Studies on the interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C solid state NMR

The effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on model phospholipid membranes were studied using 13C and 2H solid-state nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) with specific 13C and 2H labels as endogenous probes at the carbonyl and the C-7 methylene groups, respectively, of the sn-2 chain were used to study the conformational and dynamical properties of the bilayer as a function of temperature. There were no significant changes between the 13C and 2H spectra of the DPPC preparation containing the inactive steroid and that of DPPC with no drug. However, the physiologically active steroid produces significant spectral 2H and 13C changes. These changes include a reduction of the main phase transition temperature and a broadening of that transition. Alphaxalone also increases the relative number of gauche conformers in the liquid-crystalline phase of DPPC and increases the rate of axial diffusion in both the gel and liquid-crystalline phase. The thermotropic properties of the above preparations, as monitored by differential scanning calorimetry, were congruent with the spectroscopic data.     

Cells with natural killer activity in the cerebrospinal fluid of normal mice and athymic nude mice with acute Sindbis virus encephalitis

Sindbis virus causes an acute, nonfatal inflammatory encephalitis in weanling BALB/c mice. Mononuclear inflammatory cells are present in the cerebrospinal fluid (CSF) as well as in the parenchyma of the brain. Both aspects of this inflammatory response were eliminated by treatment with cyclophosphamide. Athymic nude (nu/nu) mice developed no inflammation in the brain, but did develop a CSF pleocytosis that peaked on day 2 after infection. The time course of the appearance of cells in the CSF was earlier in nu/nu mice than their heterozygote (nu/+) littermates. The pleocytosis in nu/nu mice reached a peak on day 2, whereas in nu/+ mice the peak was on day 4, as it is in normal BALB/c mice. To determine whether some of the CSF cells in nu/nu mice may be natural killer (NK) cells, NK activity was measured in a 4-hr assay by using a YAC-1 target cell. NK cell activity in the spleen and peripheral blood was induced by infection with Sindbis virus in nu/nu mice with a similar time course to that of nu/+ mice (peak 1 day after infection). CSF from nu/nu mice had NK activity present 2 days after infection that was greater than that present in either the peripheral blood or spleen. BALB/c and nu/+ mice had insufficient cells present for assay at day 2, but BALB/c mice had NK activity present in the CSF 3 and 5 days after infection that exceeded that in the peripheral blood or spleen. Brain interferon was detectable on day 1 in nu/nu mice, but not until day 2 in nu/+ mice even though the amounts of brain virus were the same in the two groups at all time points. It is concluded that cells with NK activity contribute to the CSF pleocytosis induced by acute Sindbis virus encephalitis.     

Hand-rearing baboons for laboratory investigations

To conduct laboratory studies in unsedated animals that were similar anatomically and physiologically to man, five full term baboons (four Papio cynocephalus anubis, one Papio cynocephalus cynocephalus) were hand-reared. These infants were used as unsedated animal models in short-term lung clearance studies conducted from birth to 2 years of age. The hand-rearing techniques described here encouraged the formation of an infant-human rearer bond that permitted us to control the level of expressed aggressive behavior as the infant matured. These techniques resulted in baboons which displayed subordinate behavior, showed positive reception to human contact (without evidence of negative stereotypic behavioral anomalies) and remained cooperative subjects for our investigations of short-term pulmonary clearance. The baboons generally were above average in weight in comparison to conspecifically-reared baboons of similar age, sex and species. Representative lung retention curves presented on one baboon demonstrate the feasibility of lung clearance studies in these hand-reared animals. Due to its suitability for unsedated studies, this baboon model may be considered for other types of laboratory investigations.     

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Mapping of the lipoyl-bearing domain by limited proteolysis

To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue.     

The effect of tryptase from human mast cells on human prekallikrein

Tryptase, the dominant protease in human mast cells, was examined for its effect on human prekallikrein. Tryptase in the presence and absence of heparin failed to activate prekallikrein as shown in a spectrophotometric assay for kallikrein employing benzoy 1-pro-phe-arg-p-nitroanilide. Treated prekallikrein was converted to active kallikrein by bovine trypsin. Prekallikrein cleavage products were analyzed by electrophoresis in polyacrylamide gels under denaturing conditions (+/- reduction). Tryptase caused no apparent cleavage under conditions where trypsin caused complete cleavage. Thus, tryptase, which has previously been shown to lack kallikrein and kininase activities, neither activates nor destroys prekallikrein.     

Multiple gene-like sequences related to the rabbit hepatic progesterone 21-hydroxylase cytochrome P-450 1

Rabbits exhibit phenotypic differences, 21H and 21L, in the rate of hepatic progesterone 21-hydroxylation that reflect 10-fold higher microsomal concentrations of cytochrome P-450 1 in 21H rabbits. A cDNA library in pBR322 was prepared from liver mRNA isolated from a 21H rabbit. A clone, p1-8, producing a hybrid protein resulting from the insertion of the cDNA into the beta-lactamase gene of the plasmid expressed 5 distinct epitopes that were recognized by a panel of monoclonal antibodies developed toward P-450 1. RNAs selected from total hepatic mRNA by filter hybridization with p1-8 yield at least two electrophoretically distinct proteins when translated in vitro and immunoprecipitated with the 3C3 monoclonal antibody. Only one of the two proteins is recognized by the 1F11 monoclonal antibody, which is highly specific for P-450 1, and the immunoprecipitated protein exhibits the electrophoretic mobility of P-450 1. The other protein remains unidentified. Northern blot analysis indicates that the 3' noncoding portion of p1-8 hybridizes to higher steady state concentrations of polyadenylated RNA in the 21H as compared to 21L rabbits. This correspondence in expression with that of P-450 1 in the 21H and 21L phenotypes further suggests that p1-8 encodes P-450 1 or a closely related protein. The cDNA is 1871 base pairs in length and encodes a protein of 487 amino acids. Southern blot analysis indicates that several independent, gene-like sequences hybridize with the 3' noncoding region of p1-8 under conditions of high stringency. These results indicate that P-450 1 is a member of an extensive multigene family.     

Spectra of chloroperoxidase compounds II and III

Chloroperoxidase was present as Compound II during the peroxidatic oxidation of ascorbic acid. Compound III (oxy-form) was formed when excess hydrogen peroxide was added to Compound II. By decreasing the temperature it was possible to measure the spectra of Compounds II and III in the Soret and visible regions. Each spectrum was found to resemble that of the corresponding form of lactoperoxidase. Under the experimental conditions, chloroperoxidase Compound III was apparently converted to Compound II in parallel with the decomposition of hydrogen peroxide and finally to the ferric enzyme.     

Random association of Epstein-Barr virus genomes with host cell metaphase chromosomes in Burkitt''s lymphoma-derived cell lines.

The random association of Epstein-Barr virus DNA with host cell metaphase chromosomes of all sizes in Burkitt's lymphoma-derived cell lines was demonstrated by two substantially different techniques, namely fluorescence-activated chromosome sorting and in situ hybridization. The nature and potential importance of this association are discussed.     

Comparative study of methods for the quantification of fungal spores at the leaf surfaces ofPopulus xeuramericana

Summary The removal of fungal spores, urediniospores ofMelampsora medusae and conidia ofPestalozzia sp., from the leaf surfaces ofPopulus xeuramericana (Dode) Guinier cv. I-488 was assessed using three cultural techniques conventionally employed in phylloplane studies. The method of removal and the original density of spore deposition, but not the interaction of these factors, were significant determinants of variability in spore removal. Irrespective of the original density of deposition, the leaf print method was the most, and the leaf washing technique the least, efficient means of spore removal from the leaf surface. Factors which could contribute to this difference in efficiency are discussed.     

Surface-dependent activation of human factor XII (Hageman factor) by kallikrein and its light chain

In this paper we report the effect of sulfatides on the rate constants of factor XII activation by kallikrein and its isolated light chain (the domain of kallikrein that contains the active site of the enzyme). In the absence of sulfatides, kallikrein and the light chain were equally effective in factor XII activation (k1 = 1.57 X 10(3) M-1 s-1 at pH 7.0). The pH optima were the same (pH 7.0) and the reaction was not affected by variation of the ionic strength. Sulfatides strongly increased the rate constants of factor XIIa formation. In the presence of sulfatides kallikrein was, however, much more active than its light chain. At 330 microM sulfatides, pH 7.0 and 100 mM NaCl the rate constants of factor XII activation were 5.34 X 10(6) M-1 s-1 and 4.17 X 10(4) M-1 s-1 for kallikrein and its light chain, respectively. The pH optimum of factor XII activation by kallikrein in the presence of sulfatides was shifted to pH 6.3, and the reaction became highly ionic-strength-dependent. The rate constant increased considerably at decreasing NaCl concentrations. The optimum pH for light-chain-dependent factor XII activation in the presence of sulfatides remained unaltered and the reaction was not affected by the ionic strength. Binding studies revealed that both kallikrein and factor XII bind to the sulfatide surface, whereas no binding of the light chain of kallikrein was detectable. The isolated heavy chain of kallikrein had the same binding properties as kallikrein, which indicates that the heavy-chain domain contains the functional information for kallikrein binding to sulfatides. Since the effects of pH and ionic strength on the rate constants of kallikrein-dependent factor XII activation in the presence of sulfatides correlated with effects on the binding of kallikrein, it is concluded that under these conditions surface-bound factor XII is activated by surface-bound kallikrein. Our data suggest that sulfatides stimulate kallikrein-dependent factor XII activation by two distinct mechanisms: by making factor XII more susceptible to peptide bond cleavage by kallikrein and by promoting the formation of the enzyme-substrate complex through surface binding of kallikrein and factor XII.