Proportions of diether, macrocyclic diether, and tetraether lipids in Methanococcus jannaschii grown at different temperatures.

Growth of Methanococcus jannaschii over a wide temperature range (47 to 75 degrees C) is correlated with an ability to alter dramatically the proportions of three ether lipid cores. These lipids shifted from predominantly diether (2,3-di-O-phytanyl-sn-glycerol) at the lower growth temperatures to macrocyclic diether and tetraether at near optimal growth temperatures. Lipid head groups varied as well, especially with respect to an increase in phosphate at the higher temperatures.     

Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles.

To purify and characterize the isoform of prostaglandin endoperoxide synthase (rPGSi) induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles, solubilized cell extracts were subjected to anionic exchange chromatography, column fractions were resolved by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and rPGSi was visualized by immunoblotting and silver staining techniques. Immunoreactive rPGSi and peroxidase activity co-eluted at pH 6.5 and 6.0. Amino-terminal amino acid sequence of three immunoreactive rPGSi bands (Mr = 72,000, 70,000, and 59,000) were identical, Mr = 59,000 being a proteolytic fragment. Alignment of the amino-terminal sequences of rPGSi with those of ovine PGS (oPGS) indicated that only 15 of 26 residues were identical (58% identity). In contrast, rPGSi was closely related to the deduced amino acid sequence of PGS-related cDNA clones isolated from chicken and mouse cell lines, with 77% (20/26 residues) and 96% (25/26 residues) identity, respectively. Whereas tryptic digests of oPGS generated fragments of Mr = 38,000 and Mr = 33,000, only a small peptide appeared cleaved from the carboxyl terminus of rPGSi. Peroxidase activity of partially purified rPGSi exhibited lower apparent Km and maximal velocity than that of oPGS. Collectively, these results document the existence of a novel rat PGS isoform (based on purification, enzymatic activity, and amino-terminal amino acid sequence) which is hormonally induced and obligatory for a known biological process, ovulation.     

Genetic and environmental determinants of dental occlusal variation in twins of different nationalities

We have compared 10 occlusal traits in 358 monozygous and dizygous twin pairs in 4 different samples and estimated genetic variances for these features. Variable and frequently nonsignificant genetic variance was noted across samples for incisal overbite and overjet, sagittal molar relationship, posterior crossbite, and rotations and displacements of anterior teeth. Heritability estimates (when appropriately calculated) were low in magnitude (0-40%) and erratic, emphasizing the importance of environmental influences on occlusal variation and the variability of apparent genetic determinants with respect to the environment or population in which they are measured.     

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.     

Skewed X inactivation in a female MZ twin results in Duchenne muscular dystrophy.

One of female MZ twins presented with muscular dystrophy. Physical examination, creatine phosphokinase levels, and muscle biopsy were consistent with Duchenne muscular dystrophy (DMD). However, because of her sex she was diagnosed as having limb-girdle muscular dystrophy. With cDNA probes to the DMD gene, a gene deletion was detected in the twins and their mother. The de novo mutation which arose in the mother was shown by novel junction fragments generated by HindIII, PstI, or TaqI when probed with cDNA8. Additional evidence of a large gene deletion was given by novel SfiI junction fragments detected by probes p20, J-Bir, and J-66 on pulsed-field gel electrophoresis (PFGE). Immunoblot analysis of muscle from the affected twin showed dystrophin of normal size but of reduced amount. Immunofluorescent visualization of dystrophin revealed foci of dystrophin-positive fibers adjacent to foci of dystrophin-negative fibers. These data indicate that the affected twin is a manifesting carrier of an abnormal DMD gene, her myopathy being a direct result of underexpression of dystrophin. Cytogenetic analysis revealed normal karyotypes, eliminating the possibility of a translocation affecting DMD gene function. Both linkage analysis and DNA fingerprint analysis revealed that each twin has two different X chromosomes, eliminating the possibility of uniparental disomy as a mechanism for DMD expression. On the basis of methylation differences of the paternal and maternal X chromosomes in these MZ twins, we propose uneven lyonization (X chromosome inactivation) as the underlying mechanism for disease expression in the affected female.     

Concepts of occlusion: Australian evidence

Longitudinal studies of aboriginal children over a 20-year period have drawn attention to the wide variation in morphological features of the dentition and the way in which occlusal relationships develop. This paper summarizes some important determinants of optimal occlusal development, namely, tooth size relationships within and between dentitions, the patterns of alveolar growth, and tooth migrations during the transition from primary to permanent teeth and the nature of growth changes in the dental arches. Dental occlusion constantly changes throughout life in response to changing functional requirements. Observations limited to cross-sectional material provide an incomplete, and sometimes misleading, concept of dental occlusion and masticatory function.     

Measurement of release of endogenous GABA and catabolites of [3H]GABA from synaptosomal preparations using ion-exchange chromatography

Picomole quantities of endogenous GABA in acidified superfusates of synaptosomal preparations have been measured using micro-bore ion-exchange chromatography and post-column formation of the fluorescent iso-indole derivative. Using this technique superfusates have been analyzed directly, without further manipulations, to investigate the release of endogenous GABA. Spontaneous release of GABA was 2–5 pmol/200 l superfusate increasing to 20 pmol/200 l with potassium stimulation. When -vinyl GABA (RMI 71754), an inhibitor of GABA-T was injected into rats (750 mg/kg) and synaptosomes prepared the potassium-evoked release of GABA was increased 3-fold compared to controls. Chromatographic separations and measurement of release of endogenous and radiolabeled GABA allowed the real specific activity of released GABA to be calculated. Only when 500 M amino-oxyacetic acid was added during isolation of synaptosomes was the specific activity of released GABA the same as the initial specific activity.     

Scanning electron microscopy ofMycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Summary Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth’s MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the “parent” explant was removed. Monolayer cultures infected withMycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments ofM. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or “sperules” of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites forM. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas. Electron microscopy was done with the cooperation of Dr. R. Macleod and the staff of the Center for Electron Microscopy at the University of Illinois. Critical editorial review was provided by C. Dayton. This investigation was supported in part by grants to M. G. G. from the National Institute of Allergy and Infectious Diseases (AI 12559) and the National Heart, Lung, and Blood Institute (HL 23806), Bethesda, Maryland.