Isolation and characteristics of staphylococcal alpha-toxin

A method for isolation of staphylococcal alpha-toxin preparations has been elaborated. Characteristics of the toxin isolated by the method are as follows: mol. mass = 35 Kd; HU = 0.1 microgram; DnD= 0.1 microgram; LD50 = 2 micrograms. It is for the first time that alpha-toxin was fragmented by papain and digested by alpha, gamma-chemotrypsin. The papain fragments (18.5 and 15 Kd) retained lethal activity but lost hemolytic and dermonecrotic activities. Alpha, gamma-chemotryptic digested fragments (18 and 15 Kd) retained hemolytic and lethal effects, but lost their dermonecrotic activity.     

Purification and characterization of the parvalbumin from monkey skeletal muscle

1. A high affinity Ca2+ binding and low mol. wt protein, parvalbumin, was purified from monkey skeletal muscle. 2. As compared with other animals, only one component and a lower content of monkey parvalbumin were found. 3. This may suggest that both the component and the content of parvalbumin decreases with biological evolution. 4. The parvalbumin was found to have a mol. wt of 11,400, a pI of 5.1, a high aspartic acid and lysine content, maximum absorption at around 260 nm, a blocked amino-terminal, an immunological distinction, 2 mol Ca2+ binding/mol, and a conformational change by Ca2+ binding. 5. Parvalbumin was shown to have alpha type properties.     

Chemical and Biological Properties of Lipopolysaccharide from a Marine Bacterium,Photobacterium phosphoreum PJ-1

The chemical and biological properties of the lipopolysaccharide (LPS) isolated from a marine bacterium, Photobacterium phosphoreum PJ-1, were studied. This LPS consists of 40.6% carbohydrate, 27.3% fatty acid, 0.2% 2-keto-3-deoxyoctonate (KDO) and other components. One characteristic of this LPS is its small amount of KDO, the basic component of the usual LPS. Electrophoresis in sodium dodecylsulfate polyacrylamide gel revealed at least two staining bands for carbohydrates. These bands were continuous and broad, and showed rapid electrophoretic mobility which corresponded closely to the fastest moving band of LPS from Salmonella typhimurium. This LPS preparation had adjuvant activity, lethality for ddY mice, and the ability to gel Limulus amebocyte lysate, and the strength of these activities corresponded closely to those of LPS preparations from Escherichia coli 0111:B4 and S. typhimurium. In the test for lethality of the LPS for ddY mice, the lethal action appeared in two phases depending on the dose used for intravenous (i.v.) injection : the early lethal action appeared within 30 min after injection of 250 μg or less, and the late lethal action occurred gradually after 16 hr at doses of 500 μg or more. The total (both phases) LD50 of this LPS (i.v.) for ddY mice was 265 μg per mouse and in only the late phase it was 500 μg. These results show that in spite of structual differences in regard to KDO content, LPS from P. phosphoreum PJ-1 has some biological properties similar to those of LPS from E. coli 0111:B4 and S. typhimurium but it shows no immunological cross-reaction with other LPS.     

Modulation of the phototoxic effect of hypericin in human leukemia CEM cell line by N-ethylmaleimide,amiloride and omeprazole

Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1 x 10(-5) and 1 x 10(-6) mol x l(-1) was significantly potentiated by NEM (1 x 10(-7)-1 x 10(--9) mol x l(-1)) and DMA (1 x 10(-6) and 1 x 10(-7) mol x l(-1)) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin.     

Inositol 1,4,5-trisphosphate- and arachidonic acid-induced calcium mobilization in T and B lymphocytes

Inositol triphosphate (IP3) formation and increase in intracytoplasmic calcium are mediators of signal transduction in lymphocytes. It has been proposed that IP3 induces Ca2+ release from intracellular stores. It is in order to study the relationship between these two events that we have analyzed the effect of IP3 addition on Ca2+ mobilization in permeabilized resting T and B lymphocytes, EBV-B lymphocytes, and HTLV1-T lymphocytes. IP3 induces a rapid and significant release of Ca2+ from the endoplasmic reticulum in a dose-dependent manner. Ca2+ release is more sensitive to IP3 addition in cycling cells (EBV-B lymphocytes and HTLV1-T lymphocytes) than in resting T and B lymphocytes. Arachidonic acid (AA) induces Ca2+ release from the endoplasmic reticulum (ER) in a manner similar to that of IP3. Neither component has an effect on Ca2+ accumulated in mitochondria, and they have no additive effects suggesting that they act on a similar Ca2+ pool. These results directly demonstrate that in T and B human lymphocytes IP3 mobilizes Ca2+ from ER as in other cellular systems and that other potential second messengers, namely AA, could play a significant role in the internal mobilization of calcium during T and B lymphocyte activation.     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶还原酶组分的纯化和性质

Ｍｅｔｙｌｏｍｏｎａｓｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析、ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌＨＰＬＣ分离纯化出ＭＭＯ还原酶组分．经ＨＰＬＣ分析,纯度大于９５％,纯化倍数为４．４,加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白．ＳＤＳ－ＰＡＧＥ电泳表明还原酶由一种亚基组成,分子量４２ｋＤ．ＩＣＰ－ＡＥＳ测定还原酶的Ｆｅ含量为１．８３ｍｏｌＦｅ每ｍｏｌ蛋白．ＵＶ－Ｖｉｓ光谱表明还原酶除２８０ｎｍ蛋白质特征峰外在４６０ｎｍ有最大吸收峰,且Ａ２８０ｎｍ／Ａ４６０ｎｍ为２．５０,与其它黄素一铁硫蛋白相似,推测还原酶可能含一个ＦＡＤ辅基和Ｆｅ２Ｓ２中心．在厌氧条件下,还原酶能够和ＮＡＤＨ作用,ＵＶ－Ｖｉｓ光谱分析表明还原酶４６０ｎｍ处特征吸收峰消失,说明在ＭＭＯ催化过程中还原酶接受ＮＡＤＨ的电子．ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析分离出调节蛋白Ｂ,部分纯化的调节蛋白Ｂ的分子量大约在２０ｋＤ,它能够提高ＭＭＯ比活性４０倍,ＭＭＯ还原酶和调节蛋白Ｂ单独存在时不具有ＭＭＯ     

Photosensitizing effect of 8-methoxypsoralen on bacteriophage cd

Mutagenesis in extracellular phage sd by 8-metoxypsoralen (8-MOP) and longwave (lambda greater than 310 nm) UV-irradiation has been established. The kinetics of lethal and mutagenic effects of 8-MOP+light was studied. The efficiency of mutagenesis on the first linear part of mutation curve was 0.3% per the lethal hit which is 2 times lower than that of shortwave (lambda=254 nm) UV-irradiation. The maximum yield of mutants makes up 1%, after which the mutation curve is maintained. It has been established that the main (may be the only) contribution into mutagenesis is made by monoadducts, whereas the lethal effect is conditioned by diadducts (cross-links). The comparison of the efficiency of mutagenic effects of 8-MOP+light with mutagenic effects of other kinds of irradiations was carried out. The possibility of repair of damaged 8-MOP+light phage sd DNA by transfection of Escherichia coli C (uvr+) and Cs (uvr-) lysozyme spheroplasts has been established. The repair mechanism of photodamage in intact phage sd induced with 8-MOP+light was also investigated using the method of two-step irradiation. It has been shown that 65% of photodamages are repaired in E. coli SK cells in the M9 medium, i. e. under cellular metabolism. The recovery of phage sd is completely inhibited in phosphate buffer. Unlike chloramphenicol (150 microgram/ml), 1% caffeine blocks the phage recovery only by 30%. The participation of phage sd determining enzymes in its intracellular recovery from 8-MOP+light damages is assumed.     

Role of T cell subsets in lethal graft-versus-host disease (GVHD) directed to class I versus class II H-2 differences. II. Protective effects of L3T4+ cells in anti-class II GVHD

Detailed information was sought on the capacity of purified B6 L3T4+ cells to elicit lethal graft-versus-host disease (GVHD) in irradiated class II-different class I-identical (C57BL/6 (B6) x bm 12)F1 hosts. When B6 L3T4+ cells were transferred in small doses (10(5) to 10(6) together with donor bone marrow (BM) cells, the recipients all developed acute lethal GVHD and most of the mice died within 2 wk, probably from gut damage; this syndrome was conspicuous only in mice treated with very heavy irradiation, i.e., 1000 rad. In marked contrast to L3T4+ cells given in small doses, transfer of large doses of B6 L3T4+ cells to heavily irradiated (B6 x bm 12)F1 hosts paradoxically resulted in only limited mortality: most of the recipients survived for greater than 6 mo and manifested little or no evidence of ill health. It is suggested that the capacity of large doses of L3T4+ cells to protect mice against lethal GVHD is a reflection of T helper function: the cellular immunity provided by the donor L3T4+ cells enables the host to repel pathogens entering through damaged mucosal surfaces, with the result that GVHD becomes sublethal. The protective function of L3T4+ cells in the B6----bm 12 combination was only seen in hosts given donor BM. With transfer of donor L3T4+ cells plus host BM, even lightly irradiated recipients died rapidly from hemopoietic failure. Because this syndrome failed to occur in mice given a mixture of donor and host BM, it would appear that L3T4+ cells destroyed host lymphohemopoietic cells by direct cytotoxicity rather than via a bystander effect.     

Purification and characterization of an (Na+ + K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain

Highly purified lamb kidney (Na+ + K+)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na+ + K+)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (gamma 1 and gamma 2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that gamma 1 is an aggregated form of gamma 2. The total yield was 0.8-1.5 mol of gamma component per mol of catalytic subunit recovered. This indicates that the gamma component is present in stoichiometric amounts in the Na+ + K+)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.     

Identification and catalytic properties of Mg2+-dependent ATP-hydrolase of cytoplasmic membrane of Bacillus sp. B4253 capable of gold accumulation

Ca2+-independent, Mg2+-dependent ATP-hydrolase fermentative activity consisting of two components--azide-sensitive and azide-resistant ones has been identified in cytoplasmic membrane of Bacillus sp. B4253 capable to gold accumulation in ionic and colloid forms. The authors have characterized properties of the azide-resistant component of ATP-hydrolase reaction: dynamics of accumulation of one of the reaction products--inorganic phosphate P(i); dependence of ATP hydrolysis rate on the membrane protein content; pH-dependence; sensitivity of ATP-hydrolase activity to the change of reagents (ATP, Mg2+) concentration, as well as to the effect of some specific and nonspecific inhibitors of ion-transporting Mg2+-dependent ATP-hydrolase systems (ouabain, tapsigargin, eocine Y, La ions). It is supposed that the obtained experimental data can be used for the following study of molecular and membrane mechanisms of gold accumulation in Bacillus sp. B4253.     

Proteome analysis on lethal effect of <Emphasis Type="Italic">l</Emphasis><Subscript>2</Subscript> in the sex-linked balanced lethal strains of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

The sex-linked balanced lethal (SLBL) strains of silkworm serve as an effective system for sex-control in silkworm. To gain comprehensive insight into the effect of one sex-linked balanced lethal gene l ₂, comparative proteomic analysis was carried out between the survival embryos ( W ^{+ l₁} Z^{l₁ + l₂} )\left( {W^{ + l_1 } Z^{l_1 + l_2 } } \right) and lethal embryos ( W ^{+ l₁} Z ^{+ l₁ l₂} )\left( {W^{ + l_1 } Z^{ + l_1 l_2 } } \right) before the lethal stage. The lethal stage of l ₂ was confirmed by observing the typical dead embryo morphology. The two genotype embryos before lethal stage were distinguished using polymorphic simple sequence repeats (SSR) markers closely linked to l ₂ on the sex chromosome. Finally, 11 differentially expressed protein spots were successfully identified by MALDI-TOF/TOF mass spectrometry (MS). Among them, only 1 protein identified as heat shock protein 20.4 (HSP20.4) was up-regulated in the lethal embryos, while the other 10 were down-regulated. The up-regulation of HSP20.4 suggests that there may be abnormal polypeptides produced in the lethal embryos. The gene ontology (GO) annotation indicated those down-regulated proteins are involved in important biological processes including embryo development, nucleoside metabolism, tRNA splicing, translation and protein folding. The biological pathway analysis showed that those down-regulated proteins are mainly involved in spindle assemblage and morphogenesis. Based on our results, we suggest that the l ₂ may be the mutant expressing abnormal polypeptides. Its expression has a negative effect on mitosis and morphogenesis processes. The death of the embryos may be caused by the accumulation of abnormal polypeptides and the handicap of cell proliferation and morphogenesis.     

Failure of leukotriene B4 to translocate calcium across phosphatidylcholine membranes

It has been reported that leukotriene B4 can translocate calcium across model membranes (Serhan et. al., (1982) J. Biol. Chem., 257: 4746). Such ionophoretic behavior could account for its biological effects. We have examined the effect of chromatographically pure leukotriene B4 on Ca2+ permeability when added exogenously at 3 microM to phosphatidylcholine liposomes and when incorporated at 5 mole % in the lipid mixture used to prepare liposomes. No effect was observed with either procedure. An oxidized preparation of leukotriene B4 stimulated calcium permeability, however, suggesting that oxidation may account for the previously reported ionophoretic behavior of leukotriene B4.     

一株嗜热毛壳菌β-葡萄糖苷酶的分离纯化及特性

研究了液体发酵嗜热毛壳菌Chaetomium thermophile产生的β-葡萄糖苷酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose 疏水层析、Sephacryl S-100分子筛层析等步骤后获得凝胶电泳均一的β-葡萄糖苷酶。经12.5%SDS-PAGE和凝胶过滤层析方法分别测得该酶的分子量大小约为78.4kDa和81kDa。该酶反应的最适温度和pH值分别为60℃和4.5-5.0。有较好的酸稳定性和热稳定性。金属离子对β-葡萄糖苷酶的活性影响较大, 其中Ca2+对酶有激活作用, 而Ag+、Cu2+ 、Hg2+对酶有显著的抑制作用。该酶对水杨苷具有很强的底物特异性。     

Effect of reproductive compensation and the desire to have male offspring on the incidence of a sex-linked lethal disease.

The effects of reproductive compensation on an X-linked recessive lethal are examined. Complete compensation without regard to the sex of the offspring increases the incidence of female carriers by a factor of 1.5, and of affected males by 1.33. However, if families reproduce until they have a healthy male offspring, the incidence of the X-linked lethal can be increased two or three orders of magnitude. Even only 1% of the population reproducing until a male is born can inflate the incidence of the disease by an order of magnitude, provided this pattern of family planning is culturally inherited. Similarly, reproducing until there is at least one child of each sex increases the incidence of an X-linked lethal. The impact of these types of sex-biased family planning on the fraction of new mutants among affect males is discussed.     

Induction and suppression of delayed-type hypersensitivity to non-MHC antigens during lethal graft-versus-host reaction.

A delayed-type hypersensitivity reaction (DTH) to non-MHC Ag was detected during a lethal graft-vs-host reaction (GVH) induced by incompatibility for non-MHC Ag alone. In this model, when appropriate doses of B10.D2 bone marrow and lymphoid cells are grafted to irradiated (DBA/2 x B10.D2)F1 recipients, the ensuing GVH, directed against those DBA/2 non-MHC Ag absent from the B10.D2 background, results in virtually 100% mortality in less than 3 mo; donor alloimmunization against the host histocompatibility Ag considerably reduces mortality, and survival rates of 80 to 100% are common. The experiments reported here show that: 1) the cell responsible for DTH induction expresses the CD4+ CD8- phenotype; 2) CD4+ cells likewise seem to play a predominant role in the pathology of lethal GVH in this genetic combination; 3) the alloimmunization protocol that abrogates mortality also abolishes GVH-associated DTH; and 4) this suppressive effect, as shown elsewhere for the protection against mortality, is mediated by CD4- CD8- "double negative" Thy-1+ CD3+ T suppressor cells. Thus, there is a good parallel between lethal GVH and its associated DTH as concerns both induction and suppression of the two phenomena, suggesting that mortality and DTH may represent different manifestations of a common underlying mechanism. Initiation of the effector phase of DTH in the adoptive transfer model seems to be dependent on the presence of a Thy-1+ double-negative cell in the transfer inoculum; the possible relationship of this double-negative cell to the Th-1-type CD4+ DTH-mediating cell recently shown to induce lethal GVH is discussed.     

Evaluation of colicins for inhibitory activity against diarrheagenic Escherichia coli strains, including serotype O157:H7.

Twenty-four Escherichia coli strains producing standard colicins were evaluated for inhibitory activity against 27 diarrheagenic E. coli strains of serotypes O15:H-, O26:(H11, H-), and O111:(H8, H11, H-), including O157:H7, representing diarrheagenic E. coli clones, 3, 4, 8, 9, and 10. Overlay techniques were used to assess inhibition on Luria agar and Luria agar supplemented with 0.25 micrograms of mitomycin C per ml to induce colicin production. As a group, the A colicins (Col) E1 to E8, K, and N inhibited 23 to 25 (85.2 to 92.6%) of the 27 diarrheagenic strains on mitomycin C-containing agar, whereas the most active group B colicins, Col D and Ia, inhibited 9 and 12 (33.3 and 44.4%), of the diarrheagenic strains, respectively. Col G and H and Mcc B17 inhibited 22 to 27 (81.5 to 100%) of the diarrheagenic strains on Luria agar but were suppressed on mitomycin C-containing agar medium. All O157:H7 strains evaluated were sensitive to Col E1 to E8, K, and N on mitomycin C-containing agar and to Col G and H and Mcc B17 on Luria agar. Sensitivity to colicins of the selected set of diarrheagenic strains was in the order diarrheagenic E. coli clone 9 > 4 > 3 > 10 > 8 and was not restricted to strains of a single clone or serotype. Strain 8C from clone 8 was resistant to most test colicins. There is potential for using colicins in foods and agriculture to inhibit sensitive diarrheagenic E. coli strains, including serotype O157:H7.     

Purification and characterization of a cytosolic protein-tyrosine kinase from porcine spleen

A cytosolic protein-tyrosine kinase has been highly purified from porcine spleen using [Val5]angiotensin II as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, Sephacryl S-200, casein-Sepharose 4B, heparin-Sepharose CL-6B and anti-(4-aminobenzyl phosphonic acid)--Sepharose 4B. Analysis of the most highly purified preparation by SDS/PAGE revealed a major silver-stained band of molecular mass 40 kDa. The 40-kDa cytosolic protein-tyrosine kinase was purified approximately 10,000-fold with an overall yield of about 7%. It had autophosphorylation activity which was carried out by intramolecular catalysis. The stoichiometry of phosphate incorporation was about 1 mol phosphate/mol enzyme. In the autophosphorylation reaction, the apparent Km value for ATP was relatively low, 0.35 microM; Mn2+ was slightly preferred to Mg2+ as divalent cation. [Val5]Angiotensin II phosphorylation activity of the 40-kDa kinase increased with the amount of phosphate incorporated into the enzyme. A phosphate exchange reaction was observed during the autophosphorylation. These results suggest that the 40-kDa kinase described here is a different type of protein-tyrosine kinase than the enzymes so far reported.     

Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry

The human parechovirus 1 RGD motif in VP1 was studied by mutagenesis. An RGD-to-RGE change gave only revertant viruses with a restored RGD, while deletion of GD was lethal and nonrevertable. Mutations at the +1 and +2 positions had some effect on growth properties and a +1 M-to-P change was lethal. These studies indicate that the RGD motif plays a critical role in infectivity, presumably by interacting with integrins, and that downstream amino acids can have an influence on function.     

Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus.

The genome of an isolate of tomato yellow leaf curl virus from Sardinia, Italy (TYLCV-S), a geminivirus transmitted by the whitefly Bemisia tabaci, has been cloned and sequenced. The single circular DNA molecule comprises 2770 nucleotides. Genome organisation closely resembles that of the DNA A component of the whitefly-transmitted geminiviruses with a bipartite genome. A 1.8 mer of the TYLCV-S genome in a binary vector of Agrobacterium tumefaciens is infectious upon agroinoculation of tomato plants. Typical tomato yellow leaf curl disease symptoms developed about three weeks after inoculation. The disease was transmitted by the natural vector B.tabaci from agroinfected plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-S consists of only one circular single-stranded DNA molecule. Contrary to the other whitefly-transmitted geminiviruses described so far, there is no evidence for the existence nor the necessity of a second component (B DNA) in the TYLCV-S genome.     

Determination of nystatin component composition using HPLC and TLC with densitometry]

The component composition of nystatin produced by an improved strain of Streptomyces noursei was determined by HPLC on Milichrom chromatograph (USSR). It was shown that the antibiotic consisted of nystatins A1, A2, A3 and B and admixture substances. The data appeared to be in good agreement with the results of the complex TLC investigation, by using densitometry. The component composition of the samples was evidenced by SIEAP mass spectrometry. Physiochemical and biological characteristics of separate components are presented.