Photosynthetic Prokaryotes. Cell Differentiation and Functions : Edited by G.C. Papageorgiou and L. Packer Elsevier Biomedical; New York, 1983 405 pages. $65.00

A protein (designated as luffin) with an app. M_r of 26000, which inhibits protein synthesis in rabbit reticulocyte lysate, was purified to homogeneity from the seeds of Luffa cylindria roem by extraction with 20 mM Na phosphate buffer (pH 7.2) containing 0.2 M NaCl, ammonium sulfate fractionation, and chromatography on Sephacryl S-200 and CM-Sephadex C-25. Luffin exhibited 10-times as strong inhibitory activity against protein synthesis in rabbit reticulocyte lysate (IC₅₀, 0.42 ng/ml) as that of ricin A-chain, but it showed only a weak cytotoxicity against murine leukemia L1210 cells, an activity of 1/10⁶ to 1/10⁵ that of ricin.     

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na⁺ transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na⁺-K⁺-2Cl⁻ cotransporter, NKCC2.     

Bacterial lipopolysaccharides and mycoplasmal lipoglycans: A comparison between their abilities to induce macrophage-mediated tumor cell killing and Limulus amebocyte lysate clotting

The ability of various bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) to induce macrophage-mediated tumor cell killing and $\text{Limulus}$ amebocyte lysate clotting was determined. Lipoglycans from the mycoplasma $\text{Acholeplasma}\text{axantum}$ or $\text{Acholeplasma}\text{granularum}$ had no activity or 10⁴ to 10⁵ less activity than lipopolysaccharides from $\text{Escherichia}\text{coli}$ 0128:B12, $\text{Escherichia}\text{coli}$ K235, or $\text{Salmonella}\text{minnesota}$ R595 in causing $\text{Limulus}$ lysate clotting and tumor cell killing by peritoneal macrophages from normal or bacillus Calmette-Guérin-infected mice. Previous studies have shown that the lipid A portion of bacterial lipopolysaccharide is responsible for the effects on macrophage-mediated tumor cell killing and $\text{Limulus}$ lysate clotting. The known differences in the lipid structures of bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) may account for the noted differences in the biologic potencies observed here.     

Alterations in erythrocyte membrane fluidity and Na+/K+-ATPase activity in chronic alcoholics

Ethanol disorders biological membranes causing perturbations in the bilayer and also by altering the physicochemical properties of membrane lipids. But, chronic alcohol consumption also increases nitric oxide (NO) production. There was no systemic study was done related to alcohol-induced production of NO and consequent formation of peroxynitrite mediated changes in biophysical and biochemical properties, structure, composition, integrity and function of erythrocyte membranes in chronic alcoholics. Hence, keeping all these conditions in mind the present study was undertaken to investigate the role of over produced nitric oxide on red cell membrane physicochemical properties in chronic alcoholics. Human male volunteers aged 44 ± 6 years with similar dietary habits were divided into two groups, namely nonalcoholic controls and chronic alcoholics (~125 g of alcohol at least five times per week for the past 10–12 years). Elevated nitrite and nitrate levels in plasma and lysate, changes in erythrocyte membrane individual phospholipid composition, increased lipid peroxidation, protein carbonyls, cholesterol and phospholipids ratio (C/P ratio) and anisotropic value (γ) with decreased sulfhydryl groups and Na⁺/K⁺-ATPase activity in alcoholics was evident from this study. RBC lysate NO was positively correlated with C/P ratio (r = 0.547) and anisotropic (γ) value (r = 0.428), Na⁺/K⁺-ATPase activity was negatively correlated with RBC lysate NO (r = ?0.372) and anisotropic (γ) value (r = ?0.624) in alcoholics. Alcohol-induced overproduction of nitric oxide reacts with superoxide radicals to produce peroxynitrite, which appears to be responsible for changes in erythrocyte membrane lipids and the activity of Na⁺/K⁺-ATPase.     

Insect haemolymph: Cooperation between humoral and cellular factors in Locusta migratoria

In Locusta migratoria, prophenoloxidase is present in the plasma and serum, but in reduced amounts relative to the haemocytes. This phenoloxidase activity cannot be induced by either heating or freezing and thawing and it is lost by heating at 70°C for 30 min. Both lipopolysaccharides and laminarin can elicit the prophenoloxidase activating system. These elicitors of prophenoloxidase activation are active in haemocyte lysate and in serum but never induce any phenoloxidase activity in plasma. In haemocyte lysate, the activating system is not heat resistant, and heating at 56°C for 30 min prior to incubation with laminarin or lipopolysaccharide precludes any phenoloxidase activity. Plasma contains a strong inhibitor of the prophenoloxidase activating system but serum does not. This inhibitor does not affect the phenoloxidase enzyme itself. The possible role of the activating system in immune recognition and the strategies evolved by parasites or pathogens to escape being recognized by their host are discussed.     

Probing non-specific interactions of Ca2+-calmodulin in E. coli lysate

The biological environment in which a protein performs its function is a crowded milieu containing millions of molecules that can potentially lead to a great many transient, non-specific interactions. NMR spectroscopy is especially well suited to study these weak molecular contacts. Here, non-specific interactions between the Ca²⁺-bound form of calmodulin (CaM) and non-cognate proteins in Escherichia coli lysate are explored using Ile, Leu, Val and Met methyl probes. Changes in CaM methyl chemical shifts as a function of added E. coli lysate are measured to determine a minimum ‘average’ dissociation constant for interactions between Ca²⁺-CaM and E. coli lysate proteins. ²H R ₂ and ¹³C R ₁ spin relaxation rates report on the binding reaction as well. Our results further highlight the power of methyl containing side-chains for characterizing biomolecular interactions, even in complex in-cell like environments.     

High-performance liquid chromatographic assay for methyl-β-cyclodextrin in plasma and cell lysate

This paper describes a high-performance liquid chromatographic method with fluorescence detection for the analysis of methyl-β-cyclodextrin (MEBCD) in plasma and cell lysate, after in situ complexation with 1-naphthol. The size-exclusion HPLC column packed with TSK 3000 SW gel, was equilibrated with an eluent mixture composed of methanol and purified water (2:98, v/v) containing 10⁻⁴ M 1-naphthol as a fluorophore. The detection is based on fluorescence enhancement caused by the formation of inclusion complexes and was performed at 290 and 360 nm for excitation and emission, respectively. The method involved a simple treatment of the samples with chloroform. Daunorubicin was used as internal standard. Limits of quantitation were 0.8 μM in plasma and 0.5 μM in cell lysate. Detection limits of 0.5 μM (50 pmol) and 0.3 μM (30 pmol) were obtained for MEBCD in the two media, respectively. Linear detection response was obtained for concentrations ranging from 1 to 100 μM in plasma and cell lysate. Recovery from plasma proved to be more than 40%. Precision, expressed as C.V. was in the range of 4 to 11%. Accuracy ranged from 89 to 105%.     

An Environmental Escherichia albertii Strain,DM104, Induces Protective Immunity to Shigella dysenteriae in Guinea Pig Eye Model

The environmental Escherichia albertii strain DM104, which cross-reacts serologically with Shigella dysenteriae was assessed for pathogenic properties, immunogenicity, and protective efficacy in different animal models to evaluate it as a vaccine candidate against S. dysenteriae, which causes the severe disease, shigellosis. The DM104 isolate was found to be non-invasive and did not produce any entero- or cyto-toxins. The strain also showed negative results in the mouse lethal activity assay. The non-pathogenic DM104 strain gave, however, a high protective efficacy as an ocularly administered vaccine in the guinea pig eye model against S. dysenteriae type 4 challenge. It also induced a high titer of serum IgG against S. dysenteriae type 4 whole cell lysate and lipopolysaccharide. Taken together, all these results indicate a good potential for the use of the DM104 as a live vaccine candidate against shigellosis.     

Delayed Human Neutrophil Apoptosis by Trichomonas vaginalis Lysate

Neutrophils play an important role in the human immune system for protection against such microorganisms as a protozoan parasite, Trichomonas vaginalis; however, the precise role of neutrophils in the pathogenesis of trichomoniasis is still unknown. Moreover, it is thought that trichomonal lysates and excretory-secretory products (ESP), as well as live T. vaginalis, could possibly interact with neutrophils in local tissues, including areas of inflammation induced by T. vaginalis in humans. The aim of this study was to investigate the influence of T. vaginalis lysate on the fate of neutrophils. We found that T. vaginalis lysate inhibits apoptosis of human neutrophils as revealed by Giemsa stain. Less altered mitochondrial membrane potential (MMP) and surface CD16 receptor expression also supported the idea that neutrophil apoptosis is delayed after T. vaginalis lysate stimulation. In contrast, ESP stimulated-neutrophils were similar in apoptotic features of untreated neutrophils. Maintained caspase-3 and myeloid cell leukemia-1 (Mcl-1) in neutrophils co-cultured with trichomonad lysate suggest that an intrinsic mitochondrial pathway of apoptosis was involved in T. vaginalis lysate-induced delayed neutrophil apoptosis; this phenomenon may contribute to local inflammation in trichomoniasis.     

Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes

Using a citrate-EDTA buffer as an anticoagulant it was possible to isolate intact haemocytes from the insect, Blaberus craniifer, without causing extensive degranulation and subsequent clotting. A haemocyte lysate from this insect contained prophenoloxidase (proPO), which could be activated by β 1,3-glucans. The activation process was dependent upon Ca²⁺ ions and seemed to occur by a limited proteolysis, since several serine protease inhibitors such as soybean trypsin inhibitor, benzamidine and $\text{p-}\text{nitrophenyl}\text{-p′-}\text{guanidobenzoate}$ blocked convertion of proPO to the active enzyme. Treatment of proPO with urea or heat also caused proPO activation but probably without the intervention of serine proteases, since the protease inhibitors used failed to block the activation. Within the haemocyte lysate, several endopeptidases were present, which were enhanced in activity by prior treatment with β 1,3-glucans. These endopeptidases were inhibited in activity when the haemocyte lysate was incubated with benzamidine prior to the addition of β 1,3-glucan. This provides further indications that the activation of proPO involves a limited proteolytic attack. The active phenoloxidase enzyme became strongly bound to foreign surfaces and this phenomenon may assist in providing opsonic properties for the proPO cascade.     

Biosynthesis and amino terminal acetylation of cat hemoglobin B

Acetylation of the amino-terminal serine of the β chains of cat hemoglobin B (HbB) occurs during synthesis of hemoglobin in a mRNA-dependent protein synthesizing system from rabbit reticulocyte lysate in the presence of acetyl-CoA and cat reticulocyte mRNA. The process occurs after peptide chain growth of about 30 amino acid residues. When endogenous acetyl-CoA was removed from the rabbit reticulocyte lysate by pretreatment with oxalacetate and citrate synthase, nonacetylated HbB (HbB′) was synthesized. Thus, β^B globin chain synthesis goes to completion in the absence of acetylation even though the latter normally occurs during nascent chain growth. When HbB′ was incubated with acetyl-CoA in a rabbit reticulocyte lysate, hemoglobin with properties identical to those of HbB was produced. Thus, the selective amino terminal acetylation of β^B globin also occurs in the completed hemoglobin.     

A need for careful evaluation of endotoxin contents in acellular pertussis-based combination vaccines

Two batches each of diphtheria-tetanus-acellular pertussis vaccine (DTaP) and that combined with inactivated polio vaccine purchased from foreign markets were tested by mouse body weight decreasing (BWD) toxicity test and Limulus amaebocyte lysate (LAL) test. Three out of the four imported vaccine batches showed the levels of BWD toxicity even comparable to that of DT-whole cell pertussis vaccine. BWD toxicity test is based on endotoxin dose-dependent weight loss of mice and has been used for controlling endotoxin in DTaP. Although of the strong BWD toxicity of the imported vaccines, there was no marked difference in LAL test results between the imported vaccines and Japanese DTaP. However, one imported DTaP batch showed very strong interference with LAL activity of spiked lipopolysaccharide (LPS). The batch interfered not only with LAL activity but also with pyrogenicity and prostaglandin E₂ induction activity. However, the pyrogenicity of the spiked LPS could be recovered from the precipitated fraction of the batch by treating with phosphate buffer to suggest the possibility of recovering in vivo toxicity. As an adequate in vitro test method could not be identified for controlling the safety of the interfering batch, an appropriate in vivo test would be required for testing such vaccines.     

Antimicrobial defense mechanisms in the horseshoe crab,Limulus polyphemus: Effect of sodium chloride on bactericidal activity

Bactericidal activity of heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) was found to be inhibited by low molar (10 to 100 mm) concentrations of the monovalent cations Na⁺ and K⁺. The protective effect of Na⁺ was shown to be species dependent and inversely proportional to the sensitivity of the test microorganism as determined by bactericidal titer. The effect of Na⁺ (100 mm) on bactericidal activity in whole (unheated) LAL, when assayed against a sensitive species (Escherichia coli), was negligible and in direct contrast to data obtained when using 60°C extracts of LAL. Increasing the concentration of Na⁺ to reflect levels in L. polyphemus blood (400 to 500 mm) afforded only minimal protection for sensitive bacterial species. These data suggested that whole LAL may contain a heat-labile substance (protein?) capable of binding Na⁺, obviating the inhibitory effect of Na⁺ on bactericidal activity. Further evidence for the existence of this cation-binding substance was obtained when 55°C extracts of LAL were found refractory to Na⁺ concentrations which totally abolished bactericidal activity in 60°C extracts. The mechanism of Na⁺ inhibition of bactericidal activity and the role of the cation-binding substance in L. polyphemus host defense against bacterial invasion and dissemination is discussed.     

Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

Exploring the impact of nucleic acids on protein stability in bacterial cell lysate

- Addition of salt enhanced thermal stability of model substrate proteins by reducing electrostatic repulsion between protein molecules.- However, the opposite effect was observed with bacterial cell lysate, indicating that certain molecules within the lysate could enhance protein stability via electrostatic interactions.- Such molecules present in cell lysate were found to be nucleic acids known to have a potent anti-aggregation activity toward proteins involving electrostatic interactions.- Nucleic acids showed chaperone activity in physiological salt concentration within cells and in buffer or medium commonly used in experiments.- The chaperone activity of nucleic acids should be taken into account when performing various in vitro assays using cell lysate or samples containing nucleic acids.     

Kinetic differences between two interconvertible forms of fructose-1,6-bisphosphatase from Saccharomyces cerevisiae

Newly synthesized, [³⁵S]methionine-labeled cholesterol side-chain cleavage cytochrome P-450, 11β-hydroxylase cytochrome P-450, adrenodoxin, and adrenodoxin reductase were immunoisolated from radiolabeled bovine adrenocortical cells and from rabbit reticulocyte lysate translation systems programmed with bovine adrenocortical RNA. Cholesterol side-chain cleavage cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight of 54,500 whereas this cytochrome P-450 immunoisolated from radiolabeled bovine adrenocortical cells had an apparent molecular weight of 49,000, an apparent molecular weight identical to that of the purified protein. Similarly, newly synthesized, [³⁵S]methionine-labeled 11β-hydroxylase cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight 5500 daltons larger than that immunoisolated from radiolabeled adrenocortical cells (48,000) and the authentic cytochrome (48,000). The cell-free translation products of adrenodoxin and adrenodoxin reductase were also several thousand daltons larger than the corresponding mitochondrial proteins. The apparent molecular weight of adrenodoxin immunoisolated from a reticulocyte lysate translation system was 19,000, while that of the authentic protein was 12,000. Adrenodoxin reductase immunoisolated from a lysate translation system had an apparent molecular weight of 53,400; an apparent molecular weight 2300 daltons larger than that of adrenodoxin reductase immunoisolated from radiolabeled adrenocortical cells or purified by conventional techniques. These results demonstrate that all of the components of the mitochondrial steroid hydroxylase systems of the bovine adrenal cortex are synthesized as precursor molecules of higher molecular weight. Presumably, the precursor proteins are post-translationally converted to the mature enzymes upon insertion into the mitochondrion by a process which includes the proteolytic cleavage of the precursor segments.     

Identification of true thymidine kinase in Tetrahymena pyriformis ST.

Thymidine kinase is present in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis. Previous workers have been unable to find a specific thymidine kinase activity in this organism. The cytoplasm of Tetrahymena contained a thymidine phosphorylating activity which was ATP dependent, was stimulated by Mg²⁺, and was inhibited by dTTP. This activity was also partly inhibited by dCTP. Although the mitochondrial fraction also exhibited ATP-dependent phosphorylation, it is not stimulated by Mg²⁺ and not significantly inhibited by dTTP. Nucleoside phosphotransferase activity is detectable both in cytoplasmic and mitochondrial fractions, although it is not clear whether they represent separate enzymes. Nucleoside phosphotransferase activity is inhibited both by NaF and by ATP. Thymidine kinase and nucleoside phosphotransferase activities were separated by polyacrylamide gel electrophoresis, establishing the presence of both enzymes in this organism. Both crude mitochondrial lysate and postmitochondrial supernatant samples exhibited similar gel electrophoretic patterns for thymidine kinase and nucleoside phosphotransferase activities. The former, however, exhibited a relatively small peak of thymidine kinase migrating at the same rate as that of the postmitochondrial supernatant. A separate peak of thymidine kinase was not found in the mitochondria of Tetrahymena.     

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of gram-negative bacterua

In the presence of MgCl₂, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl₂ and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I, MgCl₂ and detergent were unable to either entrap or retain [¹⁴C]-sucrose, [³H]inulin or [³H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [³H]-inulin out of Re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl₂ to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occuring in their normal intestinal habitat.     

Transfer factor—Transfer of tuberculin cutaneous sensitivity in an allogeneic and xenogeneic monkey model

Tuberculin cutaneous sensitivity can be transferred from rhesus monkeys, sensitized to tuberculin using BCG, to rhesus and cynomolgus recipients with viable or disrupted leucocytes, and with a dialysed lysate preparation from 3 × 10⁸ leucocytes. Dialysable transfer factor (TF) using the lyophilized aqueous dialysate of a leucocyte lysate prepared by the freeze-thaw method did not give an active material. Modifications to the preparative method, in that leucocytes from fresh blood were disrupted gently by mechanical shearing forces, the lysate dialysed against a balanced salt solution and injected without lyophilization, yielded an active preparation. Transferred tuberculin (PPD) skin reactivity, confirmed by biopsy, was always less than the reactivity of the donor monkey and lasted for approximately 2–3 months. Although donor monkeys showed good in vitro lymphocyte transformation response to antigen stimulation, recipient monkeys which became skin test positive did not have a concomitant blastogenic response to PPD.     

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [³H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [³H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.