Biomedical subjects

J Izard

Publications and source records attributed to J Izard.

At least 37 records · Page 2Linked to original sources

PubMed1994

A single amino acid substitution can restore the solubility of aggregated colicin A mutants in Escherichia coli.

Mutants of colicin A have been prepared in which the three tryptophan residues (Trp86, Trp130 and Trp140), localized in the C-terminal domain of the soluble wild-type protein, have been substituted by phenylalanine. The Trp140Phe single mutation had the effect of decreasing the percentage of protein that is expressed as insoluble aggregates. The created hydrophobic cavity decreased the stability of the protein during its folding, resulting in partial aggregation in the cytoplasm of the Escherichia coli-producing cells. Aggregation was increased when Trp140 was substituted by Lys, Leu or Cys, or if the Trp140 mutation was combined with the Trp86Phe and/or Trp130Phe mutations. A single mutation, Lys113Phe, however, was able to restore the solubility of the aggregated mutants in vivo. Detailed analysis of the 3-D structure of the C-terminal domain of colicin A suggests that filling of the hydrophobic cavity is responsible for this effect.

Amino Acid Sequence↗

PubMed1993

Kurloff cell ultrastructure after combined formaldehyde-cetylpyridinium chloride fixation and high-iron diamine staining.

This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosomal organelle that stains metachromatically with Toluidine Blue and which is present in Kurloff cells (a blood cell unique to the guinea pig). Splenic tissues were fixed with 1% cetylpyridinium chloride (CPC) added to 4% paraformaldehyde and examined either after Spicer's high-iron diamine staining for sulphated anionic sites followed by post-fixation with ferrocyanide-osmium tetroxide or after a simple post-fixation with ferrocyanide-osmium tetroxide. CPC-precipitated sulphated sites were preferentially located at the periphery of the Kurloff body but, unexpectedly, were absent in the central matrix. Although their electron opacity was lower, these anionic sites were readily observable in the absence of HID-staining after sole post-fixation by ferrocyanide-reduced osmium. CPC-precipitated sulphated anionic sites were either associated with the myelin figures or constituted unexpected structures. They contained (i) tightly-stacked lamellae, with a very regular 4 nm periodicity, and (ii) groups of 2, 3, 4 short dense lines with a 3-5 nm periodicity. By taking into account the susceptibility of these HID-reactive structures towards chondroitinase ABC, these different sulphated components were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell. Their colocalization with phospholipidic structures was suggested following observation of sections treated by a chloroform-methanol mixture.

Animals↗

PubMed1993

Glycoconjugates with Neu5Ac(alpha 2,6)Gal(beta 1,4)GlcNAc sequences: a selective lectin-histochemical property of Kurloff cells in guinea pig thymus.

The Kurloff cell (KC), a natural killer lymphocyte, contains a large (10-microns diameter) periodic acid-Schiff (PAS)-positive lysosome-like inclusion body called the Kurloff body (KB), which exhibits strong acid phosphatase activity. The presence of Sambucus nigra agglutinin (SNA)-reactive Neu5Ac(alpha 2,6)-D-Gal/Gal-NAc(beta 1,4)GlcNAc oligosaccharide sequences and the absence of the corresponding Neu5Ac(alpha 2,3) Maackia amurensis agglutinin (MAA)-reactive sequence in the major 35-kDa N-glycosylproteins of the complex or hybrid type extracted from purified KC were established by Western-lectin-blotting of cytosolic extracts from purified KC. Moreover, these SNA-reactive sequences, or at least part of them, were shown to be borne by sialidase-sensitive KC acid isophosphatases. Thymic sections rich in KC, from estrogenized guinea pigs were examined by affino-histochemistry with these sialic acid-reactive lectins. The SNA-reactivity of thymic sections was quasi-exclusively confined to KC clusters, whereas the whole thymic section was negative for MAA. KC were not SNA-reactive following preincubation and incubation with 200 mM lactose. When submitted to enzymatic or mild chemical desialylation processes, the SNA-reactivity of the KC clusters was enhanced. The SNA-reactivity of KC clusters was completely abolished following prolonged chemical desialylation, whereas the PAS-positivity of KB remained unchanged. Even after a prolonged sialidase treatment, this SNA-reactivity was only reduced. Moreover, after both these desialylation processes, KC developed a heavier Ricinus communis agglutinin-reactivity, thus confirming the presence of penultimate Gal residues in their abundant SNA-reactive oligosaccharide sequences Neu5Ac(alpha 2,6)Gal(beta 1,4)GlcNAc. Such a selective lectin histochemical property provides a marker for detecting KC.

Animals↗

PubMed1993

Characterization of the non-sulphated highly anionic Kurloff cell glycoconjugates by lectin- and immunoblotting: evidence for the presence of alpha (2,8)-linked polysialic acid-bearing molecules.

Urea or guanidine hydrochloride-soluble extracts from highly purified Kurloff cells (KC) radiolabelled in vitro were subjected to DEAE-cellulose chromatography. Among the three anionic peaks obtained, a major and non-sulphated peak (designated as peak IV) strongly affected by glucosamine-labelling and eluted at about 0.3 M NaCl was analyzed. Gel filtration on Sepharose CL4B and 10% SDS-PAGE indicated its heterogeneous size. Peak IV consisted mainly of N-glycans as shown by its susceptibility to tunicamycin. Further insight into its chemical nature was obtained by examining its binding capacity to different lectins and by immunodot analysis. It strongly interacted with concanavalin A (Con A) after dot-blot or Western blotting. A large amount of these glycoproteins is not of the high-mannose type since Galanthus nivalis agglutinin reacted weakly with peak IV. Moreover, bindings to Phaseolus vulgaris and to wheat germ agglutinins suggest the presence of bisecting N-acetylglucosamine residues. Bindings to Sambucus nigra and to Ricinus communis agglutinins, dramatically lessened and increased respectively after desialylation, suggest the presence of Neu5Ac alpha 2,6Gal/GalNAc sequences. The absence of outer sialic acid residues linked alpha 2,3 to galactose was demonstrated following Maackia amurensis agglutinin negativity. The use of poly(alpha 2,8-sialyl) endo-N-acylneuraminidase combined with immunodot procedure with a monoclonal antibody that specifically recognizes alpha 2,8-linked polysialic chains revealed that peak IV contains oligosaccharidic epitopes common to polysialylated neural cell adhesion molecules.

Animals↗

PubMed1993

In vitro cytotoxic effect of guinea-pig natural killer cells (Kurloff cells) on homologous leukemic cells (L2C).

The Kurloff cell (KC) of the guinea-pig develops natural killer cytotoxic activity in heterologous systems. We report in this paper the effective in vitro cytotoxic activity of the KC in a homologous guinea-pig system, i.e. against the guinea-pig target leukemic L2C cells. A dual-color flow analysis of homologous effector-target conjugates, using calcein-labeled KC and hydroethidine-labeled L2C shows a 40% frequency KC-L2C conjugation. The specific cytotoxicity of KC against L2C (78%) was estimated as the target-loss of green fluorescence due to hydrolysed carboxy-fluorescein diacetate after 4 hours at 37 degrees C. We propose that the Kurloff cell could be involved in surveillance against spontaneously arising leukemic cells, and this could be an explanation for the high degree of resistance to spontaneous or experimentally-induced cancers, in the guinea-pig.

Animals↗

PubMed1992

The major Kurloff cell glycoproteins: lectin affinities, glycosidase susceptibilities and relationship with the sialylated acid phosphatases of the Kurloff body.

Urea-soluble fractions from purified Kurloff cells (KC) were analysed by affinoblotting. Lectin reactivities were quasi-exclusively confined to the 30-35 kDa major glycoproteins (mGPs) (responsible for the PAS positivity of the Kurloff body) with strong affinities for Canavalia ensiformis lectin, Phaseolus vulgaris erythroagglutinin and Sambucus nigra (SNA), Pisum sativum, Triticum vulgaris and Ulex europeus agglutinins. These data were consistent with the presence, among the KC mGPs, of large amounts of complex or hybrid N-glycosylproteins, in particular with Neu5Ac alpha 2,6Gal/GalNAc sequences, fucosyl residues and bisected residues. Their oligosaccharide sequences belong to more than one class, since some of these lectin reactivities had to be borne by distinct N-linked oligosaccharide chains. Before further analysis, KC mGPs were separated from other highly anionic glycoconjugates, by DEAE-cellulose chromatography. Their abundant potential RCA-binding sites masked by sialic acid were then revealed after neuraminidase (sialidase) or dilute acid pre-treatment. In remaining consistent with their lectin affinities, some KC mGPs were found to be PNGase F sensitive, while, either desialylated or not, they were all O-glycanase insensitive. Finally, by combined zymography and affinoblotting, the SNA-reactive fraction of KC mGPs was shown to correspond to denatured forms of the two zymographic size populations (190 kDa and 500 kDa) of KC acid phosphatases.

Acid Phosphatase↗

PubMed1991

17 beta estradiol affects the expression of guinea pig blood leukocyte MHC antigens.

Sex hormones have an effect on various immune responses but the mechanisms of action are unknown. One of these mechanisms might be a modification of expression of major histocompatibility complex (MHC) antigens in blood leucocytes. Estradiol-induced variations of the expression of guinea pig blood leukocytes MHC antigens (GPL-A) was studied. Class I and class II MHC antigens were detected by a sensitive rosetting method using specific alloimmune sera (AIS) and staphylococcal protein A-coated sheep red blood cells (SPA-SRBC) and evaluated by counting the number of bound SPA-SRBC per 100 cells. MHC antigens decreased after estrogen treatment. Estradiol modifies the expression of GPL-A antigens on the mononuclear cells including the Kurloff cells, which are involved in immunity or in a natural killer effect, but did not affect the expression of polymorphonuclear cells, ones which are not involved in immunity.

Animals↗

PubMed1991

The Kurloff cell in estrogenized guinea pigs as a CT7+ 8BE6- CT6- MR-1- CT10- IgM- lymphocyte with natural killer activity.

The relationship of the Kurloff cell (KC), guinea pig blood mononuclear cell with natural killer (NK) activity, to a known cell lineage was established. Using indirect immunoperoxidase staining and flow cytometric analysis, numerous monoclonal antibodies directed against guinea pig macrophage antigen, Ia antigen or different T lymphocyte markers and a polyclonal anti-IgM serum were tested in unimmunized estrogenized animals. We excluded any relationship between KC and the monocytic macrophage lineage (MR-1-) and between KC and the B lymphocyte lineage (CT10- and IgM-). The KC immunophenotype was pan T CT7 positive but 8BE6 (mature thymocyte) and CT6 (cytotoxic suppressor T lymphocyte) negative. Since KC displays an NK activity, this cell may be classified among the NK effector cells exhibiting some T lymphocyte markers.

Animals↗

PubMed1989

Comparison of two techniques used to collect normal and motile sperm.

To improve the quality of sperm for intra-uterine insemination (IUI) or in-vitro fertilization (IVF) swim-up (SU) and migration-sedimentation (MS) methods were compared, for the selection of morphologically normal and motile sperm. In 74 patients consulting for couple infertility, it was shown that MS gave a better yield of motile sperm than did SU. An improvement in morphology and motility was achieved, especially in asthenospermia (motility less than 40%) and/or teratospermia (normal shape less than 40%). The percentage of midpiece and tail abnormalities was lowered by the MS technique. It is proposed that the MS method be used for IUI and IVF, adapting the number of tubes to the initial sperm concentration.

Fertilization in Vitro↗

PubMed1989

Kurloff cell proteoglycans: presence of two main size-populations of intracellular protease-resistant proteochondroitin sulphate. Effect of D-xyloside.

This paper reports that the Kurloff cell sulphated and chondroitinase AC sensitive material previously described filtered on Sepharose CL4B columns as 2 main populations with Kav of 0.25 and 0.44. Its alkaline treatment resulted in the elution of 2 peaks with Kav of 0.52 and 0.78. Their reduction in size observed after alkaline treatment and the 6-fold increase in the (35S) sulphate incorporation after addition of 0.1 mM xyloside to the incubation medium indicate that these intracellular sulphated glycosaminoglycans exist in the form of proteoglycans. They were characterized by their resistance to degradation by pronase, papain or cathepsin D, as assessed by gel filtration chromatography on Sepharose CL6B or CL4B. After the glycosaminoglycans were digested with chondroitinase AC, thin-layer chromatography analysis indicated the presence of delta di-4S and delta di-6S in a ratio of 7:1. The presence of such protease-resistant proteochondroitin sulphate in intracytoplasmic granules of both Kurloff cells and other natural killer cell types is emphasized.

Animals↗

PubMed1988

Presence of low affinity estrogen binding sites in guinea-pig Kurloff cells.

Estrogen treatment of guinea-pig leads to an increase in the number of lymphoid cells containing Kurloff inclusions. The presence of estradiol binding sites in cytosolic extracts of Kurloff cells was investigated. We confirm here our previous inability to measure the typical type I estrogen receptor by using the classical dextran-charcoal procedure to separate bound and free ligand. We report now that low affinity estrogen binding sites (Kd approximately equal to 11 nM) can be detected when Kurloff cell extracts were fractionated on hydroxylapatite or Sephadex LH-20 after binding assay. Although the real function of these binding sites remains to be defined, the question arises again whether Kurloff cell represents a target cell for estrogens.

Animals↗

PubMed1988

Cytochemical localization of arylsulfatase in guinea-pig Kurloff cells.

Kurloff cells are mononuclear cells characterized by a large metachromatic and PAS-positive inclusion called the Kurloff body. Bone-marrow and spleen Kurloff cells were incubated with p-nitrocatechol sulfate as substrate and barium chloride as capturing agent for the ultracytochemical detection of the lysosomal marker enzyme, arylsulfatase. Enzymatic reaction product was consistently found as a single spot-like deposit confined to the rim of the Kurloff body. These results, and the previously described presence of other acid hydrolases and sulfated glycosamino++glycans, emphasize the similarities between the Kurloff body and lysosomes. Reaction product could also be found occasionally in segments of the rough endoplasmic reticulum but it was absent from the Golgi apparatus. This arylsulfatase activity could be related to the natural killer activity of Kurloff cells.

Animals↗

PubMed1988

Alpha naphthyl acetate esterase activities in guinea pig Kurloff cells: a cytochemical and electrophoretic study.

We established the presence of nonspecific esterases in the Kurloff cell (KC) by cytochemical methods at both light and electron microscope levels. Acid alpha-naphthyl acetate esterase (ANAE) activities were localized on the external face of the plasma membrane and on the external surface of the membrane surrounding the Kurloff body. Different cytosoluble KC extracts were obtained from purified splenic KC suspensions. About 18 isoenzymes were observed by isoelectric focusing, whereas after polyacrylamide gradient gel electrophoresis in native conditions almost all activity was observed on a few broad bands with very high apparent molecular weights, suggesting their oligomeric arrangement. After a first aqueous extraction step which released only a few isoenzymes, the remaining pellet was subjected to Triton X-100. This released almost all the isoenzymes observed after direct Triton X-100 extraction. These data suggest that almost all the KC esterases are membrane-bound enzymes, in agreement with the subcellular enzyme distribution. Different substrates were also used to characterize the different specificities of the KC isoesterases. Weak activity was detected with alpha-naphthyl butyrate by light cytochemistry, which essentially corresponded, on zymograms, to the membrane-bound esterase activity.

Animals↗

PubMed1987

Zymograms of Kurloff cell acid phosphatases. Thin layer isoelectric focusing and native polyacrylamide 4-15% gradient gel electrophoresis.

Following previous light and ultrastructural cytochemical reports, this paper presents the first zymograms of Kurloff cell acid phosphatases extracted from highly purified cell suspensions. Using isoelectric focusing, up to 20 isoenzymes were observed both with hexazotised pararosanilin or fast garnet GBC as coupler and naphthol ASB1 phosphate as substrate. Most were tartrate-labile. After Clostridium-derived neuraminidase digestion, the highly stained bands observed at pH 4-5 disappeared and, simultaneously, a few alkaline bands were enhanced. Two main bands of Mr 190,000 and 500,000 were characterized by their acid phosphatase activity after electrophoresis on a 4-15% gradient native polyacrylamide gel.

Acid Phosphatase↗

PubMed1987

A spontaneous lymphoblastic lymphoma in a guinea-pig.

The exceptional carcinoresistance of guinea-pigs is well known and only a few cases of spontaneous leukaemia and several very rare cases of lymphoma have been reported in the literature. A spontaneous malignant tumor in an old male hybrid guinea-pig is described. The monomorphous aspect of the tumour, the cellular cohesiveness, the "starry-sky" pattern and the immunocytological characterization of tumour cells as B lymphocytes bearing IgM, lead to describe this tumour as a probable spontaneous Burkitt-type malignant lymphoblastic lymphoma.

Animals↗

PubMed1986

Kurloff cell proteoglycans. Evidence for de novo synthesis of chondroitin sulphate proteoglycans by purified Kurloff cells.

This paper reports the first direct demonstration of de novo synthesis of chondroitin sulphate proteoglycans by Kurloff cells. This was achieved using highly purified splenic Kurloff cells labelled in vitro with [35S]sulphate and D-[U-3H]glucosamine. A single population of sulphated proteoglycans was observed after dissociative extraction, DEAE-cellulose chromatography, Sepharose CL 6B chromatography and fluorography after electrophoresis. These were large, highly anionic proteoglycans and were completely digested by chondroitinase AC or ABC. Moreover, glycosaminoglycan extracted from Kurloff cells had the electrophoretic mobility of control chondroitin sulphate.

Animals↗

PubMed1986

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in Kurloff cells.

After stimulation of guinea pigs with estradiol to increase their Kurloff cell number, spleen imprints were prepared in order to detect non-specific acid phosphatase (AcPase) activity by light microscopic cytochemistry using naphthol AS-BI phosphate as substrate and pararosanilin or fast garnet GBC as coupler. For ultracytochemistry, Kurloff cells were prepared from spleens by filtration through a homogeneizer screen followed by repeated centrifugation. AcPase and trimetaphosphatase activities were tested using beta-glycerophosphate, cytidine-5'-monophosphate and inorganic trimetaphosphate as substrates. Significant enzymatic activities were demonstrated with all the substrates used in the cytoplasmic inclusion body of the Kurloff cells.

Acid Anhydride Hydrolases↗

PubMed1985

Electrophoretic characterization of the protein and glycoprotein content of purified Kurloff cell cytosol.

Proteins and glycoproteins from Kurloff cells (KC) were analyzed by SDS-polyacrylamide electrophoresis, isoelectric focusing, and two-dimensional electrophoresis, and major cytosolic glycoproteins of Mr 30,000-35,000 and pHi 5.7-6.7 were characterized. After incubation with radiolabeled amino acids (L-35S) methionine and L-(U14C) leucine) and gel autoradiography, all the proteins seemed to be labeled. D-(U14C) glucosamine-labeled proteins and periodic-acid-Schiff(PAS)-positive proteins focalized at the same pH. These data suggest that the major glycoprotein are synthesized by the KC themselves and that the PAS-positive Kurloff body has an endogenous origin. Whereas estrogens increase the KC number, 10(-6) M estradiol had no effect on the KC protein electrophoretic pattern and protein biosynthesis, in agreement with the lack of estradiol receptor in the KC cytosol.

Amino Acids↗