Cholecalciferol induces apoptosis via autocrine metabolism in epidermoid cervical cancer cells.

The anti-cancer effects of vitamin D are of fundamental interest. Cholecalciferol is sequentially hydroxylated endogenously to calcidiol and calcitriol. Here, SiHa epidermoid cervical cancer cells were treated with cholecalciferol (10-2600 nmol/L). Cell count and viability were assayed using Crystal Violet and Trypan Blue, respectively. Apoptosis was assessed using flow cytometry for early and late biomarkers along with brightfield microscopy and transmission electron microscopy. Autocrine vitamin D metabolism was analysed by reverse transcription-quantitative PCR and immunoblotting for activating enzymes: 25-hydroxylases (CYP2R1 and CYP27A1) and 1α-hydroxylase (CYP27B1), the catabolic 24-hydroxylase (CYP24A1), and the vitamin D receptor (VDR). Data were analysed using one-way ANOVA and Bonferroni post-hoc test, and p < 0.05 was considered significant. After cholecalciferol, cell count (p = 0.011) and viability (p < 0.0001) decreased, apoptotic biomarkers were positive, mitochondrial membrane potential decreased (p = 0.0145), and phosphatidylserine externalisation (p = 0.0439), terminal caspase activity (p = 0.0025), and nuclear damage (p = 0.004) increased. Microscopy showed classical features of apoptosis. Gene and protein expression were concordant. Immunoblots revealed increased CYP2R1 (p = 0.021), VDR (p = 0.04), and CYP24A1 (p = 0.0274) and decreased CYP27B1 (p = 0.031). The authors conclude that autocrine activation of cholecalciferol to calcidiol may mediate VDR signalling of growth inhibition and apoptosis in SiHa cells.

COVID-19 and Vulnerable Populations in Sub-Saharan Africa.

The novel corona virus 2019 (COVID-19) outbreak which started in Hubei province in China has now spread to every corner of the earth. While the pandemic started later in Africa, it is now found in all African countries to varying degrees. It is thought that the prevalence and severity of disease is influenced by a number of non-communicable diseases (NCDs) which are all becoming increasingly prevalent in sub-Saharan Africa (SSA). In addition, SSA bears the major burden of human immunodeficiency virus (HIV) and tuberculosis (TB) infections. While data from Europe and the United States show that children are spared severe disease, it is uncertain if the same holds true in SSA where children suffer from sickle cell disease and malnutrition in addition to other infectious diseases. There is limited data from Africa on the effects of these conditions on COVID-19. In this review, we discuss the epidemiology of some of these conditions in Africa and the possible pathogenesis for the interactions of these with COVID-19.

Glycated haemoglobin (HbA1c ) and fasting plasma glucose relationships in sea-level and high-altitude settings.

AIM: Higher haemoglobin levels and differences in glucose metabolism have been reported among high-altitude residents, which may influence the diagnostic performance of HbA1c . This study explores the relationship between HbA1c and fasting plasma glucose (FPG) in populations living at sea level and at an altitude of > 3000 m. METHODS: Data from 3613 Peruvian adults without a known diagnosis of diabetes from sea-level and high-altitude settings were evaluated. Linear, quadratic and cubic regression models were performed adjusting for potential confounders. Receiver operating characteristic (ROC) curves were constructed and concordance between HbA1c and FPG was assessed using a Kappa index. RESULTS: At sea level and high altitude, means were 13.5 and 16.7 g/dl (P > 0.05) for haemoglobin level; 41 and 40 mmol/mol (5.9% and 5.8%; P < 0.01) for HbA1c ; and 5.8 and 5.1 mmol/l (105 and 91.3 mg/dl; P < 0.001) for FPG, respectively. The adjusted relationship between HbA1c and FPG was quadratic at sea level and linear at high altitude. Adjusted models showed that, to predict an HbA1c value of 48 mmol/mol (6.5%), the corresponding mean FPG values at sea level and high altitude were 6.6 and 14.8 mmol/l (120 and 266 mg/dl), respectively. An HbA1c cut-off of 48 mmol/mol (6.5%) had a sensitivity for high FPG of 87.3% (95% confidence interval (95% CI) 76.5 to 94.4) at sea level and 40.9% (95% CI 20.7 to 63.6) at high altitude. CONCLUSION: The relationship between HbA1c and FPG is less clear at high altitude than at sea level. Caution is warranted when using HbA1c to diagnose diabetes mellitus in this setting.

Obesity risk in rural, urban and rural-to-urban migrants: prospective results of the PERU MIGRANT study.

BACKGROUND: Although migration and urbanization have been linked with higher obesity rates, especially in low-resource settings, prospective information about the magnitude of these effects is lacking. We estimated the risk of obesity and central obesity among rural subjects, rural-to-urban migrants and urban subjects. METHODS: Prospective data from the PERU MIGRANT Study were analyzed. Baseline data were collected in 2007-2008 and participants re-contacted in 2012-2013. At follow-up, outcomes were obesity and central obesity measured by body mass index and waist circumference. At baseline, the primary exposure was demographic group: rural, rural-to-urban migrant and urban. Other exposures included an assets index and educational attainment. Cumulative incidence, incidence ratio (IR) and 95% confidence intervals (95% CI) for obesity and central obesity were estimated with Poisson regression models. RESULTS: At baseline, mean age (±s.d.) was 47.9 (±12.0) years, and 53.0% were females. Rural subjects comprised 20.2% of the total sample, whereas 59.7% were rural-to-urban migrants and 20.1% were urban dwellers. A total of 3598 and 2174 person-years were analyzed for obesity and central obesity outcomes, respectively. At baseline, the prevalence of obesity and central obesity was 20.0 and 52.5%. In multivariable models, migrant and urban groups had an 8- to 9.5-fold higher IR of obesity compared with the rural group (IR migrants=8.19, 95% CI=2.72-24.67; IR urban=9.51, 95% CI=2.74-33.01). For central obesity, there was a higher IR only among the migrant group (IR=1.95; 95% CI=1.22-3.13). Assets index was associated with a higher IR of central obesity (IR top versus bottom tertile 1.45, 95% CI=1.03-2.06). CONCLUSIONS: Peruvian urban individuals and rural-to-urban migrants show a higher incidence of obesity compared with their rural counterparts. Given the ongoing urbanization occurring in middle-income countries, the rapid development of increased obesity risk by rural-to-urban migrants suggests that measures to reduce obesity should be a priority for this group.

Salivary Cortisol and Cortisone do not Appear to be Useful Biomarkers for Monitoring Hydrocortisone Replacement in Addison's Disease.

Salivary cortisol has been used to monitor hydrocortisone replacement in patients with Addison's disease (AD). Since salivary cortisol is metabolised to salivary cortisone, it may be an adjunctive analyte to assess adequacy of hydrocortisone replacement in patients with AD. We aimed to characterise the exposure of salivary cortisol and cortisone in patients and healthy controls. We measured salivary cortisol and cortisone by liquid chromatography-tandem mass spectrometry and constructed a day curve (08:00 until 24:00 h) with 16 time points in 25 AD patients taking their usual hydrocortisone dose and in 26 healthy controls. The median (interquartile range) area under the curve (AUC) for cortisol was not different for patients, compared with controls [55.63 (32.91-151.07) nmol*min*l-1 vs. 37.49 (27.41-52.00) nmol*min*l-1; p=0.098, respectively], whereas the peak cortisol Cmax was higher in patients [32.61 (5.75-146.19) nmol/l vs. 8.96 (6.96-12.23) nmol/l; p=0.013], compared with controls. The AUC for cortisone [23.65 (6.10-54.76) nmol*min*l-1 vs. 227.73 (200.10-280.52) nmol*min*l-1; p≤ 0.001, respectively], and peak cortisone Cmax was lower in patients than in controls [11.11 (2.91-35.85) nmol/l vs. 33.12 (25.97-39.95) nmol/l; p=0.002]. The AUC for salivary cortisol and salivary cortisone were not correlated with any measures of hydrocortisone dose. The time-course and AUC of salivary cortisol were similar between Addison's patients and healthy controls. Patients had substantially lower salivary cortisone AUC, compared to healthy controls. Salivary cortisol AUC and pharmacokinetics were not related to hydrocortisone dose and thus are not likely useful markers for the adequacy of hydrocortisone replacement.

Salivary cortisol day curves in Addison's disease in patients on hydrocortisone replacement.

Using salivary cortisol (SC) measurements, cortisol exposure in Addison's disease patients on hydrocortisone replacement was determined and compared with healthy controls. Cortisol pharmacokinetics was assessed in 31 patients with Addison's disease on replacement hydrocortisone doses (median daily dose 20 mg; range 5-50 mg) and 30 healthy control subjects. Saliva samples (n=16) were collected between 08:00 and 00:00 h in 1 day, using a passive drool technique. Cortisol exposure was evaluated by noncompartmental approach. In the patients, cortisol exposure was significantly higher than in controls: median inter-quartile range (IQR) peak cortisol (C(max)) 174.5 (59.3-837.0) vs. 6.50 (4.7-19.3) nmol/l, p=0.0001; area under the curve (AUC) 390.1 (177.1-928.9) vs. 21.4 (14.6-28.4) minutes*nmol/l, p=0.0001, trough cortisol level (C(min)) 0.49 (0.49-0.96) vs. 0.49 (0.49-0.49) nmol/l, p=0.02, occurring at 480.0 (0.1-660.0) vs. 405.0 (180.0-570.0) min, p=0.56. First peak cortisol was 174.5 (53.0-754.7) vs. 6.27 (3.90-8.47) nmol/l, p=0.0001 and second peak cortisol 18.90 (5.22-76.9) vs. 3.12 (1.76-4.79) nmol/l, p=0.0001. The time to first peak cortisol differed between the 2 groups, 30 (30-75) vs. 0.1 (0.1-30) minutes; p=0.0001. At doses studied, hydrocortisone replacement therapy results in cortisol pharmacokinetics being markedly different from endogenous cortisol profiles in healthy control subjects. Addison's disease patients had significantly higher SC levels compared to healthy control subjects.

The gunshot-related injuries in trauma (GRIT) study: A profile of patients affected by gunshot-related orthopaedic injuries across South Africa.

BACKGROUND: South Africa (SA) has one of the highest gun-related mortality rates in the world - 20 people per day. The available data, however, do not reflect the substantial number of patients suffering non-lethal firearm injuries. Gunshot-related injury has been recognised as a highly costly healthcare problem by individual treating centres in SA and other countries; however, no 'national picture' has been examined in detail. OBJECTIVES: To explore the burden of gunshot-related orthopaedic injuries across SA. METHODS: A multicentre research network was established in SA, and 37 orthopaedic units across 9 provinces participated. A prospective, observational cohort study was performed during a 2-week period in 2019. Patients were screened, enrolled and reported by local orthopaedic teams. Patients were included if they had at least one acute gunshot-related orthopaedic fracture referred to the orthopaedic service. Patients were asked additional questions around baseline health-related quality-of-life (HRQOL) and personal circumstances. Follow-up was at 8 weeks after injury. RESULTS: Thirty-seven centres enrolled 135 patients over the 2-week study period. Western Cape Province had the highest number of reported cases (n=52; 39%), followed by Gauteng (n=35; 26%) and KwaZulu-Natal (n=29; 21%). The median age of patients was 30.5 years and the majority were male (89%). Forty-three percent of patients had been either shot or stabbed prior to this injury. Fifty-two percent of all patients required fracture fixation surgery and 11% required wound debridement without fracture fixation. HRQOL data were collected successfully at baseline, but follow-up data were available for <25% of cases. CONCLUSIONS: Gunshot-related orthopaedic injuries represent a significant burden of disease in the SA healthcare environment. This study highlights several areas for further research in the management of the injuries and associated outcomes.

Sentinel seroprevalence of SARS-CoV-2 in Gauteng Province, South Africa, August - October 2020.

BACKGROUND: Estimates of prevalence of anti-SARS-CoV-2 antibody positivity (seroprevalence) for tracking the COVID-19 epidemic are lacking for most African countries. OBJECTIVES: To determine the prevalence of antibodies against SARS-CoV-2 in a sentinel cohort of patient samples received for routine testing at tertiary laboratories in Johannesburg, South Africa. METHODS: This sentinel study was conducted using remnant serum samples received at three National Health Laboratory Service laboratories in the City of Johannesburg (CoJ) district. Collection was from 1 August to 31 October 2020. We extracted accompanying laboratory results for glycated haemoglobin (HbA1c), creatinine, HIV, viral load and CD4 T-cell count. An anti-SARS-CoV-2 targeting the nucleocapsid (N) protein of the coronavirus with higher affinity for IgM and IgG antibodies was used. We reported crude as well as population-weighted and test-adjusted seroprevalence. Multivariate logistic regression analysis was used to determine whether age, sex, HIV and diabetic status were associated with increased risk for seropositivity. RESULTS: A total of 6 477 samples were analysed, the majority (n=5 290) from the CoJ region. After excluding samples with no age or sex stated, the model population-weighted and test-adjusted seroprevalence for the CoJ (n=4 393) was 27.0% (95% confidence interval (CI) 25.4 - 28.6). Seroprevalence was highest in those aged 45 - 49 years (29.8%; 95% CI 25.5 - 35.0) and in those from the most densely populated areas of the CoJ. Risk for seropositivity was highest in those aged 18 - 49 years (adjusted odds ratio (aOR) 1.52; 95% CI 1.13 - 2.13; p=0.0005) and in samples from diabetics (aOR 1.36; 95% CI 1.13 - 1.63; p=0.001). CONCLUSIONS: Our study conducted between the first and second waves of the pandemic shows high levels of current infection among patients attending public health facilities in Gauteng Province.

Retrograde venous bullet embolism into renal vein.

SUMMARY: The unpredictable nature and behaviour of bullet emboli can pose unique diagnostic and management challenges, related to the absence of exit wounds or variable trajectories. However, embolisation into the vascular system is an extremely unusual occurrence, with fewer than 200 such cases described since 1900. Given the relative paucity of such literature reports, it is not surprising that guidelines for the optimal management of some of these emboli are neither clear cut, nor universally accepted. We report the second case of retrograde venous bullet embolism to the right renal vein following a gunshot injury to the right chest and the surgical solution.

An audit of upper gastrointestinal endoscopy performed on patients at Prince Mshiyeni Memorial Hospital in Durban, KwaZulu-Natal.

BACKGROUND: Resource constraints have resulted in upper gastrointestinal endoscopy (UGE) being deferred where possible. However, delayed investigation is costly and leads to disease progression. This study audits the UGE done at a single institution. It was motivated by the observation that public hospitals often experience an acute shortage of endoscopes, which are prone to frequent breaks and service delivery is further compromised by an increased workload. METHOD: This was a retrospective observational descriptive study of patients aged 20 to 45 years who had undergone gastrointestinal endoscopy (UGE) at Prince Mshiyeni Memorial Hospital (PMMH) in KwaZulu-Natal, during the period January 2015 to December 2015. One hundred and ninety-four patients' charts and UGE reports were reviewed. Data were analysed using SPSS Statistics version 24. The level of significance was set at p < 0.05. Variables were expressed as mean ± standard deviation or medians (interquartile range IQR) as appropriate. Mean ± standard deviation was compared using the Student's t-test. Proportions and categorical variables were compared using the Pearson's chi-square test or Fisher's exact test as appropriate. An ethical approval was obtained from the University of KwaZulu-Natal BREC (BE 447/17) and the KwaZulu-Natal Department of Health Ethics Committee. RESULTS: Epigastric pain was found to be the most common indication for UGE, with a total of 112 (57.7%) out of total of 194 patients, followed by upper gastrointestinal bleeding (UGIB) (42) (21.6%). Amongst patients presenting with epigastric pain, only 12(10.7%) patients could be confirmed that they had received acid suppression therapy prior to the testing. In the age group 20-25 years, there was a highest number of patients presenting with corrosive substance ingestion, 11 (25.0%). The commonest finding was gastritis in 99 patients (51.0%), followed by normal findings in 50 (25.7%) patients. CONCLUSION: Epigastric pain was the most common indication and gastritis was the predominant finding.

Outcomes of lung transplantation for cystic fibrosis in a large UK cohort.

BACKGROUND: Lung transplantation is an important option to treat patients with advanced cystic fibrosis (CF) lung disease. The outcomes of a large UK cohort of CF lung transplantation recipients is reported. METHODS: Retrospective review of case notes and transplantation databases. RESULTS: 176 patients with CF underwent lung transplantation at our centre. The majority (168) had bilateral sequential lung transplantation. Median age at transplantation was 26 years. Diabetes was common pretransplantation (40%). Polymicrobial infection was common in individual recipients. A diverse range of pathogens were encountered, including the Burkholderia cepacia complex (BCC). The bronchial anastomotic complication rate was 2%. Pulmonary function (forced expiratory volume in 1 s % predicted) improved from a pretransplantation median of 0.8 l (21% predicted) to 2.95 l (78% predicted) at 1 year following transplantation. We noted an acute rejection rate of 41% within the first month. Our survival values were 82% survival at 1 year, 70% at 3 years, 62% at 5 years and 51% at 10 years. Patients with BCC infection had poorer outcomes and represented the majority of those who had a septic death. Data are presented on those free from these infections. Bronchiolitis obliterans syndrome (BOS) and sepsis were common causes of death. Freedom from BOS was 74% at 5 years and 38% at 10 years. Biochemical evidence of renal dysfunction was common although renal replacement was infrequently required (<5%). CONCLUSION: Lung transplantation is an important therapeutic option in patients with CF even in those with more complex microbiology. Good functional outcomes are noted although transplantation associated morbidities accrue with time.

Differential detection of phosphorylated glycogen synthase kinase 3 alpha and beta depending on blocking conditions.

It is generally recommended that immunoblots using phosphospecific antibodies be performed using bovine serum albumin (BSA) instead of milk. There are two subtypes of glycogen synthase kinase 3 (GSK3), GSK3 alpha and GSK3 beta, with apparent molecular weights of 51 and 49 kDa, respectively. Here we show that immunoblots performed using 5% milk allow the detection of both phosphorylated GSK3 alpha and phosphorylated GSK3 beta, whereas only phosphorylated GSK3 alpha is visible when immunoblots are performed using 3% BSA.

Clinical experience with assisted venous drainage cardiopulmonary bypass in elective cardiac reoperations.

Reoperative cardiac surgery is associated with substantial morbidity and mortality due to technical problems at sternal reentry, which can result in laceration of the right ventricle, innominate vein injury, or embolization from patent grafts. To minimize the risk associated with reentry, we adopted the method of assisted venous drainage in the cardiopulmonary bypass circuit with peripheral cannulation for cardiac reoperations. From March 1999 to May 2003, a series of 52 patients (38 males; mean age 48.7 years, range 4 months to 78 years) underwent cardiac reoperations performed with centrifugal pump venous-assisted cardiopulmonary bypass. EuroSCORE was 7.34 +/- 3.9 (range, 4-19). The reoperations were coronary artery bypass graft (25 patients), valve replacement/repair (18 patients), and complex pediatric procedures (11 patients). The studied adverse events were structural damage at reentry, mortality, blood loss, stroke, and hemolysis. Complications at sternotomy were damage to the innominate vein (1 patient) and aorta (1 patient) with blood loss of 625 and 225 mL, respectively. Four patients required intraaortic balloon pump or extracorporeal membrane oxygenation (n = 1) for hemodynamic support on weaning off cardiopulmonary bypass. Three patients died in the postoperative period. Our experience with centrifugal pump-assisted venous drainage in cardiac reoperations has shown excellent results, with reduced risk of damage to vital structures on sternal reentry. In cases in which structural damage did occur, blood loss was minimal.

Glucose-induced phosphorylation of the insulin receptor. Functional effects and characterization of phosphorylation sites.

Elevated glucose concentrations have been reported to inhibit insulin receptor kinase activity. We studied the effects of high glucose on insulin action in Rat1 fibroblasts transfected with wild-type human insulin receptor (HIRcB) and a truncated receptor lacking the COOH-terminal 43 amino acids (delta CT). In both cell lines, 25 mM glucose impaired receptor and insulin receptor substrate-1 phosphorylation by 34%, but IGF-1 receptor phosphorylation was unaffected. Phosphatidylinositol 3-kinase activity and bromodeoxyuridine uptake were decreased by 85 and 35%, respectively. This was reversed by coincubation with a protein kinase C (PKC) inhibitor or microinjection of a PKC inhibitor peptide. Phosphopeptide mapping revealed that high glucose or PMA led to serine/threonine phosphorylation of similar peptides. Inhibition of the microtubule-associated protein (MAP) kinase cascade by the MAP kinase kinase inhibitor PD98059 did not reverse the impaired phosphorylation. We conclude that high glucose inhibits insulin action by inducing serine phosphorylation through a PKC-mediated mechanism at the level of the receptor at sites proximal to the COOH-terminal 43 amino acids. This effect is independent of activation of the MAP kinase cascade. Proportionately, the impairment of insulin receptor substrate-1 tyrosine phosphorylation is greater than that of the insulin receptor resulting in attenuated phosphatidylinositol 3-kinase activation and mitogenic signaling.

Pregnancies complicated by multidrug-resistant tuberculosis and HIV co-infection in Durban, South Africa.

SETTING: Tertiary hospitals in KwaZulu Natal, South Africa. OBJECTIVE: To study the impact of multidrug-resistant tuberculosis (MDR-TB) and human immunodeficiency virus-1 (HIV-1) co-infection during pregnancy on maternal and perinatal outcome. DESIGN: Prospective study performed between 1996 and 2001. Symptomatic pregnant women were investigated for TB. Those with confirmed MDR-TB were reported on. RESULTS: Three of five pregnant women with MDR-TB were HIV-1 co-infected. One woman decided to terminate the pregnancy and one experienced pre-term labour. Two neonates had features of growth restriction. CONCLUSION: Management of pregnant women with MDR-TB in an HIV-endemic area is possible in developing countries.

A review of traumatic chylothorax.

BACKGROUND: Traumatic chylothorax is an extremely rare complication following thoracic trauma or surgery. The aetiology of traumatic chylothorax is dominated by iatrogenic causes, with a reported incidence of 0.5% to 3% following oesophageal surgery. The mortality from a chylothorax post oesophagectomy can be as high as 50%. Iatrogenic causes in total account for approximately 80% of traumatic causes. Non-iatrogenic traumatic chylothoraces are exceedingly uncommon. The complication rate in blunt thoracic trauma is said to be 0.2% to 3%, whilst in penetrating trauma, the incidence is 0.9% to 1.3%. If recognised late or managed poorly, this condition has devastating complications, including nutritional depletion, physiological derangements and immunological depression. This review revisits the anatomy of the thoracic duct, the physiology of chyle production and associated dynamics as well as the current management strategies available for traumatic chylothorax. METHODS: A review of selected English literature from 1980 to 2015 was undertaken. Databases used included Pubmed, Cochrane and Science Direct. Publications of both traumatic and postoperative chylothorax were reviewed. The appropriate literature was analysed by comparing and contrasting content with particular emphasis on management issues. Keywords and phrases were used to achieve a streamlined and focused review of the topic. CONCLUSION: Chylothorax remains a rare complication of thoracic surgery and thoracic trauma. The potential complications can result in serious morbidity and can even be fatal. Understanding the pathophysiology of a chyle leak underpins the principles of management. The overall success of conservative management ranges from 20% to 80%. The timing of surgical intervention remains debatable. Benefits of early surgical intervention are clearly documented, resulting in a gradual shift toward early operative treatment with reports suggesting thoracic duct ligation yielding a 90% success rate. Technological advances such as thoracic duct embolisation, with a potential success rate of 90%, and thoracoscopic interventions are attractive alternatives to orthodox open surgery.

Prolonged vs transient roles for early cell cycle signaling components.

Both p21ras and phosphatidylinositol 3-kinase (PI 3-k) are critical elements in signaling pathways mediating insulin/IGF-I induced cell cycle progression. For example, microinjection of antibodies, peptides, or recombinant proteins which block the interaction of the SH2 domains of the PI 3-k p85alpha subunit with tyrosine phosphorylated intracellular targets blocks insulin mediated DNA synthesis. We report here that this inhibitory phenotype is observed whether the injections are made into quiescent cells (the standard approach), or at any time point during G1 phase subsequent to stimulation. This observation is not true, however, for the major substrate of the insulin/IGF-I receptor (IRS-1) despite the well known interaction of p85 with IRS-1. Antibodies to IRS-1 are inhibitory only when injected during the first 15 min of G1 phase, as are antibodies to another major IRS-1 binding protein, the tyrosine phosphatase SHP2. We also have microinjected reagents which target proteins involved in the formation of rasGTP and which mediate some of the downstream effects of ras activation. Reagents which target the formation of rasGTP (Shc and dominant negative ras protein) inhibit DNA synthesis only at points early in G1, as do reagents which target components of the MAP kinase pathway. Injection of antibodies to p21ras itself, or a recombinant Raf-1 protein domain which binds to the effector region of ras in a GTP-dependent manner, results in the inhibition of cell cycle progression throughout G1 phase. The results point to a continuous requirement for both PI 3-k and ras activity until cellular commitment to DNA synthesis, although some of the molecules which are both upstream and downstream of these activities are only required transiently. Our results are also consistent with a Raf-1 independent ras activity late in G1, as well as IRS-1 independent effects of PI 3-kinase.

Insulin regulates protein kinase CbetaII alternative splicing in multiple target tissues: development of a hormonally responsive heterologous minigene.

Cells respond to external signals like insulin to alter metabolic pathways in response to varying physiological environments. Insulin stimulates the protein kinase C beta (PKCbeta) isozymes and preferentially switches the expression to PKCbetaII isozyme, which is shown to have a crucial role in glucose uptake, cellular proliferation, and differentiation. We have developed an insulin-responsive PKCbetaII heterologous minigene to identify cis-elements in vivo in eukaryotes by cloning the PKCbetaII exon and its flanking intronic sequences into the splicing vector pSPL3. The transfected minigene mimicked the endogenous insulin response of PKCbetaII alternative splicing in five distinct cell types, i.e. L6 skeletal muscle, 3T3-L1 pre-adipocytes, HepG2 human hepatoma cells, A10 vascular smooth muscle cells, and murine embryonic fibroblasts within 30 min of insulin stimulation. Sequential deletions of the flanking introns in the minigene demonstrated that insulin regulated elements within the 5'-intron flanking the PKCbetaII exon. Mutational studies indicated the SRp40 binding site promotes splice site selection. In these cases, splicing appears to be regulated by a phosphatidylinositol 3-kinase signaling pathway because LY294002 and wortmannin, its specific inhibitors, blocked exon inclusion. Cotransfection with constitutively active Akt2 kinase mimicked insulin action. Signal-dependent regulation of splicing by insulin is unique from tissue-specific and developmentally regulated mechanisms previously reported and serves as a prototype for studies of alternative splicing involving protein phosphorylation.

The HIV-1 exposed neonate: outcome of intensive care management in the first week of life.

A prospective study was carried out to determine if the outcome in HIV-exposed neonates requiring intensive care (n=30) is different from that in HIV-unexposed neonates (n=40) requiring intensive care in the first week of postnatal life. It was noted that the outcome in terms of incidence of death and intensive care stay do not differ significantly in these two groups although some hematological parameters may be significantly different. Considering the fact that the outcome is not worse in HIV-exposed babies and that most of these babies ultimately turn out to be HIV-uninfected, these babies should not be deprived of intensive care, whenever necessary.