Complexes of human papillomavirus type 16 E6 proteins form pseudo-death-inducing signaling complex structures during tumor necrosis factor-mediated apoptosis

High-risk strains of human papillomavirus (HPV) such as HPV type 16 (HPV16) and HPV18 are causative agents of most human cervical carcinomas. E6, one of the oncogenes encoded by HPV16, possesses a number of biological and transforming functions. We have previously shown that the binding of E6 to host apoptotic proteins such as tumor necrosis factor (TNF) R1, the adaptor protein FADD, and procaspase 8 results in a significant modification of the normal flow of apoptotic events. For example, E6 can bind to and accelerate the degradation of FADD. In addition, full-length E6 binds to the TNF R1 death domain and can also bind to and accelerate the degradation of procaspase 8. In contrast, the binding of small splice isoforms known as E6* results in the stabilization of procaspase 8. In this report, we propose a model for the ability of HPV16 E6 to both sensitize and protect cells from TNF as well as to protect cells from Fas. We demonstrate that both the level of E6 expression and the ratio between full-length E6 and E6* are important factors in the modification of the host extrinsic apoptotic pathways and show that at high levels of E6 expression, the further sensitization of U2OS, NOK, and Ca Ski cells to TNF-mediated apoptosis is most likely due to the formation of a pseudo-death-inducing signaling complex structure that includes complexes of E6 proteins.     

Position of anthers at plating and its influence on anther callusing in rice

Effect of position of anthers at plating in relation to their ability to form callus was studied in rice (Oryza sativa var. Taipei 309). Among the callusing anthers, about 60% were those positioned on edge with one lobe in contact with the medium while the rest were flat with both lobes touching the medium. Anthers in both positions produced calli and regenerated green plants.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA Naphthalene acetic acid - IAA Indole acetic acid - BAP Benzyl aminopurine - K Kinetin     

A new series of trpE vectors that enable high expression of nonfusion proteins in bacteria.

Expression of recombinant proteins in bacteria has facilitated the characterization of many gene products. However, the biochemical characterization of recombinant proteins is limited since the bacterially expressed proteins are often synthesized as fusion polypeptides. The presence of bacterial sequences in fusion proteins further limits the use of these proteins for generating antibodies since the bacterial sequences are also antigenic. We describe two new bacterial expression vectors based on the pATH series of plasmids. These vectors were made by precisely deleting all of the trpE coding sequences found in pATH. The new vectors have enabled us to express eukaryotic genes as nonfusion polypeptides. These altered plasmids can be used to insert any DNA sequence of interest through a multiple cloning site located just 3' of an ATG start codon. Protein expression is still under the control of the trp operon and is carried out at great efficiency when the bacteria are tryptophan deprived. Studies presented here test the expression system with neurofilament subunits, NF-L and NF-H. Large amounts of recombinant nonfusion proteins were produced. Also, a time course of induction shows that the production of the nonfusion proteins was under the control of the trp operon which is readily inducible after tryptophan starvation and addition of indoleacrylic acid. These vectors may be useful for the overexpression of many proteins in a form closely approximating their native state.     

Glucosylceramide synthase is essential for alfalfa defensin-mediated growth inhibition but not for pathogenicity of Fusarium graminearum

Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of a fungal pathogen, Fusarium graminearum, at micromolar concentrations. However, molecular mechanisms by which they inhibit the growth of this fungus are not known. We have characterized a functional role of the fungal sphingolipid glucosylceramide in regulating sensitivity of the fungus to MsDef1 and MtDef4. A null mutation of the FgGCS1 gene encoding glucosylceramide synthase results in a mutant lacking glucosylceramide. The DeltaFggcs1-null mutant becomes resistant to MsDef1, but not to MtDef4. It shows a significant change in the conidial morphology and displays dramatic polar growth defect, and its mycelia are resistant to cell wall degrading enzymes. Contrary to its essential role in the pathogenicity of a human fungal pathogen, Cryptococcus neoformans, GCS1 is not required for the pathogenicity of F. graminearum. The DeltaFggcs1 mutant successfully colonizes wheat heads and corn silk, but its ability to spread in these tissues is significantly reduced as compared with the wild-type PH-1 strain. In contrast, it retains full virulence on tomato fruits and Arabidopsis thaliana floral and foliar tissues. Based on our findings, we conclude that glucosylceramide is essential for MsDef1-mediated growth inhibition of F. graminearum, but its role in fungal pathogenesis is host-dependent.     

Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation

Mitochondrial DNA (mtDNA) is packaged into DNA-protein assemblies called nucleoids, but the mode of mtDNA propagation via the nucleoid remains controversial. Two mechanisms have been proposed: nucleoids may consistently maintain their mtDNA content faithfully, or nucleoids may exchange mtDNAs dynamically. To test these models directly, two cell lines were fused, each homoplasmic for a partially deleted mtDNA in which the deletions were nonoverlapping and each deficient in mitochondrial protein synthesis, thus allowing the first unequivocal visualization of two mtDNAs at the nucleoid level. The two mtDNAs transcomplemented to restore mitochondrial protein synthesis but were consistently maintained in discrete nucleoids that did not intermix stably. These results indicate that mitochondrial nucleoids tightly regulate their genetic content rather than freely exchanging mtDNAs. This genetic autonomy provides a molecular mechanism to explain patterns of mitochondrial genetic inheritance, in addition to facilitating therapeutic methods to eliminate deleterious mtDNA mutations.     

Adequacy of control of cardiovascular risk factors in ambulatory patients with type 2 diabetes attending diabetes out-patients clinic at a county hospital,Kenya

Background

Type 2 diabetes is associated with substantial cardiovascular morbidity and mortality arising from the high prevalence of cardiovascular risk factors such as hypertension, dyslipidaemia, obesity, poor glycaemic control and albuminuria. Adequacy of control of these risk factors determines the frequency and outcome of cardiovascular events in the patients. Current clinical practice guidelines emphasize primary prevention of cardiovascular disease in type 2 diabetes. There is scarce data from the developing countries, Kenya included, on clinical care of patients with type 2 diabetes in the regions that are far away from tertiary health facilities. So we determined the adequacy of control of the modifiable risk factors: glycaemic control, hypertension, dyslipidemia, obesity and albuminuria in the study patients from rural and peri-urban dwelling.

Methods

This was a cross-sectional study on 385 randomly selected ambulatory patients with type 2 diabetes without overt complications. They were on follow up for at least 6 months at the Out-patient diabetes clinic of Nyeri County Hospital, a public health facility located in the central region of Kenya.

Results

Females were 65.5%. The study subjects had a mean duration of diabetes of 9.4 years, IQR of 3.0–14 years. Their mean age was 63.3 years, IQR of 56-71 years.Only 20.3% of our subjects had simultaneous optimal control of the three (3) main cardiovascular risk factors of hypertension, high LDL-C and hyperglycaemia at the time of the study. The prevalence of cardiovascular risk factors were as follows: HbA1c above 7% was 60.5% (95% CI, 55.6–65.5), hypertension, 49.6% of whom 76.6% (95% CI, 72.5–80.8) were poorly controlled. High LDL-Cholesterol above 2.0 mmol/L was found in 77.1% (95% CI 73.0–81.3) and Albuminuria occurred in 32.7% (95% CI 27.8–37.4). The prevalence of the other habits with cardiovascular disease risk were: excess alcohol intake at 26.5% (95% CI 27.8–37.4) and cigarette-smoking at 23.6%.A modest 23.4% of the treated patients with hypertension attained target blood pressure of <140/90 mmHg. Out of a paltry 12.5% of the statin-treated patients and others not actively treated, only 22.9% had LDL-Cholesterol of target <2.0 mmol/L.There were no obvious socio-demographic and clinical determinants of poor glycaemic control. However, old age above 50 yrs., longer duration with diabetes above 5 yrs. and advanced stages of CKD were significantly associated with hypertension. Female gender and age, statin non-use and socio-economic factor of employment were the significant determinants of high levels of serum LDL-cholesterol.

Conclusion

The majority of the study patients attending this government-funded health facility had high prevalence of cardiovascular risk factors that were inadequately controlled. Therefore patients with type 2 diabetes should be risk-stratified by their age, duration of diabetes and cardiovascular risk factor loading. Consequently, composite risk factor reduction strategies are needed in management of these patients to achieve the desired targets safely. This would be achieved through innovative care systems and modes of delivery which would translate into maximum benefit of primary cardiovascular disease prevention in those at high risk. It is a desirable quality objective to have a higher proportion of the patients who access care benefiting maximally more than the numbers we are achieving now.

     

Nonhomologous end joining of complementary and noncomplementary DNA termini in mouse testicular extracts

Mammalian somatic cells are known to repair DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) and homologous recombination (HR); however, how male germ cells repair DSBs is not yet characterized. We have previously reported the highly efficient and mostly precise DSB joining ability of mouse testicular germ cell extracts for cohesive and blunt ends, with only a minor fraction undergoing terminal deletion [Mutat. Res. 433 (1999) 1]; however, the precise mechanism of joining was not established. In the present study, we therefore tested the ability of testicular extracts to join noncomplementary ends; we have also sequenced the junctions of both complementary and noncomplementary termini and established the joining mechanisms. While a major proportion of complementary and blunt ends were joined by simple ligation, the small fraction having noncleavable junctions predominantly utilized short stretches of direct repeat homology with limited end processing. For noncomplementary ends, the major mechanism was "blunt-end ligation" subsequent to "fill-in" or "blunting", with no insertions or large deletions; the microhomology-dependent joining with end deletion was less frequent. This is the first functional study of the NHEJ mechanism in mammalian male germ cell extracts. Our results demonstrate that testicular germ cell extracts promote predominantly accurate NHEJ for cohesive ends and very efficient blunt-end ligation, perhaps to preserve the genomic sequence with minimum possible alteration. Further, we demonstrate the ability of the extracts to catalyze in vitro plasmid homologous recombination, which suggests the existence of both NHEJ and HR pathways in germ cells.     

Prevalence of Babesia microti in free-ranging baboons and African green monkeys

Babesia microti-like parasites have been reported to infect captive non-human primates (NHPs). However, studies on the prevalence of Babesia spp. in free-ranging NHPs are lacking. This investigation aimed at determining the prevalence of B. microti in wild-caught Kenyan NHPs. In total, 125 animals were studied, including 65 olive baboons (Papio cynocephalus anubis) and 60 African green monkeys ([AGMs] Chlorocebus aethiops). Nested polymerase chain reaction targeting Babesia β-tubulin genes was used to diagnose infection prevalence. Results indicated a prevalence of 22% (27/125) B. microti infection in free-ranging NHPs in Kenya. There was no statistically significant difference in B. microti infection prevalence between baboons and AGMs or male and female animals. This is the first report of the presence and prevalence of B. microti in free-ranging Kenyan NHPs.     

Influence of Boar and Semen Parameters on Motility and Acrosome Integrity in Liquid Boar Semen Stored for Five Days

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16–18°C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables.     

Moving towards Routine Evaluation of Quality of Inpatient Pediatric Care in Kenya

Background

Regular assessment of quality of care allows monitoring of progress towards system goals and identifies gaps that need to be addressed to promote better outcomes. We report efforts to initiate routine assessments in a low-income country in partnership with government.

Methods

A cross-sectional survey undertaken in 22 ‘internship training’ hospitals across Kenya that examined availability of essential resources and process of care based on review of 60 case-records per site focusing on the common childhood illnesses (pneumonia, malaria, diarrhea/dehydration, malnutrition and meningitis).

Results

Availability of essential resources was 75% (45/61 items) or more in 8/22 hospitals. A total of 1298 (range 54–61) case records were reviewed. HIV testing remained suboptimal at 12% (95% CI 7–19). A routinely introduced structured pediatric admission record form improved documentation of core admission symptoms and signs (median score for signs 22/22 and 8/22 when form used and not used respectively). Correctness of penicillin and gentamicin dosing was above 85% but correctness of prescribed intravenous fluid or oral feed volumes for severe dehydration and malnutrition were 54% and 25% respectively. Introduction of Zinc for diarrhea has been relatively successful (66% cases) but use of artesunate for malaria remained rare. Exploratory analysis suggests considerable variability of the quality of care across hospitals.

Conclusion

Quality of pediatric care in Kenya has improved but can improve further. The approach to monitoring described in this survey seems feasible and provides an opportunity for routine assessments across a large number of hospitals as part of national efforts to sustain improvement. Understanding variability across hospitals may help target improvement efforts.