Quantitation of mitochondrial injury in cultured rat heart endothelioid cells with nitroblue tetrazolium

Synopsis A sensitive method is presented for measurement of changes in the permeability of mitchondria in cultured cells. Rat heart endothelioid cells were used to determine the penetration rate of nitroblue tetrazolium (NitroBT) or other reactants into mitochondriain situ. Nitroblue formazan, produced as a consequence of succinate dehydrogenase activity in the mitochondria, was eluted and measured with a spectrophotometer. Prior injury of cells with hypo-osmolar solutions increased the rate of formazan production. Several methods are described or suggested for the statistical analysis of the data.     

Prophylactic immunization against experimental leishmaniasis. IV. Subcutaneous immunization prevents the induction of protective immunity against fatal Leishmania major infection

Durable immunity against fatal L. major infection in genetically susceptible mice can be induced by immunization with 150,000-rad irradiated or heat-killed promastigotes administered i.v. or to a lesser extent i.p. Conversely, subcutaneous (s.c.) and intramuscular (i.m.) injections are not only totally ineffective but generally increase susceptibility to and enhance the progression of the disease, leading to earlier mortality. This detrimental effect is particularly evident with lower infecting challenge doses. Disease exacerbation is apparent in mice given 4 X s.c. injections of as few as 2 X 10(4) irradiated promastigotes, but it appears most potent after doses of 2 X 10(7). When mice given 4 X s.c. injections were subsequently immunized i.v. with 2 X 10(7) irradiated promastigotes, they failed to develop any evidence of protection against infection with 2 X 10(5) promastigotes, whereas mice given i.v. immunization alone were strongly protected. Thus, s.c. injections are capable of blocking the prophylactic effect of i.v. immunization with irradiated parasites. This inhibitory effect can be achieved with a single s.c. injection, although rather less potently than with four, and is even effective against four repeated weekly i.v. immunizations. Once induced, the effect persists undiminished after 100 days. A weaker effect is also inducible by s.c. injection given after i.v. immunization. The blocking effect of s.c. injection is not dependent on continuing viability of the promastigotes, as it can be induced equally readily with heat-killed, formalin-fixed, or sonicated parasites. The phenomenon extends to mouse strains genetically resistant as well as susceptible to L. major infection and, in congenic mice of BALB background, is independent of the major histocompatibility (H-2) gene complex.     

REGIONAL ISOLATION PERFUSION IN THE TREATMENT OF ADVANCED MALIGNANT DISEASE

Regional isolation perfusion with an alkylating agent was carried out in a small community hospital, using readily available equipment, ten times in nine cases of advanced malignant disease. Visible regression of tumor, either grossly or histologically, occurred in seven patients and decided subjective improvement occurred in five patients.The long range effect of the procedure could not be determined in the present series, not enough time having elapsed.Side effects included jaundice in four patients, massive necrosis of the tumor in one patient, nausea and vomiting in three patients, postoperative edema in five patients, wound infection in two patients, Horner''s syndrome in one patient, temporary alopecia in three patients, and depression of hemoglobin level in seven patients, of the leukocyte count in four patients and of the platelet count in four patients.A feature of the procedure used in the present series was a system of monitoring the escape of alkylating agent into the systemic circulation.     

Analysis of nonmethylated GATC sites in the Escherichia coli chromosome and identification of sites that are differentially methylated in response to environmental stimuli.

Seven GATC sites that are nonmethylated in logarithmic growth phase cells using glycerol as a carbon source were isolated from the Escherichia coli chromosome. Three of these GATC sites are located upstream of the operons gut, mtl, and ppiA, whereas DNA sequences adjacent to three other nonmethylated GATC sites are not homologous to previously identified genes. The seventh nonmethylated GATC site is located downstream of uspA. The protection of this site from DNA methylation requires leucine-responsive regulatory protein and is leucine responsive. The carbon source and the growth phase influenced the protection of the GATC site 5' of the ppiA gene. The other five sites were protected under all the environmental conditions examined.     

Hydrolase and serum treatment of normal chick embryo cells: effects on hexose transport.

We have asked whether treatment of normal cultured cells with proteases, other hydrolytic enzymes, or serum can convert them into transient phenocopies of transformed cells with respect to the very high rate of hexose transport characteristic of transformed cells. Treatment of density-inhibited cultures of normal chick embryo fibroblasts with trypsin, plasmin, neuraminidase, or hyaluronidase stimulated their rate of 2-deoxyglucose uptake to a level only marginally higher than that seen in normal exponentially growing cultures, and only 35-45% of that seen in transformed cultures. Addition of the hydrolytic enzymes to growing cell cultures had little effect on 2-deoxyglucose uptake. Serum, however, could stimulate 2-deoxyglucose uptake all the way up to the transformed level. Even though the hydrolases and serum differed in their ability to stimulate 2-deoxyglucose uptake, both reagents were capable of stimulating cell division equally well. Evidence is presented suggesting that the hexose transport rate is controlled by serum factors, and that proteolysis can affect the response of the cells of these factors.