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S Engelbrecht

Publications and source records attributed to S Engelbrecht.

At least 19 recordsLinked to original sources
PubMed2006

Serotyping and genotyping of HIV-1 infection in residents of Khayelitsha, Cape Town, South Africa.

It is estimated that between 5.5 and 6.1 million people are infected with HIV/acquired immunodeficiency syndrome (AIDS) in South Africa, with subtype C responsible for the majority of these infections. The Khayelitsha suburb of Cape Town has one of the highest HIV prevalence rates in South Africa. Overcrowding combined with unemployment and crime in parts of the area perpetuates high-risk sexual behavior, which increases exposure to infection by HIV. Against this background, the objective of this study was to characterize HIV-1 in residents confirmed to be seropositive. Serotyping was performed through a competitive enzyme-linked immunosorbent assay (cPEIA). Genotyping methods included RNA isolation followed by RT-PCR and sequencing of the gag p24, env gp41 immunodominant region (IDR), and env gp120 V3 genome regions of HIV-1. With the exception of a possible C/D recombinant strain, all HIV-1 strains were characterized as HIV-1 group M subtype C. One individual was shown to harbor multiple strains of HIV-1 subtype C. In Southern Africa, the focus has been to develop a subtype C candidate vaccine, as this is the major subtype found in this geographical area. Therefore, the spread of HIV-1 and its recombinant strains needs to be monitored closely.

Adult↗
PubMed2005

Microbial content of commercial South African high-moisture dried fruits.

AIM: The aim was to evaluate commercially available South African high-moisture dried fruits (HMDF) for the microbial, moisture and SO2 contents, as well as aw and pH. METHODS AND RESULTS: The microbial content of commercially available HMDF was evaluated using nine different growth media. The moisture content, aw) SO2 and pH of each product were determined using standard analytical methods. It was found that the highest total aerobic counts were generated from high-moisture dried (HMD) prunes and raisins. The most frequent spoilers were members of the genus Bacillus. Fungal counts were also very high in the apricot products, exceeding the limit of 1000 CFU g(-1) as set by HMDF producers. Members of the genus Staphylococcus were found in the HMD raisins and Salmonella and thermoduric organisms were isolated from the HMD prunes. CONCLUSIONS: The microbial levels of South African HMDF were within the limits set, with the exception of apricots. SIGNIFICANCE AND IMPACT OF STUDY: The study shows the presence of Salmonella, Staphylococcus and Clostridium in South African HMDF. The presence of thermoduric organisms indicated that the current pasteurization process is not adequate and that the addition of preservatives would be an additional method to ensure safety and quality.

Bacillus↗
PubMed2003

Molecular characteristics of human immunodeficiency virus type 1 subtype C viruses from KwaZulu-Natal, South Africa: implications for vaccine and antiretroviral control strategies.

The KwaZulu-Natal region of South Africa is experiencing an explosive outbreak of human immunodeficiency virus type 1 (HIV-1) subtype C infections. Understanding the genetic diversity of C viruses and the biological consequences of this diversity is important for the design of effective control strategies. We analyzed the protease gene, the first 935 nucleotides of reverse transcriptase, and the C2V5 envelope region of a representative set of 72 treatment-naïve patients from KwaZulu-Natal and correlated the results with amino acid signature and resistance patterns. Phylogenetic analysis revealed multiple clusters or "lineages" of HIV-1 subtype C that segregated with other C viruses from southern Africa. The same pattern was observed for both black and Indian subgroups and for retrospective specimens collected prior to 1990, indicating that multiple sublineages of HIV-1 C have been present in KwaZulu-Natal since the early stages of the epidemic. With the exception of three nonnucleoside reverse transcriptase inhibitor mutations, no primary resistance mutations were identified. Numerous accessory polymorphisms were present in the protease, but none were located at drug-binding or active sites of the enzyme. One frequent polymorphism, I93L, was located near the protease/reverse transcriptase cleavage site. In the envelope, disruption of the glycosylation motif at the beginning of V3 was associated with the presence of an extra protein kinase C phosphorylation site at codon 11. Many polymorphisms were embedded within cytotoxic T lymphocyte or overlapping cytotoxic T-lymphocyte/T-helper epitopes, as defined for subtype B. This work forms a baseline for future studies aimed at understanding the impact of genetic diversity on vaccine efficacy and on natural susceptibility to antiretroviral drugs.

AIDS Vaccines↗
PubMed2001

Genetic analysis of the complete gag and env genes of HIV type 1 subtype C primary isolates from South Africa.

South Africa has one of the fastest growing HIV-1 epidemics, with an estimated 4.7 million people infected. To better understand the genetic diversity of this epidemic and its potential impact on vaccine development, we have cloned and sequenced the complete gag and env genes of 13 primary virus isolates. Phylogenetic analysis of our sequences and 69 complete env genes from the Los Alamos and GenBank databases revealed multiple subclusters within subtype C. The V3 loop region was relatively conserved in all our strains when compared with other subtypes, but the region immediately downstream was highly variable. No intersubtype recombinant forms were observed when comparing the gag and env sequences. Characterization of the complete gag and env genes enabled us to select specific strains for further vaccine development.

Amino Acid Sequence↗
PubMed2001

Inter-subunit rotation and elastic power transmission in F0F1-ATPase.

ATP synthase (F-ATPase) produces ATP at the expense of ion-motive force or vice versa. It is composed from two motor/generators, the ATPase (F1) and the ion translocator (F0), which both are rotary steppers. They are mechanically coupled by 360 degrees rotary motion of subunits against each other. The rotor, subunits gamma(epsilon)C10-14, moves against the stator, (alphabeta)3delta(ab2). The enzyme copes with symmetry mismatch (C3 versus C10-14) between its two motors, and it operates robustly in chimeric constructs or with drastically modified subunits. We scrutinized whether an elastic power transmission accounts for these properties. We used the curvature of fluorescent actin filaments, attached to the rotating c ring, as a spring balance (flexural rigidity of 8.10(-26) N x m2) to gauge the angular profile of the output torque at F0 during ATP hydrolysis by F1. The large average output torque (56 pN nm) proved the absence of any slip. Angular variations of the torque were small, so that the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the three-fold stepping and high activation barrier (>40 kJ/mol) of the driving motor (F1) itself, the rather constant output torque seen by F0 implied a soft elastic power transmission between F1 and F0. It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate under load of the two counteracting and stepping motors/generators.

Actins↗
PubMed2001

F-ATPase: forced full rotation of the rotor despite covalent cross-link with the stator.

In ATP synthase (F(O)F(1)-ATPase) ion flow through the membrane-intrinsic portion, F(O), drives the central "rotor", subunits c(10)epsilongamma, relative to the "stator" ab(2)delta(alphabeta)(3). This converts ADP and P(i) into ATP. Vice versa, ATP hydrolysis drives the rotation backwards. Covalent cross-links between rotor and stator subunits have been shown to inhibit these activities. Aiming at the rotary compliance of subunit gamma we introduced disulfide bridges between gamma (rotor) and alpha or beta (stator). We engineered cysteine residues into positions located roughly at the "top," "center," and "bottom" parts of the coiled-coil portion of gamma and suitable residues on alpha or beta. This part of gamma is located at the center of the (alphabeta)(3) domain with its C-terminal part at the top of F(1) and the bottom part close to the F(O) complex. Disulfide bridge formation under oxidizing conditions was quantitative as shown by SDS-polyacrylamide gel electrophoresis and immunoblotting. As expected both the ATPase activities and the yield of rotating subunits gamma dropped to zero when the cross-link was formed at the center (gammaL262C <--> alphaA334C) and bottom (gammaCys(87) <--> betaD380C) positions. But much to our surprise disulfide bridging impaired neither ATP hydrolysis activity nor the full rotation of gamma and the enzyme-generated torque of oxidized F(1), which had been engineered at the top position (gammaA285C <--> alphaP280C). Apparently the high torque of this rotary engine uncoiled the alpha-helix and forced amino acids at the C-terminal portion of gamma into full rotation around their dihedral (Ramachandran) angles. This conclusion was supported by molecular dynamics simulations: If gammaCys(285)-Val(286) are attached covalently to (alphabeta)(3) and gammaAla(1)-Ser(281) is forced to rotate, gammaGly(282)-Ala(284) can serve as cardan shaft.

Adenosine Triphosphate↗
PubMed2001

Characterization and phylogenetic analysis of South African HIV-1 subtype C accessory genes.

To acquire new knowledge about the genetic diversity and potential impact on vaccine strategies of HIV-1 subtype C in South Africa, we have characterized the vif, vpr, and vpu genes of 15 isolates. Phylogenetic analysis of the genomic fragment encompassing these genes revealed subtype C subclusters, suggesting close relatedness with subtype C strains from other geographic locations and excluded isolation of South African strains. The putative T155 phosphorylation site in the C terminal of Vif was absent in all subtype C sequences. Variation in the predicted amino acid sequences of the three genes further showed strong correlation with other subtype C sequences.

AIDS Vaccines↗
PubMed2001

Viscoelastic dynamics of actin filaments coupled to rotary F-ATPase: angular torque profile of the enzyme.

ATP synthase (F(O)F(1)) operates as two rotary motor/generators coupled by a common shaft. Both portions, F(1) and F(O), are rotary steppers. Their symmetries are mismatched (C(3) versus C(10-14)). We used the curvature of fluorescent actin filaments, attached to the rotating c-ring, as a spring balance (flexural rigidity of 8. 10(-26) Nm(2)) to gauge the angular profile of the output torque at F(O) during ATP hydrolysis by F(1) (see theoretical companion article (. Biophys. J. 81:1234-1244.)). The large average output torque (50 +/- 6 pN. nm) proved the absence of any slip. Variations of the torque were small, and the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the threefold stepping and high activation barrier of the driving motor proper, the rather constant output torque implied a soft elastic power transmission between F(1) and F(O). It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate of the two counteracting and stepping motor/generators.

Actin Cytoskeleton↗
PubMed2000

F-ATPase: specific observation of the rotating c subunit oligomer of EF(o)EF(1).

The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, F(o), of ATP synthase, F(o)F(1), is still under contention. It was studied with EF(o)EF(1) (Escherichia coli) using microvideography with a fluorescent actin filament. To overcome the limited specificity of actin attachment through a Cys-maleimide couple which might have hampered the interpretation of previous work, we engineered a 'strep-tag' sequence into the C-terminal end of subunit c. It served (a) to purify the holoenzyme and (b) to monospecifically attach a fluorescent actin filament to subunit c. EF(o)EF(1) was immobilized on a Ni-NTA-coated glass slide by the engineered His-tag at the N-terminus of subunit beta. In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 microm(2) size, in some cases yielding a proportion of 5% rotating over total filaments. The rotation was unequivocally attributable to the ring of subunit c. The new, doubly engineered construct serves as a firmer basis for ongoing studies on torque and angular elastic distortions between F(1) and F(o).

Actins↗
PubMed1999

Rotation of Escherichia coli F(1)-ATPase.

By applying the same method used for F(1)-ATPase (TF(1)) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302), we observed ATP-driven rotation of a fluorescent actin filament attached to the gamma subunit in Escherichia coli F(1)-ATPase. The torque value and the direction of the rotation were the same as those observed for TF(1). F(1)-ATPases seem to share common properties of rotation irrespective of the sources.

Actin Cytoskeleton↗
PubMed1999

Functional identification of alpha 1-giardin as an annexin of Giardia lamblia.

A protein with a relative molecular mass of 31 kDa was specifically extracted by EGTA from a detergent-insoluble fraction of Giardia lamblia. N-terminal sequencing showed this protein to be identical to alpha 1-giardin, a component of the ventral disc which, based on its predicted amino acid sequence, has been classified as annexin XIX. Purified alpha 1-giardin associated with multilamellar phosphatidyl serine-containing vesicles in a Ca(2+)-dependent manner, confirming that it is a functional annexin. Molecular modelling of the amino acid sequence of the giardial annexin into the X-ray structure of annexin V suggests that the Ca(2+)-binding sites, which, as in other annexins, are all located on the convex surface of the molecule, are of the low-affinity type III.

Amino Acid Sequence↗
PubMed1999

HIV type 1 V3 domain serotyping and genotyping in Gauteng, Mpumalanga, KwaZulu-Natal, and Western Cape Provinces of South Africa.

More than 20.8 million people are living with HIV/AIDS in sub-Saharan Africa, with southern Africa the worst affected area and accounting for one of the fastest growing AIDS epidemics worldwide. Samples from 81 patients, including 25 from KwaZulu-Natal, 26 from Gauteng, 5 from Mpumalanga, and 25 from Western Cape Province, were serotyped using a competitive V3 peptide enzyme immunoassay (cPEIA). Viral RNA was also isolated from serum and the V3 region amplified by reverse transcriptase polymerase chain reaction (RT-PCR) to obtain a 240-bp product for direct sequencing of 29 samples. CLUSTAL W was used to make multiple sequence alignments. Distance calculation, tree construction methods, and bootstrap analysis were done using TREECON. Subtype C-like V3 loop sequences predominate in all provinces tested in South Africa. Discordant sero- and genotype results were observed in one patient only. The correlation between sero- and genotyping was 96% (24 of 25) in KwaZulu-Natal and 100% in Gauteng and Mpumalanga. In Western Cape Province 18% of patients were identified as sero/genotype B and 82% as sero/genotype C. Our data show that results of the second-generation V3 cPEIA correlated well with V3 sequencing and would be a rapid and affordable screening test to monitor the explosive southern African HIV-1 epidemic.

Adolescent↗
PubMed1999

Immunohistochemical evaluation of Fhit protein expression in oral squamous cell carcinomas.

The expression of Fhit (fragile histidine triad) protein in oral squamous cell carcinoma (OSCC) and adjacent oral epithelium was evaluated by immunohistochemistry on formalin-fixed paraffin-embedded blocks of 32 cases of OSCC. Rabbit polyclonal anti-GST-Fhit antiserum at 1:600 was used, after antigen enhancement in a microwave pressure cooker, in a saturated lead thiocyanate solution. This antiserum has been shown specifically to detect human Fhit by immunohistochemistry at dilutions up to 1:10,000. The Fhit protein expression was evaluated using both the intensity and extent of staining. Normal stratified squamous epithelium showed strong positivity, especially in the stratum spinosum and areas of keratinisation. Basal and parabasal cells were negative or expressed low levels of Fhit relative to the squamous epithelium. Mild and moderate epithelial dysplasia showed Fhit expression in the superficial layers, while Fhit expression was absent from severely dysplastic lesions. A reduction or loss of Fhit expression was found in 21 (66%) of the OSCC. The alterations in Fhit protein expression in OSCC, and not in normal tissues, are consistent with the proposal that Fhit inactivation plays a role in oral carcinogenesis.

Acid Anhydride Hydrolases↗
PubMed1998

Detection and subtyping of human herpesvirus-8 in renal transplant patients before and after remission of Kaposi's sarcoma.

BACKGROUND: Kaposi's sarcoma (KS) is a complication of renal transplantation. If the human herpesvirus-8 (HHV-8) causes KS, the virus should be present in all KS lesions and be drastically reduced or cleared from involved tissue on remission of the KS. METHODS: Fourteen renal transplant patients with cutaneous KS, including autopsy material from two cases, were investigated for the presence of HHV-8. A second skin biopsy was taken from 11 survivors, after remission of KS, from normal skin in the same anatomical region as the first biopsy. Remission was induced by reduction or cessation of immunosuppression. A peripheral blood sample was collected simultaneously with the repeat biopsy. A nested polymerase chain reaction assay was used to detect HHV-8 DNA in the biopsy tissue and peripheral blood mononuclear cells followed by direct sequencing of polymerase chain reaction product to detect any nucleotide changes. RESULTS: HHV-8 DNA was detected in all the cutaneous KS and all the visceral KS samples, as well as a number of KS-free organs including the thyroid, salivary gland, and myocardium that have not been described before. Mutations in the viral DNA could be demonstrated in all patients. The mutations found were related more to that seen in AIDS-KS cases than that found in African endemic KS cases. HHV-8 sequences could be detected in follow-up frozen skin biopsies of five patients but were negative in the equivalent formalin-fixed specimens. Viral DNA was also detected in 2 of 11 peripheral blood mononuclear cell samples collected at the time of the follow-up skin biopsies. CONCLUSION: Reduction or withdrawal of immunosuppression allows the immune system to recover sufficiently to reduce viral replication with subsequent viral persistence and low grade viral replication that coincides with clinical remission of the KS lesions. This provides further evidence for the important etiological role played by HHV-8 in the pathogenesis of posttransplant KS.

Adult↗