Phenotype-dependent inhibition of human alpha 1-antitrypsin by 1,6-hexane diamine in vitro

Blood donors and patients from pulmonary wards gave serum specimens for the assay of their alpha 1-antitrypsin activity and phenotype. The same specimens were then incubated at the room temperature overnight with increasing concentrations of 1,6-hexane diamine. The 5 mM amine concentration caused a significant decrease in heterozygous antitrypsins (M1M2, M1M3, M2M3 and M1S) activity while it was less in the homozygous (M1M1 and M2M2) antitrypsin phenotypes. The SS and ZZ phenotypes showed a very low initial activity which was, however, further reduced. Analysis for the antitrypsin protein showed a simultaneous loss of its activity. The data suggest that heterozygous antitrypsin carriers may be more prone to the effects of inhaled amines despite their 'normal' phenotypic constitution.     

Identification and characterization of Epp, the secreted processing protease for the Vibrio anguillarum EmpA metalloprotease

The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an approximately 10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.     

Isomerization of an Antimicrobial Peptide Broadens Antimicrobial Spectrum to Gram-Positive Bacterial Pathogens

The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.     

Lipoprotein lipase-like activity in the liver of mice with Sarcoma 180

The triglyceride lipase (TGL) activity of liver homogenates of mice with Sarcoma 180 was measured. The liver homogenate of normal or tumor-bearing mice was treated with 0.25% Triton X-100 and centrifuged at 100,000 g for 60 min, and the supernatant was applied to a heparin-Sepharose column. In normal mice, most of the TGL activities in the supernatant was eluted with 0.75 M NaCl from the column. In mice with Sarcoma 180, the TGL gave two peaks on heparin-Sepharose column chromatography, which were eluted with 0.75 M and 1.5 M NaCl, respectively. The activity in the first peak (0.75 M NaCl eluate) decreased; that in the second peak (1.5 M NaCl eluate) increased, and the ratio of the second peak to the first peak increased during tumor development. The livers of normal mice and mice on day 10 after tumor inoculation were perfused with heparin. The highest rate of the TGL release occurred within 1 min of heparin perfusion, and the bulk of heparin-releasable activity appeared within 2 min of perfusion in both normal and tumor-bearing mice. The TGL activity in liver perfusate of tumor-bearing mice, as well as that of liver homogenate, was resolved on a heparin-Sepharose column into two peaks, which were eluted with 0.75 M and 1.5 M NaCl, and most of the activity was eluted with 1.5 M NaCl. The nature of the TGL activity eluted from a heparin-Sepharose column was investigated. In both liver homogenates and liver perfusates, the first peak did not require serum for maximal activity and was relatively resistant to a high concentration of NaCl or protamine sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

毕赤酵母木糖还原酶定点突变改善其对双辅酶的亲和力

通过毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase, XR)基因定点突变,获得NADH高亲和力的毕赤酵母木糖还原酶(PsXR),改善了辅酶不同而导致的酿酒酵母胞内氧化还原失衡.同时克隆了PsXR编码基因,通过BLAST工具进行同源性搜索,并用生物软件进行序列比对和结构分析,确定突变位点.用融合PCR方法进行定点突变,并在大肠杆菌表达系统中进行融合表达,且对表达产物进行HIS-TAG亲和纯化,分光光度法检测酶活性,计算比活力.本研究成功获得突变XR编码基因,并收集了纯化的突变蛋白.酶活性检测和比活力计算显示,3种突变酶对2种辅酶的亲和力在一定程度上都发生了变化.与未突变的PsXR相比,3种突变酶对辅酶NADPH的亲和力均显著下降,突变酶M3对辅酶NADH的亲和力未发生变化,而突变酶M1和M4对辅酶NADH的亲和力显著升高,其中突变酶M1对NADH的亲和力明显提高,对NADPH的亲和力明显下降,其活性主要依赖辅酶NADH,提示K270R位点在XR与辅酶结合中起关键作用.     

Separation of soluble amoebicidal and tumoricidal activity of activated macrophages.

Macrophage-conditioned medium (M phi CM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in M phi CM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in M phi CM. Anti-TNF alpha antiserum inhibited tumoricidal activity in M phi CM. The antiserum reduced amoebicidal activity in BCG-activated M phi CM but had no effect on amoebicidal activity in P. acnes-activated M phi CM. Recombinant TNF alpha, rIL-1 alpha, or rIL-1 beta independently did not affect cytolysis of amoebae. Also, rTNF alpha had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated M phi CM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF alpha antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF alpha and anti-IL-1 alpha antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in M phi CM affected cytolysis of several species of amoebae.     

Primary structure of colicin M, an inhibitor of murein biosynthesis.

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.     

Functional analysis of activated C1s, a subcomponent of the first component of human complement, by monoclonal antibodies

Three mouse monoclonal antibodies (M365, M81, and M241) directed against human C1s were used to analyze the structure of C1s related to the enzymatic activity. M365 and M81 recognized different epitopes on the heavy chain of C1s and could bind to C1s, as well as to C1s. The C4 cleaving activity of C1s was completely blocked by M81 and was partially blocked by M365. Although the C2 cleaving activity of C1s was partially inhibited by M81, no blocking was observed with M365. Both antibodies had no effect on the esterolytic activity of C1s. These results indicate that the C4 and C2 binding sites on C1s reside in the heavy chain, and they are distinct from each other. M241 could bind only to C1s, an active form of C1s. After reduction of C1s, M241 could not react with either heavy or light chain of C1s. The esterolytic activity of C1s was markedly reduced by M241. Furthermore, M241 blocked not only the cleavage of C4 and C2 by C1s but also the complex formation of C1s and C1 inactivator. From these observations, we suggest that M241 reacts with the active site of C1s, and both heavy and light chains of C1s participate in the composition of the active site.     

Upregulation of macrophage plasma membrane and nuclear phospholipase D activity on ligation of the alpha2-macroglobulin signaling receptor: involvement of heterotrimeric and monomeric G proteins

The effect of ligating the alpha2-macroglobulin signaling receptor (alpha2MSR) with receptor-recognized forms of alpha2M (alpha2M*) was studied with respect to phospholipase D (PLD) activity in murine macrophages, their plasma membranes, and nuclei. PLD activity in plasma membranes and nuclei increased linearly up to a ligand concentration of about 100 pM of either alpha2M* or a cloned and expressed receptor binding fragment (RBF). The RBF binding site mutant K1370A, which binds with high affinity to alpha2MSR, also increased nuclear PLD activity comparable to RBF and alpha2M*. Phorbol dibutyrate caused a two- to threefold stimulation of membrane and nuclear PLD activity, whereas PLD activity was nearly abolished by downregulation of protein kinase C; prior treatment with staurosporin, genestein, cyclosporin A, actinomycin D; or chelation of intracellular Ca2+. In permeabilized macrophages, isolated plasma membranes, and nuclei, GTP-gamma-S increased alpha2M*-stimulated PLD activity via a pertussis toxin-insensitive G protein and this effect was abolished on preincubation with GDP-beta-S. Incubation of plasma membranes with polyclonal antibody against sARFII, or the addition of cytosol which was immunoprecipitated with antibody against sARFII, greatly reduced alpha2M*-stimulated PLD activity in the presence of GTP-gamma-S. Preincubation of plasma membranes with GDP-beta-S prior to the addition of GTP-gamma-S and recombinant ARF1 significantly inhibited alpha2M*-stimulation of PLD activity. Nuclear PLD activity was maximally stimulated in the presence of both GTP-gamma-S and rARF1, whereas plasma membrane PLD activity was maximally stimulated in the presence of rARF1, GTP-gamma-S, RhoA, and ATP. In contrast, nuclear PLD activity was not affected by RhoA either alone or in combination with GTP-gamma-S or ATP.     

Inhibitory effect of alpha 2-macroglobulin on Vibrio vulnificus protease

Vibrio vulnificus, an etiologic agent of wound infections and septicemia in humans, elaborates a metalloprotease which is known to be an important virulence factor of the Vibrio. The proteolytic activity of V. vulnificus metalloprotease (VVP) toward casein and elastin was inhibited by alpha 2-macroglobulin (alpha 2 M) at the molar ratio of 1:1, although partial activity was maintained. Permeability-enhancing and hemorrhagic activities were also inhibited, but the peptidase activity toward Z-Gly-Phe-NH2 was not reduced, even by an excess amount of alpha 2 M. VVP formed a complex with alpha 2 M through cleavage of the bait regions of all four alpha 2 M subunits and elicitation of conformational change of the alpha 2 M molecule, which resulted in entrapment of VVP in the alpha 2 M molecule. The peptidase activity of alpha 2 M-VVP complex was inhibited by low-molecular-weight inhibitors such as phosphoramidon, but IgG antibody against VVP failed to neutralize its peptidase activity. Of human plasma proteins, alpha 2 M was the only inhibitor for VVP. These findings indicate that VVP produced during V. vulnificus infection is inactivated by plasma alpha 2 M that leaks from the vascular system.     

Inhibition of the protease activity of influenza virus RNA polymerase PA subunit by viral matrix protein

Influenza virus PA is a subunit of RNA-dependent RNA polymerase. We demonstrated that PA has a unique chymotrypsin-like serine protease activity with Ser624 as an active site. To obtain further insight into the role of the protease activity of PA in viral proliferation, we examined the interaction between PA and matrix protein (M1). Both M1 purified from virion and hexa-histidine-tagged M1 expressed in Escherichia coli bound to PA. Hexa-histidine-tagged M1 pulled down PA. The interaction of PA with M1 was sensitive to ionic strength, suggesting that the interaction is formed by electrostatic force. Using Suc-Leu-Leu-Val-Tyr-MCA, a specific substrate for PA protease, M1 was demonstrated to inhibit the amidolytic activity of PA, whereas M1 did not inhibit that of chymotrypsin or trypsin at all. These results suggest that M1 binds to and inhibits the amidolytic activity of PA.     

Induction and function of 2',5'-oligoadenylate synthetase in differentiation of mouse myeloid leukemia cells

The activity of 2′,5′-oligoadenylate synthetase (2-5A synthetase), known to be induced by interferon, was detected in mouse myeloid leukemic M1 cells only when they differentiated to phagocytic cells after incubation with conditioned medium (CM) from rat embryo cells. However, no interferon activity occurred in culture fluids of CM-treated M1 cells, although some activity was detected in the cell extracts. When anti-interferon serum was added to M1 cell cultures, the induction of 2-5A synthetase by CM was suppressed. These results suggest that CM stimulated the M1 cells to produce a minute amount of interferon, which was reponsible for induction of the 2-5A synthetase activity. On the other hand, development of the phagocytic activity of M1 cells could not be influenced by addition of antiserum. Interferon added exogenously per se neither induced phagocytic activity of M1 cells, nor did it enhance the CM-induced differentiation of the cells. Moreover, dexamethasone, which induced differentiation of M1 cells, was not capable of inducing 2-5A synthetase. These results indicate that interferon and/or 2-5A synthetase plays no essential part in the differentiation of M1 cells.     

Spontaneous cytotoxicity mediated by human monocyte-macrophages against human fibroblasts infected with herpes simplex virsu--augmentation by interferon

Human monocyte-macrophages (MΦ) displayed spontaneous cytotoxicity for human fibroblasts infected with herpes simples virus (HSV). The HSV-immune status of the MΦ did not affect the spontaneous cytotoxicity. Significant cytotoxicity required an effector cell to target cell ratio of at least 50: 1, and was first observed 12 hr after incubation of MΦ with HSV-infected cells. A 12- to 18-hr preincubation of MΦ with interferon enhanced the cytotoxic activity. Cytotoxicity activity was also observed with cytomegalovirus-infected HF implying that the cytotoxic activity of the MΦ was nonspecific for viral-infected cells.     

Effects of ganglioside GM1 on DNA synthesis in isolated nuclei and on the activity of DNA polymerase α derived from S-phase HeLa cells

Ganglioside G_M1 inhibited either DNA synthesis in isolated nuclei or the activity of DNA polymerase α fractionated from S-phase HeLa cells. The concentrations of G_M1 necessary for 50% inhibition were about 5 μM and 10 μM for nuclei and DNA polymerase α, respectively. The G_M1 inhibition of the enzyme activity was suppressed by the addition of 0.05% Triton X-100. Neither gangliotetraosylceramide (asialo-G_M1) nor free N-acetylneuraminic acid inhibited the enzyme activity. These facts suggest that G_M1, probably in the form of micelles, could influence the enzyme activity by behaving as a polyanionic macromolecule. The kinetic studies indicate that the G_M1 inhibition of the enzyme activity was not competitive with the substrate, deoxythymidine triphosphate, but rather with the template DNA. Binding of G_M1 and DNA polymerase α was suggested by the cocentrifugation of G_M1 and the enzyme fraction after their preincubation. It was also observed that other acidic glycolipids, i.e., brain sulphatide and seminolipid, also inhibited the enzyme activity, whilst neutral galactosylceramide did not. The inhibitory influences of these sulphate esters of glycolipids were, similarly to G_M1, suppressed by the addition of 0.05% Triton X-100.     

Purification and Properties of Lytic Enzymes from Streptomyces globisporus 1829

Two lytic enzymes capable of lysing Streptococcus mutans have been purified to give a single band on disc-gel electrophoresis, respectively. The M–1 and M–2 enzymes were both proved to be N-acetylmuramidases. However, these enzymes were entirely different on their enzymatic properties. The molecular weights were about 20,000 and 11,000 for M–1 and M–2 enzymes, respectively, The maximal lytic activity of M–1 enzyme was obtained at ionic strength 0.05, while lytic activity of M–2 enzyme did not change within the ionic strength range of 0 to 0.05. The M–1 enzyme constituted the majority of the total lytic activity against the cell walls of Streptococcus mutans BHT of cultured filtrate. The M–2 enzyme showed less specific lytic activity on the cell walls of Streptococcus mutans BHT than M–1 enzyme.     

A bacterial-induced lectin which triggers hemocyte coagulation in Manduca sexta

An inducible hemagglutinin termed M13, was purified from M. sexta hemolymph. M13 is a glucose-specific lectin which in addition to erythrocyte agglutination, can activate dedifferentiation of various hemocytes into a filamentous coagulation network. When lectin activity was inhibited with glucose or antiserum, neither erythrocyte agglutination or hemocyte coagulation occurred. When M13 was boiled or trypsin treated, hemocyte activation was lost, but erythrocyte agglutination remained. Hence M13 activity appears to be bimodal, possessing both a lectin activity and a hemocyte-coagulating activity.     

通过定点突变提高乳链菌肽对热及pH的稳定性

【目的】通过定点突变技术改变乳链菌肽(nisin)特定位置氨基酸,获得性质改善的nisin突变体,为扩大其应用范围提供依据。【方法】在抑菌谱扩大的nisin单突变体M21K nisinZ的基础上,对M21K nisZ基因第29位丝氨酸密码子进行定点突变;将其克隆至乳酸菌表达载体pMG36e,并在Lactococcus lactis NZ9800中进行表达;双突变体M21K/S29K nisinZ经分离纯化后检测其在抑菌活性、抑菌谱和稳定性等方面的变化。【结果】与单突变体M21K nisinZ及野生型nisinZ(wild-type,WT)相比,双突变体M21K/S29K nisinZ对指示菌的抑菌活性虽有所下降,但其对温度及pH值的稳定性有显著提高。同时其抑菌谱与M21K nisinZ相同,可抑制革兰氏阴性菌,扩大了WT的抑菌谱。【结论】通过改变nisin分子特定位置的氨基酸可以改善nisin分子的理化性质,有可能得到应用范围更广的nisin品种。     

Bacteriostatic and bactericidal activity of antituberculosis drugs against Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium intracellulare complex and Mycobacterium kansasii in different growth phases.

Bacteriostatic and bactericidal activities of rifampicin, isoniazid, streptomycin, enviomycin and ethambutol against Mycobacterium tuberculosis, Mycobacterium avium--M. intracellulare complex and Mycobacterium kansasii were studied in different growth phases. Bacteriostatic activities of the drugs were similar in different growth phases, except isoniazid. M. tuberculosis was much less susceptible to isoniazid in the lag phase than in the log and the stationary phases. In contrast, bactericidal activity was influenced by the growth phase. M. tuberculosis was killed by isoniazid, streptomycin and rifampicin. The bactericidal activity of isoniazid was strongest. The bactericidal activity of isoniazid and streptomycin was most marked in the log phase. M. avium complex and M. kansasii resisted the bactericidal activity, but some strains of M. avium complex were killed by streptomycin and enviomycin, and the activities of these two drugs were most marked in the lag phase.     

Enzymes for biosynthesis de novo and elongation of fatty acids in mycobacteria grown in host cells: is Mycobacterium leprae competent in fatty acid biosynthesis?

Fatty acid synthetase activity in extracts of Mycobacterium leprae was equivalent to 1.7 pmol malonyl-CoA incorporated into fatty acid min-1 (mg protein)-1. This activity--if representative of living M. leprae organisms--is insufficient to enable them to synthesize their lipid requirements rapidly enough to support growth. The major activity for scavenging fatty acids in extracts of Mycobacterium microti and Mycobacterium avium, as well as in extracts of M. leprae, was acetyl-CoA-dependent fatty acyl-CoA 'elongase'. This activity was about four times higher in M. avium and M. microti grown in a medium which contained lipids, or when grown in mice, than in medium without added lipids. In contrast, the de novo fatty acid synthetase activity was repressed in M. avium and M. microti when grown in medium that contained lipids, or when grown in mice. These results are consistent with the hypothesis that mycobacteria grown in vivo preferentially scavenge lipids from the host cells, and suggest that a source of lipid should be included in media for attempted axenic isolation of M. leprae.     

Effects of irradiation on RNA synthesis primed by calf thymus deoxyribonucleoprotein treated with salt and salt-urea.

1. The effects of ionizing radiation on the activity of calf thymus templates were examined in a Escherichia coli RNA polymerase system. 2. The template activity of native and 2 M NaCl-5M urea-treated deoxyribonucleoproteins was enhanced by relatively low doses of irradiation, while that of 2 M NaCl-treated deoxyribonucleoprotein was not enhanced by irradiation. 3. The template activity of purified DNA was markedly decreased by irradiation, while that of native deoxyribonucleoprotein, 2 M NaCl-treated, and 2 M NaCl-5 M urea-treated ones were slightly decreased at a higher dose range. The doses for 50% inactivation of these templates were 1.3, 210, 140, and approximately 200 krad, respectively.