Complete amino acid sequence of a variant surface glycoprotein (VSG 117) from Trypanosoma brucei

The amino acid sequence of a variant surface glycoprotein (VSG 117) of Trypanosoma brucei has been determined by manual sequencing of tryptic. staphylococcal protease and cyanogen bromide peptides and fragments derived from these peptides. Some overlaps needed for completion of the sequence were deduced from the nucleotide sequence of complementary DNA derived from messenger RNA coding for VSG 117. The glycoprotein consists of 470 amino acid residues with two carbohydrate chains attached at Asn420 and Asp470. No pronounced hydrophobic regions, which are characteristic of many membrane proteins, are present in the isolated glycoprotein, and the carboxy-terminal region, which is close to the membrane, is remarkably hydrophilic. These observations indicate that the molecule probably does not penetrate the lipid bilayer of the plasma membrane. The high proportion of charged residues in the carboxyterminal region is more consistent with electrostatic interaction with the polar head groups of the phospholipids.     

Retention of human skin fibroblast fatty acid modifications during maintenance culture

Summary The fatty acid composition of cultured human skin fibroblasts was modified by adding either oleic or linoleic acid to the growth medium. After the cultures became confluent, they were washed and transferred to different maintenance media in order to determine the stability of the various fatty acyl modifications. Some changes in fatty acid composition occurred under all conditions. When the maintenance medium was supplemented with fatty acid, the cellular neutral lipid and phospholipid fatty acyl composition were altered markedly within 16 to 24 hr. If no supplemental fatty acid was available during the maintenance period, however, the modified fatty acyl compositions were sufficiently retained so that appreciable differences between the cells enriched with oleate and linoleate persisted for at least 48 to 72 hr. This considerable degree of stability occurred when either 10% delipidized fetal bovine serum or 10% fetal bovine serum containing its inherent lipids were present in the maintenance medium. Although the triglyceride content of the fatty acid-modified cells was quite labile, neither the cholesterol nor phospholipid content changed appreciably during culture in any of the maintenance media. Since the fatty acid compositional differences persisted during several days of maintenance under certain conditions, these modified cultures appear to be a useful experimental system for assessing the effect of lipid structure on fairly long-term cellular functions. This work was supported by Arteriosclerosis Specialized Center of Research Grant HL14230 from the National Heart, Lung and Blood Institute, National Institutes of Health.     

Five-year perspective of the large-scale growth of mammalian cells in suspension culture

The Cell Culture Center at the University of Alabama in Birmingham was set up to produce large quantities of cells and their products from suspension cultured cell lines. This system has now been in operation for over five years and has been effective in producing large quantities of mammalian cells of murine and human origin. This article describes the system and some growth parameters which have been of importance for large-scale mammalian cell growth.     

Purification and preliminary characterization of two asclepains from the latex of Asclepias syriaca L. (milkweed).

Two groups of asclepains have been isolated from Asclepias syriaca L. (milk-weed) latex and a representative of each has been purified. Asclepains A3 and B5 are homogeneous proteins with molecular weights of 23 000 and 21 000, respectively. Both require a reducing and chelating agent for maximum activity and hydrolyze ester, amide and peptide bonds. The optimum pH for hydrolysis of casein is 7.5 to 8.5 for asclepain A3 and 7.0 to 7.5 for asclepain B5. Both enzymes are autolytic when active and are inhibited by p-chloromercuribenzoate, iodoacetic acid and sodium tetrathionate. Asclepains A3 and B5 each contain one titratable SH group per molecule and no bound carbohydrate. Each of the two enzymes has leucine as the N-terminal amino acid. There are notable differences in their amino acid compositions.     

Role of bovine serum albumin in the nutrition of Mycobacterium tuberculosis.

Bovine serum albumin promotes the growth of small inocula of Mycobacterium tuberculosis in media containing unesterified fatty acids. Albumin binds fatty acids present in concentrations toxic for the organisms. In the present study, additional roles of albumin were investigated. When present in a basal medium, fatty acid-free albumin could be utilized by M. tuberculosis as a sole source of carbon. Since albumin could not substitute for the amino acids in basal medium as a nitrogen source, it was concluded that the protein component in albumin was not utilized as a nutrient by the organisms. An ether extract of fatty acid-free albumin supported a small but significant amount of growth. Analysis of the lipids in fatty acid-free albumin by gas chromatography revealed the presence of 686 microgram of fatty acid per g of albumin. Although a small amount of growth occurred when a lipid extract of albumin was present in the medium, growth stimulation was dependent in major part on the presence of undenatured albumin in the medium. Lipids, when bound to albumin, can serve as a nontoxic source of carbon and energy.     

Genetic influences in self-reported symptoms of obstructive sleep apnoea and restless legs: a twin study.

Sleep disorders, such as obstructive sleep apnoea (OSA) and restless legs syndrome (RLS), are very common. The relative importance of genetic and nongenetic (environmental) influences on the symptomatology of these conditions has not been well studied. This study uses the twin design to examine this by evaluating OSA and RLS symptoms in monozygotic (MZ) and dizygotic (DZ) twins. Six thousand six hundred unselected female twin pairs, identified from a national volunteer twin register, were asked to complete a medical questionnaire. This questionnaire included questions on OSA and RLS symptoms, as well as questions on subject demographics, past medical history, smoking history and menopausal status. Responses were obtained from 4503 individuals (68% response rate). A total of 1937 twin pairs were evaluable: 933 MZ pairs (mean [range] age 51 [20-76] years) and 1004 DZ pairs (age 51 [20-80] years). Concordance rates were higher for MZ than DZ twins for OSA and RLS symptoms. Multifactorial liability threshold modeling suggests that additive genetic effects combined with unique environmental factors provide the best model for OSA and RLS symptoms. Heritability was estimated to be 52% (95% confidence interval 36% to 68%) for disruptive snoring, 48% (37% to 58%) for daytime sleepiness, 54% (44% to 63%) for restless legs, and 60% (51% to 69%) for legs jerking. These estimates dropped only slightly after adjustment for potential confounding influences on the symptoms of snoring and daytime sleepiness. These results suggest a substantial genetic contribution to the symptomatology of OSA and RLS. More research is needed to identify the genes responsible, and may ultimately lead to new therapies.     

New circuits for old memories: the role of the neocortex in consolidation

Studies of learning and memory have provided a great deal of evidence implicating hippocampal mechanisms in the initial storage of facts and events. However, until recently, there were few hints as to how and where this information was permanently stored. A recent series of rodent molecular and cellular cognition studies provide compelling evidence for the involvement of specific neocortical regions in the storage of information initially processed in the hippocampus. Areas of the prefrontal cortex, including the anterior cingulate and prelimbic cortices, and the temporal cortex show robust increases in activity specifically following remote memory retrieval. Importantly, damage to or inactivation of these areas produces selective remote memory deficits. Additionally, transgenic studies provide glimpses into the molecular and cellular mechanisms underlying cortical memory consolidation. The studies reviewed here represent the first exciting steps toward the understanding of the molecular, cellular, and systems mechanisms of how the brain stores our oldest and perhaps most defining memories.     

Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation

The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. We set out to study the actin cross-linking activity of Kelch and how Kelch function is regulated. Biochemical studies using purified, recombinant Kelch protein showed that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Two-dimensional electrophoresis demonstrated that Kelch is tyrosine phosphorylated in a src64-dependent pathway. Site-directed mutagenesis determined that tyrosine residue 627 is phosphorylated. A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescued the actin disorganization phenotype of kelch mutant ring canals, but failed to produce wild-type ring canals. Electron microscopy demonstrated that phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the nonphosphorylatable KelY627A protein phenocopied src64 ring canals. KelY627A protein in ring canals also dramatically reduced the rate of actin monomer exchange. The phenotypes caused by src64 mutants and KelY627A expression suggest that a major function of Src64 signaling in the ring canal is the negative regulation of actin cross-linking by Kelch.     

<Emphasis Type=Italic>Aulonemia cochabambensis</Emphasis> (Poaceae: Bambusoideae: Bambuseae: Arthrostylidiinae), an anomalous new species from Bolivia

Aulonemia cochabambensis (Poaceae: Bambusoideae: Bambuseae: Arthrostylidiinae), a new species from the Department of Cochabamba, Bolivia, is described and illustrated. It has foliage leaves with delicate fimbriae, no sheath auricles, narrow blades, an abaxial dark marginal stripe, and intercostal sclerenchyma; few-flowered paniculate synflorescences; and robust, awned spikelets. The new species is compared with its putative relatives Aulonemia laxa and Arthrostylidium schomburgkii. A key to the species of Aulonemia in Bolivia is also included.