Treatment of SR membranes with mild acid (pH 5.6) (Berman, M.C., McIntosh, D.B. and Kench, J.E. (1977) J. Biol. Chem. 252, 994-1001) or incubation with millimolar concentrations of ethylene glycol bis (β-aminoethyl ether)-N ,N'-tetraacetic acid (EGTA) at neutral pH and 37°C (McIntosh, D. B. and Berman, M. C. (1978) J. Biol. Chem. 253, 5l40-5146) results in a progressive irreversible inhibition of calcium transport while (Ca²⁺, Mg²⁺)-ATPase activity is unimpaired. Possible conformational changes associated with this uncoupling were monitored by following alterations in kinetic mobility of sulphydryl (-SH) groups either by using 5, 5'-dithiobis- (2-nitrobenzoate) (DTNB) and stopped flow analysis or 1-¹⁴C-N-ethylmaleimide (NEM). Kinetic reactivity with DTNB revealed a total of 20 thiol groups/1.5 x 10⁵ g of SR protein (rontaining 1 mole of ATPase protein) in the presence of sodium dodecyl sulphate, which constitute four kinetic classes. In native control vesicles 4.5 thiol groups were unreactive, 0.4 represented the fast reacting class, 0.8 the moderately fast reacting class and 14.4 the slowly reacting class, displaying pseudo-first order rate constants, k, of 159.0-, 22.0- and 6.23 x 10⁻² sec⁻¹, respectively. Inactivation of calcium transport to the extent of 90%, using mild acid conditions, increased the number of fast and moderately fast reacting groups, each by 1.0 - 1.5 sulphydryl groups / mol ATPase. The number of slowly reacting groups decreased by approximately 3 .0 thiol groups/mol ATPase. The kinetics of the reaction with 1-¹⁴C-NEM was essentially similar to that with DTNB. EGTA inactivation of calcium transport, to the extent of 90% and subsequent 1-¹⁴C-NEM modification, resulted in an increase in the number of fast reacting thiol groups by 0.5-1.0 thiol groups/mol ATPase. The total number of reactive thiol groups decreased by 1.0 -2.0 thiol groups/ mol ATPase, probably due to autoxidation of the newly exposed sulphydryl group. Inactivation of transport carried out in the presence of N-ethylmaleimide to prevent autoxidation resulted in an increase of approximately one thiol group/mol ATPase. The rate constant for the increase in reactivity of this group was 1.45 min⁻¹. This thiol group was localized on the ATPase protein of molecular weight approximately 100 000 daltons. Trypsinization of the ATPase produced four fragments of molecular weights 55 000, 45 000, 30 000 and 20 000. More extensive cleavage resulted in a significant decrease in the 55 000 dalton fragment and increased amounts of the 30 000 and 20 000 dalton subfragments. There was increased labelling on all subfragments of EGTA-treated vesicles compared to control, untreated vesicles. However, the greatest relative increase in labelling appeared to be localized on the 55 000 dalton and 20 000 dalton subfragments. Peptide mapping of the purified ATPase revealed 24 ninhydrin-positive peptides. Five of these were labelled in control and EGTAtreated vesicles, four of which showed increased labelling in the latter preparation. Random labelling of the nonoverlapping fragments may be due to the enzyme being "trapped" in a number of intermediate conformations or due to heterogeneity within the ATPase populations. NEM modification of SR membranes did not affect the tryptic cleavage pattern or the mobilities of the tryptic subfragments. It did however, affect the extent of tryptic cleavage resulting in solubilization of NEM-labelled protein into the medium following centrifugation. This protein fraction was identified as consisting largely of the 55 000 dalton molecular weight species on sodium dodecyl sulphate gel electrophoresis. It is concluded that occupancy of high affinity K₀.₅(Ca²⁺)≈10⁻⁶M) calcium binding sites maintain the (Ca²⁺, Mg²⁺)- ATPase in a stable, coupled conformation. Displacement of this calcium induces a conformational change in the protein which results in the loss of the vectorial component of calcium transport.

No description available.

The goal of this study is to develop a biodegradable bone wax that will allow the control of sternal bleeding during cardiothoracic procedures without adverse effects. Twenty dogs were anesthetized and prepared for the sterile surgical procedure. After median sternotomy and anticoagulation (3 mg heparin/kg), biodegradable bone wax made of tocopheryl polyethylene glycol succinate (TPGS) and oxidized cellulose (20% or 52% by weight) was evaluated in 10 dogs. The 20% oxidized cellulose in TPGS: H2O = 60% : 40% mixture resulted in complete hemostasis and was used for chronic evaluation. Four dogs each were subjected to biodegradable and normal bone wax with two additional dogs serving as controls (no bone wax). At 6 weeks after operation, the sterna were atraumatically harvested en bloc and radiographed immediately. The histological specimens were transversely sectioned across the manubrium to evaluate the healing and new formation of bone. The control and biodegradable bone wax groups were markedly better than the normal bone wax group. The strength of healed sternal segments (3 per dog) were measured. The control (9.2 ± 1.7 kg) and biodegradable bone wax (10.1 ± 1.6 kg) groups were significantly (P < .01) stronger than the normal bone wax (2.4 ± 1.0 kg) group. A biodegradable bone wax was developed to effectively maintain sternal hemostasis during cardiothoracic procedures without hindering the healing of the sternum. In addition, antibiotics, growth factors, and pharmacologic agents can also be included to prevent infection, enhance bone healing and subdue inflammation.

This study examined the effect of TLR2 activation by its specific ligand, Pam3CSK4, on cerebral ischemia/reperfusion (I/R) injury. Mice (n = 8/group) were treated with Pam3CSK4 1 h before cerebral ischemia (60 min), followed by reperfusion (24 h). Pam3CSK4 was also given to the mice (n = 8) 30 min after ischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The morphology of neurons in brain sections was examined by Nissl staining. Pam3CSK4 administration significantly reduced infarct size by 55.9% (p < 0.01) compared with untreated I/R mice. Therapeutic treatment with Pam3CSK4 also significantly reduced infarct size by 55.8%. Morphologic examination showed that there was less neuronal damage in the hippocampus of Pam3CSK4-treated mice compared with untreated cerebral I/R mice. Pam3CSK4 treatment increased the levels of Hsp27, Hsp70, and Bcl2, and decreased Bax levels and NF-κB-binding activity in the brain tissues. Administration of Pam3CSK4 significantly increased the levels of phospho-Akt/Akt and phospho-GSK-3β/ GSK-3β compared with untreated I/R mice. More significantly, either TLR2 deficiency or PI3K inhibition with LY29004 abolished the protection by Pam3CSK4. These data demonstrate that activation of TLR2 by its ligand prevents focal cerebral ischemic damage through a TLR2/PI3K/Akt-dependent mechanism. Of greater significance, these data indicate that therapy with a TLR2-specific agonist during cerebral ischemia is effective in reducing injury.

INTRODUCTION: Constitutional Mismatch Repair Deficiency (CMMR-D) syndrome is a rare tumour predisposition and polyposis syndrome that presents in childhood. It is caused by mutations in mismatch repair (MMR) genes that result in a tumour spectrum including colorectal cancers, high-grade gliomas, non-Hodgkin T-cell lymphomas and leukaemias. It is characterized by biallelic germline mutation of one of four possible MMR genes resulting in loss of protein expression that can be identified by applying immunohistochemistry to formalin fixed paraffin embedded tissue sections. Use of MMR immunohistochemistry is established in the setting of Lynch syndrome (LS); however, the pattern of loss of staining in the background, non-tumour tissue is unique to CMMR-D syndrome. CMMR-D syndrome is seen in LS families and occurs as a result of consanguinity or founder effect. The South African population has LS families concentrated in the Western Cape and Northern Cape Provinces and the mixed ancestry population shows a unique MLH1 c1528C>T mutation which may have implications on the incidence, penetrance and severity of CMMR-D syndrome seen in our population. The diagnosis of CMMR-D syndrome includes clinical findings outlined in the European Consortium's Care of CMMRD document and confirmation of the biallelic mutation in one of the MMR genes. MMR immunohistochemistry can be used in the diagnosis of CMMR-D syndrome by identifying cases for targeted molecular genetic tests. However, MMR immunohistochemical staining patterns are not usually described in detail, particularly the loss of staining of the affected gene in the background, non-tumour tissue, the key feature of CMMR-D syndrome. METHODS: We performed a retrospective analysis of archival formalin fixed paraffin embedded tissue of children attending Red Cross Children's Hospital with tumours that form part of the CMMR-D spectrum, outlined by the Care for CMMRD criteria. We used the criteria of high-grade gliomas (WHO Grade III or IV) occurring before 25 years of age, cutaneous lesions suggestive of CMMR-D syndrome and patients with a first or second degree relative diagnosed with LS. MMR immunohistochemistry was applied, and the staining pattern was documented in terms of proportion of tumour staining and intensity of staining using a modified Allred Scoring system. Specific attention was given to the characterization of the staining pattern of the background normal tissue. RESULTS: 21 samples taken from 18 patients were evaluated. 16 samples represented brain tumours, predominantly high-grade gliomas. Three samples were excluded due to suboptimal staining despite positive external controls. 12 samples showed intact staining of all four MMR stains. Two samples showed staining of unknown significance. Four samples from 3 different patients showed staining patterns compatible with MMR deficiency. This included two patients, each with a biopsy showing high-grade glioma and two samples of the same patient taken at a 1-year interval of a Burkitt lymphoma. Of these four samples, three samples showed loss of staining in background non-tumour tissue with positive external control, the unique staining pattern for CMMR-D syndrome. These cases will be referred for confirmatory testing by molecular genetic techniques. CONCLUSION: MMR immunohistochemistry can be used in the evaluation of CMMR-D syndrome, but care is needed in evaluating adequacy of staining, the pattern and scoring of staining of both the tumour and the background non-tumour tissue. Endothelial cells are easy to identify and evaluate as background tissue which is useful in extra-intestinal tumours. Neurons and choroid plexus can also be evaluated as background tissue in brain tumour samples. Selection bias in this study resulted in the underrepresentation of lymphomas and colorectal carcinomas. Improved characterization and search for Non-Hodgkin T-cell lymphomas and inclusion of samples of colorectal carcinomas of adolescents and adults would be needed to include these tumours. Use of MMR immunohistochemistry in postmortem tissue samples is not recommended because of suboptimal staining, even with a short post-mortem interval of 1 day. The diagnosis of CMMR-D syndrome depends on clinical application of Care for CMMRD criteria, MMR immunohistochemistry in conjunction with molecular genetic testing. It is important to identify cases of CMMR-D syndrome and offer cancer screening to prevent development of other cancers in the index patient. It also provides an opportunity for genetic counselling and testing of the parents and at-risk siblings.

Introduction: Renal cell carcinoma (RCC) accounts for 87% of all kidney cancers. Despite advances in diagnostic techniques and management, renal cell carcinoma remains a lethal tumour accounting for substantial mortality and morbidity. The poor prognosis arises from metastasis, chemoradiation resistance and disease relapse. Cancer stem cells, a subpopulation of tumour cells with capacity to self-renew and reconstitute tumour heterogeneity have been implicated as the root cause of poor prognosis. Therefore, a better understanding of biomarkers of cancer stem cells will be useful for risk stratification, prognostication and may lead to novel targeted therapies that will ultimately alter the management of many patients. Aims and objectives: To review the morphological subtypes of renal cell carcinomas diagnosed in the Division of Anatomical Pathology, National Health Laboratory Service, Groote Schuur Hospital over a 10-year period. To identify cancer stem cells in various histopathological subtypes of renal cell carcinoma using immunohistochemical markers (CD133 and CD105). To review the WNT signalling pathway in renal cell carcinomas using selected protein expression by immunohistochemistry (β-Catenin).Materials and methods: Ten-year retrospective study in which sixty-four cases of renal cell carcinoma were retrieved and reviewed. Four immunohistochemical stains (β-catenin, HIF-1α, CD133 and CD105) were performed and scored in tumour tissue. Data were analysed to determine if there was any correlation between expression of the biomarkers and the histopathological subtypes of renal cell carcinoma. Results: The mean age of the patients was 56-years (range, 35 to 81 years). Females constituted just over half (52%, n = 33) of the study patients. All 64 cases were confirmed as renal cell carcinomas, with 29 (45%) clear cell renal cell carcinomas, 14 (22%) papillary renal cell carcinomas, 9(14%) chromophobe renal cell carcinomas, 9 (14%) multicystic renal cell carcinomas and 3 (5%) sarcomatoid renal cell carcinomas. Ten (16%) cases showed abnormal β-Catenin cytoplasmic localisation. The majority of cases (n=6, 60%) showing abnormal β-Catenin localisation were clear cell renal cell carcinomas. However, there was no significant correlation between abnormal and normal β-Catenin localisation and RCC histopathological subtype (p = 0.766). CD133 immunohistochemical studies showed low expression in 52 (81 %) cases and high expression in 12 (19 %) cases. There was no correlation between low and high CD133 expression and histopathological RCC subtype (P = 0.800). CD105 immunostaining showed tumour cell immunopositivity in one case of clear cell renal cell carcinoma whilst the rest of the cases were negative. The low, moderate and high microvascular density categories had 24, 10 and 32 cases respectively. There was no significant correlation between low, moderate, and high microvascular densities and the histopathological RCC subtype (P = 0.320). HIF-1α immunohistochemical studies showed low expression in 39 (61 %) cases and high expression in 25 (39 %) cases. There was no significant correlation between levels of HIF-1α expression and the histopathological RCC subtype (P =0.972).Conclusion: Within the power limitations of this small study,β-catenin abnormal expression, microvascular densities and levels cytoplasmic CD133 and HIF-1α were not associated with any histopathological subtype of renal cell carcinoma.

Introduction: Perinatal mortality is regarded as an indicator of the social status and obstetrical care within a given community. The developed world has witnessed a dramatic decline in perinatal mortality as standards of living improved. Unfortunately, this turn of events has not been seen in the Third World where mortality remains very high. When improved perinatal autopsy techniques are applied the causes of perinatal deaths are readily appreciated. No previous autopsy study has been conducted at our hospital. The application of new techniques has stimulated the present study, which is also intended to monitor current and future clinical practice. Problem formulation: What are the common causes of death in the perinatal period at Ga-Rankuwa Hospital? Aims of the study: a. To assess the common causes of fetal and neonatal deaths at our hospital. b. To determine those causes which are preventable and propose specific obstetric interventions. c. To obtain a baseline for future studies along the same line. d. To lay a foundation for clinicopathologic discussion with clinical colleagues. Research Methodology: An autopsy study is to be conducted on each and every stillbirth and neonatal death that occurs during the period of study. The technique will be discussed in detail in Chapter 3.

Includes bibliography. / Between 1985 and 2000, 43 children (age range 6 months-13 years) underwent liver transplantation at Red Cross Children's Hospital. In 46% of these cases, viral infections resulted in considerable morbidity and mortality. Included in this group were: de novo hepatitis B (5 patients, 2 deaths), EBV-related post-transplantation lymphoproliferative disease (6 patients, 4 deaths) and CMV disease (9 patients, 4 deaths).

Bibliography: p. 83-94. / The number of albatrosses that are killed on longlines in the Southern ocean is conservatively estimated to be 44 000 birds per annum. These numbers are biologically significant since albatrosses are a prime example of an extreme K-selected species. Ongoing long line fishing in the Southern ocean could lead to a decrease in the size of breeding colonies, and is a cause for major concern as it may impact the long-term survival of these birds. Quantifying genetic variation in threatened populations is a valuable application of molecular biology in conservation. In this study genetic variation was quantified using microsatellite analysis in order to investigate the effects of the longline fisheries on seabird populations. In addition, the feasibility of developing diagnostic markers for determining the provenance of birds forming part of the bycatch was also investigated. The inter-population genetic variance of three species of albatross from four distinct breeding colonies is described. Microsatellite markers were found to be highly variable and provided an assessment of the heterzygosity in the distinct populations, and a measure of the gene flow between these populations. Despite the extreme fidelity that adult albatrosses show to their breeding colonies, relatively low levels of genetic differentiation were observed between the colonies. This suggests that an integrated conservation management strategy could be undertaken successfully.

Background: Nucleoside reverse transcriptase inhibitors (NRTIs) interfere with mitochondrial DNA polymerase gamma causing significant toxic effects, including fatal lactic acidosis. Little is known about mitochondrial DNA (mtDNA) in human immunodeficiency virus (HIV) infected children who face a lifetime exposure to these agents. We performed a cross sectional observation of mtDNA levels in whole blood in a pediatric population to ascertain the relationship between mtDNA, NRTI regimens and parameters of HIV-infection severity. Methods: Whole blood mt:nDNA ratios were determined by real-time PCR in three groups: 27 presumed HIV-negative, 89 HIV-infected, NRTI-treated and 62 HIV-infected treatment-naive children. Multivariate analysis was used to identify variables independently associated with mtDNA depletion. Results: Mean mt:nDNA ratios were lower (P < 0.001) at 77% of control in the HIVinfected antiretroviral treatment (ART) Naïve group and 73% of control in the ART group, but not different between the two HIV-infected groups. Mt:nDNA ratios were negatively associated with age (P = 0.029), HIV status (P < 0.0001) and Log10 of the HIV-1 viral load (P = 0.035) and positively associated with CD4 % (p = 0.032). A 6 stavudine vs zidovudine based regimen was associated with lower but not significant levels of mtDNA (P = 0.1). Conclusions: Depletion of whole blood mtDNA in children is associated independently with HIV-infection and markers of HIV infection severity, and does not improve with either stavudine or zidovudine based ART despite virological control, suggesting that these agents also deplete mtDNA.