The surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a. Analysis of its glycosylation.

Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall. In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques. The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1. Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n = 13-18), with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains. The glycan chains are bound via the disaccharide core -->3)-alpha-l-Rhap-(1-->3)-alpha-L-Rhap-(L--> and the linkage glycose beta-D-Galp in O-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794. This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized.     

Accumulation of cardiolipin and lysocardiolipin in fibroblasts from Tangier disease subjects

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. Not only cholesterol efflux, but also phospholipid efflux was shown to be impaired in TD cells. By use of thin layer chromatography, high performance liquid chromatography and time-of-flight secondary ion mass spectrometry, we characterized the cellular phospholipid content in fibroblasts from three homozygous TD patients. The cellular content of the major phospholipids was not found to be significantly altered in TD fibroblasts. However, the two phospholipids cardiolipin and lysocardiolipin, which make up minute amounts in normal cells, were at least 3–5-fold enriched in fibroblasts from TD subjects. A structurally closely related phospholipid (lysobisphosphatidic acid) has recently been shown to be enriched in Niemann–Pick type C, another lipid storage disorder. Altogether these data may indicate that the role of these phospholipids is a regulatory one rather than that of a bulk mediator of cholesterol solubilization in sterol trafficking and efflux.     

Functional Interactions of Human Immunodeficiency Virus Type 1 Integrase with Human and Yeast HSP60

Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the nuclear genome is catalyzed by the retroviral integrase (IN). In addition to IN, viral and cellular proteins associated in the high-molecular-weight preintegration complex have been suggested to be involved in this process. In an attempt to define host factors interacting with IN, we used an in vitro system to identify cellular proteins in interaction with HIV-1 IN. The yeast Saccharomyces cerevisiae was chosen since (i) its complete sequence has been established and the primary structure of all the putative proteins from this eucaryote has been deduced, (ii) there is a significant degree of homology between human and yeast proteins, and (iii) we have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype. Strong evidences suggest that this lethality is linked to IN activity in infected human cells where integration requires the cleavage of genomic DNA. Using IN-affinity chromatography we identified four yeast proteins interacting with HIV-1 IN, including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60. Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was coexpressed, therefore suggesting that both proteins interact in vivo. Besides interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro processing and joining activities of IN and protected this enzyme from thermal denaturation. In addition, the functional human HSP60-HSP10 complex in the presence of ATP was able to recognize the HIV-1 IN as a substrate.     

Mutual Mate Assessment in Wolf Spiders: Differences in the Cues Used by Males and Females

When males engage in conspicuous courtship displays, it seems obvious that females would use characteristics of that display in mating decisions. However, males must also have a way to identify and evaluate females prior to engaging in what might be a costly mating ritual. Although it was known that female wolf spiders of the species Pardosa milvina (Araneae; Lycosidae) attract males using volatile chemical cues, the nature of the cues used by males and females in mate selection had not been investigated. Specifically we determined whether males could detect the mating status of the female and if chemotactile cues from the female played a role in that process. In addition, we quantified conspicuous aspects of the male courtship (leg raises and body shakes) to determine if courtship intensity was related to female choice. Although repeated mating occurred in our studies, males were more likely to court and mate with virgin females. Males used substrate‐borne cues deposited by females to discriminate between mated and virgin females. Females used the conspicuous behaviors of males during courtship, body shakes and leg raises, in mate selection. Thus males and females use different kinds of information and different sensory modalities to assess the suitability of a potential mate.     

Female-Directed Aggression in Free-Ranging Ateles geoffroyi

In a study of the reproductive biology and behavior of black-handed spider monkeys (Ateles geoffroyi) on Barro Colorado Island, Panama, 107 instances of female directed male aggression (male attacks) occurred during >1,000 h of observation. Two hypotheses might explain this behavior. First, males may attack to induce defecation/urination by target females, from which they can gain reproductive information. Rates of defecation and urination did not increase subsequent to attacks; however, there was an association between attacks and investigative behaviors such as place sniffing. Alternatively, the attacks might be a form of sexual coercion. My results indicate that female reproductive state, as determined by fecal steroid metabolites (E1C and PdG), had an inconsistent effect on the frequency of attacks received. In addition, when females were in the peri-ovulatory period of the ovarian cycle they did not receive more attacks than at other times. Nor were attacks associated with observed copulations, suggesting that sexual coercion does not sufficiently explain the behavior. Some support is provided for the notion that the attacks allow males to dominate equally-sized females.     

Retinal Dystrophy Due to Paternal Isodisomy for Chromosome 1 or Chromosome 2, with Homoallelism for Mutations in RPE65 or MERTK, Respectively

Uniparental disomy (UPD) is a rare condition in which a diploid offspring carries a chromosomal pair from a single parent. We now report the first two cases of UPD resulting in retinal degeneration. We identified an apparently homozygous loss-of-function mutation of RPE65 (1p31) in one retinal dystrophy patient and an apparently homozygous loss-of-function mutation of MERTK (2q14.1) in a second retinal dystrophy patient. In both families, the gene defect was present in the patient's heterozygous father but not in the patient's mother. Analysis of haplotypes in each nuclear kindred, by use of DNA polymorphisms distributed along both chromosomal arms, indicated the absence of the maternal allele for all informative markers tested on chromosome 1 in the first patient and on chromosome 2 in the second patient. Our results suggest that retinal degeneration in these individuals is due to apparently complete paternal isodisomy involving reduction to homoallelism for RPE65 or MERTK loss-of-function alleles. Our findings provide evidence for the first time, in the case of chromosome 2, and confirm previous observations, in the case of chromosome 1, that there are no paternally imprinted genes on chromosomes 1 and 2 that have a major effect on phenotype.