Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis

The polysaccharide chains of enterobacterial common antigen (ECA) consist of linear trisaccharide repeat units with the structure -->3)- alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1--> 4)-alpha-d-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido-4, 6-dideoxy-d-galactose, ManNAcA is N - acetyl-d- mannosaminuronic acid, and GlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phospholipid moiety functions to anchor molecules in the outer membrane. The ECA trisaccharide repeat unit is assembled as a polyisoprenyl-linked intermediate which has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc- pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a block-polymerization mechanism. However, the identity of the polyisoprenoid carrier-lipid has not been established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier involved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNAcA-GlcNAc- pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol, undecaprenol. These analyses also resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be 1,2-diacyl- sn -glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP- disaccharide). An examination of purified ECAPGby phosphorus-31 nuclear magnetic resonance spectroscopy confirmed that the polysaccharide chains are linked to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does not appear to be an intermediate in ECAPGsynthesis. Nevertheless, although the available evidence clearly indicate that lipid II is a precursor of DGP-disaccharide, the function of this novel glycolipid is not yet known, and it may be an intermediate in the biosynthesis of a molecule other than ECAPG.      

Effect of burning on selected soil parameters in a grass fallow shifting cultivation system in Bhutan

Grass fallow shifting cultivation is an important land use practice in the highlands of Bhutan. Part of the nutrient pool contained in soil organic matter is made available for the traditional buckwheat (Fagopyrum tataricum) crop through a highly labor intensive system exposing 250–500 MT soil ha⁻¹ to temperatures of 500°C and above. Dry topsoil is collected in mounds and burned using plant biomass or manure and soil organic matter as fuel. Labor input ranged from 150–401 days ha⁻¹ with land preparation accounting for 65–85% of the total requirement. The burning increased soil pH from 6.0 to 6.9 and available K from 34 to 69 mg kg⁻¹. Soil organic C and total N were reduced from 3.3 and 0.17% to 0.8 and 0.08%, respectively. Release of P from soil organic matter and plant material and reduction in C/N ratio resulting in increased N availability are considered the most essential effects required for good crop yields. Through the burning about 16 MT of C and 470 kg N ha⁻¹ are released into the atmosphere. Fallow periods of 15–20 years are required for the system to be sustainable. The research was supported by the Department of Agriculture. Royal Government of Bhuttan and the Swiss Association for Technical Assistance The research was supported by the Department of Agriculture. Royal Government of Bhuttan and the Swiss Association for Technical Assistance     

Selection of a chemically defined medium for culturing fetal mouse small intestine

Summary We evaluated six commercially available tissue culture media in their capacity to support villi morphogenesis and enterocyte differentiation during duodenal development of the fetal mouse in vitro: McCoy's 5A, Medium 199, Swim's S77, Trowell T8, Leibovitz L-15, and RPMI-1640. The duodenal segments were resected at 15 d gestation, before the formation of intestinal villi. In the segments cultured with the first four media, no villi differentiated even at 72 h culture. The number of epithelial cells per transverse section of the explants did not increase at 24 h and thereafter the number of epithelial cells decreased, except with McCoy's 5A. With the Leibovitz and RPMI media, rudimentary villi differentiated at 24 h of culture and they attained their longest length at 48 h. With the RPMI medium, the number of epithelial cells doubled at 24 h of culture and with Leibovitz medium it doubled at 48 h. At the fine structural level absorptive cells remained poorly differentiated with all the media studied. Goblet cells were easily identified after 24 h culture; they had a well developed rough endoplasmic reticulum and numerous mucous granules. Endocrine cells differentiated in culture and they were loaded with secretion granules. It was concluded that the small intestine of the fetal mouse can be kept in organ culture for at least 72 h. Full maturation of absorptive cells seemed to require some additional factor(s) as they remained poorly differentiated with all the media studied. Because well differentiated endocrine cells were present in all the explants, it appeared that gastrointestinal hormones do not affect villi morphogenesis and absorptive cells differentiation. This investigation was supported by Grant MA-6069 from the Medical Research Council of Canada. Mr. P. A. Micheletti was supported by a studentship from the FCAC.     

Bacteriochlorophyll and Photosynthetic Reaction Centers in Rhizobium Strain BTAi 1

Rhizobium strain BTAi 1, which nodulates both stems and roots of Aeschynomene indica L., formed bacteriochlorophyll and photosynthetic reaction centers resembling those of purple photosynthetic bacteria when grown aerobically ex planta under a light-dark cycle. Bacteriochlorophyll formation was not observed under continuous dark or light growth conditions. The amount of pigment formed was similar to that previously found in aerobic photosynthetic bacteria. Stem nodules appear to fix nitrogen photosynthetically, as illumination of A. indica stem nodules with near-infrared light resulted in an enhanced rate of acetylene reduction. Near-infrared light did not enhance acetylene reduction when either A. indica or soybean root nodules were illuminated. The BTAi 1 isolate can be differentiated from members of the family Rhodospirillaceae by several criteria.     

Further freeze-fracture studies on the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela) using the antibiotic filipin

Summary The apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipinsterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.We are indebted to Mrs. K. Ott for excellent technical assistance, to Miss E.S. MacLure for linguistic corrections and to Dr. J.E. Grady of the Upjohn Co., Kalamazoo, Michigan USA, for kindly providing the filipin     

Antenatal screening by measurement of symphysis-fundus height.

A study was undertaken to assess the value of symphysis-fundus measurement as a screening procedure for intrauterine growth retardation. The reproducibility of this measurement was investigated in two groups of six patients, each measured six times by six different observers. The intraobserver coefficient of variation was 4.6% and the interobserver coefficient of variation 6.4%. There was no evidence that experience aided consistency. A chart of symphysis-fundus measurements derived from Cardiff data was found to be similar to others previously published, and one measurement below the 10th centile identified 64% of pregnancies in which the eventual birth weight was below the 10th centile for gestational age. Symphysis-fundus measurement is a useful screening test; one chart could be used for any Caucasian population and should be incorporated into the maternity services "co-operation card."     

Klinik und Genetik des Joubert-Syndroms

Joubert syndrome generally represents an autosomal recessive and rarely X-linked disorder characterized by hypotonia, an irregular breathing pattern, abnormal eye movements, ataxia, developmental delay and a complex mid-hindbrain malformation causing the molar tooth sign on magnetic resonance imaging (MRI). Many patients have additional features, with nephronophthisis, retinal dystrophy, coloboma and hepatic fibrosis representing the most frequent features. Due to its clinical variability and overlap with other syndromes, the term “Joubert syndrome and related disorders” (JSRD) was proposed. To date 10 genes are known to cause JSRD. The encoded proteins are localized to cilia, linking JSRD to other human ciliopathies.     

Conformationally-restricted vigabatrin analogs as irreversible and reversible inhibitors of gamma-aminobutyric acid aminotransferase

Compounds that inhibit gamma-aminobutyric acid aminotransferase exhibit anticonvulsant activity; vigabatrin is a known irreversible inhibitor of this enzyme and anticonvulsant drug. Conformationally-restricted, five-membered- and six-membered-ring vigabatrin analogs were synthesized and tested as inhibitors of gamma-aminobutyric acid aminotransferase. Two monofluorinated compounds, 4 and 5, are time-dependent inhibitors of the enzyme, and their potencies are comparable to that of vigabatrin. Compounds 6 and 7 are weak reversible inhibitors.     

Pharmacologic enhancement of rat skin flap survival with topical oleic acid

This study was instituted to investigate in a rat model the effect of topical coadministration of the penetration enhancer oleic acid (10% by volume) and RIMSO-50 (medical grade dimethyl sulfoxide, 50% by volume) on rat skin flap survival. A rectangular abdominal skin flap (2.5 x 3 cm) was surgically elevated over the left abdomen in 40 nude rats. The vein of the flap's neurovascular pedicle was occluded by placement of a microvascular clip, and the flap was resutured with 4-0 Prolene to its adjacent skin. At the end of 8 hours, the distal edge of the flap was reincised to gain access to the clips and the clips were removed. After resuturing of the flap's distal edge to its adjacent skin, the 40 flaps were randomly divided into four groups. Group 1 (control) flaps were treated with 5 g of saline, group 2 (dimethyl sulfoxide) flaps were treated with 2.7 g of dimethyl sulfoxide (50% by volume), group 3 flaps (oleic acid) were topically treated with 0.45 g of oleic acid (10% by volume), and group 4 (dimethyl sulfoxide plus oleic acid) flaps were treated with a mixture of 0.45 g of oleic acid (10% by volume) and 2.7 g of dimethyl sulfoxide (50% by volume) diluted in saline. Each flap was topically treated with 5 ml of drug-soaked gauze for 1 hour immediately after clip removal to attenuate reperfusion injury. Thereafter, drug was applied topically once daily for 4 more days. Digital photographs of each flap were then taken on day 6 and the flaps were then harvested. The percentage of skin survival in each flap was determined by computerized morphometry and planimetry. The mean surviving area of group 3 (oleic acid-treated flaps) was 23.60 +/- 4.19 percent and was statistically higher than that in group 1 (control, saline-treated flaps) at 7.20 +/- 2.56 percent. The mean surviving area of group 2 (dimethyl sulfoxide-treated flaps) at 18.00 +/- 5.23 percent and group 4 (oleic acid- and dimethyl sulfoxide-treated flaps) at 9.90 +/- 3.44 percent did not achieve statistically higher mean surviving areas than controls. A topical solution of oleic acid (10% by volume) caused a statistically significant increase in the survival of rat abdominal skin flaps relative to controls. Dimethyl sulfoxide and the two experimental drugs together did not increase the percentage of flap survival when given as a single 5-ml dose released from a surgical sponge at reperfusion for 1 hour and then daily for a total of 5 days. The reasons for the lack of response are unknown but may have included the technical difficulty of delivering an adequate dose of dimethyl sulfoxide topically and immiscibility between dimethyl sulfoxide and oleic acid. Further studies may be warranted.