Cryptococcal meningitis in a tertiary hospital in Pretoria, mortality and risk factors - A retrospective cohort study.

Aim This retrospective cohort study analyzes the impact of possible risk factors on the survival chance of patients with cryptococcal meningitis. These factors include the patient's socio-economic background, age, gender, presenting symptoms, comorbidities, laboratory findings and, in particular, non-adherence versus adherence to therapy. Methods Data were collected from all adult patients admitted to Kalafong Hospital with laboratory confirmed cryptococcal meningitis over a period of 24 months. We analyzed the data by the presentation of descriptive summary statistics, logistic regression was used to assess factors which showed association between outcome of measure and factor. Furthermore, multivariable logistic regression analysis using all the factors that showed significant association in the cross tabulation was applied to determine which factors had an impact on the patients' mortality risk. Results A total of 87 patients were identified. All except one were HIV-positive, of which 55.2% were antiretroviral therapy naïve. A history of previous tuberculosis was given by 25 patients (28.7%) and 49 (56.3%) were on tuberculosis treatment at admission or started during their hospital stay. In-hospital mortality was 31%. Statistical analysis showed that antiretroviral therapy naïve patients had 9.9 (CI 95% 1.2-81.2, p < 0.0032) times greater odds of dying compared to those on antiretroviral therapy, with 17 from 48 patients (35.4%) dying compared with 1 out of 21 patients (4.8%) on treatment. Defaulters had 14.7 (CI 95% 1.6-131.6, p < 0.016) times greater odds of dying, with 9 from 18 patients dying (50%), compared to the non-defaulters. In addition, patients who presented with nausea and vomiting had a 6.3 (95% CI 1.7-23.1, p < 0.005) times greater odds of dying (18/47, 38.3%); this remained significant when adjusted for antiretroviral therapy naïve patients and defaulters. Conclusion Cryptococcal meningitis is still a common opportunistic infection in people living with HIV/AIDS resulting in hospitalization and a high mortality. Defaulting antiretroviral therapy and presentation with nausea and vomiting were associated with a significantly increased mortality risk.

Monitoring gene expression using DNA microarrays.

The concurrent development of high-density array technologies and the complete sequencing of a number of microbial genomes is providing the opportunity to comprehensively and efficiently survey the transcription profile of microorganisms under different conditions and well-defined genotypes. Microarray-based studies are uncovering broad patterns of genetic activity, providing new understanding of gene functions and, in some cases, generating unexpected insight into transcriptional processes and biological mechanisms. One topic that has come to the forefront is how best to effectively manage and interpret the large data sets being generated. Although progress has been made, this remains a challenging opportunity for functional genomics research.

FSP95, a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa.

Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of approximately 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.

Characterization of the serogroup O11 O-antigen locus of Pseudomonas aeruginosa PA103.

We previously cloned a genomic DNA fragment from the serogroup O11 Pseudomonas aeruginosa strain PA103 that contained all genes necessary for O-antigen synthesis and directed the expression of serogroup O11 antigen on recombinant Escherichia coli and Salmonella. To elucidate the pathway of serogroup O11 antigen synthesis, the nucleotide sequence of the biosynthetic genes was determined. Eleven open reading frames likely to be involved in serogroup O11 O-antigen biosynthesis were identified and are designated in order as wzzPaO111 (wzz from P. aeruginosa serogroup O11), wzxPaO11, wbjA, wzyPaO11, wbjB to wbjF, wbpLO11 and wbpMO11 (wbpL and wbpM from serogroup O11). Consistent with previous descriptions of O-antigen biosynthetic gene loci, the entire region with the exception of wbpMO11 has a markedly reduced G+C content relative to the chromosomal average. WzyPaO11 shows no significant similarity at the protein or DNA sequence level to any database sequence and is very hydrophobic, with 10 to 12 putative transmembrane domains, both typical characteristics of O-antigen polymerases. A nonpolar chromosomal insertion mutation in wzyPaO11 in P. aeruginosa PA103 confirmed the identity of this gene. There is striking similarity between WbjBCDE and Cap(5/8)EFGL, involved in type 5 and type 8 capsule biosynthesis in Staphylococcus aureus. There is nearly total identity between wbpMO11 and wbpMO5, previously shown by others to be present in all 20 P. aeruginosa serogroups. Using similarity searches, we have assigned functions to the proteins encoded by the PA103 O-antigen locus and present the potential steps in the pathway for the biosynthesis of P. aeruginosa serogroup O11 O antigen.

Panning for genes--A visual strategy for identifying novel gene orthologs and paralogs.

We have developed a rapid visual method for identifying novel members of gene families. Starting with an evolutionary tree, 20-50 protein query sequences for a gene family are selected from different branches of the tree. These query sequences are used to search the GenBank and expressed sequence tag (EST) DNA databases and their nightly updates using the tfastx3 or tfasty3 programs. The results of all 20-50 searches are collated and resorted to highlight EST or genomic sequences that share significant similarity with the query sequences. The statistical significance of each DNA/protein alignment is plotted, highlighting the portion of the query sequence that is present in the database sequence and the percent identity in the aligned region. The collated results for database sequences are linked using the WWW to the underlying scores and alignments; these links can also be used to perform additional searches to characterize the novel sequence further. With traditional "deep" scoring matrices (BLOSUM50) one can search for previously unrecognized families of large protein superfamilies. Alternatively, by using query sequences and EST libraries from the same species (e. g., human or mouse) together with "shallow" scoring matrices and filters that remove high-identity sequences, one can highlight new paralogs of previously described subfamilies. Using query sequences from the glutathione transferase superfamily, we identified two novel mammalian glutathione transferase families that were recognized previously only in plants. Using query sequences from known mammalian glutathione transferase subfamilies, we identified new candidate paralogs from the mouse class-mu, class-pi, and class-theta families.

Novel allergen structures with tandem amino acid repeats derived from German and American cockroach.

Cockroaches produce potent allergens that are an important cause of asthma. The two principal domiciliary cockroach species, Blattella germanica and Periplaneta americana, secrete major allergens, Bla g 1 and Per a 1. Here, we report the molecular cloning of three Bla g 1 cDNA clones, which showed 70% amino acid sequence identity with Per a 1. Plaque immunoassays with human IgE antibodies or murine monoclonal antibodies showed that these allergens were antigenically cross-reactive. The Bla g 1 sequences also showed homology to five previously undefined cockroach allergen sequences. An unusual feature of all these sequences was that they contained multiple tandem amino acid repeats of approximately 100 amino acid residues. Between one and seven repeat units were identified by dot-plot matrix analysis. The sequences also showed homology to a mosquito protein involved in digestion (ANG12 precursor) and to mitochondrial energy transfer proteins. High levels of Bla g 1 were found in cockroach hindgut and proventriculus. Amino acid sequencing of natural Bla g 1 and Per a 1 suggested that these allergens are cleaved by trypsin-like enzymes following secretion into the digestive tract. The repeat sequences appear to have evolved by duplication of an ancestral amino acid domain, which may have arisen from the mitochondrial energy transfer proteins.

Soluble expression in E. coli of a functional interphotoreceptor retinoid-binding protein module fused to thioredoxin: correlation of vitamin A binding regions with conserved domains of C-terminal processing proteases.

The exchange of all-trans retinol and 11-cis retinal between the photoreceptors and retinal pigmented epithelium is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP contains binding sites for retinoids, docosahexaenoic acid and probably cell surface and matrix receptors. IRBP arose through the quadruplication of an ancient protein, represented by its carboxy-terminal module (module 4 in amphibians and mammals). Module 4 has retinol binding activity and is composed of regions coded for by each of IRBP's four exons. Determining the function of the exons has been hampered by insoluble expression of module 4 in Escherichia coli. Here, we found that module 4 of Xenopus IRBP (X4IRBP), as well as its exon segments, can be expressed in a soluble form as thioredoxin fusion proteins. The recombinant proteins were purified by ion exchange and arsenical-based affinity chromatography. Liquid chromatography/mass spectrometry confirmed that the sequence of X4IRBP is correct. All-trans retinol binding was characterized by monitoring enhancement of retinol fluorescence, quenching of intrinsic protein fluorescence, and transfer of energy to the bound retinol. Retinol bound to X4IRBP at 2.20+/-0.29 sites with a KD=1.25+/-0.39. One of the two sites was localized to Exons(2+3) and had a KD=0.26+/-0.13 micron. This site, which supported protein quenching and energy transfer, probably contains at least one of the two conserved tryptophans present in this segment. The second site was localized to Exon 4. This site supported the enhancement of retinol fluorescence but not protein quenching or energy transfer and had a KD=1.94+/-0.20 micron. Exon 1 had no retinol binding activity. The location of the retinol binding regions correlated with the distribution of domains conserved between IRBPs and the newly recognized family of C-terminal processing proteases (CtpAs), proteins which bind and cleave non-polar carboxy termini.

Sebaceous carcinoma. Tumor progression through mutational inactivation of p53.

BACKGROUND: Sebaceous carcinoma may masquerade for years as an inflammatory condition. In many cases, this may be because of the presence of longstanding intraepithelial disease (e.g., dysplasia or carcinoma in situ), which eventually progresses to invasive carcinoma recognized through tumefaction and a worsening clinical presentation. The mechanism for this tumor progression is unknown. In the Far East, human papilloma virus (HPV) has been suggested to play a role in the development of sebaceous carcinoma by inactivating tumor suppressor gene p53. Here, the authors explore the molecular basis of the progression of ocular sebaceous carcinoma. METHODS: Cases of sebaceous carcinoma seen at the University of Virginia, Department of Ophthalmology, during the period from 1989 to 1996 were analyzed for HPV infection by in situ hybridization and polymerase chain reaction. The expression of p53, p21WAF-1, Bcl-2, and epithelial membrane antigen was examined by immunohistochemistry. In one of the cases, frozen tumor was available, allowing exons 5 through 9 of the p53 gene to be sequenced. RESULTS: Seven cases were identified, all of which were from women. All were negative for HPV. In cases in which disease was restricted to dysplasia (carcinoma in situ), p53 but not p21WAF-1 was negative. In contrast, cases that contained a component of invasive or metastatic carcinoma showed striking hyperexpression of nuclear p53 in all of the malignant cells. In one of these cases, a G:C-->T:A transversion was found in the p53 gene. This mutation, characteristic of bulky carcinogens, substituted phenylalanine for cysteine 277, a residue that participates in hydrogen bonding to the p53 DNA binding consensus sequence. CONCLUSIONS: Mutational inactivation of p53 may be involved in the progression of sebaceous carcinoma.

The evolution of protamine P1 genes in dasyurid marsupials.

We report the complete DNA sequences of the protamine P1 gene and flanking regions for 13 species of the marsupial family Dasyuridae. The structure of the protamine locus is conserved in dasyurids and consists of two exons (of lengths 142-151 and 47 bp) separated by an intron (208-240 bp). A key feature of the dasyurid intron is a 38-40 bp duplication found in all species examined to date. This duplication apparently predates the radiation of modern dasyurid lineages and may be homologous to a similar feature in the marsupial mole (Notoryctes). Sequences from a species of Planigale demonstrate that this genus is unique among marsupials in possessing cysteine residues in its protamine P1 molecules. Cysteines may provide enhanced chemical stability for condensed sperm nuclei, a physiological feature that would converge on the common eutherian pattern. Phylogenetic analysis of the protamine genes yields a tree that is largely congruent with previous molecular systematic studies in two areas: (1) There are three main dasyurid lineages corresponding to the Sminthopsinae. Dasyurinae, and Phascogalinae; (2) Dasyurinae and Phascogalinae are sister groups. This study is the first estimate of dasyurid relationships based on a nuclear DNA sequence.

Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales.

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.

cDNA sequence and structure of a trout HMG-2 gene. Evidence for a trout-specific 3'-untranslated region.

A complete cDNA sequence (1026 bp) and a partial structure of a gene encoding the trout testis chromosomal HMG (high mobility group) 2 protein (HMG-T2) is presented. The deduced protein consists of 214 amino acids and shares over 80% similarity to the trout HMG-1 protein (HMG-T1) as well as to the mammalian or avian HMG-2 proteins. Northern blot analysis revealed two transcripts, a major one of 1.2 kb and a minor one of 1.6 kb. Southern analysis and polymerase chain reaction of trout genomic DNA indicated that the HMG-T2 gene is encoded by several introncontaining genes. The 5'-UTR (untranslated region) of the HMG-T2 is interrupted by an intron and the coding region of the HMG-T2 is divided into four exons by three relatively short introns (173, 91 and 78 bp). The exon/intron boundaries of trout HMG-2 are identical to those of human HMG-2, as reported earlier [Shirakawa and Yoshida, J. Biol. Chem. 267 (1992) 6641-6645], suggesting the evolution of the HMG-1/2 family genes from a common ancestor. Phylogenetic analysis indicated that the common ancestor of trout HMG-1/2 genes very likely diverged from the ancestor of the mammalian (or avian) HMG-1/2 genes before its separation into two distinct mammalian or (avian) HMG-1 and HMG-2 genes. Sequence comparisons of the 3'UTR of trout HMG-2 cDNA with the corresponding regions in the mammalian (or avian) HMG-2 revealed that the trout 3'-UTR was clearly distinct from the 3'-UTR of the mammalian or avian HMG-2 cDNAs which were otherwise remarkably well conserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Evolution of protamine P1 genes in mammals.

Protamine P1 genes have been sequenced following PCR amplification from 11 mammals representing five major mammalian orders: Rodentia (rat and guinea pig), Carnivora (cat and bear), Proboscidea (elephant), Perissodactyla (horse), and Artiodactyla (camel, deer, elk, moose, and gazelle). The predicted amino acid sequence for these genes together with previously reported sequences results in a data set of 25 different P1 genes and 30 different P1 amino acid sequences. The alignment of all these sequences reveals that protamines are amongst the most rapidly diverging proteins studied. In spite of the large number of differences there are conserved motifs that are also common to birds such as the N-terminal ARYR followed by the triple alternating SRSRSR phosphorylation site. The central region contains 3 arginine clusters consisting of 5-6 arginines each. The C-terminus appears to be the most variable region of the protamines. Overall the molecular evolution of P1 genes is in agreement with the expected species evolution supporting that these genes have evolved vertically.

Molecular phylogeny and evolution of marsupial protamine P1 genes.

We report the DNA sequences of protamine P1 genes and flanking regions from 21 mammalian species, including representatives of all extant marsupial orders. The protamine P1 locus in marsupials displays highly conserved 5' and 3' flanking sequences, as well as a highly variable intron. Marsupial protamines examined are distinct from those of eutherian mammals in lacking cysteine residues, a feature that may be correlated with lower stability of marsupial sperm nuclei during chromatin condensation. Phylogenetic analysis of protamine sequences leads to the following conclusions: (i) the microbiothere Dromiciops is part of a clade that includes dasyurids, Notoryctes and diprotodontians but not bandicoots; (ii) dasyurids are the closest living relatives of Notoryctes; (iii) macropodids, phalangerids and pseudocheirids form a clade apart from the phascolarctids; and (iv) the closest living relatives of caenolestids are didelphids.

Significance of adenosine deaminase activity and its isoenzymes in tuberculous effusions.

Adenosine deaminase (ADA) activity is increased in effusions caused by certain clinical conditions including tuberculosis and bacterial infections. In this study, the ADA isoenzyme patterns in tuberculous and parainfective effusions were investigated to determine the isoenzyme responsible for this increase in activity. Fifty-one tuberculous effusions and six parainfective effusions were investigated. All effusions had increased ADA activity (median values of 126 and 127 units/L, respectively). In the tuberculous effusions, ADA2 isoenzyme was found to be primarily responsible for total activity, with a median contribution of 88 percent. The ADA1 (both ADA1m and ADA1c isoenzymes) was the major isoenzyme in the parainfective effusions with a median contribution of 70 percent. The ADA2 isoenzyme activity most likely reflects monocyte-macrophage turnover or activity, while ADA1 probably originates from lymphocytes or neutrophils. It is therefore essential to determine the isoenzyme profile when interpreting ADA activity levels in effusions. The measurement of the individual isoenzymes will enhance the diagnostic utility of ADA activity determinations in pleural effusions.

Evolution of the monotremes. The sequences of the protamine P1 genes of platypus and echidna.

The protamine P1 genes from two monotremes, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) were isolated after polymerase-chain-reaction amplification then cloned and sequenced. The two protamine P1 genes are of 290 bp and 311 bp for platypus and echidna, respectively, and are clearly orthologous to the published sequences of protamine P1 genes of eutherian mammals and birds. Both genes contain an intron, like the mammals and marsupials and unlike the bird P1 genes that are intronless. The deduced protein sequences from the coding areas of the platypus and echidna protamine P1 genes do not contain any cysteine residues. This absence of cysteine residues leaves the sperm nuclei susceptible to disruption in vitro by exposure to increasing ionic strength and is a characteristic of fish, birds and marsupials. In contrast, the P1 protamines of placental mammals invariably contain 6-9 cysteine residues that, as a result of the formation of intermolecular and intramolecular disulfide bridges, significantly increase the stability of the sperm nuclei that can only be disrupted following disulfide-bond cleavage. Phylogenetic analysis of the protamine P1 gene sequences indicates that the monotremes occupy a position half-way between the eutherian mammals and birds. From the DNA sequences we estimate the time of divergence of the platypus and the echidna to be around 22 million years ago. This date agrees very well with the published estimates of divergence based on other criteria.

Evolution of protamine P1 genes in primates.

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine P1s indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10-13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24-29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39-44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest-neighbor matrices and trees based on maximum parsimony indicates that P1 genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine P1s are expressed in germ line rather than somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)