immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Development of an improved medium for the isolation of 'Haemophilus somnus'

A medium containing lincomycin (3 μg/ml), cycloheximide (100 μg/ml) and chloral hydrate (0–1 %) was superior to all others examined for the isolation of 'Haemophilus somnus' from material contaminated with Proteus species.     

Accessibility of phosphatidylethanolamine in bacteriophage PM2 and in its gram-negative host.

The reaction of trinitrobenzenesulfonic acid with phosphatidylethanolamine in the cytoplasmic membrane of Alteromonas espejiana suggests that 50% of this lipid occupies the outer lamella. In PM2, similar analysis suggests that 56% of this lipid populates the outer lamella of the membrane, the surface of which accounts for 60% of the membrane area.     

Mutant ts1 of bacteriophage PM2 is defective in the major capsid protein and fails to package its DNA.

Infection of Alteromonas espejiana at restrictive temperature with mutant ts1 of bacteriophage PM2 resulted in the intracellular accumulation of virus-sized empty-appearing membrane vesicles. The DNA associated with purified vesicles was fully susceptible to digestion with DNase. Sedimentation analysis and electron microscopy suggested a full-length linear form of the normally circular viral genome. A pulse-chase-shift experiment suggested that [3H]thymidine-labeled DNA made under restrictive conditions is assembled into virions after shift to permissive temperature. A defective structural protein in the ts1 virion appears to be the cause of a rapid rate of thermal inactivation of infectivity. Analysis of the proteins of ts1 by isoelectric focusing indicated a more alkaline isoelectric mobility of the major capsid protein, sp27. Six spontaneous revertants of ts1 showed reversion to the wild-type isoelectric form of sp27. These results identify sp27 as the defective gene product of ts1. Taken together, these results suggest that the membrane of PM2 is formed without the aid of an inner core or an outer scaffolding.     

Apolipoprotein A-I isoforms in human lymph: effect of fat absorption

The effect of fat feeding (100 g of cream) on the apoA-I isoproteins distribution has been analyzed by two-dimensional gel electrophoresis in the chylomicrons, VLDL, LDL, and HDL isolated from the thoracic duct lymph of patients undergoing lymph drainage for immunosuppression, Isoforms apoA-I3 and apoA-I4 are the most abundant apoA-I isoproteins in plasma lipoproteins as well as in lymph lipoproteins collected in the fasting state. Fat feeding, on the other hand, results in a marked change in the apoA-I isoform pattern in lymph chylomicrons and VLDL, with a significant increase in the relative concentration of the apoA-I1 isoform. As a result the total concentration of this isoprotein in the lymph increased. The data indicate that fat feeding is associated with major changes in the distribution of the apoA-I isoforms in the lymph (d less than 1.006 g/ml lipoproteins), which may be of significance in their plasma catabolism.     

Studies of activating and nonactivating metal ion binding to yeast enolase

Measurements of binding of certain divalent cations to yeast apoenolase were made using a pH-meter, chromatography, a divalent cation electrode, and ultrafiltration. The binding of the activating metal ions Mg2+ and Co2+ and the nonactivator Ca2+ were studied as functions of the presence or absence of substrate/product, phosphate, and fluoride or level of Tb3+. The data suggest phosphate and fluoride increase Mg2+ binding but not Ca2+ binding. Substrate/product appears to increase Ca2+ binding as well as that of Mg2+ and Co2+. In the presence of substrate, Co2+ binding was 5-6 mol/mol dimer. In the absence of substrate/product, Tb3+ reduced Co2+ binding from 4 mol/mol to 2. These data are interpreted in terms of binding to "conformational," "catalytic" (substrate/product dependent), and "inhibitory" sites. Measurements of Tb3+ fluorescence quenching by Co2+ suggested that the distance between "conformational" sites on the two subunits was large, while the distance between "conformational" and "inhibitory" sites was ca. 17 +/- 4 A. Potentiometric titrations of apoenzyme with Ca2+ and Mg2+ showed that the metal ions produced the same proton release in the presence or absence of substrate/product. If phosphate and fluoride were present, then more protons were released if Ca2+ was the titrant rather than Mg2+, suggesting a difference in ionization state in the complex with the activating metal. Electron paramagnetic resonance studies of Co2+ binding to the various sites in the enzyme are presented. The Co2+ bound to all three sites appears to be high spin, consistent with a preponderance of oxyligands in an octahedral environment. Substrate, citrate, and a strongly binding substrate analogue strongly enhance the hyperfine structure of conformational Co2+. This is interpreted as the result of a change in interaction of an axial ligand to conformational Co2+ produced by carbon-3 of substrate or analogue.     

Effect of zinc on increasing oxygen affinity of sickle and normal red blood cells

We have hypothesized a state of zinc deficiency in sickle cell disease (SCD). This could at least partially explain the growth problems, hypogonadism, and slow healing leg ulcers associated with SCD. Preliminary findings revealed abnormally low red blood cell zinc levels in 10 of 16 patients studied. Before suggesting zinc supplementation in SCD we thought it important to look at the effect of zinc on red cell metabolism and function. It was found that zinc chloride added to normal and SCD blood to a final concentration of 1.5 × 10^?3 M caused a left-shift of the blood oxygen affinity curve (increased oxygen affinity) varying from 1.5 to 3.5 mm Hg change in half saturation (p50). This curve shifting property has important implications for SCD since recent work with cyanate suggests that such shifts are very beneficial in treatment of SCD. Thus zinc supplementation in SCD, in addition to its potential role in correcting wound healing and growth problems, may have a beneficial effect on the basic pathological process. Data are given which suggest that zinc and 2, 3-diphosphoglycerate may not be competing for the same site on the hemoglobin molecule.     

Non-protein components of the cell surface of Staphylococcus aureus

1. pH–mobility curves of various laboratory strains of Staphylococcus aureus are non-sigmoid in shape, and all pass through a maximum value in the range pH4–5. 2. The maxima in the curves are not due to incomplete washing of the cells, adsorption of buffer components or irreversible surface damage. 3. Mild oxidation of the cell-surface teichoic acid with sodium metaperiodate gives cells that have typical sigmoid pH–mobility curves, characteristic of either a simple carboxyl surface or a mixed carboxyl–amino surface. 4. The results are discussed in terms of a pH-dependent change in the configuration of the teichoic acid molecules at the surface.