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S Engelbrecht

Publications and source records attributed to S Engelbrecht.

71 records · Page 4Linked to original sources
PubMed1986

Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0.

Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].

Chloroplasts↗
PubMed1985

The importance of disulfide bridges in human endopeptidase (enkephalinase) after proteolytic cleavage.

The neutral endopeptidase (NEP) is a membrane-bound enzyme, which is solubilized by treatment with the protease, papain. Papain did not affect the apparent catalytic activity or the molecular mass of the purified human enzyme in SDS-PAGE. When NEP was treated with a reducing agent after papain digestion, it dissociated into smaller, lower molecular mass fragments. Amino acid analysis and s-carboxymethylation of the half cystine residues indicated that NEP contains four S-S bridges. We concluded that, although covalent bonds appear to be cleaved in NEP by papain, its activity and structure are sustained by S-S bridges.

Amino Acids↗
PubMed1985

Quaternary structure of chloroplast F1-ATPase in solution. Conformational changes in spatial arrangement of subunits upon activation.

The hydrodynamic properties of isolated ATPases were studied via their rotational diffusion in buffer solution. Chloroplast F1-ATPase (CF1) and Escherichia coli F1-ATPase (EF1) were covalently labeled with eosinisothiocyanate and then investigated by polarized laser spectroscopy. The rotational correlation time in aqueous buffer of latent (five-subunit) CF1 was 390 ns. Four-subunit (delta-deficient) CF1 showed the same correlation time, however, for three-subunit (delta, epsilon-deficient) CF1 the rotational correlation time was more than eight times larger (3200 ns). The rotational correlation time of activated CF1 was three times larger than the one of latent CF1. These large changes in the rotational correlation times are directly related to changes in the quaternary structure of CF1 upon activation. EF1 was found to behave essentially as activated CF1. Based on the observed rotational correlation times we concluded that the mass distributions of latent CF1 and of delta-deficient CF1 resemble a dimeric arrangement. The structure of delta, epsilon-deficient CF1 more likely resembles a hexagon, the mass centers of the six main subunits lie in one plane. The structure of the activated forms of CF1 can be described best as an intermediate between the dimeric arrangement of latent CF1 and an octahedron. The large changes in the quaternary structure of isolated CF1 are reversed when the activation of the enzyme is reversed.

Chemical Phenomena↗
PubMed1985

Rapid procedure for the preparation of ferredoxin-NADP+ oxidoreductase in molecularly pure form at 36 kDa.

Ferredoxin-NADP+ oxidoreductase (FNR, EC 1.18.1.2) was purified to molecular homogeneous form as judged by regular and sodium dodecyl sulfate (SDS)-electrophoresis using EDTA extraction of spinach thylakoids, followed by anion exchange on DEAE-cellulose, Procion Red HE 3B dye-ligand chromatography, and hydroxyapatite chromatography. By this procedure, within 1 week approx 7.5 mg of pure FNR, starting from 1 kg of spinach leaves, could be routinely obtained. By comparison with commercially available FNR and with aged preparations two different molecular forms of the enzyme were observed in SDS-electrophoresis. FNR prepared according to the described procedure revealed an apparent molecular mass of 36,000 Da, whereas all other tested preparations showed molecular masses of 3000 Da smaller. Migration in regular gel electrophoresis was the same for all preparations and zymogram stain indicated similar diaphorase activity of both the smaller and the larger forms.

Chromatography↗
PubMed1982

Separation of the human leucocyte enzymes alanine aminopeptidase, cathepsin G, collagenase, elastase and myeloperoxidase.

A simple and rapid procedure is described for the separation of the human leucocyte enzymes alanine aminopeptidase, cathepsin G, collagenase, elastase and myeloperoxidase. The enzymes are prepared from leucocytes, obtained from buffy coat, by repeated extraction with buffer A(1 M salt concentration). The pooled extracts are successively subjected to batch adsorption on concanavalin A-Sepharose, gel filtration on Sephacryl S-300, affinity chromatography on collagen-Sepharose 4-B, batch adsorption on CM-Sephadex C-50 and adsorption chromatography on hydroxyapatite. The yields of the isolated enzymes of a typical preparation are 47% alanine aminopeptidase, 9% cathepsin G, 90% latent and active collagenase, 23% elastase and approximately 100% myeloperoxidase with respect to the pooled extracts. The cathepsin G, collagenase and elastase preparations are essentially free from other proteolytic enzymes and may be used without further purifications.

Aminopeptidases↗
PubMed1980

Synthesis and analytical use of 3-carboxypropionyl-alanyl-alanyl-valine-4-nitroanilide: a specific substrate for human leukocyte elastase.

A simple synthesis is described for 3-carboxypropionyl-Ala-Ala-Val-4-nitroanilide, a convenient and very specific substrate for human leukocyte elastase (Km = 1.0mM, kcat = 8.7 s-1). The substrate does not undergo appreciable spontaneous hydrolysis. It is not cleaved by trypsin or chymotrypsin and only rather slowly by porcine pancreatic elastase (Km = 9.1mM, kcat = 1.4 s-1).

Humans↗
PubMedDate not supplied

Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase.

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Subunit delta of chloroplast F0F1-ATPase and OSCP of mitochondrial F0F1-ATPase: a comparison by CD-spectroscopy.

CD spectra have been recorded with subunit delta from chloroplast CF0CF1 and with OSCP from mitochondrial MF0MF1. These subunits are supposed to act similarly at the interface between proton transport through the F0-portion and ATP-synthesis in the F1-portion of their respective F0F1-ATPase. Evaluation of the data for both proteins revealed a very high alpha-helix content of approximately 85% and practically no beta-sheets. Despite their low homology on the primary structure level (23% identity) and their different electrostatic properties (pI-values differ by 3 units), spinach delta and porcine OSCP are indistinguishable with respect to their secondary structure as measured by CD. Prediction and analysis of consensual alpha-helices even in poorly conserved regions indicate a high degree of structural similarity between chloroplast delta and OSCP. In view of the topology and function of delta and OSCP in intact F0F1 these findings are interpreted to indicate the dominance of secondary and tertiary structure over the primary structure in their supposed function between proton flow and ATP-synthesis.

Adenosine Triphosphatases↗
PubMedDate not supplied

Epstein-Barr virus strain characterisation in South African patients with nasopharyngeal carcinomas.

UNLABELLED: Epstein-Barr virus (EBV) has been implicated in various diseases, among others, nasopharyngeal carcinoma (NPC). In this study we investigated the frequency and subtype distribution of EBV in 39 NPCs. The presence of EBV was detected by using a nested PCR to amplify the Bam Hl W-fragment of the genome. Two regions were targeted for subtype analysis, namely the EBNA-2A and EBER regions. PCR was used to amplify these regions, and the EBER region was sequenced to detect subtype specificity. The results showed that EBV could be detected in 82% (31/38) of the tumours. In 15 of these, EBNA subtypes could be identified of which 14 were subtype A and one tumour had both subtypes A and B present. The EBER region was amplified in 21 samples. The majority of cases (18/21) demonstrated a mutation profile which consisted of 5 type B and one type A mutations. The consensus type is therefore type B. IN CONCLUSION: a strong association was found between EBV and NPCs in our group of patients and their "consensus" genotype was A/B based on the two genome areas investigated.

Adolescent↗
PubMedDate not supplied

FHIT RNA and protein expression in oral squamous cell carcinomas.

BACKGROUND: To investigate the possible role of FHIT, a possible tumour suppressor gene, in oral carcinogenesis, we examined 17 oral squamous cell carcinomas (OSCCs) for genetic alterations. MATERIALS AND METHODS: Fresh tissue was obtained during surgery, snap-frozen in liquid nitrogen and stored at -70 degrees C. Nested PCR amplification to examine the integrity of FHIT mRNA was performed on the reverse transcribed complementary DNA obtained from the frozen normal and tumour tissue. Immunohistochemistry was done on formal in-fixed paraffin-embedded tissue protein from the same cases using a polyclonal antiserum against the full length Fhit. RESULTS: Twelve out 17 (71%) OSCCs showed reduced or absent Fhit protein and half of the cases with reduced Fhit protein exhibited aberrant RT-PCR products. CONCLUSION: Immunohistochemical detection of Fhit protein expression in OSCCs is the more sensitive method to determine the status of Fhit in these tumours, in agreement with previous studies of other tumour types.

Acid Anhydride Hydrolases↗
PubMedDate not supplied

[Dynamics in the 3d stage of labor observed by ultrasound].

In two groups, with 50 pregnant women each, changes in the third stage of labour were followed up by a real-time scanner. In the experimental group the umbilical cord on the mother's side as not tied up, while in the control external uterine wall to the chorionic line in the separated placenta immediately before a cord traction intervention amounted to 5.45 cm, while in the control group, under the same conditions, it amounted to 8.61 cm and was caused by the intensified development of the retroplacental hematoma. The difference was statistically significant (P < 0.001). In the way presented, along with the shortening of the third stage of labour, the authors prevent the development of anemia in parturients.

Delivery, Obstetric↗
PubMedDate not supplied

Detection of EBV DNA in oral squamous cell carcinomas in a black African population sample.

The purpose of this study was to determine the presence of Epstein-Barr virus (EBV) DNA in oral squamous cell carcinoma (OSCC) patients from a Black African population. Formalin fixed paraffin embedded blocks of OSCC of two randomly selected groups were investigated. Group 1 consisting of 57 blocks containing OSCC with a fragment of normal appearing adjacent/overlying epithelium. Group 2 consisted of 48 blocks containing only OSCC tissue without any normal appearing epithelium. The control group consisted of 38 non-malignant, non-viral associated lesions. A standard polymerase chain reaction (PCR) was used to amplify the Bam HI W-fragment using a nested primer set. EBV DNA was demonstrated in 14/57 (25%) blocks from Group 1, in 13/48 (27%) blocks from Group 2 and in 16/38 (42%) blocks from the control group. No evidence for a direct role of EBV in the process of malignant transformation of intraoral epithelial cells was found in this study.

Africa↗
PubMedDate not supplied

Prevalence of EBV in oral squamous cell carcinomas in young patients.

BACKGROUND: Recent studies reported a difference in the age distribution of oral squamous cell carcinoma (OSCC) between Black and White South Africans with OSCC more prevalent in Black patients under the age of 50 compared to Whites. MATERIALS AND METHODS: Paraffin embedded blocks of OSCC were divided into two groups: one with a mean age of 56.2 years and the second group all younger than 40 years of age. A control group of 30 non-neoplastic intraoral lesions were selected. A standard PCR reaction was used to amplify the BAM H1 W-fragment of the EBV. RESULTS: EBV DNA was demonstrated in 11/45 (24%) cases from the first group and in 11/45 (24%) cases from the second group. EBV DNA was present in 11/30 (37%) cases from the control group. CONCLUSIONS: This study showed that the prevalence of EBV in OSCC was not influenced by the age of the patient.

Adult↗
PubMedDate not supplied

Human papillomavirus DNA in oral squamous cell carcinomas from an African population sample.

BACKGROUND: The incidence of oral squamous cell carcinoma (OSCC) is on the increase in developing countries. MATERIALS AND METHODS: Formalin fixed paraffin embedded blocks of OSCCs from a Black South African population sample of peri-urban and rural origin were selected as follows: Group 1 - 57 OSCCs with a mean age of 59 years; Group 2 - 43 OSCCs all cases younger than 40 years; Group 3 - 46 OSCCs with blocks containing only tumour tissue without any normal epithelium and Group 4, a control group of 38 non-neoplastic epithelial lesions. Type specific primers were used in a standard PCR to amplify a segment of the E6 region of HPV 6, 11, 16 and 18. RESULTS: HPV 11 and 16 DNA were found in one sample each from groups 1 and 2 respectively. CONCLUSION: HPV is not an etiologic factor in the development of OSCC in the population studied.

Adult↗
PubMedDate not supplied

Correlation between p53 gene mutation, p53 protein labeling and PCNA expression in oral squamous cell carcinomas.

BACKGROUND: The prevalence of oral squamous cell carcinoma (OSCC) among the Black community in South Africa is unacceptably high. The association between p53 protein, and PCNA overexpression and the presence of p53 gene mutations was evaluated. MATERIALS AND METHODS: One hundred and ten formalin-fixed, paraffin-embedded blocks of OSCC were selected for immunohistochemical studies for p53 protein and PCNA expression using the DO-7 and PC10 monoclonal antibodies, respectively. DNA was extracted from fifty-five blocks and exons 5 to 9 of the p53 gene were amplified with nested primers, thereafter sequencing was performed to confirm the presence of mutations detected by single stranded conformational polymorphism. RESULTS: Fifty-six cases (51%) showed p53 expression, while fourteen mutations (25%) were detected. A significant difference was found between the PCNA index in p53 positive and p53 negative tumors while the mean PCNA index for the tumors with p53 mutations was not significantly different from the tumors without mutations. CONCLUSIONS: No association between p53 protein overexpression and p53 gene mutations could be demonstrated.

Adult↗