An adenosinetriphosphate-activated hemolytic system. II. Utilization of adenosinetriphosphate in the reaction

Failure to demonstrate ATP¹ utilization in an ATP-activated hemolytic system had been previously reported. In the present study ATP utilization is shown to be associated with the hemolytic reaction and also with the ATP-dependent inactivation of the hemolytic factor of the system by crude, washed, human red cell stroma. Using crude stroma, relatively large ATP utilization occurs and continues, but at a decreasing rate, after inactivation of the hemolytic factor is complete. With purified stroma there is very slight uptake of ATP by the stroma in the presence of hemolytic factor and Mg⁺⁺. This uptake can only be demonstrated by radioactive ATP. Both C¹⁴- and P³²—labelled nucleotide were used for this purpose. In the presence of an excess of stroma the uptake seems to be dependent on the amount of hemolytic factor used. Evidence is given contraindicating the possibility that this uptake is non-specific.     

Sequencing of the variant thyroxine-binding globulin (TBG)-San Diego reveals two nucleotide substitutions.

Thyroxine-binding globulin (TBG) is a liver glycoprotein that transports thyroid hormone in serum. In 1989, a variant TBG was reported with reduced binding affinity for thyroxine (T4) and triiodothyronine (T3) which results in low serum T4 and T3 levels. This variant, TBG-San Diego (TBG-SD), also displays reduced heat stability but has a normal isoelectric focusing pattern. We now report the sequence of the entire coding region of TBG-San Diego. It reveals two nucleotide substitutions: one located in exon 1 which results in the replacement of the normal Ser-23 (TCA) with threonine (ACA) and the other, located in exon 3, changes the normal codon 283 of TTG (leucine) with that of TTT, (phenylalanine). Allele specific amplification was used to search for both nucleotide substitutions in four affected members of the family. Results confirmed the co-segregation of these nucleotide substitutions with the TBG-SD phenotype. The substitution in codon 283 has been previously described and exists as a polymorphism in some ethnic groups or in combination with other TBG variants with different physical characteristics. Thus, it appears that the replacement of Ser-23 with threonine is responsible for the observed alterations in physical properties of TBG-San Diego.     

Molecular characterization of a Campylobacter jejuni lipoprotein with homology to periplasmic siderophore-binding proteins.

A genomic library of Campylobacter jejuni (NCTC 11351) was used to identify genes which could confer a hemolytic phenotype to Escherichia coli. Accordingly, when transformants were screened on blood plates, hemolytic colonies appeared at a frequency of 3 x 10(-4). The gene conferring the hemolytic activity was identified by subcloning and was found to be responsible for the phenotype of all hemolytic transformants isolated. The open reading frame conferring this activity encodes a protein of 36,244 Da with a typical endopeptidase type II leader sequence. The protein is modified with palmitic acid when it is processed in E. coli, confirming that it is a typical lipoprotein. The deduced gene product of 329 amino acids has significant homology to the group of solute binding proteins from periplasmic-binding-protein-dependent transport systems for ferric siderophores, including the FatB protein from Vibrio anguillarium and the FhuD protein from Bacillus subtilis. In particular, the protein contained the signature sequence for siderophore-binding proteins, suggesting that the protein may be the siderophore-binding protein component of an iron acquisition system of C. jejuni.     

Critical micelle concentration and hemolytic activity--a correlation suggested by the marine sterol, halistanol trisulfate.

The marine natural product, halistanol trisulfate, has a relatively low critical micelle concentration of 0.001% m/v (14.5 microM) and strong hemolytic potency with an EC50 of 0.00046% m/v (6.67 microM). As expected of a detergent, it inhibits the growth of gram-positive but not gram-negative bacteria. The hemolytic activity of halistanol trisulfate and other detergents has been shown to correlate with critical micelle concentration. This correlation may have important implications in the mechanism of membranolytic bioactivity.     

Hemolytic Interaction of Newcastle Disease Virus and Chicken Erythrocytes. II. Determining Factors

Standard procedures have been described for measuring paramyxovirus-induced hemolysis. The choice of these procedures is based on the analysis of the behavior of eight different strains of Newcastle disease virus. Significant strain-specific differences in hemolytic activity have been found. The presence of at least two kinds of inhibitors of hemolysis in virus preparations necessitates the use of purified virus when comparisons of hemolytic activities are to be made. In addition, it has been stressed that both hemagglutination titers and hemolysis determinations provide only relative values. Thus, quantitative comparisons can be made only with results obtained on the same day and with the same erythrocyte preparation.     

Barriers to prescribing the Copper T 380A intrauterine device by physicians.

From a questionnaire sent to all obstetricians and gynecologists and all family and general practitioners in San Diego County, California, regarding the Copper T 380A intrauterine device, substantial barriers to prescribing it were identified. Of all physicians responding, 40% reported that they were not recommending the Copper T 380A to anyone, the single most common reason given being concern about medical liability. A lack of knowledge about the new device, a lack of intrauterine device insertion skills, and certain medical practice settings were also important barriers to prescribing it. The new intrauterine device is considered in the context of innovation-diffusion theory. Substantial amounts of education and training and improvement in the medical-legal climate are needed before current barriers to prescribing the new device are removed.     

Acinetobacter sp. HM746599 isolated from leatherback turtle blood

A newly described bacterial isolate, Acinetobacter sp. HM746599, has been obtained from leatherback sea turtle hatchling blood. The implication is that the hatchling was infected during development in the egg, which is substantiated by other studies to be reported by us in the future. The 16S rRNA gene sequence of the bacterium (GenBank accession number: HM746599) showed the greatest similarity to the identified species, Acinetobacter beijerinckii (97.6-99.78%) and Acinetobacter venetianus (99.78%). Acinetobacter sp. HM746599 are gram-negative, rod-shaped coccobacilli and are hemolytic/cytotoxic to human and sea turtle red blood cells (RBCs). Hemolysis is not the result of any detectable soluble toxin. Acinetobacter beijerinckii and A. venetianus hemolyze sheep RBCs while Acinetobacter sp. HM746599 does not, and unlike A. venetianus, the growth of Acinetobacter sp. HM746599 and A. beijerinckii is not supported by l-arginine. Many Acinetobacter species, especially hemolytic ones, are pathogenic to immunologically compromised humans and it is possible that, in addition to sea turtles, this bacterium might also be a danger to susceptible humans who handle infected hatchlings. The bacteria are available from CCUG (Culture Collection, University Gothenburg, G?teborg, Sweden) and from NRRL (Agricultural Research Service Culture Collection, Peoria, IL).     

Molecular Cloning and Expression of Mn2+-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium, Pseudomonas sp. Strain TK4

We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn(2+). The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.     

Iron Deficiency Anemia in Two Prehistoric American Indian Skeletons: a Dietary Hypothesis

Abstract

Two prehistoric adult skeletons exhibiting pathological bone changes resembling those of hemolytic anemia were examined to determine the nature of these changes. Our findings show that abnormal hemoglobin variants responsible for these disorders do not exist in modem day American Indian natives. On this basis, it is very unlikely that their predecessors possessed the genes for these variants. A condition known as porotic hyporostosis similarto those bony changes produced by hemolytic disorders occur in high incidence in neighboring Anasazi people, andit has been found that the condition results from a lack of iron and protein in their diet. Since the Evans Mound people (reported on here) subsisted on a similar diet, it is suggested that these pathological changes are also the result of a dietary deficiency     

Studies on the terminal stages of antibody-complement-mediated killing of a tumor cell. I. Evidence for the existence of an intermediate, T.

The mechanism of the terminal steps in the lysis of antibody-sensitized tumor cells by complement (TAC) was studied. It was shown that once complement has reacted, lysis proceeded even in the absence of fluid-phase complement. Transformation of TAC to dead cells was found to be at least a two-step process: one of the steps was temperature dependent whereas the other was reversibly inhibited by EDTA. In analogy to the hemolytic system, TAC has been designated T.     

A parallel workload model and its implications for processor allocation

We develop a workload model based on the observed behavior of parallel computers at the San Diego Supercomputer Center and the Cornell Theory Center. This model gives us insight into the performance of strategies for scheduling moldable jobs on space-sharing parallel computers. We find that Adaptive Static Partitioning (ASP), which has been reported to work well for other workloads, does not perform as well as strategies that adapt better to system load. The best of the strategies we consider is one that explicitly reduces allocations when load is high (a variation of Sevcik's (1989) A+ strategy). This revised version was published online in July 2006 with corrections to the Cover Date.     

A UNIQUE COLONIAL MARINE CENTRIC DIATOM,COENOBIODISCUS MURIFORMIS GEN. ET SP. NOV.1

A new colonial centric marine diatom, Coenobio-discus muriformis gen. et sp. nov. (family Coscinodiscaceae) has been found in San Diego Bay, San Diego, California. The colony is composed of a single layer of 200-530 cells linked girdle to girdle by a compartmentalized matrix. The colony has the shape of a slightly concavo-convex disk approximately 400 μ. in diameter. Each cell is about 10 μ, in diameter and the valves have many structural features in common with those of Thalassiosira. The organic matrix contains 6-11 partitions that radiate from each cell, forming a number of extracellular chambers. The organic matrix does not contain chitin or cellulose as a major constituent. Histochemical similarities are found with the capsular and tubular material of previously described diatoms. The colonies reproduce without passing through a unicellular vegetative stage.     

Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains.

Identification of 12 strains originally characterized as nonpathogenic Listeria monocytogenes was reassured following the evaluation of their hemolytic capability with a newly developed horse blood agar plate. Seven of the strains were observed consistently to be hemolytic and confirmed as L. monocytogenes with the use of two commercial systems: the Gene-Trak L. monocytogenes-specific colorimetric DNA hybridization assay and the API Listeria system. Except for one strain that formed typical smooth colonies, these hemolytic strains formed rough colonies on a selective medium, lithium chloride-ceftazidime agar. The rest of the strains were nonhemolytic and did not hybridize with the DNA probe; they were identified as Listeria innocua on the basis of their API Listeria system biochemical profile. All but one of these nonhemolytic strains formed smooth colonies on lithium chloride-ceftazidime agar.     

Studies on the toxin of Aspergillus fumigatus. VII. Purification and some properities of hemolytic toxin (asp-hemolysin) from culture filtrates and mycelia.

A hemolytic toxin has been obtained from mycelia and culture filtrates of Aspergillus fumigatus by the procedures that included precipitation with ammonium sulfate, chromatography of DEAE-Sephadex, affinity chromatography on Concanavalin A-Sepharose and gell filtration on Sephadex G-50, G-100 AND G-150. The purified homolytic toxin was homogeneous on immunological and disk electrophoretic analysis, and the toxin from culture filtrates was identical with that from mycelia by the immunodiffusion technique. The hemolytic toxin was obtained for the first time from fungi and designated as Asp-hemolysin. The molecular weight of Asp-hemolysin was estimated to be appoximately 30,000 by the gel-filtration technique and its isoelectric point was found to be around pH 4.0. This Asp-hemolysin contained large amounts of protein and very small amounts of carbohydrate. The UV absorption spectrum of Asp-hemolysin showed a maximum absorption at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm, E 1% 1CM, was 12.4 and the ratio of absorbance at 280 nm to that at 260 nm was 2.3. The optimum pH for the hemolytic activity of the toxin toward chicken erythrocytes was 5.0 at room temperature and it was active in the pH range of 3.5 to 10.5. The optimum temperature was 21 C and about 50% of the activity was lost by incubation at 50 C for 5 min or 45 C for 23 min. The hemolytic activity was remarkably inhibited by Hg2+, Cu2+, Fe2+, Ag1+, iodine and p-CMB, but enhanced slightly by Zn2+ and Co2+.     

Genetic markers and malaria. Observations in Gujarat, India

189 healthy controls and 175 patients suffering from malaria vivax have been investigated with regard to associations between this disease and 22 genetic polymorphisms of the blood (ABO, MN, Ss, Rh, Kell, P, Lutheran, Kidd, Duffy, Diego, Xg; ABH-Secretor; Hp, Gc, Gm, Km; aP, AK, PGM1, 6-PGD, EsD; Hb variants) Significant associations could be demonstrated only for P and Hp systems, though in accordance with other investigations it cannot be excluded that the ABO system plays also a role in this connection.     

A festival of cell-cycle controls.

The second biennial Salk Cell Cycle meeting convened on 22 June 2001 in San Diego, California. Organized by Tony Hunter and Susan Forsburg of the Salk Institute, the five-day conference was highlighted by enlightening science and plenty of San Diego sunshine. Presentations covered a broad range of contemporary cell-cycle topics, ranging from regulation of DNA replication and mitosis to DNA damage recognition and checkpoint control.     

Simultaneous monitoring of pituitary hormone secretion and gene expression within individual cells.

We have recently developed a method to simultaneously quantitate the level of gene expression and the level of secretion of a peptide from individual cells. Our approach has been to combine the reverse hemolytic plaque assay sequentially with in situ hybridization. We present data to show how we have used the pituitary lactotroph as a model to demonstrate the power of this technique. However, we are particularly excited about the potential application of this strategy to approach a broad spectrum of questions regarding the cellular and molecular mechanisms that regulate the coupling of peptide secretion and gene expression at the single cell level. The method can be used in any system in which an appropriate antibody for the reverse hemolytic plaque assay and probes complementary to the mRNA of interest are available.     

A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage.

A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular level. Nucleotide sequence determination revealed the presence of two open reading frames. Both open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.     

Cryptococcosis in columbiformes at the San Diego Zoo.

Two cases of cryptococcosis in columbiformes exhibited at the San Diego Zoo are described. The organism isolated from the first case had morphological, chemical and temperature growth characteristics of C. neoformans. The culture from case 2 died before it could be examined biochemically or by mouse inoculation.     

Activation of therminal components of human complement by a trypsin-activated complex of human factor B and cobra venom factor.

Cleavage of C3 by CVF-B was demonstrated by hemolytic, immunoelectrophoretic and immune adherence reactions. No cleavage of C5 was detected by immunoelectrophoresis, but C5 hemolytic activity, assayed with EAC1423, decreased although less than C3 hemolytic activity. The co-existence of C3 with limiting amounts of C5 did not reduce the final degree of hemolysis of guinea pig erythrocytes (GPE) induced by late-acting components C6 through C9 and CVF-B. Thus, a CVF-B hemolytic system composed of GPE, C5 through C9 and CVF-B provided a method for titration of terminal components of human complement. CVF-B was able to generate hemolytically active sites of C567 on GPE by activation of C5, C6 and C7. The complex C567 in the fluid-phase decayed within 1 min but C567 on GPE was quite stable. Originally insensitive sheep erythrocytes became sensitive to the CVF-B hemolytic system if C3b sites were present, suggesting that cell-bound C3b played a role in orienting the positions of C567 to be fixed. CVF-B could be recovered quantitatively from the supernatant of the reaction mixture in which the hemolytically active intermediate GPEC-5678 had been formed through the interaction between C5 to C8 and CVF-B.