Identification and disruption of the gene encoding the third member of the low-molecular-mass rhoptry complex in Plasmodium falciparum

The low-molecular-mass rhoptry complex of Plasmodium falciparum consists of three proteins, rhoptry-associated protein 1 (RAP1), RAP2, and RAP3. The genes encoding RAP1 and RAP2 are known; however, the RAP3 gene has not been identified. In this study we identify the RAP3 gene from the P. falciparum genome database and show that this protein is part of the low-molecular-mass rhoptry complex. Disruption of RAP3 demonstrated that it is not essential for merozoite invasion, probably because RAP2 can complement the loss of RAP3. RAP3 has homology with RAP2, and the genes are encoded on chromosome 5 in a head-to-tail fashion. Analysis of the genome databases has identified homologous genes in all Plasmodium spp., suggesting that this protein plays a role in merozoite invasion. The region surrounding the RAP3 homologue in the Plasmodium yoelii genome is syntenic with the same region in P. falciparum; however, there is a single gene. Phylogenetic comparison of the RAP2/3 protein family from Plasmodium spp. suggests that the RAP2/3 duplication occurred after divergence of these parasite species.     

Polyglutamine-expanded androgen receptors form aggregates that sequester heat shock proteins, proteasome components and SRC-1, and are suppressed by the HDJ-2 chaperone

Spinal bulbar muscular atrophy is a neurodegenerative disorder caused by a polyglutamine expansion in the androgen receptor (AR). We show in transiently transfected HeLa cells that an AR containing 48 glutamines (ARQ48) accumulates in a hormone-dependent manner in both cytoplasmic and nuclear aggregates. Electron microscopy reveals both types of aggregates to have a similar ultrastructure. ARQ48 aggregates sequester mitochondria and steroid receptor coactivator 1 and stain positively for NEDD8, Hsp70, Hsp90 and HDJ-2/HSDJ. Co-expression of HDJ-2/HSDJ significantly represses aggregate formation. ARQ48 aggregates also label with antibodies recognizing the PA700 proteasome caps but not 20S core particles. These results suggest that ARQ48 accumulates due to protein misfolding and a breakdown in proteolytic processing. Furthermore, the homeostatic disturbances associated with aggregate formation may affect normal cell function.     

T cell subsets in idiopathic glomerulonephritis

Determination of T lymphocyte subsets using monoclonal antibodies revealed a deficiency of suppressor cells and an elevated helper:suppressor T cell ratio in 7 patients with untreated idiopathic immune complex glomerulonephritis and nephrotic syndrome. Possible pathogenetic mechanisms of immunoregulatory cell imbalance and their implications specifically in reference to idiopathic glomerulonephritis are discussed.     

Monoclonal antibodies to human secretory "pregnancy-associated endometrial alpha 1-globulin," an insulin-like growth factor binding protein: characterization and use in radioimmunoassay, Western blots, and immunohistochemistry

Monoclonal antibodies were raised against pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 32 KD insulin-like growth factor binding protein (IGF-BP), which represents a major secretory product of the human decidualized endometrium during pregnancy. This class of IGF-BP has been implicated in the modulation of action, inhibitory and stimulatory, of insulin-like growth factors. Immunization with the protein purified from pregnancy endometrium resulted after myeloma fusion in the isolation of six hybridoma clones and the antibodies produced were characterized. The Ka of the antibodies ranged between 4.75 x 10(9) M-1 and 0.7 x 10(8) M-1. In Western blots all monoclonal antibodies reacted with purified protein of molecular weight 32 KD and specifically detected this IGF-BP species in culture medium and cytosolic extracts of pregnancy endometrium and amniotic fluid. The monoclonal antibodies appear to define three epitope-bearing regions as evidenced by their reactivity to polypeptide fragments of the protein. After synthesis and secretion by tissue explants in vitro the protein is susceptible to cleavage into fragments possessing different monoclonal antibody-defined reactivity. Employing immunohistochemical techniques the protein was principally localized to decidual cells in tissue sections of pregnancy endometrium and solely to these cells after enzymic digestion of the tissue. The implications of these results are discussed with respect to potential role of IGF-BP in the action of IGF upon the IGF-1 receptor-bearing populations, including lymphocytes and trophoblast cells, D in the decidua.     

On the ultrastructural diversity and essence of residual bodies in neuronal ceroid-lipofuscinosis.

In 4 patients with neuronal ceroid-lipofuscinoses (NCL) (3 patients with the junvenile type, 1 patient with the late infantile type), the ultrastructural spectrum of residual bodies in the central and peripheral nervous system presented curvilinear profiles in all cases and regions investigated and many more ultrastructural patterns within and beyond regions commonly accessible to biopsy, probably due to age dependence, local tissue and cellular biochemical factors. Sampling from basal ganglia especially yielded combined curvilinear-fingerpint bodies, from peripheral ganglia additional membranous bodies. Residual bodies in NCL were present in almost every cell type, similar to the distribution of regular lipofuscin. Although the classical subgroups of NCL contain electronmicroscopically well defined residual bodies, permitting distinction of the late infantile type from the juvenile type, the ultrastructural differences are more of a quantitative than of a qualitative nature. However, they are not pathognomonic. N.m.r. spectra of ceroid and lipofuscin support the concept of their biochemical similarity, and argue against the proposition that they contain a single major component.     

Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.

The gene coding for protein A (spa) of Staphylococcus aureus 8325-4 has been inactivated by substituting part of the spa coding sequence for a DNA fragment specifying resistance to ethidium bromide. The in vitro-constructed spa::EtBrr substitution mutation was introduced into the S. aureus chromosome by recombinational allele replacement. Southern blot hybridization showed that the in vitro-constructed mutation was present in the chromosomal spa locus. We have previously reported the inactivation of the alpha-toxin gene (hly) by allele replacement with an in vitro-constructed hly::Emr (erythromycin resistance) mutation (M. O'Reilly, J.C.S. de Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathogen. 1:125-138, 1986). A double Spa- Hly- mutant was constructed by transduction. The virulence of Spa- and Hly- mutants was tested by experimental infection of mice. When subcutaneous injections were given, Hly- mutants formed a flat, darkened lesion, whereas Hly+ strains caused a raised, cream lesion. Alpha-toxin was shown to be a major factor in forming subcutaneous lesions and in causing the death of mice injected intraperitoneally. Spa- mutants were slightly less virulent than their Spa+ counterparts, which suggests that protein A is also a virulence factor of S. aureus.     

Thyroid hormone and development of the rat hippocampus: cell acquisition in the dentate gyrus

A quantitative autoradiographic histological study was carried out to examine mechanisms underlying the reduction in the rates of growth and of cell acquisition, including that of granule cells, in the dentate gyrus of hypothyroid rats. Thyroid deficiency in early life had no effect on the replication of intrinsic cells present in the polymorph and granular layers. The pyknotic index was also normal in the "proliferative zone", polymorph layer and granule cell layer, indicating that thyroid hormone had no effect on the survival of replicating, migrating or maturing granule cells. By contrast, the arrival of migrating cells from the "proliferative zone" to the granular layer was severely retarded in thyroid deficiency. This deficit was rapidly restored after a physiological dose of thyroxine given to hypothyroid rats. The present findings are consistent with our previous proposal that the role of thyroid hormone in the formation and/or the maintenance of nerve cells is related to changes in either cell migration or maturation, rather than to alterations in the replication of germinal cells.     

Fine mapping of a gene responsible for regulating dietary cholesterol absorption; founder effects underlie cases of phytosterolaemia in multiple communities

Sitosterolaemia (also known as phytosterolaemia, MIM 210250) is a rare recessive autosomal inherited disorder, characterised by the presence of tendon and tuberous xanthomas, accelerated atherosclerosis and premature coronary artery disease. The defective gene is hypothesised to play an important role in regulating dietary sterol absorption and biliary secretion, thus defining a molecular mechanism whereby this physiological process is carried out. The disease locus was localised previously to chromosome 2p21, in a 15 cM interval between microsatellite markers D2S1788 and D2S1352 (based upon 10 families, maximum lodscore 4.49). In this study, we have extended these studies to include 30 families assembled from around the world. A maximum multipoint lodscore of 11.49 was obtained for marker D2S2998. Homozygosity and haplotype sharing was identified in probands from non-consanguineous marriages from a number of families, strongly supporting the existence of a founder effect among various populations. Additionally, based upon both genealogies, as well as genotyping, two Amish/Mennonite families, that were previously thought not to be related, appear to indicate a founder effect in this population as well. Using both homozygosity mapping, as well as informative recombination events, the sitosterolaemia gene is located at a region defined by markers D2S2294 and Afm210xe9, a distance of less than 2 cM.