminal Cys residue found in the native 19Ag, and is not glycosylated, but is otherwise indistinguishable from 19Ag isolated from Mt culture supernatant.     

The association of leukocytes with secondary brain injury

Shock increases mortality from brain injuries, but the mechanism is poorly understood. We hypothesized that brain injury followed by shock and resuscitation leads to a secondary reperfusion injury mediated in part by polymorphonuclear leukocytes (PMNs). To validate this hypothesis, we studied cerebral perfusion pressure (CPP), intracranial pressure (ICP), cerebral blood flow (CBF), cortical water content (CWC), and hemodynamic variables in a porcine model of focal cryogenic brain injury and hemorrhagic shock. Cerebral PMN accumulation (CPMN) in the injured and uninjured hemispheres was determined histologically from the total PMNs in five high-power fields (400x). Twenty-nine mature swine were randomized to four groups. Group 1, the control group, was instrumented only. Group 2 animals had a brain injury alone and were studied for 24 hours. Group 3 animals had a brain injury and hemorrhagic shock. Group 4 animals had hemorrhagic shock alone. Brain injury followed by shock caused a significantly greater ICP and a significantly lower CBF than brain injury or shock alone. There was no significant difference in CPP between groups after resuscitation. The CWC of the lesioned area was similar in both brain-injured groups but was significantly increased when compared with the controls and the shock-only group. The CWC of the nonlesioned hemisphere was higher in group 3 than in group 2. The CPMN in both hemispheres in group 3 was significantly greater than in either group 2 or group 4. There was a significant positive correlation between CPMN and both ICP and CWC, and a significant negative correlation between CPMN and CBF. These data suggest an association between CPMN accumulation and secondary brain injury.     

The putative tumor suppressor gene on chromosome 5q for hepatocellular carcinoma is distinct from the MCC and APC genes

We have previously shown that the tumor suppressor gene for hepatocellular carcinoma (HCC) without cirrhosis may be located on chromosome 5q35-qter. In this study, we analyzed nine cases of primary HCC without cirrhosis using probes from the MCC and APC genes, which are in the region 5q21-22. None of the informative cases had allele loss detected by these probes, whereas the probe lambda MS8 for the region 5q35-qter showed allele loss in six out of six informative cases. The results confirm that the putative tumor suppressor gene for HCC without cirrhosis on chromosome 5q is distinct from the MCC and APC genes.     

Gel-entrapment of perfluorocarbons: a fluorine-19 NMR spectroscopic method for monitoring oxygen concentration in cell perfusion systems

Oxygenation is a major determinant of the physiological state of cultured cells. 19F NMR can be used to determine the oxygen concentration available to cells immobilized in a gel matrix by measuring the relaxation rate (1/T1) of perfluorocarbons (PFC) incorporated into the gel matrix. In calcium alginate gel beads without cells the relaxation rate (1/T1) of the trifluoromethyl group of perfluorotripropylamine (FTPA) varies linearly with oxygen concentration, with a slope of 1.26 +/- 0.15 x 10(-3) s-1 microM-1 and an intercept of 0.50 +/- 0.04 s-1. During perfusion with medium equilibrated with 95%/5% O2/CO2, changes in PFC T1s indicate that the average oxygen concentration was reduced from 894 +/- 102 microM in the absence of cells to 476 +/- 65 microM and 475 +/- 50 microM in the presence of 0.7 x 10(8) EMT6/Ro and RIF-1 murine tumor cells per milliliter of gel, respectively. The presence of 0.2 microliters of FTPA/ml of gel had no effect on the energy status of the cells as indicated by 31P NMR spectra. To calculate oxygen gradients within the beads from the average PFC T1 of the sample, a mathematical model was used assuming that oxygen is the limiting nutrient for cell metabolism and that the cellular oxygen consumption rate is independent of oxygen concentration. Data for EMT6/Ro cells were fit using experimentally determined perfusion parameters together with literature values for cell volume and oxygen consumption rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Timing of reproductive life stages

We have investigated lysis of cultured human glial cells by non-major histocompatibility complex (MHC)-restricted or 'promiscuous' CD(4+)-T lymphocytes, activated either under relatively long-term limiting dilution culture conditions in the presence of phytohemagglutinin (PHA) and interleukin (IL)-2, or under short-term PHA-activated bulk culture conditions. Specific effector:target cell ratio-dependent lysis of oligodendrocytes (OGCs) by CD4+ T lymphocytes, generated under both of the above conditions, was observed in an 18-h 51Cr-release assay, but not in a 5-h assay. The extent of CD4 T-cell-mediated OGC lysis was less than for the tumor necrosis factor (TNF)-alpha-sensitive cell line U937, but greater than for TNF-resistant cell lines (K562, EL4). The effect could not be reproduced by T-cell culture supernatants or by high concentrations of recombinant TNF-alpha or beta. Anti-TNF-alpha antibody did not inhibit CD4-mediated lysis of OGC or U937 cells, but did inhibit U937 lysis induced by recombinant TNF-alpha, added in amounts exceeding those secreted by CD4 T cells. Human astrocytes and microglia were also susceptible to CD4+ T-cell-mediated lysis. Our results suggest that non-antigen non-MHC-restricted CD4+ T-cell-mediated injury of human glial cells can occur and may be dependent or enhanced by effector:target cell contact.