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.

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.

Comparison of equations for the calculation of LDL-cholesterol in hospitalized patients.

BACKGROUND: The Friedewald equation is widely used to calculate LDL-C for cardiovascular risk prediction but is less accurate with comorbidities and extreme lipid values. Several novel formulae have been reported to outperform the Friedewald formula. METHODS: We examined 14,219 lipid profiles and evaluated four formulae (Friedewald, Chen, de Cordova, Hattori) and compared these to direct measurement of LDL-C across various triglyceride (TG), total cholesterol (TC) and HDL-cholesterol (HDL-C) ranges using Beckman reagents and instruments. Linear regression and ROC analysis were performed. RESULTS: The de Cordova formula showed a high correlation with directly measured LDL-C (r=0.90, P<0.001), comparable to the Friedewald calculated values for directly measured LDL-C (r=0.95, P<0.001). The de Cordova formula was favorable in some ranges of HDL, TC and the lowest TG range (r=0.97, P<0.001) but performed least well in comparison with the three other LDL-C calculations (AUC=0.8331), demonstrating inconsistent bias. The Chen formula performed better than Friedewald (AUC=0.9049). The Hattori formula outperformed all formulae including Friedewald over various ranges of lipid values (AUC=0.9097). CONCLUSIONS: We observe favorable correlations of the de Cordova formula with Friedewald at low TG values. However, the Hattori formula appears to be best for application in hospitalized patients, even at extreme lipid values.

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.

Investigation of glucocorticoid receptor polymorphisms in relation to metabolic parameters in Addison's disease.

BACKGROUND: Uncertainty exists whether glucocorticoid receptor (GCR) polymorphisms play a role in steroid-related side effects in Addison's disease (AD) patients on hydrocortisone. The polymorphisms Bcll and N363S appear to increase sensitivity to cortisol, while the ER22/23EK polymorphism has been associated with resistance to cortisol. METHOD: One hundred and forty seven AD patients, and gender, and ethnicity-matched controls were recruited in South Africa. Three polymorphisms in the GCR were studied, using PCR followed by restriction fragment length analysis. Associations with BMI, lipids, glucose and inflammatory markers were investigated. RESULTS: In both patients and controls, the Bcll polymorphism occurred more frequently in whites than in other ethnic groups studied but was not associated with any of the metabolic parameters tested. The ER22/23EK polymorphism was associated with an increased BMI in both patients (29.4 vs 24.7  kg/m²) and control subjects (26.3 vs 24.2  kg/m²). The ER22/23EK polymorphism was also associated with lower LDL cholesterol in control subjects (3.46 vs 3.93  mmol/l) and in patients (3.52 vs 4.10  mmol/l). N363S was associated with increased BMI in controls 29.9  kg/m² vs wild type 24.8  kg/m². Median hydrocortisone doses were greater in patients heterozygous for either ER22/23EK 30.0  mg or N363S 25.0  mg polymorphisms than in wild type patients 20.0  mg (both comparisons). CONCLUSION: Alterations in lipids, BMI and hydrocortisone dose were associated with two polymorphisms. Further larger studies are warranted to corroborate these findings.

Plasma free fatty acid reference interval in South African neonates in the first week of life.

BACKGROUND: Non-esterified fatty acid (NEFA) levels are an important diagnostic tool in the investigation of neonatal hypoglycaemia. NEFA reference intervals have not been reported for neonates previously. METHODS: The objective of this study was to determine an NEFA reference interval for neonates. RESULTS: Heparinized plasma obtained from 106 healthy neonates in the first week of life was analysed using the Roche "Free fatty acid, Half-micro test" kit. Results were then analysed statistically for normality (Shapiro-Wilk test) and reference interval determined non-parametrically (bootstrap method). CONCLUSIONS: NEFA levels displayed a non-Gaussian distribution and the reference interval (2.5th and 97.5th percentiles) was 0.2-1.5 mmol/L (90% confidence intervals 0.1-0.3 and 1.4-2.0 mmol/L, respectively). The NEFA reference interval in South African neonates less than a week old is similar to that described in infants (1-12 months), indicating that this reference range can be used over the entire neonatal period.

Insulin Resistance Induced by Antiretroviral Drugs: Current Understanding of Molecular Mechanisms

The increase in incidence of HIV infection continues to be a major public health problem across the world; but more especially in sub- Saharan Africa. Treatment with highly active antiretroviral therapy (HAART) has improved the prognosis of patients with AIDS; but it has also increased the incidence of various metabolic disorders; in particular insulin resistance accompanied by dyslipidaemia; hyperglycaemia and lipodystrophy. This is often accompanied by frank type 2 diabetes and increased mortality from cardiovascular disease. It is important to understand the mechanistic basis for these side-effects as the incidence of these is likely to increase as the rollout of antiretroviral drugs continues

Quality of teaching in chemical pathology: ability of interns to order and interpret laboratory tests.

BACKGROUND AND OBJECTIVE: There has been a steady decline in the overt teaching of many basic and pathology sciences in the medical curriculum worldwide. As interns are the doctors most likely to request and act on tests, an assessment of their confidence in dealing with laboratory investigations was undertaken. METHODS: Interns at two hospitals in Cape Town, South Africa, were asked to complete a structured questionnaire designed to assess their confidence in ordering and interpreting a number of tests. The questionnaire also probed their desire for further teaching and the preferred delivery vehicle for such teaching. RESULTS AND CONCLUSIONS: 61 out of 117 questionnaires were returned. Interns were confident in the use of common tests, but 23% were not confident in interpreting a test that they were confident in ordering. All respondents felt they would benefit from teaching in at least one area and lectures were the preferred method, although the majority felt it very likely that they would complete an online tutorial if available. The results suggest that institutions need to devise strategies to fulfil the learning needs of new graduates in the area of chemical pathology and clinical biochemistry.

Relationship between vitamin D, calcium and parathyroid hormone in Cape Town.

AIM: The aim of this study was to test the hypothesis that vitamin D deficiency is associated with abnormal levels of calcium and parathyroid hormone (PTH). METHODS: Vitamin D requests at a tertiary hospital in South Africa over 2 years were retrospectively analysed along with calcium and PTH levels. RESULTS: Only when the 25-hydroxyvitamin D (25(OH)D) level dropped below 25 nmol/l, was there a significant rise in PTH. A subnormal 25(OH)D level was also not always related to hypocalcaemia, as more than half of patients with their 25(OH)D level below 25 nmol/l had calcium levels in the reference range. However, all patients with calcium levels below 1.8 mmol/l were shown to have vitamin D insufficiency. CONCLUSION: Hypovitaminosis D may co-exist with a blunted PTH response. Therefore, assumptions about vitamin D status should not be made based on PTH and calcium values. 25(OH)D measurements should be requested when vitamin D deficiency is clinically suspected, irrespective of biochemical results.

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.

Asb6, an adipocyte-specific ankyrin and SOCS box protein, interacts with APS to enable recruitment of elongins B and C to the insulin receptor signaling complex.

The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport. Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor. Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed. However, Asb6 expression appears to be restricted to adipose tissue. Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts. In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction. In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6. Asb6 did not appear to undergo tyrosine phosphorylation. Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes. In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C. Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6. We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC.

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.

A fisherman's tale: Phage display as a discovery tool.

Extract: Genome sequencing efforts have produced a wealth of information that remains to be exploited at the protein level. A major area of drug discovery involves the identification of novel peptide ligands which could provide leads for the development of new drugs that mimic the structure and function of proteins. These drugs could be used to modulate the activity or interaction of proteins in cells. In biochemistry, ligand refers to a small molecule that binds to a larger macromolecule. Phage surface display, using viruses that infect bacteria called bacteriophages, is a technique where fragments of foreign peptides are joined to the virus proteins and displayed on the phage surface coat, thus maintaining the spatial structure and activity of the protein, and allowing for its exposure to potential interactors or ligands. Both the genetic information and the phenotype are contained within the phage particle allowing for the peptide sequence to be deduced from the DNA sequence. The technique can be used to analyze the molecular recognition properties of peptides and proteins and to identify new ligands for a protein of interest. This would allow for the study of how proteins recognize each other, such as antibody-antigen epitopes (binding sites) and the isolation of biologically active molecules or novel peptide drugs. It can also be used to isolate new antibodies from antibody libraries or identify new substrates for enzymes.

Functional effects of APS and SH2-B on insulin receptor signalling.

APS [for 'adapter protein with a pleckstrin homology (PH) and Src homology 2 (SH2) domain'] belongs to a family of adapter proteins involved in signalling by the receptors for insulin, insulin-like growth factor 1, platelet-derived growth factor and nerve growth factor. Other members include alternatively spliced SH2-B isoforms (SH2Balpha, SH2-Bbeta and SH2-Bgamma) and Lnk. These have a C-terminal SH2 domain, a central PH domain and an N-terminal proline-rich region. SH2Balpha, APS and Lnk have a conserved C-terminal tyrosine phosphorylation site, whereas the alternatively spliced SH2-Bbeta and SH2-Bgamma have distinct C-termini. There is considerable sequence similarity between APS, SH2-B and Lnk, particularly in the SH2 domain. Both APS and SH2-Balpha interact with the insulin-receptor activation loop phosphorylation sites and undergo insulin-stimulated tyrosine phosphorylation, although the phosphorylation of SH2-B is considerably weaker. APS couples c-Cbl to the insulin receptor, resulting in ubiquitination of the insulin receptor. We established cell lines [Chinese hamster ovary (CHO). T-APS and CHO. T-SH2-B cells] overexpressing APS and SH2-Balpha to study their roles in insulin receptor signalling. Either adapter protein enhances insulin receptor and ERK (extracellular-signal-regulated kinase) phosphorylation. In CHO. T-APS cells, Akt phosphorylation is observed earlier than in CHO.T-SH2-B cells. Both enhance insulin-stimulated Akt activation but APS seems to cause greater activation. Thus APS and SH2-B have similar effects on insulin receptor signalling, although the effects of SH2-B are independent of its phosphorylation.

The APS adapter protein couples the insulin receptor to the phosphorylation of c-Cbl and facilitates ligand-stimulated ubiquitination of the insulin receptor.

The APS adapter protein is rapidly tyrosine-phosphorylated following insulin stimulation. In insulin-stimulated 3T3-L1 adipocytes, APS co-precipitated with phosphorylated c-Cbl. In CHO.T-APS cells overexpressing the insulin receptor and APS, APS co-precipitated with c-Cbl but not in CHO.T cells which do not express APS. APS-mediated recruitment of c-Cbl to the insulin receptor led to rapid ubiquitination of the insulin receptor beta-subunit in CHO. T-APS but not in parental CHO.T cells. These results suggest that the function of APS is to facilitate coupling of the insulin receptor to c-Cbl in order to catalyse the ubiquitination of the receptor and initiation of internalisation or degradation.

APS, an adapter protein with a PH and SH2 domain, is a substrate for the insulin receptor kinase.

APS (adapter protein with a PH and SH2 domain) is the newest member of a family of tyrosine kinase adapter proteins including SH2-B and Lnk. We previously identified SH2-B as an insulin-receptor-binding protein and substrate [Kotani, Wilden and Pillay (1998) Biochem J. 335, 103-109]. Here we show that APS interacts with the insulin receptor kinase activation loop through its SH2 domain and insulin stimulates the tyrosine-phosphorylation of APS. Furthermore, the phosphorylation of activation-loop tyrosine residues 1158 and 1162 are required for this interaction.

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.