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Using two-dimensional electrophoresis, we have investigated the responses of human cells in culture to heat shock and to various chemical agents producing a similar effect. These treatments result in the induction of increased synthesis of several specific proteins. One (HShock:1, SDS-molecular mass about 65000) is increased by about 350-fold over the amount in untreated cells. Computer analysis of time-course studies indicates, however, that rates of synthesis of various proteins other than the classical "heat shock proteins" are affected, some of these alterations following time courses quite different from the main (HShock) inductions. The heat shock effect is thus much more complicated than previously realized. We purified the HShock:1 protein from heat-shocked human lymphoblastoid cells, and prepared a rabbit antiserum specific for HShock:1 on nitrocellulose two-dimensional gel transfers of total lymphoblastoid cell protein. A survey of mouse tissues shows high concentrations of an HShock:1-like protein in the testis, and human testes also appears to contain substantial (though lower) concentrations. These results are consistent with the hypothesis (derived from the tissue-culture studies) that the heat shock effect is a general response to the need for increased protein catabolism within the cell. Increased concentrations of HShock:1 are also observed in preparations of blood leukocytes collected from patients after surgery, indicating that some types of physiological trauma may induce the heat shock proteins in man. Using the antiHShock:1 antibody in an immunoassay, it will be possible to systematically examine HShock:1 concentrations in plasma and leukocytes, thereby opening up the possibility of a clinical test based for the first time upon an inducible aspect of cellular gene expression.

American tegumentary leishmaniasis is caused by a diverse group of Leishmania classified within two species complexes: L. mexicana and L. braziliensis. Because distinct disease forms are associated with certain species or subspecies, prognosis requires taxonomic identification of the pathogen. Biological criteria allow identification only to the species level; thus, biochemical and immunological markers are eagerly sought. We have used two-dimensional electrophoresis to examine the relationships among reference strains of New World Leishmania and stocks isolated from Colombian patients. The L. mexicana and L. braziliensis species complexes are shown to be extremely disparate, and the relative affinities of the subspecies reference strains and Colombian Leishmania stocks are documented. The latter displayed a variety of patterns--some clearly identifiable with a particular L. braziliensis subspecies reference strain, others intermediate between those of L.b. panamensis and L.b. guyanensis. Such comparisons are useful both in establishing relatedness and identifying subspecies and variant marker proteins, which may have biological significance.

Human erythrocyte lysate proteins were resolved into over 250 discrete spots by two-dimensional electrophoresis using isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate, (SDS) in the second. The overwhelming excess of hemoglobin has made such analyses difficult in the past. However, with the ISO-DALT two-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of hemoglobin. When hemoglobin and several other major proteins are removed by adsorption to DEAE-cellulose, additional minor components are seen, giving a total of over 275. With the use of purified preparations, the map positions of five cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, and carbonic anhydrase. The mapping techniques described complement and extend those traditionally used to find human red cell protein variants.

During the first 3 days of exposure of rats to 5°C, the nitrogen concentration of interscapular brown fat increased by 50% and remained at this elevated level for the duration of the 8-wk observation period, while the mass of tissue increased fourfold. The concentration of both DNA and RNA per unit nitrogen reached a maximum after 3 days, then declined; however, the total quantity of each continued to rise. The concentration of various respiratory enzymes decreased during the first few days and then increased, but at different rates. The morphological changes in mature brown fat cells during cold acclimation were observed to be: a reduction in fat droplet size during the first 3 days, followed by a gradual increase in size through 6 wk in the cold; a continual increase in the amount of intermitochondrial ground substance during the first 3 wk, with increased granularity and glycogen content after 1 wk; initial disappearance of glycogen between mitochondria, followed by the reappearance of a few isolated particles in the intermitochondrial ground substance after 1 wk in the cold; initial increase in the density of intramitochondrial matrix for the first 3–4 days, followed by a gradual return to the control density; loss in integrity of mitochondrial outer membranes during the first 4 days, followed by gradual but incomplete restoration; temporary loss of the dense material in lipid droplets during the first 24 hr, with return after 1 wk in the cold; and a 40% increase in mitochondrial diameter within 1 day, followed by a decrease in diameter within 1 wk to a constant value about 15% larger than the controls.

The sedimentation characteristics of mitochondria from livers of mice treated with various radioprotective aminothiols were measured by techniques of density-gradient and zonal centrifugation. Trea...

The extent of the effects of ionizing radiation on structure and function of liver mitochondria has been the subject of a large number of communications. For example, Noyes and Smith (1) reported that within 15 minutes after exposure of male rats to 1000 R of Co60 y-radiation there was a marked depression of mitochondrial numbers, with a concomitant increase in unit mass per mitochondrion. This effect persisted about 90 minutes and then diminished, and by 6 hours after irradiation both the number and the size of mitochondria were normal. Hall et al. (2) described a similar effect in male rats receiving 840 R of X-radiation; although the decrease in number was not so marked, the recovery was more protracted, and the increase in total nitrogen per mitochondrion observed by Noyes and Smith was not noted in these studies. Hall et al. (2) also observed significant changes in oxidative phosphorylation during the first 12 hours after exposure, the P/O ratios (micromoles of orthophosphate esterified per microatom of oxygen consumed) falling to 70 to 75 % of the control values, then returning to normal at 24 hours. This decrease appeared to represent a true uncoupling, oxygen consumption increasing and phosphate esterification decreasing. Inhibition of oxidative phosphorylation in rat liver mitochondria was observed 24 hours after irradiation by Ryser et al. (3) and by Yost and his co-workers (4, 5); the former group observed no effect 2 hours after exposure, whereas the latter made no mention of experiments at earlier times. Nitz-Litzow and Biihrer also reported a 30% decrease in P/O ratios 12 hours after 800 R (6). Profound but transitory changes in phosphorylation, respiration, and swelling characteristics of mouse liver mitochondria 6 hours after total-body irradiation with 500 R have been described by Urakami (7). By the use of density-gradient centrifugation of rat liver homogenates, Thomson and Rahman (8) were unable to detect any changes in size distribution of mito

When rats are transferred to a 5°C environment immediately after exposure to 400 R of x-irradiation, the rate of growth of interscapular brown fat is significantly inhibited, in comparison with that in pair-fed unirradiated cold-exposed controls. The most striking effect is on the early hyperplasia of the tissue, which in the controls is maximal after 3 days in the cold. After 3 weeks, the mass of interscapular brown fat in the irradiated animals is only slightly less than that in control rats, but the total amounts of various components of the tissue (total nitrogen, nucleic acids, phospholipids, respiratory enzymes) remain below the control level for at least 5 weeks. It seems unlikely that decreased thermogenic activity is of major importance in reducing the survival of cold-exposed irradiated rats.