Purification and properties of a plasminogen activator from cultured rat prostate adenocarcinoma cells

Zymographic analysis of the supernates from confluent cultures of a rat prostate adenocarcinoma cell line, PA-III, revealed the existence of two molecular forms of specific plasminogen activators, one of molecular weight of approximately 80 000 and another of approximate molecular weight of 45 000, in sodium dodecyl sulfate. The low molecular weight form has been purified 364-fold in 66% yield from the culture medium by a combination of gel filtration on Sephacryl S-200 and affinity chromatography on Sepharose 4B-benzamidine. The purified material possessed a specific activity of 192 000 urokinase CTA units mg-1. This enzyme displayed activity toward human Glu1-plasminogen, characterized by a Km of 1.7 +/- 0.2 microM and a Vmax of 0.53 +/- 0.1 pmol of plasmin min-1 unit-1. A synthetic chromogenic substrate, H-D-Ile-Pro-Arg-p-nitroanilide (S-2288), was found for the activator. The enzyme possessed a Km of 0.33 mM and a kcat of 55 s-1 for S-2288. The activator was found to be a serine protease, inhibited by diisopropyl fluorophosphate (iPr2PF). At a concentration of 1 mM iPr2PF, and 30 nM enzyme, the half-time of this inhibition was 3.8 min. The 45 000 molecular weight enzyme was found to be inhibited by rabbit antibodies to human urokinase, thus characterizing the activator as a member of the urokinase class. The 80 000 molecular weight enzyme was not neutralized by anti-human urokinase but was neutralized by rabbit anti-human melanoma activator, likely allowing it to be classified as the tissue activator type.     

Discrete conductance fluctuations in lipid bilayer protein membranes

Discrete fluctuations in conductance of lipid bilayer membranes may be observed during the initial stages of membrane interaction with EIM ("excitability inducing material"), during destruction of the EIM conductance by proteolysis, and during the potential-dependent transitions between low and high conductance states in the "excitable" membranes. The discrete conductance steps observed during the initial reaction of EIM with the lipid membranes are remarkably uniform, even in membranes of widely varying lipid composition. They range only from 2 to 6 x 10_-10 ohm_-1 and average 4 x 10_-10 ohm_-1. Steps found during destruction of the EIM conductance by proteolysis are somewhat smaller. The transition between high conductance and low conductance states may involve steps as small as 0.5 x 10_-10 ohm_-1. These phenomena are consistent with the formation of a stable protein bridge across the lipid membrane to provide a polar channel for the transport of cations. T6he uniform conductance fluctuations observed during the formation of these macromolecular channels may indicate that the ions in a conductive channel, in its open state, are largely protected from the influence of the polar groups of the membrane lipids. Potential-dependent changes in conductance may be due to configurational or positional changes in the protein channel. Differences in lipid-lipid and lipid-macromolecule interactions may account for the variations in switching kinetics in various membrane systems.     

Cancer of the Nasopharynx

Of 75 consecutive patients with nasopharyngeal cancer treated at UCLA Center for Health Sciences, 42 were eligible for long-term study. Fourteen (33 percent) survived five years, and one survived ten years. Since the management of nasopharyngeal cancer is primarily irradiation, a well-planned aggressive treatment program using supravoltage therapy is recommended. In spite of cranial nerve involvement or skull erosion, radiation therapy occasionally may offer long term control of the disease.     

Chromosome Replication in Salmonella typhimurium

The replication of the Salmonella typhimurium chromosome was studied. As with E. coli 15T(-), replication was sequential. After amino acid starvation, replication proceeded from a unique and heritable region of the chromosome. 5-Bromouracil, when substituted for thymine, did not disturb the sequence of replication nor did it initiate extra replication cycles. By labeling the origin and the terminus of the chromosome with (3)H- and (14)C-thymine, respectively, it was possible to determine that the rate of chain elongation decreases as the growth rate decreases. No gap in the replication cycle could be observed.     

Seasonal changes in the distribution of gonadal lipids and spermatogenetic tissue in the male phase of Monopterus albus (Pisces: Teleostei)

The gonad of Monopterus albus undergoes cyclical changes after the reversal of sex from female to male. The seasonally variable events include a prenuptial accumulation of cholesterol-positive lipid droplets in the cytoplasm of the interstitial cells when spermatogenetic activity is resumed in late February and early March. The development of the interstitial Leydig cells reaches a maximum in May just before spawning. There occurs a sudden depletion of the interstitial lipids during the breeding season in June at a time when the male animals exhibit active nuptial behaviour. After spermiation, the old interstitial cells degenerate, and during the succeeding phase of gonadal inactivity, become replaced by a new generation from connective tissue cells in the interstitium of the gonadal lamellae which gradually accumulate lipoidal material.
The lobular cycle comprises a postnuptial accumulation of amorphous intralobular lipids which become completely cleared in February when active spermatogenesis is restored. Spermatogenesis resumes shortly after spawning, but only advances as far as primary spermatocytes during the postnuptial period of inactivity.
The authors conclude that, as far as the seasonal variations in gonadal lipid distribution is concerned, the cycles in the gonad of the hermaphroditic teleost, M. albus , conform to the same pattern as those of the gonochoristic seasonal breeders studied.     

Magnetic resonance study of renal transplantation

In the last few years, we have focused our research effort on the magnetic resonance spectroscopic (NMR) studies of organ transplantation in the rat. P-31 NMR was employed to study changes in high-energy phosphates, intracellular pH in vivo of transplanted kidneys either during normal function, while undergoing the rejection process or subjected to other insults (e.g. ischemia, cyclosporine nephrotoxicity, urinary obstruction) which may also cause graft dysfunction. Nuclear magnetic resonance (NMR) parameters, specifically relative peak areas and intracellular pH, accurately distinguished among the different causes of graft dysfunction. Ureteral obstruction was clearly identified by elevations in the phosphodiester/urine phosphate peak. Ischemia and rejection were both associated with increases in inorganic phosphates and phosphomonesters and decreases in the beta-phosphate peak of adenosine triphosphate but were distinguishable from each other by differences in intracellular pH which was normal in rejected allografts (7.33 +/- 0.07, n = 3) and low in ischemic allografts (7.00 +/- 0.05, n = 3, p less than 0.05). Grafts insulted with cyclosporine toxicity were not distinguishable from normal allografts by any of the parameters studied. To determine the temporal relationship of NMR changes in allograft rejection, similar studies were performed serially in a group of rejecting (R) kidneys (n = 7) and compared with a control group of nonrejecting (NR) kidneys (n = 7). Major decrease in adenosine triphosphate (ATP) with increases in Pi and a marked increase in the Pi/ATP ratio were noted in the R allografts over time. The R allografts could be completely segregated from the NR allografts on the basis of the Pi/ATP ratio by day 7. These data suggest that 31P NMR spectroscopy may have potential clinical application in differentiating among the causes of graft failure of human renal allografts.     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

Vasoactive properties of dihomo-gamma-linolenic acid and series one prostaglandins in a freshwater teleost, Channa maculata

1. Prostaglandins A1, B1, E1 and F1 alpha (2-120 micrograms/kg), arachidonic acid and dihomo-gamma-linolenic acid (0.1-2 mg/kg) were injected intravenously into Channa maculata and changes in arterial blood pressure were recorded. 2. Injection of PGF1 alpha had no significant effect on arterial blood pressure. Injection of PGA1 and PGE1 was followed by dose-dependent hypotension whereas injection of PGB1 elicited significant dose-dependent increase in arterial blood pressure. 3. Both dihomo-gamma-linolenic acid and arachidonic acid were also depressor agents but dihomo-gamma-linolenic acid was more potent. 4. A single bolus intravenous injection of indomethacin (5 mg/kg) or 4 daily intraperitoneal injections (4 x 10 mg/kg) significantly lowered arterial blood pressure. One hour after pre-treatment of indomethacin, the vascular effects of both prostaglandin precursors were abolished. 5. It appears that the vascular effects of prostaglandins in Channa maculata are qualitatively different from those reported for mammals.