Lymphocyte granule-mediated cytolysis requires serine protease activity

We show that chymotrypsin-like, as well as trypsin-like, proteases are in granules isolated from cytolytic lymphocytes by the capacity of the granules to hydrolyze the peptide substrates Z-Phe-Leu-Phe-SBzl and Z-Ala-Gly-Arg-SBzl, respectively. We report protease inhibitors that can abrogate or delay granule-mediated cytolysis. Two mechanism-based isocoumarin serine protease inhibitors and Z-Gly-Leu-Phe-CH2Cl completely abrogated granule cytolysis. Lima bean and soybean trypsin inhibitors and chymostatin delayed but did not prevent this cytolysis. These data represent the first use of the powerful isocoumarin inhibitors as biological probes and indicate that lymphocyte serine proteases participate in the granule cytolytic process.     

Kinetic properties of the insulin receptor tyrosine protein kinase: activation through an insulin-stimulated tyrosine-specific, intramolecular autophosphorylation

The insulin receptor is an insulin-activated, tyrosine-specific protein kinase. Previous studies have shown that autophosphorylation of tyrosine residues on the Mr 95,000 is associated with an activation of the protein kinase activity toward exogenous protein substrates. We have employed the highly purified insulin receptor, immobilized on insulin-Sepharose or eluted in an active form, to define the metal/ATP requirements for kinase activation, the relationship of receptor autophosphorylation to activation, and the kinetic properties of the autophosphorylated, activated receptor kinase. Prior incubation of the immobilized receptor with 2 mM ATP, 10 mM Mg (or 10 mM Mn), followed by removal of these reactants, served to abolish the upward curvilinearity in the rate of histone 2b (tyrosine) phosphorylation measured subsequently. This treatment also markedly increased the rate of histone 2b phosphorylation as compared to that observed with the unmodified, immobilized receptor, as estimated under conditions that per se minimized further activation. The extents of maximal activation of receptor histone 2b (tyrosine) kinase obtained on preincubation with MgATP or MnATP are identical; however, the affinity of the receptor for MnATP is approximately 10-fold higher than that for MgATP. The higher affinity of the receptor for MnATP is observed for both autophosphorylation/autoactivation and histone 2b tyrosine kinase activity (Km MnATP approximately 0.01 mM; Km MgATP approximately 0.1 mM). Autophosphorylation/autoactivation per se does not significantly alter the apparent affinity for MeATP (or protein substrate, as previously reported) but increases Vmax. Activation of receptor histone 2b (tyrosine) kinase is due to tyrosine-specific autophosphorylation of the Mr 95,000 (beta) subunit; thus the extent of total 32P incorporation into the beta subunit correlates precisely with the extent of kinase activation, both over time and at a wide variety of Me2+ ATP concentrations. Sequential treatment of the autophosphorylated receptor with elastase and trypsin yields a single, basically charged 32P-peptide, Mr less than 2000. The functional properties of the unphosphorylated and fully phosphorylated receptor were compared after elution from insulin-Sepharose. The insulin binding characteristics of the two forms of the receptor were indistinguishable; the kinase properties differed greatly; whereas the histone 2b activity of the unphosphorylated receptor was low in the basal state, and activated 10-fold by insulin, the fully autophosphorylated receptor exhibits maximal histone 2b kinase in the basal state and is unaffected by insulin addition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet β-Cell Function

Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet β-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.     

Monodomain samples of dipalmitoyl phosphatidylcholine with varying concentrations of water and other ingredients.

Methods are presented for the preparation of large monodomain phospholipid bilayer arrays containing variable amounts of water approaching the two-phase limit. The optical birefringence of these lamellar phases of dipalmitoyl phosphatidylcholine (DDPC) is measured over a range of temperature and water content, and phase transitions are observed. The techniques employed for pure DPPC and water are extended in order to produce macroscopically aligned samples containing varying concentrations of cholesterol, inorganic salts, antibiotics, and chlorophyll a. Polarization studies of the 670-nm band of chlorophyll a indicate macroscopic orientational order in the chromophore under the same conditions.     

The co‐evolution of social institutions,demography, and large‐scale human cooperation

Human cooperation is typically coordinated by institutions, which determine the outcome structure of the social interactions individuals engage in. Explaining the Neolithic transition from small‐ to large‐scale societies involves understanding how these institutions co‐evolve with demography. We study this using a demographically explicit model of institution formation in a patch‐structured population. Each patch supports both social and asocial niches. Social individuals create an institution, at a cost to themselves, by negotiating how much of the costly public good provided by cooperators is invested into sanctioning defectors. The remainder of their public good is invested in technology that increases carrying capacity, such as irrigation systems. We show that social individuals can invade a population of asocials, and form institutions that support high levels of cooperation. We then demonstrate conditions where the co‐evolution of cooperation, institutions, and demographic carrying capacity creates a transition from small‐ to large‐scale social groups.     

Plasmodium knowlesi: Morphology and course of infection in rhesus monkeys treated with clindamycin and its N-demethyl-4′-pentyl analog

The clinical and morphologic effects of clindamycin and N-demethyl-4′-pentyl clindamycin were evaluated using Plasmodium knowlesi in rhesus monkeys. Both compounds cured blood-induced infections when administered daily for five consecutive days. When the rapidity of action of these antibiotics was compared with chloroquine it was evident that although they were able to control fulminating infections in all treated monkeys their effect was about 2 days slower than chloroquine in decreasing parasitemias and 3 to 4 days slower in clearing parasites from the blood. Morphologic changes within the parasite associated with drug action were studied in time sequences by light and electron microscopy. Changes were observed in the parasite ribosomes 24 hr after drug administration. Affected ribosomes showed electron-lucent zones measuring ~50 Å in the center. During the following 24 hr these changes became more prominent with foci in which disintegrated ribosomes were replaced by fine fibrillar material and the cisternae of the endoplasmic reticulum became dilated. By 72 hr this dilation was more apparent and resulted in abundant coalescence of vacuoles in the cytoplasm. Mitochondria became dilated with fibrils in the matrix, although the degree of swelling was not a conspicuous and constant feature. The nucleus presented a fine fibrillar appearance and fewer granules were seen than in normal parasites. The latter two observed changes appear to be secondary to changes in the ribosomes and probably are not directly related to the action of the antibiotics. These studies indicate that clindamycin and its analog affect primarily the ribosomes and their mode of action is different from that of the commonly used antimalarials.     

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.