Itzhak Kahane

In Vivo and In Vitro Impairment of Human and Ram Sperm Nuclear Chromatin Integrity by Sexually Transmitted Ureaplasma urealyticum Infection

Abstract The incidence of Ureaplasma urealyticum infection in the semen of infertile men is variable (7%–42%). Evidence has accumulated through routine semen analysis to suggest that this infection can cause embryo loss without necessarily affecting sperm quality. The aim of this study was to specifically investigate the effects of U. urealyticum infection on sperm chromatin stability and DNA integrity, which are known to be correlated to pregnancy outcome. Sperm cells isolated from human semen infected in vivo with U. urealyticum exhibited a low percentage of stable chromatin as determined by nuclear chromatin decondensation assay (42% ± 4.8%, n = 8) and a high percent of denatured DNA as determined by sperm chromatin structure assay (60.9% ± 9.1%, n = 7). After doxycyclin treatment, a significant improvement in both parameters was observed (73.7% ± 3.6%, P < 0.001 and 30.1% ± 3.5%, P < 0.008, respectively). Sperm cells infected in vitro exhibited higher rates of viability and motility than uninfected cells. In contradistinction, U. urealyticum caused significant dose- and time-dependent chromatin decondensation and DNA damage. The percentage of human sperm cells with denatured DNA increased significantly by 54.9% ± 23.9% and 47.9% ± 12.1%, after 30 min infection with serotypes 8 and 3, respectively, at a multiplicity of infection of 100 ureaplasmas per sperm compared with uninfected control cells. The damage to DNA was significantly more pronounced in infected ram sperm (180.9% ± 21.5%). These results indicate that preserved sperm activity post U. urealyticum infection resulted in damage to paternal DNA, although a high fertilization rate was maintained, and embryonic development may, therefore, be impaired.

Distinguishing between Mn-containing and Fe-containing superoxide dismutases in crude extracts of cells.

Abstract Superoxide dismutases containing manganese or iron can be resolved by exposure to low pH in the presence of guanidinium chloride. Apoenzymes so produced are inactive and are reactivated only by the metal characteristic of the native enzymes. In crude extracts of procaryotes, which may contain the iron enzymes or the manganese enzyme or both together, one can, by resolution followed by treatment with Fe(II) or Mn(II), identify and distinguish these enzymes. This method was validated with extracts of Escherichia coli , known to contain both types of enzymes, and was then used to demonstrate the presence of an iron superoxide dismutase in Alcaligenes faecalis and of manganese superoxide dismutases in Streptococcus sanguis, S. lactis, Bacillus megaterium , and Acholeplasma laidlawii . The A. laidlawii enzyme was found to have a molecular weight of ~41,000.

Cross‐linking of red blood cell membrane proteins induced by oxidative stress in β thalassemia

The structure-function relationship of the normal red blood cell (RBC) membrane has been extensively studied [ 1] and in many ways the RBC membrane served as a model for the understanding of membrane physiology in general. Recently there is a growing interest in characterizing RBC membrane defects in several congenital hemoltyic disorders where changes in the membranes are not considered to be the primary defect of the disease, but nevertheless contribute to the pathophysiology of the hemolytic process. Alterations in membrane structure were reported in hemoglobinopathies such as sickle cell anemia [2] and in thalassemia [3,4]. In the latter syndrome, direct and indirect evidence indicate that the pathological RBC are exposed to higher oxidative stress when compared to normal RBC [5] , as a result of increased production of ‘activated oxygen’, such as superoxide, peroxide, singlet oxygen and hydroxyl radicals [S] . These radicals eventually oxidize various RBC components including membrane proteins and lipids, as indicated by the significantly lower titratable SH groups (about 50% normal) [3] , while lipid oxidation was suggested by the lower ratio of unsaturated to saturated fatty acids of membrane lipids [4] . In addition, malonildialdehyde (MDA), a secondary breakdown product of lipid peroxidation, was generated in excess amounts in thalassemic RBC,

Characterization of the mycoplasma membrane proteins II. Solubilization and enzymic activities of Acholeplasma laidlawii membrane proteins

Abstract Treatment of Acholeplasma laidlawii membranes with EDTA in low-ionic strength media released about 11% of the total membrane protein in a water-soluble form. The released protein fraction had no NADH oxidase, ATPase and p -nitrophenylphosphatase activities. The strongly ionic detergents sodium dodecyl sulfate and cetyltrimethylammonium bromide were more effective in the solubilization of A. laidlawii membranes than the nonionic detergents Triton X-100, Lubrol W or Brij 58. Sodium deoxycholate occupied an intermediate position. The solubilization of the membranes by detergents affected their NADH oxidase, ATPase and p -nitrophenylphosphatase activities in two antagonistic ways: activation and inactivation. The balance of these processes depended on the type and concentration of the detergent used and on the enzymic activity tested. The activation effect was most pronounced with low concentrations of the nonionic detergents and with p -nitrophenylphosphatase activity. Inactivation of the enzymes was most pronounced with sodium dodecyl sulfate and cetyltrimethylammonium bromide. The results of the present study favor the use of nonionic detergents for the solubilization and further fractionation of mycoplasma membrane proteins.

Synthesis and turnover of membrane protein and lipid in Mycoplasma laidlawii

Abstract The relatioship of lipid to protein synthesis in Mycoplasma laidlawii membranes was studied in heavy suspensions of the organisms in complex or partially defined media. Under the test conditions used the organisms did not multiply, and the amount of total cell protein remained constant. Lipid synthesis was measured by the incorporation of [3H]oleic acid into membrane lipids, while protein synthesis was measured by the incorporation of l -[14C]phenylalanine into membrane proteins. Though the ability of the cells to synthesize both membrane proteins and lipids declined steeply with age their protein-synthesizing ability was impaired at a much earlier stage. When membrane protein synthesis was arrested by treating the cells with chloramphenicol, the membrane lipid synthesis was not affected, so that the cell membranes formed had a density of 1.158 g/cm3 as against 1.170 g/cm3 for untreated cell membranes. The total membrane proteins labeled with l -[14C]phenylalanine turned over at a relatively high rate, having a half life of approx. 3 h. Turnover of the total membrane lipids labeled with [3H]oleic acid became apparent only after a lag period of several hours after the beginning of the chase. It was thus found that in M. laidlawii membrane lipid synthesis can be uncoupled from the synthesis of the membrane protein, so that the two processes are not necessarily synchronized. The possibility of varying the lipid to protein ratio in biological membranes without affecting their function is discussed.

Characterization of the myoplasma membrane proteins: III. Gel filtration and immunological characterization of Acholeplasma laidlawii membrane proteins

Abstract Acholeplasma laidlawii (formerly Mycoplasma laidlawii) membranes solubilized by ionic and nonionic detergents were fractionated on Sephadex G-200 columns containing the detergent used for solubilization. When sodium dodecyl sulfate or sodium deoxycholate were present, membrane lipids were resolved as a single elution peak while membrane proteins formed several reproducible peaks. Gel filtration in the presence of the nonionic detergents Triton X-100, Brij 58 and Lubrol W was usually inferior. The proteins were eluted as two broad peaks, one of which included the void volume. Since the NADH oxidase, ATPase and p- nitrophenylphosphatase activities of the solubilized membranes could be detected in the first peak, containing little or no lipid, it appears that these enzymes do not depend on membrane lipids for activity. Serological activity could be demonstrated in membrane fractions isolated by gel filtration, even when sodium dodecyl sulfate was used. However, the use of deoxycholate enabled the separation of a membrane protein fraction highly enriched in antigens which elicited the production of antibodies inhibiting A. laidlawii metabolism and growth. A high content of antigens located on the outer membrane surface was evidenced by the high agglutination titer of A. laidlawii cells exhibited by antisera to this fraction.

Caenorhabditis briggsae and C. elegans: partial characterization of cuticle surface carbohydrates.

Abstract The presence of galactose, glucose, mannose, and N-acetylglucosamine on the exposed surface of the nematodes Caenorhabditis briggsae and C. elegans was indicated by specific binding of three iodinated plant lectins. Proteolysis experiments suggested the absence of digestible glycoproteins on the exposed surfaces of the two nematode species. High resolution micrographs of cuticle surface preparations labeled with cationized ferritin indicated that the negative charge-bearing molecules are more densely packed on the nematode surface than on animal plasma membranes.

Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.

Phagocytosis of Nucleated and Mature β Thalassaemic Red Blood Cells by Mouse Macro-phages in Vitro

Summary. Physiological or experimental decrease in sialic acid (SA) content on the red blood cell (RBC) membrane is believed to play an important role in the recognition of these cells by macrophages. Since there is a 20–30% decrease in the SA content on the membrane of thalassaemic RBC, the interaction between macrophages and these RBC was studied in vitro. Using mouse peritoneal macrophages, it was found that these macrophages ‘recognize’and phagocytize thalassaemic RBC while RBC from normal donors are hardly phagocytized. The average level of phagocytosis of thalassaemic RBC from splenectomized patients was found to be 22‐fold higher than that of RBC from normal donors. The phagocytized cells consisted of both mature and nucleated RBC. Mouse peritoneal macrophages seem to be a useful in vitro system for the study of the accelerated sequestration and shortened life span of thalassaemic RBC.

The erythrocyte membranes in β-thalassemia. Lower sialic acid levels in glycophorin

The sialic acids content of glycophorin of thalassemic erythrocyte membranes is about 25% lower than in glycophorin of normal erythrocyte membranes. Glycophorin extracted from old thalassemic erythrocytes separated by density centrifugation, has about half the sialic acids content found in glycophorin extracted from young thalassemic erythrocytes. Possible sialidase activty was sought in the plasma and erythrocyte membranes of thalassemic erythrocytes. No increased sialidase activity was detected in the plasma of the patients as compared to that of normal donors. Thus, other sites for sialidase activity, or other possibilities have to be explored to account for the increased sialic acid hydrolysis of glycophorin of the thalassemic erythrocytes.