Egr-1 functions as a master switch regulator of remote ischemic preconditioning-induced cardioprotection.

Despite improved treatment options myocardial infarction (MI) is still a leading cause of mortality and morbidity worldwide. Remote ischemic preconditioning (RIPC) is a mechanistic process that reduces myocardial infarction size and protects against ischemia reperfusion (I/R) injury. The zinc finger transcription factor early growth response-1 (Egr-1) is integral to the biological response to I/R, as its upregulation mediates the increased expression of inflammatory and prothrombotic processes. We aimed to determine the association and/or role of Egr-1 expression with the molecular mechanisms controlling the cardioprotective effects of RIPC. This study used H9C2 cells in vitro and a rat model of cardiac ischemia reperfusion (I/R) injury. We silenced Egr-1 with DNAzyme (ED5) in vitro and in vivo, before three cycles of RIPC consisting of alternating 5 min hypoxia and normoxia in cells or hind-limb ligation and release in the rat, followed by hypoxic challenge in vitro and I/R injury in vivo. Post-procedure, ED5 administration led to a significant increase in infarct size compared to controls (65.90 ± 2.38% vs. 41.00 ± 2.83%, p < 0.0001) following administration prior to RIPC in vivo, concurrent with decreased plasma IL-6 levels (118.30 ± 4.30 pg/ml vs. 130.50 ± 1.29 pg/ml, p < 0.05), downregulation of the cardioprotective JAK-STAT pathway, and elevated myocardial endothelial dysfunction. In vitro, ED5 administration abrogated IL-6 mRNA expression in H9C2 cells subjected to RIPC (0.95 ± 0.20 vs. 6.08 ± 1.40-fold relative to the control group, p < 0.05), resulting in increase in apoptosis (4.76 ± 0.70% vs. 2.23 ± 0.34%, p < 0.05) and loss of mitochondrial membrane potential (0.57 ± 0.11% vs. 1.0 ± 0.14%-fold relative to control, p < 0.05) in recipient cells receiving preconditioned media from the DNAzyme treated donor cells. This study suggests that Egr-1 functions as a master regulator of remote preconditioning inducing a protective effect against myocardial I/R injury through IL-6-dependent JAK-STAT signaling.

The nitroxide 4-methoxy TEMPO inhibits neutrophil-stimulated kinase activation in H9c2 cardiomyocytes.

After acute myocardial infarction (AMI), neutrophils are recruited to the affected myocardium. Hypochlorous acid (HOCl) produced by neutrophil myeloperoxidase (MPO) damages cardiomyocytes and potentially expands the primary infarct. Rat cardiomyocyte-like cells were incubated with isolated human neutrophils treated with chemical activators in the absence or presence of nitroxide 4-methoxy-Tempo (MetT; 25 µM) for 4, 6 or 24 h; studies with reagent HOCl served as positive control. Treating cardiomyocytes with activated neutrophils or reagent HOCl resulted in a marked increase in protein tyrosine chlorination and a decline in protein tyrosine phosphatase (PTP) activity. On balance our data also supported an increase in phosphorylation of MAPK p38 and ERK1/2 suggestive of an intracellular hyperphosphorylation status and this was accompanied by decreases in cell viability, as judged by assessing caspases-3/7 activity. For cells exposed to activated neutrophils receptor-mediated uptake of transferrin decreased although total matrix metalloproteinase (MMP) activity was unaffected. Addition of MetT ameliorated protein tyrosine chlorination, decreased MAPK activity and restored receptor-mediated transferrin uptake and PTP activity in cardiomyocytes. Overall, adverse effects of neutrophil-derived HOCl on cultured cardiomyocytes were ameliorated by MetT suggesting that nitroxides may be beneficial to inflammatory pathologies, where neutrophil recruitment/activation is a prominent and early feature.

Clinicopathological insights into lupus nephritis in South Africans: a study of 251 patients.

There are few published studies on biopsy proven lupus nephritis (LN) from sub-Sahara Africa, mainly due to lack of expertise and pathology back-up for performing and interpreting renal biopsies in many centres. The purpose of this study was to document factors associated with biopsy proven LN and to determine clinical and laboratory models that best predict proliferative LN in South Africans. Of the 251 patients studied, 84.1% were females and 79.3% were of mixed ancestry. There were more observed cases of proliferative LN (63%) than non-proliferative LN. Factors associated with proliferative LN were male gender (p = 0.049), haematuria on dipstix (p < 0.0001), proteinuria on dipstix (p = 0.042), low serum albumin (p = 0.032), low complement C3 (p < 0.0001), low complement C4 (p = 0.009) and positive double-stranded DNA (p = 0.039). Using four models designed from various combinations of the factors associated with proliferative LN, the specificity and positive predictive values were highest for the model that combined gender (male), presence of dipstix haematuria and proteinuria, hypoalbuminaemia, low C3 and low C4 and positive double-stranded DNA (100% respectively). Further study is recommended to identify the value of using these demographic and laboratory parameters in identifying patients with proliferative LN in resource limited centres where the performance of a biopsy is not possible.

Resistance mechanisms in BRAFV600E paediatric high-grade glioma and current therapeutic approaches.

Paediatric high-grade gliomas (pHGG) are aggressive central nervous system tumours with a poor prognosis. BRAFV600E mutant pHGGs can be treated with targeted BRAF inhibitors, which have shown both preclinical activity and potent clinical efficacy. Unfortunately, the development of drug resistance results in disease relapse or progression and is the primary cause of treatment failure. While there is a lot of data to explain mechanisms of resistance in other BRAFV600E tumours, comparatively little is known about the mechanisms of BRAF inhibitor resistance in BRAFV600E pHGG. Recent literature has identified aberrations in members of the RAS/RAF/ERK pathway, the PI3K/AKT/MTOR pathway and the cell cycle as major contributors to the resistance profile. A range of novel therapies have been suggested to overcome BRAF inhibitor drug resistance in BRAFV600E pHGG. This review will discuss the current literature available for BRAF inhibitor resistant BRAFV600E pHGGs and provide an overview of the currently available and proposed therapies.

Hypertension in the South African public healthcare system: a cost-of-illness and burden of disease study.

OBJECTIVES: To quantify the health and economic burden of hypertension in the South African public healthcare system. SETTING: All inpatient, outpatient and rehabilitative care received in the national public healthcare system. PARTICIPANTS: Adults, aged ≥20 years, who receive care in the public healthcare system. OUTCOMES: Worksheet-based models synthesised data from multiple sources to estimate the burden of disease, direct healthcare costs, and societal costs associated with hypertension. Results were disaggregated by sex. RESULTS: Approximately 8.22 million (30.8%, 95% CI 29.5% to 32.1%) South African adults with no private health insurance have hypertension. Hypertension was estimated to cause 14 000 (95% CI 11 100 to 17 200) ischaemic heart disease events, 13 300 (95% CI 10 600 to 16 300) strokes and 6100 (95% CI 4970 to 7460) cases of chronic kidney disease annually. Rates of hypertension, hypertension-related stroke and hypertension-related chronic kidney disease were greater for women compared with men.The direct healthcare costs associated with hypertension were estimated to be ZAR 10.1 billion (95% CI 8.98 to 11.3 billion) or US$0.711 billion (95% CI 0.633 to 0.793 billion). Societal costs were estimated to be ZAR 29.4 billion (95% CI 26.0 to 33.2 billion) or US$2.08 billion (95% CI 1.83 to 2.34 billion). Direct healthcare costs were greater for women (ZAR 6.11 billion or US$0.431 billion) compared with men (ZAR 3.97 billion or US$0.280 billion). Conversely, societal costs were lower for women (ZAR 10.5 billion or US$0.743 billion) compared with men (ZAR 18.9 billion or US$1.33 billion). CONCLUSION: Hypertension exerts a heavy health and economic burden on South Africa. Establishing cost-effective best practice guidelines for hypertension treatment requires further research. Such research will be essential if South Africa is to make progress in its efforts to implement universal healthcare.

Prophylaxis is the new standard of care in patients with haemophilia.

Randomised controlled clinical trial evidence on prophylaxis as optimal care for patients with haemophilia was generated more than a decade ago. However, this knowledge has not translated into clinical practice in South Africa (SA) owing to many barriers to prophylaxis. These include the high treatment burden imposed by prophylaxis (frequent injections two to four times a week), the need for intravenous access to administer replacement clotting factor therapies, and the higher volume of clotting factor required compared with episodic treatment. The recently introduced non-factor therapies in haemophilia care have addressed many of these barriers. For example, emicizumab, which is currently the only globally approved non-factor therapy, can be administered subcutaneously less frequently (weekly, fortnightly or every 4 weeks) and has led to global adoption of prophylaxis as the standard of care in haemophilia by the bleeding disorders community. Haemophilia A is the most prevalent clotting factor deficiency in SA, with >2 000 people diagnosed to date. However, only a few of these patients are currently on prophylaxis. In this 'In Practice' article, we review the rationale for prophylaxis, outline its goals and benefits, and provide evidence-based guidance on which haemophilia patients should be prioritised for emicizumab prophylaxis. This consensus guidance facilitates the adoption of prophylaxis as a national policy and the new standard of care in haemophilia in SA.

COVID-19 complications in a small-town hospital in South Africa.

South Africa has experienced three deadly waves of the COVID-19 pandemic with devastating consequences, but little is known about the experiences in small-town hospitals in the country. Between May 2020 and June 2021, author GC treated ~100 confirmed COVID-19 cases. This retrospective case series report describes 10 of these cases, 7 with unusual complications and 3 with sudden death.

PASCAR commentary on the International Society of Hypertension global guidelines 2020: relevance to sub-Saharan Africa.

Hypertension guidelines have been based on country-specific data until the publication of the International Society of Hypertension (ISH) global guidelines. The major differences between the ISH global guidelines and other international guidelines are the stratified recommendations to accommodate differences in available resources between countries and within countries. This is a key and novel proposal in the new ISH guidelines. There is the separation of optimal versus essential criteria for diagnosis and treatment according to availability of resources. This guideline includes recommendations for sub-Saharan Africa. The Pan-African Society of Cardiology (PASCAR) continues to promote awareness and recommendations on hypertension in Africa. This commentary provides a summary and discussion of the global guidelines in order to clarify the position of PASCAR.

A multicentre cross-sectional descriptive study evaluating the cardiovascular risk profile of preoperatively identified patients with hypertension.

BACKGROUND: The prevalence of hypertension in adults in South Africa (SA) is 35%. Hypertension is the most important modifiable risk factor for cardiovascular (CV) and chronic kidney disease (CKD) in sub-Saharan Africa. However, 49% of people are unaware of their blood pressure status. Screening for hypertension prior to surgery provides a unique opportunity to diagnose and treat affected individuals. Furthermore, assessing overall CV risk identifies patients at highest risk for complications, and improves the utilisation of scarce resources. OBJECTIVES: To evaluate the CV risk profile of hypertensive patients in the adult population of the Western Cape Province presenting for elective non-cardiac, non-obstetric surgery. METHODS: This report documents the CV risk profile of patients recruited to the HASS-2 study (Hypertension and Surgery Study 2), which was undertaken in seven Western Cape hospitals. Patients were screened for hypertension and pharmacological treatment was initiated or adjusted in patients with stages 1 and 2 disease. Stage 3 patients were referred to a physician. In the present substudy, patients with stages 1 and 2 hypertension were assessed for associated CV risk factors, the presence of target organ damage, and documented CV or kidney disease; they received an overall risk stratification according to the 2018 European Society of Cardiology and the European Society of Hypertension Guidelines. RESULTS: Sixty-one patients with stage 1 and 12 with stage 2 hypertension were analysed. Established CV disease was present in 13.7% of the study population, and CKD (eGFR <60 mL/min) in 10.8%. Seventy-one percent of the study group had a raised body mass index, and 55.9% underlying metabolic syndrome. Prediabetes and diabetes were present in 16.1% and 14.5%, respectively. According to the 2018 European guidelines, 34.7% were at moderate, 33.3% at high and 16.7% at very high risk for a CV event in the following 10 years. CONCLUSIONS: The perioperative period is a critical time during which surgeons, nurses and anaesthetists can influence patients' CV risk of adverse events. This involves appropriate screening, education and treatment. In this study population, nearly 9 out of 10 elective surgical patients with stage 1 or 2 hypertension had CV risk factors placing them at moderate to very high risk. The simultaneous assessment of these additional CV risk parameters, in addition to diagnosis and management of hypertension, may further decrease the health and financial burden in resource-limited facilities in SA, and improve CV outcomes.

Hypertension in pregnancy: A future risk for chronic kidney disease in South Africa.

BACKGROUND: Hypertension in pregnancy is a risk factor for end-stage chronic kidney disease (ESKD) and is particularly common in South Africa (SA). There are no data for the risk of developing chronic kidney disease (CKD). OBJECTIVES: To conduct a study of all female patients who presented to the renal replacement programme at Groote Schuur Hospital, Cape Town, SA. METHODS: This was a retrospective study of female patients with ESKD who were presented to renal replacement meetings between 2007 and 2017. For each patient who was assessed, there was a comprehensive letter detailing patient demographics, as well as psychosocial and medical history, which served as the source data. Patients with a history of hypertension in pregnancy were identified as the case group and those without the condition were the control group. Patient demographics, causes of CKD, kidney function and outcome of the meeting were documented. RESULTS: Of the 415 female patients with ESKD, 70 (16.9%) had a history of hypertension in pregnancy. The ethnic breakdown was as follows: 132 (42.44%) black, 172 (55.3%) mixed ancestry and 7 (2.25%) white. Compared with the control group, the patients were younger, with a median age of 33 v. 41 years (p<0.001), higher serum creatinine 1 045 v. 751 µmol/L (p=0.017) and lower estimated glomerular filtration rate (eGFR) 4.0 v. 5.1 mL/min (p=0.029). Patients were more likely to abuse methamphetamine (5.7 v. 1.7%; p=0.049), and less likely to be diabetic (1.4 v. 20.9%; p<0.001) or HIV-positive (2.9 v. 12.5%; p=0.019). There were no ethnic differences between patients and controls. Underlying causes of renal disease showed significant differences, as patients were more likely to have hypertensive nephropathy (57.1 v. 22.9%; p<0.0001), and less likely to have diabetic kidney disease (1.4 v. 20.4%; p<0.001), HIV-associated nephropathy (HIVAN) (1.4 v. 6.4%) or polycystic kidney disease (1.4 v. 7.0%). There was no difference in acceptance to the dialysis and transplant programme (53 v. 47%). CONCLUSIONS: This study suggests an important link between hypertension in pregnancy and ESKD. The patients were significantly younger, presented later and were more likely to have hypertensive nephropathy. Methamphetamine abuse appears to be a risk factor. The study suggests that all women with hypertensive disorders during pregnancy need further evaluation and follow-up postpartum.

Life-threatening hyperkalaemia due to trimethoprim in a patient treated for Pneumocystis jirovecii pneumonia.

Hyperkalaemia is a potentially life-threatening condition frequently encountered in hospitalised patients. Among the many causes of hyperkalaemia, drugs have often been implicated. In the South African context, with the high burden of HIV, there is an increased incidence of opportunistic infections such as Pneumocystis jirovecii pneumonia (PJP), and consequently many patients receive high doses of trimethoprim-sulfamethoxazole. A lesser-known side-effect of the trimethoprim component of this combination antibiotic is hyperkalaemia. We report a case in which life-threatening hyperkalaemia developed after institution of high-dose co-trimoxazole for PJP.

Registration of amiloride in South Africa: Cutting the Gordian knot.

Amiloride is an antagonist of the renal tubular epithelial sodium channel (ENaC). As such, it is a diuretic that is both potassium and magnesium sparing. It is used for the treatment of potassium depletion and hypertension, and is the specific therapy for hypertension due to overactivity of the ENaC (Liddle syndrome and several additional genetic causes of the Liddle phenotype - low renin and low aldosterone). It is listed as a World Health Organization essential drug, but has never been registered in South Africa (SA) and can therefore only be prescribed under a Section 21 application to the SA Health Products Regulatory Authority (SAHPRA) on a case-by-case basis. In SA, >50% of patients treated for hypertension are not controlled. In the USA, the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study reported that African Americans are more likely to be diagnosed with hypertension, more likely to be treated, more likely to be treated intensively, and less likely to achieve blood pressure (BP) control. Although the reasons are complex, studies show that 10 - 20% of blacks may carry the Liddle phenotype. Observational data and a controlled clinical trial done in three African countries have shown that these patients respond to amiloride and not to conventional guideline-based antihypertensive treatment. The former is likely to result in a significant reduction in cardiovascular, stroke and kidney morbidity and mortality, because of improved BP control. Amiloride is very unlikely to ever be registered in SA, as it was first developed >50 years ago, and SAHPRA regulations prevent widespread prescription of this essential drug. This is a classic Gordian knot that requires a novel approach from authorities to sever the knot and improve the health of many South Africans.

Polymorphonuclear leukocyte phagocytic function increases in plasminogen knockout mice.

BACKGROUND: Mice lacking plasminogen (PG-/-) require alternative pathways of fibrinolysis for survival. This may depend on polymorphonuclear leukocytes (PMN) that can clear soluble and insoluble fibrin(ogen) through PG-independent processes. Our objective was to demonstrate that PMNs from PG-/- mice exhibit increased Mac-1 dependent phagocytic activity, which may explain their increased fibrin(ogen)lytic activity compared with wild type (PG+/+) mice. METHODS: Phagocytic activity of PMNs from PG-/- and PG+/+ mice was compared following exposure to Staphylococcus aureus (S. aureus) particles and the expression of Mac-1 was assessed in parallel by flow cytometric analysis. Resistance to phorbol-12-myristate-13-acetate (PMA)-induced cell death was compared between PMNs from the different genotypes. RESULTS: Stimulation of PG-/- PMNs by opsonized S. aureus diluted in PG-/- plasma significantly increased phagocytosis (15-fold) compared with stimulation of PG+/+ PMNs in PG+/+ plasma. Incubation of PG-/- PMNs with PG+/+ plasma (control) or PG-/- plasma supplemented with human PG inhibited this increased phagocytic activity. Mac-1 cell surface density increased 6.2+/-1.0-fold in PG-/- PMNs versus 2.9+/-0.6-fold in PG+/+ PMNs (P < 0.01) indicating that Mac-1 may be associated with increased phagocytic activity. Supporting this, treatment of PG-/- PMNs with an anti-Mac-1 antibody in PG-/- plasma inhibited phagocytic activity. In addition, physiologic PG blocked Mac-1 accessibility at the surface of PMNs. Addition of PMA resulted in 33% death of PMNs from PG-/- mice versus 68% in PG+/+ controls (P < 0.001). CONCLUSIONS: PMNs from PG-/- mice exhibit a Mac-1 dependent increase in phagocytic activity that is suppressed with human PG, an anti-Mac-1 antibody or the plasma from PG+/+ mice. The propensity for PMNs from PG-/- mice to be activated in response to PMA together with their relative resistance to PMA-toxicity may contribute to increased PMN half-life and enhanced fibrin(ogen) clearance in the setting of PG deficiency.

Remote ischemic preconditioning attenuates EGR-1 expression following myocardial ischemia reperfusion injury through activation of the JAK-STAT pathway.

BACKGROUND/OBJECTIVES: Remote ischemic preconditioning (RIPC) protects the myocardium from ischemia/reperfusion (I/R) injury however the molecular pathways involved in cardioprotection are yet to be fully delineated. Transcription factor Early growth response-1 (Egr-1) is a key upstream activator in a variety of cardiovascular diseases. In this study, we elucidated the role of RIPC in modulating the regulation of Egr-1. METHODS: This study subjected rats to transient blockade of the left anterior descending (LAD) coronary artery with or without prior RIPC of the hind-limb muscle and thereafter excised the heart 24h following surgical intervention. In vitro, rat cardiac myoblast H9c2 cells were exposed to ischemic preconditioning by subjecting them to 3cycles of alternating nitrogen-flushed hypoxia and normoxia. These preconditioned media were added to recipient H9c2 cells which were then subjected to 30min of hypoxia followed by 30min of normoxia to simulate myocardial I/R injury. Thereafter, the effects of RIPC on cell viability, apoptosis and inflammatory markers were assessed. RESULTS: We showed reduced infarct size and suppressed Egr-1 in the heart of rats when RIPC was administered to the hind leg. In vitro, we showed that RIPC improved cell viability, reduced apoptosis and attenuated Egr-1 in recipient cells. CONCLUSIONS: Selective inhibition of intracellular signaling pathways confirmed that RIPC increased production of intracellular nitric oxide (NO) and reactive oxygen species (ROS) via activation of the JAK-STAT pathway which then inactivated I/R-induced ERK 1/2 signaling pathways, ultimately leading to the suppression of Egr-1.

High prevalence of "non-dipping" blood pressure and vascular stiffness in HIV-infected South Africans on antiretrovirals.

BACKGROUND: HIV-infected individuals are at increased risk of tissue inflammation and accelerated vascular aging ('inflamm-aging'). Abnormal diurnal blood pressure (BP) rhythms such as non-dipping may contribute to an increased risk of cardiovascular and cerebrovascular events in HIV infected individuals. However, little data exists on ambulatory blood pressure (ABP) and measures of vascular stiffness in the black African HIV infected population. METHODS: This is a cross-sectional analysis of otherwise well, HIV infected outpatients on ART for >5 years. Study assessments included: 24hr ABP monitoring, pulse wave velocity (PWV) and central aortic systolic pressure (CASP) using a AtCor Medical Sphygmocor device, fasting lipogram, oral glucose tolerance test, high-sensitivity C-reactive protein (hsCRP) and anthropometric data. Patients completed a questionnaire of autonomic symptoms. CD4+ counts and viral loads were obtained from the National Laboratory results system. RESULTS: Sixty seven black participants were included in the analysis of whom 91% (n = 61) were female with a mean age of 42.2 ± 8.6 years. The median duration on ART was 7.5 years (IQR = 6-10), 84% were virally supressed and the median CD4 count was 529.5cells/mm3 (IQR = 372.0-686.5). The majority (67%) were classified as overweight and 76% had an increased waist circumference, yet only 88% of participants were normotensive. A hsCRP level in the high cardiovascular risk category was found in 68% of participants. The prevalence of non-dipping BP was 65%. Interestingly, there was no association on multivariable analysis between dipping status and traditional risk factors for non-dipping BP, such as: obesity, autonomic dysfunction and older age. CONCLUSION: This relatively young cross-sectional sample of predominantly normotensive, but overweight black women on effective ART >5 years showed: a high prevalence of non-dipping BP, inflammation and vascular stiffness. Causality cannot be inferred but cardiovascular risk reduction should be emphasized in these patients.

Therapeutic drug monitoring of amlodipine and the Z-FHL/HHL ratio: Adherence tools in patients referred for apparent treatment-resistant hypertension.

BACKGROUND: Non-adherence to antihypertensives is a cause of 'pseudo-treatment-resistant' hypertension. OBJECTIVE: To determine whether monitoring plasma amlodipine concentrations and inhibition of angiotensin-converting enzyme (ACE) can be adjunct adherence tools. METHODS: Patients with hypertension who were prescribed enalapril and amlodipine were enrolled. Blood pressures (BPs) were monitored and an adherence questionnaire was completed. Steady-state amlodipine was assayed using liquid chromatography-mass spectrometry and degree of ACE inhibition using the Z-FHL/HHL (z-phenylalanine-histidine-leucine/hippuryl-histidine-leucine) ratio. RESULTS: One hundred patients (mean (standard deviation) age 50.5 (12) years, 46% male) were enrolled. Based on plasma assays, 26/97 patients (26.8%) were unsuppressed by enalapril and 20/100 (20%) were sub-therapeutic for amlodipine. There were significant BP differences based on plasma levels of the medication: 21/20 mmHg lower in the group with suppressed ACE and 26/20 mmHg in the group with steady-state amlodipine concentrations. CONCLUSIONS: Monitoring antihypertensive adherence by assaying plasma medication concentrations is a feasible option for evaluating true v. pseudo-resistant hypertension.

Chiral and steric effects in the efficient binding of alpha-anomeric deoxyoligonucleoside N-alkylphosphoramidates to ssDNA and RNA.

We report hybridization properties of new phosphate-modified alpha-oligonucleoside analogs with non-ionic or cationic internucleotide linkages such as methoxy-ethylphosphoramidate (PNHME), phosphoromorpholi-date (PMOR) and dimethylaminopropylphosphor-amidate (PNHDMAP). First we evaluated the chirality effect of the phosphorus atom on the affinity of alpha- or beta-dodecanucleoside phosphodiesters containing one chirally enriched N -alkylphosphoramidate linkage located in the middle of the sequence d(TCTT-AA*CCCACA). As for P-substituted beta-oligonucleo-tides, a difference in binding behavior between the two diastereoisomers (difference in Delta T (m)) exists in the hybridization properties of alpha-analogs when DNA was the target but this effect was not detrimental to duplex stability. This effect was considerably reduced when RNA was the target. Secondly we studied the effect of steric hindrance around phosphorus on the affinity of fully modified beta- and alpha-oligonucleoside N -alkylphosphoramidates for their DNA and RNA targets. This effect was very weak with alpha-analogs whereas it was more pronounced with beta-oligos. PNHME-modified alpha-oligonucleosides formed more stable duplexes with DNA (Delta T (m)+9.6 degrees C) and RNA (Delta T (m)+1.4 degrees C) targets than the 'parent' phosphodiester. Finally, base pairing specificity of these alpha-oligonucleo-side N -alkylphosphoramidates for their targets was found to be as high as for natural oligonucleoside phosphodiesters.

Lipophilic pro-oligonucleotides are rapidly and efficiently internalized in HeLa cells.

Model t-Bu-SATE pro-dodecathymidines labeled with fluorescein and exhibiting various lipophilicities were evaluated for their uptake by cells in culture. Pro-oligonucleotides with appropriate lipophilicity were found to permeate across the HeLa cell membrane much more extensively than the control phosphorothioate oligo or than the hydrophilic pro-oligos. Fluorescence patterns of internalization were consistent with a diffusion mechanism resulting in the appearance of a uniform cytoplasmic distribution and nuclear accumulation, as confirmed by confocal microscopy.

Stereochemical control of DNA biosynthesis.

Stereochemical control of DNA biosynthesis was studied using several DNA-synthesizing complexes containing, in each case, a single substitution of a 2'-deoxy-D-nucleotide residue by an enantiomeric L-nucleotide residue in a DNA chain (either in the primer or in the template) as well as 2'-deoxy-L-ribonucleoside 5'-triphosphates (L-dNTPs) as substrates. Three template-dependent DNA polymerases were tested, Escherichia coli DNA polymerase I Klenow fragment, Thermus aquaticus DNA polymerase and avian myeloblastosis virus reverse transcriptase, as well as template-independent calf-thymus terminal deoxynucleotidyl transferase. Very stringent control of stereoselectivity was demonstrated for template-dependent DNA polymerases, whereas terminal deoxynucleotidyl transferase was less selective. DNA polymerase I and reverse transcriptase catalyzed formation of dinucleoside 5',5'-tetraphosphates when L-dTTP was used as substrate. Comparison between models of template-primer complexes, modified or not by a single L-nucleotide residue, revealed striking differences in their geometry.

A two B-Z junction containing DNA resolves into an all right-handed double-helix.

Natural and artificial oligonucleotides are capable of assuming many different conformations and functions. Here we present results of an NMR restrained molecular modelling study on the conformational preferences of the modified decanucleotide d((m)C1G2(m)C3G4C5(L)G6(L)(m)C7G8(m)C9G10) .d((m)C11G12(m)C13G14C15(L)G (L)16(m)C17-G18(m)C19G20 ) which contains L deoxynucleotides in its centre. This chimeric DNA was expected to form a right-left-right-handed B-type double-helix (BB*B) at low salt concentration. Actually, it matured into a fully right-handed double helix with its central C(L)pG(L) core forming a right-handed Z-DNA helix embedded in a B-DNA matrix (BZ*B). The interplay between base-base and base-sugar stackings within the core and its immediately adjacent residues was found to be critical in ensuring the stabilisation of the right-handed helix. The structure could serve as a model for the design of antisense oligonucleotides resistant to nucleases and capable of hybridising to natural DNAs and RNAs.