Effect of the interferon inducer tilorone in inbred CBA mice.

Tilorone hydrochloride (200 mg/kg) was administered intragastrically to inbred CBA mice. After 5, 18 and 48 hr the number of circulating leucocytes and peritoneal cells as well as the migration of unstimulated peritoneal cells, the blood corticosteroid level and interferon production were investigated. In spite of the considerable decrease of the number of mononuclear cells in the blood and polynuclear ones in the peritoneal exudate, the drug induced production of circulating interferon and stimulated its synthesis by peritoneal cells. The blood corticosteroid level and the mast cell count in the peritoneal cavity were significantly elevated, but the migration of peritoneal cells in antigen-free medium decreased.     

Do fish lymphocytes secrete interferon-γ?

Macrophage activating factor (MAF)-containing supernatants, generated by mitogen (Con A/PMA) stimulation of rainbow trout leucocytes, were found to confer viral resistance (interferon, IFN, activity) on a rainbow trout epithelial cell line challenged with infectious pancreatic necrosis virus. Both the MAF and IFN activities co-eluted by HPLC size exclusion chromatography and showed similar sensitivities to acid (pH 2), temperature (60° C) and trypsin. The mode of induction of this IFN, its acid and temperature sensitivity and its possible MAF activity suggest that fish leucocytes can secrete an IFN-γ like molecule.     

The mechanism of the movement of leucocytes

In a study of the movement of human leucocytes it was clarified that characteristic contraction waves were observed on the cell surface during movement and an initial morphological change directly related to the appearance of the wave originated in the surface of the granuloplasm and not in the cell membrane. From these findings, together with physicochemical properties of the contractile protein from equine leucocytes, it was proposed that the wave observed in moving leucocytes might be conducted, in some way, by contraction and relaxation of the contractile protein in the cells. Myosin A and actin as constituents of the contractile protein were extracted separately from leucocytes in polymerized form, which resemble myosin aggregate and F-actin from muscle, respectively. The thick and thin filaments of about 150 and 80 Å in diameter were observed in glycerinated leucocytes with electron microscopy. When glycerinated leucocytes were incubated with heavy meromyosin (HMM) from rabbit skeletal myosin A, the thin filaments developed a structure resembling the ‘arrowhead structure’ of the HMM F-actin complex in vitro. The thick filaments seemed to correspond to myosin aggregates and the thin ones to filaments containing F-actin.     

Interferon production by human leukaemic leucocytes.

The Sendai virus-induced interferon (IF) production by partially purified human leucocyte suspensions of normal donors and of leukaemic patients have been investigated in vitro. (i) An increased production was observed with leucocytes taken from patients suffering from chronic myelogenous leukaemia (CML) during exacerbation, but the production was approximately normal with cells taken during remission. (ii) IF production was not influenced by large doses of cytostatics (DBM, 5-FU, FUDR, 5-HU, 6-MP, cyclophosphamide) irrespective of whether normal or CML leucocytes were used. (iii) In contrast, production was inhibited in both systems by inhibitors of RNA or protein synthesis (actinomycin D, puromycin, cycloheximide). (iv) CML leucocytes produced IF for a prolonged period of time as compared to normal leucocytes. (v) Leucocytes from children with acute blastic leukaemia and those from adults with chronic lymphoid or acute paramyeloblastic leukaemia produce, in contrast to normal leukocytes, approximately as much IF in the absence as in the presence of serum. It is concluded that the Sendai virus-induced IF synthesis in CML leucocytes requires de novo protein synthesis.     

Are there pathological defects of interferon secretion in man?

Interferon is now recognized as an important biological mediator with both antiviral and non-antiviral (including immunoregulatory) functions. In some patients with repeated and severe infections, leucocytes appear to be unable to produce normal levels of interferon after stimulation with soluble antigens or allogeneic lymphoblastoid cells. This defect contrasts with normal immune functions, with the exception of "natural killer" non-specific cytotoxic activity which is usually impaired. This selective defect of interferon secretion may result in a special type of immuno-deficiency with multiple biological consequences, some of which can be reversed by interferon therapy.     

A mannose-receptor is possibly involved in the phagocytosis of Saccharomyces cerevisiae by seabream (Sparus aurata L.) leucocytes

In this paper the possible involvement of the mannose-receptor on the non-specific recognition and phagocytosis of heat killed yeast cells (Saccharomyces cerevisiae) by gilthead seabream (Sparus aurata L.) head-kidney leucocytes was established by studying the ability of different sugars to inhibit the uptake of the yeast cells by leucocytes. Leucocytes were preincubated for 30min with different concentrations of sugar (alpha-mannan, d-mannose, d-fucose, l-fucose, d-glucose, d-glucosamine and n-acetyl-glucosamine, all of them described as specific ligands of the vertebrate mannose-receptor) and afterwards incubated with FITC-labelled yeast cells for phagocytosis assays. The phagocytic ability (percentage of cells with one or more ingested yeast cells within the total cell population) and capacity (number of ingested yeast cells per cell) of leucocytes was analysed by flow cytometry. The results demonstrate the potential existence of a specific receptor-sugar or receptor-yeast cell binding process, which was saturable, specific and dose-dependent. More specifically, when leucocytes were preincubated with appropriate doses of d-mannose, d- or l-fucose, d-glucose or n-acetyl-glucosamine the phagocytosis of yeast cells by head-kidney leucocytes was partially blocked. Seabream leucocytes were also preincubated with chloroquine, a lysosomotropic drug which downregulates (in a nonspecific manner) the expression of mannose-receptors in mammals, before phagocytosis assays were performed. The results demonstrated that the phagocytosis of yeast was completely blocked by this substance. The overall results seem to corroborate the presence of the mannose-receptor in seabream phagocytes, which is involved in the non-specific binding and phagocytosis of yeast cells by head-kidney leucocytes.     

An electron microscope study of the circulating leucocytes of the marine mussel,Mytilus edulis

Circulating leucocytes of the mussel, Mytilus edulis, were studied by electron microscopy. Based on morphological criteria, the leucocytes were classified as agranular and granular leucocytes, dependent upon the presence or absence of specific granules in their cytoplasm. Furthermore, the existing literature is being critically revised, and it is suggested that agranular and granular leucocytes might belong to the same cell line.     

Isolation of leucocytes from the anterior kidney and spleen of rainbow trout in a self-generating density gradient

Separation of leucocytes from tissues and enrichment of specific types of leucocytes are essential first steps in studies of leucocyte function. We describe a simple and rapid method for separating and enriching leucocytes from the anterior kidney and spleen of rainbow trout. Leucocytes were separated on self-generating density gradients of Sepracell-MN, a colloidal silica-based medium. Recovery of leucocytes from the gradients was near 100% and was not affected by procedural variables such as cell suspension: Sepracell-MN ratio (separation ratio), initial temperature, centrifugation time, or number of cells per gradient. Two bands of leucocytes formed at separation ratios of 1 : 1, 1:1.5, 1:2 and 1 : 3. Recovery of selected leucocyte types could be maximized, and contamination by other cell types reduced, by selection of the appropriate separation ratio and cell band. Recovered leucocytes were responsive in assays of functional capability (adherence, phagocytosis, superoxide anion production). Leucocyte populations that adhered to glass were enriched for macrophages, but some neutrophils and lymphocytes (paricularly spleen lymphocytes) were also adherent. The most abundant leucocytes in the anterior kidney and spleen of the experimental fish were lymphocytes (respectively, 47 and 88%, including thrombocytes), neutrophils (35 and 7%), and macrophages (11 and 3%).     

The action of streptomycin in a mutant of Escherichia coli with increased sensitivity to the antibiotic

A mutant of Escherichia coli with increased sensitivity to streptomycin has been studied. This strain differed from a normal str(s) strain in that streptomycin produced inhibition of protein synthesis and loss of viability with almost no lag period. Chloramphenicol protected a normal str(s) strain but not the mutant against the bactericidal action of streptomycin. The results obtained support the idea that access of streptomycin to its site of action in a normal cell is restricted, and that this restriction, which is much less effective in the mutant, probably involves a permeability barrier. Comparison of the inhibition of protein synthesis by streptomycin with concomitant changes in the distribution of polyribosomes in both strains suggested that the antibiotic can directly inhibit the translation of mRNA.     

Naphthol-AS-D-chloroacetate esterase activity in globule leucocytes in the tracheal epithelium of rats

The enzyme naphthol-AS-D-chloroacetate esterase is histochemically demonstrated in the granules of globule leucocytes in the tracheal epithelium of rats. The enzyme reactivity may be used as a cytochemical marker of these cells. The previously postulated mast cell origin of globule leucocytes is doubted, and the possibility that globule leucocytes belong to the group of natural killer cells is discussed. The biological role of the located esterase and the function of the globule leucocytes are also briefly discussed.     

The mechanism of resistance to streptomycin inEscherichia coli. Functional analysis of the permeability barrier of cells harbouring the Rldrd-19Km plasmid

The mechanism of phenotypically altered SM resistance in mutants of Escherichia coli JC5455 (Rldrd-19Km-) lrs and JC5455 (pON5300) was compared with that of the standard strain JC5455 (Rldrd-19Km-). On analyzing the membrane polypeptides in polyacrylamide gel both mutants were found to possess a protein spectrum different from that of ths standard strain. Transport of D-xylose and L-arginine was the same in all strains, transport of L-proline was decreased in JC5455 (pON5300) which may indicate a mutational interference with energy metabolism. The basic uptake of dihydrostreptomycin was the same in all strains but there were differences after preincubation of cells with streptomycin or glucose. The increased resistance of JC5455 (Rldrd-19Km-) lrs may be due to observed quantitative differences in membrane polypeptides that might play a role in the binding and functional expression of aminoglycoside-3'adenylyl transferase which modifies streptomycin. The increased sensitivity toward streptomycin in JC5455 (pON5300) can be explained by a mutation due to N-methyl-N'-nitro-N-nitrosoguanidine in the host cell since this change of sensitivity to streptomycin could not be transferred by transformation into a nonmutagenized strain. The coincidence of inducibility of increased transport of streptomycin by this antibiotic and the altered frequency of reversion to high levels of streptomycin resistance in JC5455 (pON5300) and in the transformant JC5455 (pON5302) may indicate that the altered reversibility toward phenotypically high resistance to streptomycin is a property of pON5300 and is transferred by transformation.     

Effect of aminoglycoside antibiotics on the autolytic enzyme of Streptomyces griseus

The isolated cell wall of Streptomyces griseus 52–1 strain labelled with fluorescein isothiocyanate (FITC) and containing wall-bound autolytic enzyme was lysed as a function of different cations. The autolysis was accelerated by aminoglycoside antibiotics (streptomycin and the structurally closely related neomycin) which have a polycationic character. Since this strain is a streptomycin producer it is suggested that streptomycin may have a regulatory function on autolysis.     

Induction of streptomycin resistance in the wild tomato Lycopersicon peruvianum

Summary A protoplast mutagenesis and cell selection system was used for the isolation of streptomycin resistant Lycopersicon peruvianum colonies. Protoplasts were treated with the mutagen N-nitroso-methylurea and could be regenerated into fertile plants, carrying the streptomycin resistant character. Several classes of streptomycin resistance could be distinguished. Reciprocal crosses between streptomycin resistant and sensitive plants showed a non-Mendelian transmission of the resistance trait. Streptomycin resistance is the first selectable and maternally inherited cell organelle marker described in tomato.     

Molecular cloning and expression in Streptomyces lividans of a streptomycin 6-phosphotransferase gene from a streptomycin-producing microorganism

The gene encoding streptomycin 6-kinase involved in the self-resistance of the streptomycin-producing Streptomyces griseus HUT 6037 was cloned in the plasmid vector pIJ703. The resulting plasmid, pSP6, contained 2.5 kb inserts of S. griseus DNA. When streptomycin-susceptible S. lividans 1326 was retransformed with pSP6, all transformants produced streptomycin 6-kinase. Addition of streptomycin to the culture medium of S. lividans carrying pSP6 plasmid brought about a remarkable increase in streptomycin 6-kinase activity in the cell extracts. It is suggested from the results that the production of streptomycin 6-kinase in streptomycin producer was induced by streptomycin accumulated during cultivation.     

链霉素对鸟听觉毒性作用的研究

目的：了解链霉素对鸟听觉毒性的作用。方法：选33只健康成年虎皮鹦鹉,不同剂量的链霉素肌肉注射15日,脑干听觉诱发电位测试外周的听觉敏度和听觉通路中神经的传导和传递能力。结果：链霉素对鸟听觉有毒性作用。结论：用链霉素处理鸟可以制作聋鸟模型。     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.     

Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response

The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers. In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy. The antibody reacted with 40%-50% of head-kidney and peritoneal exudate leucocytes and 10%-20% of spleen and peripheral blood leucocytes. More importantly, G7(+) acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum. The results are discussed in relation to the role played by this cell type in fish immune responses.     

The polyphosphoinositide content of the leucocyte, erythrocyte and macrophage

1. The polyphosphoinositide content of macrophages and the cell membranes of leucocytes and erythrocytes was determined by an extension of the `acid-hydrolysis' procedure of Dawson & Eichberg (1965). The estimation was controlled by adding a little highly radioactive polyphosphoinositide to the tissue extracts before fractionation. Several standard methods for determining polyphosphoinositides gave low recoveries when applied to leucocytes, and it is suggested that these cells contain materials that form complexes with the polyphosphoinositides and interfere with the assay. 2. The method for the preparation of leucocyte cell surface membranes has been modified.     

Characteristics of the transport of ascorbic acid into leucocytes

The degree and the mode of association of [14C]-ascorbic acid with leucocytes are examined. The degree of association of ascorbic acid with polymorphonuclear leucocytes (1-3%) is dependent on cell type, extracellular concentration of ascorbic acid, incubation temperature, intactness of the cells and the extracellular pH. All experiments are performed according to strict protocols as these compounds are labile in aqueous solutions. Further it is noticed that in all experiments an outward gradient of leucocyte endogenic ascorbic acid exists. The results suggest that the association process comprises at least one saturable pathway. The activation of polymorphonuclear leucocytes by phorbol myristate acetate increases the accumulation of ascorbic acid threefold.     

Effect of streptomycin on lipid composition with particular reference to cyclic depsipeptide biosynthesis in Serratia marcescens and other micro-organisms

The addition of low concentrations of streptomycin (5-10mug/ml of medium) to Serratia marcescens caused significant alterations in the lipid composition of this organism, but neither growth nor pigmentation was affected. The acetone-soluble cyclic depsipeptides, which comprise on average 15% of the total lipid, were decreased almost to zero and the total lipid phosphorus was more than doubled in the presence of streptomycin. Most of the phospholipid increase was due to an increase in phosphatidylethanolamine. Cyclic depsipeptides were not leached from the cell in the presence of streptomycin, indicating a definite inhibition of the biosynthetic pathway. The effect of streptomycin on the reported peptidolipids of Rhodopseudomonas spheroides, Halobacterium halobium, Nocardia asteroides and Pseudomonas tabaci was investigated. In the case of the only strictly comparable cellular cyclic depsipeptide (that of N. asteroides) the biosynthesis was strongly inhibited by streptomycin, but cell weight was maintained or even slightly increased. A possible mode and site of action of low concentrations of streptomycin on bacterial lipids is discussed.