Genetic evidence for a product of the Fv-1 locus that transfers resistance to mouse leukemia viruses.

Extracts of mouse cells have been shown to transfer to N- or B-trophic host range types of mouse leukemia viruses. The genetic specificity of the inhibition was tested in two ways: (i) by correlating the Fv-1 genotype of a number of mouse strains with the restriction-transferring activity of extracts of the respective embryo cell cultures, and (ii) by correlating the Fv-1 genotype of BLC3F2 (C57BL/6 female [Fv-1bb] by C3H male [Fv-1nn] parental strains) mouse embryos, which segregate the Fv-1 alleles in a 12:1 ratio, with the inhibitor activity of extracts of the cells from each embryo. Five independent matings, totaling 45 individual embryos, were tested. Each embryo was cultured, and the Fv-1 genotype was determined independently by titration of N- and B-tropic viruses; the extracts of replicate secondary cultures were tested for their effect on infection of permissive cells by N- and B-tropic viruses. The specific-restriction-transferring activity of the embryos was found to segregate with the appropriate Fv-1 genotype. These res-lts confirm the suggestion that the inhibitor of the leukemia virus host range types in the cellular extracts is a product of the Fv-1 locus.     

A rapid, colourimetric assay for cytotoxin activity in Campylobacter jejuni

Abstract Cell extracts and culture supernates of Campylobacter jejuni NCTC 11168 and three isolates from faecal samples from patients with enteritis were tested for cytotoxic activity on HeLa and Vero cells using a sensitive and rapid dye reduction assay which represents a simple assay for cytotoxin activity that can be assessed visually or spectrophotometrically in the wells of microplates. The assay was as sensitive as trypan blue exclusion and did not require the use of radioisotopes. A low level of cytotoxin activity, compared to that produced by a control verotoxin 2-producing Escherichia coli strain, was detected in cell extracts of all four strains, but no activity was detected in culture supernates. Production of an enterotoxin was evaluated by reverse passive latex agglutination with anti-cholera toxin antibody, a procedure which also represents a rapid and simple assay for this toxin. No enterotoxin activity was detected in cell extracts or culture supernates from any of the isolates.     

Avian parasympathetic neurotrophic factors: Age-related increases and lack of regional specificity

Parasympathetic neurons from 8-day-old chick embryo ciliary ganglia were grown in culture for 24 hr in the presence of extracts of chick heart from animals aged 7 days in ovo to 8 days posthatching. The biological activity of these cardiac extracts with respect to both neuronal survival and nerve fiber production increased with age of the donor animal to reach a plateau around hatching. Following polyacrylamide gel isoelectric focusing of posthatching chick heart and eye, a peak in activity that supports parasympathetic neuronal survival was found associated with eluates of gel slices of pH between 4.5 and 5.5 for both tissues. Our results suggest that the factor responsible for parasympathetic neuronal survival is common to at least two parasympathetic target organs.     

The distribution of agglutinins and lytic activity against Trypanosoma rangeli and erythrocytes in Rhodnius prolixus and Triatoma infestans tissue extracts and haemolymph.

Haemolymph, heads, salivary glands, crops, midguts, hindguts, and Malpighian tubules from Rhodnius prolixus and Triatoma infestans were extracted in phosphate or Tris buffer saline with calcium, and tested for agglutination and lytic activities by microtitration against both vertebrate erythrocytes and cultured epimastigote forms of Trypanosoma rangeli. Haemagglutination activity against rabbit erythrocytes was found in the crop, midgut and hindgut extracts of T. infestans but only in the haemolymph of R. prolixus. Higher titers of parasite agglutinins were found in R. prolixus haemolymph than T. infestans, whilst the converse occurred for the tissue extracts. In addition, the extracts of T. infestans salivary glands, but not those of R. prolixus, showed a trypanolytic activity that was heat-inactivated and was not abolished by pre-incubation with any of the sugars or glycoproteins tested. T. infestans, which is refractory to infection by T. rangeli, thus appears to contain a much wider distribution of agglutinating and trypanolytic factors in its tissues than the more susceptible species, R. prolixus.     

Neurotrophic and hepatotrophic stimulation of proliferation of embryonic chick muscle cells in vitro: Assay and partial characterization of mitogenic activity in chick embryonic organ and tissue extracts

Whole embryo extract is routinely employed as a growth-promoting supplement in chick embryonic muscle cell cultures. In assessing the effect of the extract on muscle cell cultures, extracts of various embryonic tissues and organs were substituted for whole embryo extract and the effects on proliferation of dissociated 12-day chick embryonic leg muscle cells were observed. The effects were measured according to [³H]thymidine incorporation into deoxyribonucleic acid (DNA) and were confirmed with total cell counts. Brain and liver extracts were found to be especially effective in stimulating muscle cell proliferation. The extracts were found to be heat and trypsin labile. Further analysis of activity in the extracts by dialysis and Sephadex G-25 fractionation revealed the presence of at least two classes of activity—one of high molecular weight (>5000) and one of low molecular weight (<5000)—which must be present together to yield the full activity of crude extracts from embryonic liver and brain. The results are discussed against the background of our interest in the neurotrophic phenomenon.     

A new survey of Brazilian marine algae for agglutinins

Aqueous protein extracts from 30 Brazilian marine algae were examined for haemagglutinating activity using native and enzyme-treated rabbit, chicken, sheep and human erythrocytes. Most extracts agglutinated at least one of the blood cells used. Sheep and rabbit erythrocytes were more suitable for detection of the agglutinating activity. The minimum protein concentration necessary to produce positive agglutination was usually lower with enzyme-treated erythrocytes than native ones. The five algal protein extracts showing the greatest haemagglutination titre were tested for sugar-binding specificity. Only the activity present in the green alga Cauler pacupressoides was inhibited by simple sugars and not by the glycoproteins tested. The activity of the other four extracts was inhibited by at least one of the glycoproteins utilised. This revised version was published online in August 2006 with corrections to the Cover Date.     

Serological reactions associated with the clumping factor of Staphylococcus aureus

Rotter, Joan (University of Oklahoma Medical Center, Oklahoma City), and Florene C. Kelly. Serological reactions associated with the clumping factor of Staphylococcus aureus. J. Bacteriol. 91:588-594. 1966.-Evidence that the substance which causes staphylococci to clump in the presence of fibrinogen (clumping factor) is antigenically similar in strains which are serologically diverse according to agglutination reactions has been obtained from fibrinogen-cell clumping inhibition tests. Antisera for clumping factor (CF)-positive strains inhibited the clumping reaction of all strains tested. After adsorption with homologous cells or with cells of other CF-positive strains, the antisera no longer inhibited clumping. When antisera were adsorbed with trypsin-treated, CF-positive cells, or with cells of CF-negative mutants, the ability to inhibit the clumping reaction persisted. Antibody to CF activity was not associated with coagulase. Latex coated with extracts derived from the cells of five CF-positive and six CF-negative strains was, in each instance, agglutinated by sera from rabbits immunized with CF-positive cells. After adsorption with trypsinized, CF-positive cells, antisera still agglutinated latex which had been treated with the CF-positive extracts, but not with the CF-negative extracts. Similar results were obtained after antisera were adsorbed with the cells of CF-negative mutants. Cell agglutination titers of sera from rabbits immunized with CF-negative staphylococci were significantly lower than those produced in response to CF-positive cells, regardless of their coagulase activity. If the CF-inhibiting antibody also functions as an agglutinin, it apparently is not solely responsible for this difference.     

Distinct lectin activities from six species of cellular slime molds.

Extracts of cohesive cells of four species of cellular slime mold, D. mucoroides, D. purpureum, D. rosarium and P. violaceum agglutinate erythrocytes in a manner that is similar to that previously observed with extracts of D. discoideum and P. pallidum. We determined inhibitory activity of a series of sugars on the agglutination activity of each of these extracts, using both semiquantitative and quantitative agglutination assays. The inhibitory potency of this series of sugars was distinct for each extract, although only slight differences were found between several species, especially D. discoideum and P. violaceum. A possible role of these agglutinins in species-specific cell cohesion is considered.     

Cryptococcal capsular polysaccharide clearance in nonimmune mice

Clearance of cryptococcal polysaccharide (CP) from tissues and body fluids of nonimmune mice was studied. Mice were injected intravenously one time only with one mg of purified CP, and serum, urine and tissues were obtained from each animal at various intervals for a period of 84 days. Tissue extracts, serum and urine were tested for CP content by enzyme-linked immunosorbent assay (ELISA) and latex agglutination. High concentrations of CP were detected by both assays one-half hour after injection in blood (serum), liver, spleen, kidney and lung (extracts). The duration of ELISA detectable CP was longest (70 days) in liver and spleen and shortest (14 days) in lung extract. By 14 days after injection, concentration of CP in the blood fell below that found in the liver and spleen. CP remained detectable (titers 32–64) after all other extracts became negative. These results indicate that CP is stored in tissues (binding mechanism and site unknown), and that the liver and spleen possess greater storage capacity than other tissues. Antibody (IgM) to CP appeared in low titer on the 14th day and thereafter.     

The use of methanol extract of Leptospira interrogans in complement fixation tests for leptospirosis

Methanol extracts were obtained from L. interrogans serovars icterohaemorrhagiae and canicola and L. biflexa serovar patoc. Human sera from 167 normal individuals and 40 patients with different infectious diseases tested by complement fixation tests showed negative reactions. Sera from 100 patients with a suspicion of leptospirosis were tested by complement fixation tests and microscopic agglutination reactions. Agreement of 84% was found for those two reactions. Positive microscopic agglutination tests at a dilution 1:20-1:400 with negative complement fixation tests were observed in 5% of patients and negative microscopic agglutination with complement fixation tests in the range of 1:20-1:1280 were observed in 11% of the cases.     

Late hexokinase activity expression during Bufo bufo development.

Hexokinase activity was detected in cytosols and homogenates from different developmental stages of Bufo bufo embryos starting from stage 17. Free glucose was measured in the embryo cytosol and was detected at each stage tested. At stage 15, a large increase of glucose content of the embryo cytosol occurs. Hexokinase expression in the embryo thus occurs after the increase of cytosol glucose content occurring at stage 15. The findings rule out that glucose by itself is the hexokinase inducer in vivo. The very low glucose utilization found by many authors during early amphibian development may be related to the late hexokinase expression during Bufo bufo development.     

Tissue Specific Distribution of Pyrimidine Deoxynucleoside Salvage Enzymes Shed Light on the Mechanism of Mitochondrial DNA Depletion

Deficiency in thymidine kinase 2 (TK2) activity due to genetic alterations caused tissue specific mitochondrial DNA (mtDNA) depletion syndrome with symptoms resembling these of AIDS patients treated with nucleoside analogues. Mechanisms behind this mitochondrial effects is still not well understood. With rat as a model we isolated mitochondrial and cytosolic fractions from major organs and studied enzymes involved in thymidine (dT) and deoxycytidine (dC) phosphorylation by using ionic exchange column chromatography. A cytosolic form of TK2 was identified in all tested tissues in addition to mitochondrial TK2. TK1 was detected in liver and spleen cytosolic extracts while dCK was found in liver, spleen and lung cytosolic extracts. Thus, the nature of dT and dC salvage enzymes in each tissue type was determined. In most tissues TK2 is the only salvage enzyme present except liver and spleen. These results may help to explain the mechanisms of mitochondrial toxicity of antiviral nucleoside analogues and mtDNA depletion caused by TK2 deficiency.     

Fixation of carbon dioxide in the dark by the malic enzyme of bean and oat stem rust uredospores

Malic enzyme was found in both bean rust and cat stem rust uredospores. In bean rust uredospores it was shown to catalyze the formation of pyruvic acid from l-malic acid and to synthesize malic acid from pyruvic acid and CO₂. The malic enzyme from bean rust uredospores was specific for NADP and dependent on manganous ions for activity. The specific activity of the bean rust malic enzyme in crude extracts of ungerminated uredospores was approximately 6 times greater than that found in crude extracts obtained from germinated uredospores. The malic enzyme was also found in extracts obtained from healthy and rust-infected bean leaves. The specific activity of the enzyme was approximately 2 to 5 times greater in partially purified extracts obtained from the infected bean tissue at 6 days after inoculation. The specific activity of the malic enzyme in crude extracts obtained from oat stem rust uredospores was 2 times greater than the specific activity of this enzyme in crude extracts obtained from bean rust uredospores. Phosphoenolpyruvate carboxylase activity could not be demonstrated in crude extracts obtained from the ungerminated uredospores of the bean rust fungus.     

Metabolic regulation in C4 photosynthesis: Phosphoenol pyruvate carboxylase and 3C intermediates of the photosynthetic carbon reduction cycle

Summary Gibberellins and auxins were extracted from embryos and suspensors of Phaseolus coccineus L. at two stages of development: A) heart-shaped embryo and B) cotyledonary embryo with suspensor in the initial stage of degeneration. The time interval between the two stages was 5–6 days.In both embryos and suspensors, gibberellin (GA)-like activity was found in three fractions: F-1 (ethyl acetate fraction at pH 8.0), F-2 (free GAs) and F-3 (bound GAs). At stage A, the total GA activity in the suspensor was about 30 times greater than in the embryo and the bound GAs contributed by about 90% to the total GA content. A dramatic decrease in level of bound GA-like substances was found in suspensors at stage B, when the level of total GAs in the embryo had increased to 10 times that at stage A. This might suggest a transport of GAs from the suspensor to the embryo. In both embryo and suspensor, qualitative changes in GAs with shift in activity of the fractions tested occurred at the two developmental stages.The methanolic extracts of stage A suspensors showed two inhibitors, one much more active than the other, and two large peaks of growth promoting activity at Rf 0.4–0.7; in stage A embryos, the general activity of the extracts was lower and the promoting effect was spread over Rf 0.3–0.9.The present results seem to support the view that the suspensor plays a role in embryogenesis by acting as a site of synthesis of growth regulators needed by the embryo.Abbreviations F-1 ethyl acetate fraction at pH 8.0 - F-2 free gibberellins - F-3 bound gibberellins - GA gibberellic acid - Stage A heart-shaped embryo - stage B cotyledonary embryo with suspensor in the initial stage of degeneration     

Late hexokinase activity expression during Bufo bufo development

Hexokinase activity was detected in cytosols and homogenates from different developmental stages of Bufo bufo embryos starting from stage 17. Free glucose was measured in the embryo cytosol and was detected at each stage tested. At stage 15, a large increase of glucose content of the embryo cytosol occurs. Hexokinase expression in the embryo thus occurs after the increase of cytosol glucose content occuring at stage 15. The findings rule out that glucose by itself is the hexokinase inducer in vivo. The very low glucose utilization found by many authors during early amphibian development may be related to the late hexokinase expression during Bufo bufo development.     

Antibacterial activity in Strongylocentrotus droebachiensis (Echinoidea), Cucumaria frondosa (Holothuroidea), and Asterias rubens (Asteroidea)

A search for antibacterial activity in different body parts of the green sea urchin Strongylocentrotus droebachiensis, the common starfish Asterias rubens, and the sea cucumber Cucumaria frondosa was conducted. Antibacterial activity was detected in extracts from several tissues in all species tested, but mainly in the coelomocyte and body wall extracts. Relatively high antibacterial activity could also be detected in gastrointestinal organs and eggs from A. rubens and in eggs from C. frondosa. Differences between active extracts regarding hydrophobicity and sensitivity to heat and proteinase K treatment indicated that several different compounds were responsible for the antibacterial activities detected. Lysozyme-like activity could be detected in several tissues from A. rubens. Haemolytic activity could be detected in all species tested, especially in the body wall extracts. Results from the current study suggest that marine echinoderms are a potential source for the discovery of novel antibiotics.     

The appearance of 1,25-dihydroxyvitamin D3 receptor during chick embryo development

The appearance of the 1,25-dihydroxyvitamin D3 receptor in intestine, kidney, and chorioallantoic membrane of chick embryo was followed by sucrose density gradient sedimentation analysis and Scatchard plot analysis. The receptor from each of these organs sediments as a single 3.7S component. At 19 days of embryonic life, intestine had the highest specific 1,25-dihydroxyvitamin D3 binding activity followed by kidney and chorioallantoic membrane. The 1,25-dihydroxyvitamin D3 binding activity increased gradually at 12-15 days and rapidly until 20 days in intestine. In kidney, this protein increased rapidly from 12 to 16 days and did not change subsequently. In chorioallantoic membrane, the receptor increased slowly from 8 through 15 days, rapidly until 19 days, and decreased at 20 days. The injection of hydrocortisone into the chick embryo at 10 days increased receptor number in intestine, kidney, and chorioallantoic membrane by a factor of 2 at 12 days. Injection of this hormone after this time had little or no effect.     

Immunological properties of an L-form of a group A beta hemolytic streptococcus

Antisera were prepared by injecting several groups of rabbits with antigen preparations of the parent streptococcus and its derived L-form. Serum was obtained from each rabbit by cardiac puncture prior to and at intervals after injection of the organisms. To measure the immune response of all animals a modified latex agglutination test was used. This test was found to be specific and easily reproducible. Animals immunized intravenously with the heat-killed streptococci and those immunized intramuscularly with the L-form produced the highest level of antibodies against their respective antigen preparations. Lancefield extracts of the streptococcus reacted with the streptococcal antisera but not with antisera to the L-form. In the antisera to the L-form we found specific antibodies which could not be adsorbed with the parent streptococcus.A portion of this study formed part of a thesis submitted by Sister M. B. Muellenberg to the University of South Dakota, Vermillion, S.D. in partial fulfillment for the requirement for the degree of Master of Arts.     

Hemagglutinins extracted from locks of unopened cotton bolls

Hemagglutinins, which may mediate plant-fungal interactions, were detected in water soluble extracts from locks of unopened cotton bolls. Hemagglutinating activity was nondialyzable, and thus the agglutinating components possess molecular weights greater than 6 000 daltons. Five of 23 tested sugars inhibited agglutination. The inhibitory sugars did not possess common stereochemical features and included mono-, di-, and trisaccharides.     

A new lectin from the tuberous rhizome of Kaempferia rotunda: isolation, characterization, antibacterial and antiproliferative activities

A lectin (designated as KRL) was purified from the extracts of Kaempferia rotunda Linn. tuberous rhizome by glucose-sepharose affinity chromatography. KRL was determined to be a 29.0 ± 1.0 kDa polypeptide by SDS-PAGE under both reducing and non-reducing conditions. KRL was a divalent ion dependent glycoprotein with 4% neutral sugar which agglutinated different groups of human blood cells. Methyl-α-D-mannopyranoside, D-mannose and methyl-α-D-glucopyranoside were the most potent inhibitors. N-terminal sequence of KRL showed similarity to some mannose/ glucose specific lectins but the main differences with their molecular masses and sugar content. KRL lost its activity markedly in the presence of denaturants and exhibited high agglutination activity from pH 6.0 to 8.2 and temperature 30 to 60° C. The lectin showed toxicity against brine shrimp nauplii with the LC50 value of 18 ± 6 μg/ml and strong agglutination activity against seven pathogenic bacteria. KRL inhibited the growth of six bacteria partially and did not show antifungal activity. In addition, antiproliferative activity against Ehrlich ascites carcinoma (EAC) cells showed 51% and 67% inhibition in vivo in mice administered 1.25 mg/kg/day and 2.5 mg/kg/day of KRL respectively by injection for five days.