Zvi Ne'eman

Cholesterol in mycoplasma membranes. Composition, ultrastructure and biological properties of membranes from Mycoplasma mycoides var. capri cells adapted to grow with low cholesterol concentrations.

Abstract 1. 1. Serial passages of the sterol-requiring Mycoplasma mycoides var. capri in a serum-free medium supplemented with decreasing concentrations of cholesterol resulted in the adaptation of the organisms to grow with no added cholesterol. The cells of the adapted strain were osmotically more fragile than cells of the native strain and were more permeable to erythritol. 2. 2. The cell membrane of the adapted strain contained low amounts of cholesterol (up to 3% of the total membrane lipid as against 22–25% in the native strain) but its polar lipids were more saturated than those of the native strain, as reflected by the preferential incorporation of [14C]palmitic acid when added to the growth medium together with [14C]oleic acid. 3. 3. The native strain was capable of growing at temperatures as low as 25°C, whereas the adapted strain could not. The lowering of the growth temperature of the native strain resulted in a decrease in the cholesterol content of the membrane (from 24–15% of the total lipid) but had no effect on the fatty acid composition of the membrane polar lipids. On the other hand, aging of the culture increased the ratio of saturated to unsaturated fatty acids in both adapted and native strains, and decreased the cholesterol content of the native strain. 4. 4. Freeze-etching of cells of the native and adapted strains, kept at 37°C prior to freezing, showed a random distribution of particles on the fracture faces of the cell membranes. Keeping the cells at 4°C prior to freezing caused the aggregation of particles on the fracture faces of the adapted strain but had no effect on the distribution of particles in the native strain. 5. 5. Our data support the thesis that cholesterol functions as a regulator of membrane fluidity and that changes in the fatty acid composition of membrane lipids may act to compensate for the lack of cholesterol.

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.

Selective reaggregation of solubilized mycoplasma-membrane proteins and the kinetics of membrane reformation

Abstract 1. 1. Proteins and lipids of mycoplasma membranes solubilized in sodium dodecyl sulfate reaggregated to membraneous structures when the detergent was diluted by dialysis against a Mg 2+ -containing buffer. The Mg 2+ concentration determined the degree of reaggregation, the lipid-to-protein ratio in the reaggregate and, as shown by electrophoretic and enzymic analyses, the species of membrane proteins aggregated. 2. 2. The reassembly of the solubilized membrane components to membraneous structures proceeded rapidly. After 40 min of dialysis against 20 mM Mg 2+ , the reaggregate already contained membranes of a similar triple-layered structure and thickness as the original ones, but with a smaller number of protein species and a higher lipid-to-protein ratio. 3. 3. On density-gradient analysis reaggregates of Mycoplasma laidlawii membranes were found to be heterogeneous, while at 5 mM Mg 2+ only a “light” lipid-rich band ( d = 1.140) was obtained. At higher Mg 2+ concentrations this was accompanied by one or two heavier bands. The “light” band was transformed into a heavier one when the Mg 2+ concentration was increased. 4. 4. Our data suggest that the reaggregated membranes are assembled by a multi-step process and not by the single-step assembly of lipoprotein subunits. 5. 5. The application of the selective aggregation of solubilized membrane proteins to the fractionation and characterization of membrane enzymes and antigens is suggested and 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.

Osteoporosis in the Cohen Diabetic Rat: Correlation Between Histomorphometric Changes in Bone and Microangiopathy

Osteoporosis is well documented in type I diabetes, but its occurrence is controversial in type II diabetes. Microangiopathy is a major complication of type I and type II diabetes. We studied bone and microvascular changes in the Cohen diabetic rat, a unique nonobese model of noninsulin-dependent diabetes mellitus. The aim of this study was to find whether there is a temporal correlation between the onset of these two complications. The diabetic rats were divided into three groups (A, B, and C) according to duration of diabetes (2 months, 3 months, and 7 to 8 months, respectively). Trabecular bone area was assessed by computerized image analysis and microangiopathy by means of renal function tests, histologic examination of the kidneys, and ultrastructural measurement of the width of capillary basement membranes. Bone density of the distal femur and vertebra was significantly reduced in the diabetic rats relative to the control rats in all three groups (Group A femur: 11.5 ± 1.6% versus 21.8 ± 3.0%, p < 0.02; Group A vertebra: 15.9 ± 1.6% versus 28.5 ± 2.0%, p < 0.02; Group C femur: 7.9 ± 1.1% versus 29.6 ± 3.5%, p < 0.001; Group C vertebra: 11.4 ± 0.7% versus 37.1 ± 1.9%, p < 0.002). Renal function tests were normal in the Group A diabetic rats and there was marked albuminuria in the Group C diabetic rats. Histologic changes in the kidneys were seen only in the Group C diabetic rats. Five of 15 Group C diabetic rats showed no albuminuria or histologic evidence of kidney damage. The bone density in this subgroup was reduced relative to controls to the same degree as that of the rats with renal damage. There was no evidence of capillary basement membrane thickening in the Group A diabetic rats. Our findings indicate that in the Cohen diabetic rat, osteoporosis precedes the onset of microangiopathy. Microangiopathy probably does not play an important role in the pathogenesis of osteoporosis in this animal model.

Characterization of the mycoplasma membrane proteins: V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50% of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group. Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [125-I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core. Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.

Childhood macrophagic myofasciitis—consanguinity and clinicopathological features

Macrophagic myofasciitis has been almost exclusively detected in adults only. We describe six children of Arab Moslem origin with this disorder. Three presented with hypotonia, developmental delay and seizures and were evaluated for a mitochondrial disorder. The other three children had hypotonia and predominantly motor delay. Five of the six families were consanguineous. A massive collection of macrophages was present in the fascia and adjacent epimysium in all biopsies. The macrophages were periodic-acid-Schiff positive and immunoreactive for CD68. One biopsy which was evaluated by electron microscopy and energy-dispersive X-ray microanalysis showed crystalline structures containing aluminum in macrophages. Two children with motor delay and hypotonia were treated with oral prednisone for 3 months with no clinical improvement. Genetic predisposition probably accounts for the variability in the prevalence of macrophagic myofasciitis in different populations. At least in childhood, there seems to be no connection between macrophagic myofasciitis as a pathological entity and the clinical symptoms and signs.

Alveolar soft-part sarcoma of the female genital tract

SummaryAn alveolar soft-part sarcoma of the uterine cervix observed in a 26-year-old women is described. One year after extended radical hysterectomy there was no evidence of tumor recurrence. To the best of our knowledge, this is the second reported case of alveolar soft-part sarcoma arising in the female genital tract and the first description of this tumor in the uterine cervix.

Localization of glycogen in the placenta of diabetic rats: a Light and electron microscopic study

The ultrastructure of the placentae on day 20 of gestation was studied in rats made diabetic by streptozotocin injection on day 13 of gestation. In the placentae of control rats most of the glycogen was found in the glycogen cells, while some of it was localized to the labyrinth trophoblastic layers. In the diabetic rats a marked increase in glycogen content, together with higher numbers of glycogen cells in the junctional zone, was seen. Glycogen was also stored in other cell types of this zone, as well as in all cell types of the placental labyrinth of the diabetic animals.

Prenatal diagnosis of oculocutaneous albinism type I: review and personal experience.

ABSTRACT Oculocutaneous albinism type I (OCA I) comprises autosomal recessive syndromes of hypopigmentation and low vision, caused by the lack of tyrosinase activity. Affected families seek genetic counseling and prenatal diagnosis as preventive measures. Until recently, prenatal diagnosis of OCA I was achieved by histologic and electron microscopic examination of fetal skin biopsies. Lately, a molecular genetic approach has become possible by the identification of the two mutated copies of the TYR gene, coding the tyrosinase, in which over 60 mutations have been identified.We report here our experience in prenatal diagnosis of OCA I using the two strategies. Thirty-four prenatal tests were performed in fetuses at risk for OCA I. In 31 cases the diagnosis was made in fetal scalp biopsies using the histological approach. The microscopic observations revealed normal melanogenesis in 26 biopsies. Five albino fetuses were diagnosed by the demonstration of arrest of melanogenesis in early stages I and II. In three pregnancies, molecular genetic tests were performed on DNA extracted from amniocytes, using direct mutation analysis (in one), and complemented by linkage analysis (in two). One albino and two normally pigmented fetuses were diagnosed.The prenatal molecular genetic test can be applied to families when at least one mutation is diagnosed in the albino patient. The histological approach is applicable in all families at risk for OCA I.