An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 x 107. The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.

The osmotic properties of human lymphocytes isolated from 15 ml of venous blood were examined. Measurements of the permeability of the membrane to water under an osmotic gradient were also made. The Boyle-Van't Hoff relation held very well for the human lymphocyte when the cells were shrunken in hyperosmotic media to concentrations twice isosmotic. The volume of osmotically inactive material or "b" value averaged 32% of the mean corpuscular volume. These values were independent of temperature. Ponder's R ranged between 0.8 and 0.9. The average value for Lp, the hydralic coefficient was 0.46 mu/min atm +/- 0.02 (S.E.M.) at 25 degrees C. No significant effect of age, sex, or race was noted. The effect of temperature between 10 degrees C and 37 degrees C was measured and heats of activation between 11.1 and 17.4 kcal/mole were calculated with a mean of 14.1 kcal/mole +/- 1.6 (S.E.M.). Concanavalin A at 10 microgram/1.5 X 10(6) lymphocytes produced blastogenesis of 25% or more of the lymphocytes without clumping, agglutination, or toxicity. The mean corpuscular volume increased by 21% after 72 hours due to an increase in the "b" value which increased by 80%. The volume of free water remained constant. Histograms of the distribution of cell volumes showed that volume changes were uniform throughout the population with no evidence of agglutination of clumping. The significance of these results is discussed in the context of membrane fluidity and the state of intracellular water.

Pasteurized concentrated skim milk containing 35 to 40% total solids was inoculated with cultures of Bacillus subtilis, Micrococcus flavus, or Escherichia coli to contain 1 × 106 organisms per g and spray-dried to determine the effect of exit air temperatures of 93.3, 82.2, and 71.1 C on survival of the organisms and moisture content of the finished product. The numbers of survivors increased as the drying temperature decreased. The percent survivors varied from 27.57 in the product made from milk inoculated with M. flavus and dried at 71.1 C to 0.02 in the product made from milk inoculated with E. coli and dried at 93.3 C. The organism most resistant to drying and most persistent during storage was B. subtilis, followed by M. flavus and E. coli, with the latter showing low survival during drying and abrupt die-off during the first 4 weeks of storage. The moisture content of the dry milks varied from 2.75 to 4.80% with low moisture associated with high drying temperature.

Specific high affinity binding sites for CRH have been identified and characterized in the pituitary and central nervous system as well as in peripheral tissues. We recently identified and characterized a specific CRH receptor in human myometrium that changes to a high affinity state before term. In view of this, we searched for receptor heterogeneity in the pregnant and nonpregnant human myometrial CRH receptor. Myometrial membranes were prepared by differential centrifugation from either pregnant (cesarian section) or nonpregnant (hysterectomy) myometrium. Using a specific RRA followed by isoelectric focusing and autoradiography, multiple isoforms of the human myometrial CRH receptor were identified that were identical in both pregnant and nonpregnant myometrium. Five isoforms were identified (pI 4.65, 4.8, 4.95, 5.1, and 5.2). Reduction of disulfide bridges with reducing agents (dithiothreitol and cysteine) increased the specific binding of CRH to its myometrial receptor, and the action of dithiothreitol affected the two most basic receptor isoforms. These results suggest the presence of multiple isoforms of CRH receptors that may have different properties and functions and the presence of disulfide bridges within the myometrial CRH receptor, which are important, but not critical, for the receptor binding.

Pseudomonas fragi is one of several pseudomonads known to produce proteolytic enzymes. During growth of P. fragi in brain heart infusion broth (BHI) at 10°C, the bacterial population increased from 107 to over 1010 CFU/ml after 130 h, with a concurrent increase in pH from 7.4 to 8.5. Maximal extracellular protease activity occurred after 60 to 72 h. Ultrastructural examination of cells grown in BHI showed the presence of bleb-like evaginations of the cell wall. Similar structures were not detected when P. fragi was grown in Koser citrate broth, a medium which was unsuitable for supporting protease production by P. fragi.