Risk factors for premature death in middle aged men

The causes of premature death and the associated risk factors were analysed in a cohort of 7935 middle aged men participating in a preventive population programme in Malmö. They were screened when aged 46-48 and then followed up for 3½-8 years. Two hundred and eighteen died, of whom 181 (83%) underwent necropsy. Three major causes of death were established: cancer in 61 (28%), deaths related to consumption of alcohol in 55 (25%), and coronary heart disease in 50 (23%).Distinctly different patterns of risk factors were found to be associated with each of the three main causes of premature death. In death due to coronary heart disease smoking (p=0·0062), serum cholesterol concentration (p=0·00014), serum triglyceride concentration (p=0·00013), systolic blood pressure (p=0·000012), and diastolic blood pressure (p=0·0021) were the strongest single determinants but diastolic blood pressure ceased to be a predictive factor in a multivariate analysis whereas all the other variables could be combined in a highly predictive logistic model. In death related to consumption of alcohol equal or even stronger associations were found for serum γ glutamyltransferase activity (p<0·0001), points scored in a questionnaire screening for alcoholism (p<0·0001), and, inversely, serum cholesterol (p=0·0046) and serum creatinine (p<0·0001) concentrations both when applied independently and when combined in a logistic model. In death due to cancer significant associations were found for serum urate concentration (p=0·023) and, inversely, serum cholesterol concentration (p=0·056-0·031).Malignant diseases and diseases related to consumption of alcohol were at least as prominent as cardiovascular disorders in causing premature death in the cohort of men studied. All three types of conditions are potentially avoidable and seem to be associated with significant and distinctive patterns of risk factors. These patterns should be used, as blood pressure and serum lipid concentrations already are, to predict the risk of premature death and indicate preventive measures.     

Influence of Pregnancy Spacing on Outcome of Pregnancy

To assess the significance of the length of time between two pregnancies on the outcome of the second we used information collected by the British Perinatal Mortality Survey of 1958. From questionnaires on the 16,994 singleton births in the first week of March 1958 and the 7,117 singleton stillbirths and neonatal deaths in March, April, and May 1958 we abstracted information on the date and outcome of any preceding pregnancy. The interpregnancy interval was taken as the length of time between this preceding pregnancy and the last menstrual period before the index pregnancy. The most important factors influencing pregnancy spacing were outcome of the preceding delivery, social class, and maternal age. When these variables had been taken into account we found that the length of interpregnancy interval had little effect on stillbirth rates. High neonatal death rates, however, occurred when interpregnancy intervals were less than six months (P <0·005), though longer intervals had no significant effects.     

Psychrotolerant Bacteria Isolated from Arctic Soil That Degrade Polychlorinated Biphenyls at Low Temperatures

Psychrotolerant polychlorinated biphenyl (PCB)-degrading bacteria were isolated at 7°C from PCB-contaminated Arctic soil by using biphenyl as the sole organic carbon source. These isolates were distinguished from each other by differences in substrates that supported growth and substrates that were oxidized. 16S ribosomal DNA sequences suggest that these isolates are most closely related to the genus Pseudomonas. Total removal of Aroclor 1242, and rates of removal of selected PCB congeners, by cell suspensions of Arctic soil isolates and the mesophile Burkholderia cepacia LB400 were determined at 7, 37, and 50°C. Total removal values of Aroclor 1242 at 7°C by LB400 and most Arctic soil isolates were similar (between 2 and 3.5 μg of PCBs per mg of cell protein). However the rates of removal of some individual PCB congeners by Arctic isolates were up to 10 times higher than corresponding rates of removal by LB400. Total removal of Aroclor 1242 and the rates of removal of individual congeners by the Arctic soil bacteria were higher at 37°C than at 7°C but as much as 90% lower at 50°C than at 37°C. In contrast, rates of PCB removal by LB400 were higher at 50°C than at 37°C. In all cases, temperature did not affect the congener specificity of the bacteria. These observations suggest that the PCB-degrading enzyme systems of the bacteria isolated from Arctic soil are cold adapted.     

Rates of entry and oxidation of acetate,glucose, d(?)-β-hydroxybutyrate,palmitate, oleate and stearate,and rates of production and oxidation of propionate and butyrate in fed and starved sheep

1. Rates of entry and oxidation of a range of metabolites have been measured in tracheostomized sheep (diet, 800g. of lucerne chaff and 100g. of maize/day) by combining isotope-dilution techniques with the continuous measurement of total respiratory gas exchange, and ¹⁴CO₂ production during the intravenous or intraruminal infusion of ¹⁴C-labelled substrates. 2. Mean entry rates in fed and starved (24hr.) sheep respectively, expressed as mg./min./kg. body wt.^0·75, were: glucose, 5·0 (range 4·8–5·1, 2 observations) and 3·8 (3·2–4·2, 4); acetate, 10·8 (9·1–13·5, 4) and 5·8 (1); d(−)-β-hydroxybutyrate, 1·4 (1) and 1·5 (0·8–2·4, 4); palmitate, oleate and stearate (starved sheep only) 1·0 (0·6–1·9, 7), 0·9 (0·2–1·6, 10) and 0·9 (0·5–1·1, 11) respectively. 3. Production rates of propionate and butyrate in continuously feeding sheep were 6·4 (4·7–8·3, 4) and 4·3 (3·4–6·1, 4) mg./min./kg.^0·75 respectively, and in starved (24hr.) sheep were 2·5 (2·2–2·9, 2) and 1·0 (0·8–1·2, 2) mg./min./kg.^0·75 respectively. 4. Calculated terminal values for the specific radioactivity of respiratory ¹⁴CO₂ during measurements of entry rates and production rates were used to calculate the contributions of individual substrates to overall oxidative metabolism. Mean values for fed and starved sheep respectively were: glucose, 9·1 (8·6–9·6, 2) and 11·2 (5·9–15·1, 4)%; acetate, 31·6 (26·8–38·1, 4) and 22·1 (1)%; d(−)-β-hydroxybutyrate, 10·4 (1) and 4·8 (1·9–7·7, 4)%; propionate, 23·0 (13·8–29·9, 4) and 7·1 (6·8–7·4, 2)%; butyrate, 16·5 (13·7–20·5, 4) and 5·3 (5·2–5·3, 2)%; palmitate, oleate and stearate (starved sheep only), 4·7 (2·0–7·7, 7), 4·0 (1·2–6·6, 10) and 4·4 (3·8–5·8, 9)% respectively. The sum of these values for individual substrates in fed and starved sheep, excluding that of β-hydroxybutyrate and after correction of the glucose value for the known interrelations of this substrate with propionate, accounted for 76% and 58% respectively of total production of carbon dioxide. 5. Calculations based on the proportion of substrate entry directly oxidized indicated that the substrates studied accounted for 63% (fed sheep) and 43% (starved sheep) of total energy expenditure measured by oxygen uptake. The contribution of β-hydroxybutyrate was excluded, and corrections were made for glucose–propionate interrelations, and for the different rates of oxidation of the methyl and carboxyl fragments of acetate. 6. The present results have been combined with those obtained earlier in this Laboratory to examine the relationships between rates of substrate entry and oxidation, and concentrations of substrate in blood. Rates of entry of acetate, glucose, d(−)-β-hydroxybutyrate, palmitate and oleate (but not stearate) were well correlated with concentration in blood, and substrate contribution to production of carbon dioxide showed a similar correlation to blood concentration, except with glucose. 7. It was concluded that the general technique is of potential value in providing valid quantitative parameters of animal metabolism.     

Factors protective against retinopathy in insulin-dependent diabetics free of retinopathy for 30 years

To investigate which factors might protect against the development of retinopathy 40 insulin-dependent diabetics who had remained free from retinopathy despite diabetes of long duration (mean±1 SD 30±10 years) were compared with 40 patients who had background and 47 who had proliferative retinopathy (mean durations of disease 16±5 and 19±5 years respectively). The three groups had had similar mean ages at onset of diabetes. The mean of all postprandial blood glucose measurements at hospital clinics from diagnosis of diabetes to detection of retinopathy, or to the most recent negative eye examination, was 9·9±2·1 mmol/l (178±38 mg/100 ml) in the group with no retinopathy, 11·8±2·1 mmol/l (213±38 mg/100 ml) in those with background retinopathy, and 12·4±2·1 mmol/l (223±38 mg/100 ml) in those with proliferative retinopathy (p <0·0001). This difference was not reflected in present concentrations of haemoglobin A_1C, probably because glycaemic control had been improved after the development of retinopathy. In the groups with background and proliferative retinopathy there were significant negative correlations between mean blood glucose concentrations and the number of years that had elapsed from diagnosis of diabetes to detection of retinopathy, suggesting that the development of both grades of retinopathy depends on the degree and duration of glycaemic exposure.The patients with no retinopathy had attended clinic more frequently (p <0·025), more of them had required emergency hospital treatment for hypoglycaemia (p <0·0025), and they tended to have had a lower prevalence of hyperglycaemic coma than the other groups. Although mean percentage ideal body weight and diastolic blood pressure were lower in the patients with no retinopathy at the time of study, mean body weight, blood pressure, and the prevalence of smoking in the years before the development of retinopathy had been similar in all groups, suggesting that these did not influence the development of retinopathy.     

Small-intestinal Cell Turnover in Patients with Parasitic Infections

Small-intestinal deoxyribonucleic acid (DNA) loss rates were measured in six patients with Strongyloides stercoralis hyperinfestation, in four patients with hookworm disease, and in eight normal controls. In the four patients with strongyloidiasis having weight loss, hypoproteinaemia, and oedema the mean DNA loss rates were 73·9, 51·6, 58·0, and 62·2 ng atoms DNA-P/min respectively, which was significantly higher than that of patients with hookworm disease (mean 17·3, S.D. 6·6) or in eight control subjects (mean 14·5, S.D. 7·5). In two of three patients with strongyloidiasis the high DNA loss rates fell to normal after treatment, and in two others investigated only after treatment the rates were normal. It is suggested that the high epithelial cell turnover in these patients may result in excessive loss of endogenous substances and that this may be an important mechanism in causing malnutrition and hypoproteinaemia in patients with S. stercoralis hyperinfestation.     

Influence of spatial distribution and size of clones on the realized outcrossing rate of the marsh cinquefoil (Comarum palustre)

Background and Aims

Clonal growth is a common feature in flowering plants. As clone size increases, the selfing rate in self-compatible species is likely to increase due to more frequent geitono-pollination events (i.e. pollination among flowers within the same genet). This study investigated the breeding system of the marsh cinquefoil (Comarum palustre) and assessed spatial distribution of clones, clone size and architecture, and their effects on realized outcrossing rates. In addition, pollen dispersal was investigated in two patchy populations.

Methods

The species'' breeding system was investigated under controlled conditions through hand pollinations (self- vs. cross-pollination). Using microsatellite markers, an assessment was made of the realized outcrossing rates and the genetic diversity in four natural populations, the clonal structure in two populations within five 15 × 15 m sampling plots following 0·5 × 0·5 m grids, and the pollen dispersal through paternity assignment tests in those two populations.

Key Results Comarum palustre

is a self-compatible species but only presents a low rate of spontaneous self-pollination. The occurrence of inbreeding depression was not detected at the seed set stage (δ_SS = 0·04). Clones were spatially clumped (A_C = 0·60–0·80), with intermediate to no intermingling of the ramets (D_C = 0·40–1·00). Genet size ranged from one to 171 ramets. Patchy populations had low outcrossing rates (t_m = 0·33–0·46). Large clones showed lower outcrossing rates than small clones. Pollen dispersal mainly occurred within patches as only 1–7 % of the pollination events occurred between patches of >25 m separation. Seedling recruitment events were detected.

Conclusions

Genet size together with distances between patches, through increasing geitono-pollination events, appeared to be important factors influencing realized outcrossing rates. The study also revealed seed flow allowing seedling recruitment, which may contribute to increasing the number of new patches, and potentially further enhance gene flow within populations.     

Fat Embolism in Patients with Fractured Hips

Fat embolism was assessed at necropsy and correlated with clinical findings in the patients who died among 854 with fractured hips admitted to hospital between 1967 and August 1971. Sixteen cases of clinical importance were found, eight of which were judged to have been fatal or to have seriously contributed to death. Frequencies were as follows: 2·4 to 3·3% among 424 patients with subcapital fractures; 0·7 to 0·8% in the 405 with trochanteric fractures; 4·1 to 7% among subjects treated without operation, representing 30% of those who died within seven days; and 0·9 to 1·1% among patients treated by pinning, nailing, or nail-plating. The higher frequency in the conservatively treated group is probably related to selection of poor-risk subjects. Fat embolism was found in 6·8 to 8·0% of those with subcapital fractures treated by primary Thompson''s arthroplasty which utilizes acrylic cement, and in none of those given Moore''s prostheses for which cement is not used. Study of a larger group after Moore''s prosthesis is required to establish its lack of special risk. Fat embolism accounted for all the deaths within seven days of Thompson''s arthroplasty and for most within 14 days; it was clearly related to surgery in some cases.A possible explanation of the hazard of Thompson''s arthroplasty is that fat globule entry is enhanced by a rise of intramedullary pressure due to proximal occlusion of the reamed marrow cavity. A controlled trial of the effect of venting the marrow cavity on the frequency of fat embolism is warranted. It is possible that the acrylic monomer may also contribute to venous entry of medullary fat. The higher-age group of those with subcapital fractures and associated chronic cardiac and pulmonary disease might make them more susceptible to fat embolization than those in whom arthroplasty is also carried out for chronic hip disease.     

Positronium Formation in Muscle: An Investigation of the Structure of Cell Water

Positronium formation in muscle at +4°C and -4°C was examined by the measurement of the angular correlation of positron annihilation radiation. Since the positronium formation rate in ice is considerably higher than it is in water, there should be a comparable increase in the positronium formation rate in muscle tissue if recent speculation that cellular water is ordered in a semicrystalline icelike state is correct. Comparison of the angular correlation from muscle at +4°C with that from water at +4°C shows no enhancement of the positronium formation rate. Frozen muscle at -4°C shows an enhancement of the positronium formation rate of approximately half that found in ice at -4°C, indicating that most cellular water undergoes a normal water-ice transition when frozen. It is concluded therefore that cell water in muscle is not ordered in a hexagonal icelike structure. While the results are consistent with the hypothesis that cell water is in the liquid state, the hypothesis that cell water is ordered in an undetermined close packed structure which transforms to the hexagonal ice structure at or near 0°C cannot be ruled out.     

Adaptive phenotypic plasticity and local adaptation for temperature tolerance in freshwater zooplankton

Many organisms have geographical distributions extending from the tropics to near polar regions or can experience up to 30°C temperature variation within the lifespan of an individual. Two forms of evolutionary adaptation to such wide ranges in ambient temperatures are frequently discussed: local adaptation and phenotypic plasticity. The freshwater planktonic crustacean Daphnia magna, whose range extends from South Africa to near arctic sites, shows strong phenotypic and genotypic variation in response to temperature. In this study, we use D. magna clones from 22 populations (one clone per population) ranging from latitude 0° (Kenya) to 66° North (White Sea) to explore the contributions of phenotypic plasticity and local adaptation to high temperature tolerance. Temperature tolerance was studied as knockout time (time until immobilization, T_imm) at 37°C in clones acclimatized to either 20°C or 28°C. Acclimatization to 28°C strongly increased T_imm, testifying to adaptive phenotypic plasticity. At the same time, T_imm significantly correlated with average high temperature at the clones’ sites of origin, suggesting local adaptation. As earlier studies have found that haemoglobin expression contributes to temperature tolerance, we also quantified haemoglobin concentration in experimental animals and found that both acclimatization temperature (AccT) and temperature at the site of origin are positively correlated with haemoglobin concentration. Furthermore, Daphnia from warmer climates upregulate haemoglobin much more strongly in response to AccT, suggesting local adaptation for plasticity in haemoglobin expression. Our results show that both local adaptation and phenotypic plasticity contribute to temperature tolerance, and elucidate a possible role of haemoglobin in mediating these effects that differs along a cold–warm gradient.     

Australia (Hepatitis-Associated) Antigen in Patients Attending a Venereal Disease Clinic

A total of 1,650 patients attending the venereal disease department at St. Mary''s Hospital, London, have been tested for Australia antigen. Twenty-three positive results were obtained, or 1·39%, which is more than 10 times the rate noted by others in blood donor populations in the U.K. and U.S.A. The positive rates among female patients and European male heterosexual patients were 0·36% and 0·19% respectively. High rates were obtained for homosexual patients (3·8%) and non-European heterosexual patients (3·1%). The reasons for the higher rates found in these groups merit further study.     

Heat Resistance and Mechanism of Heat Inactivation in Thermophilic Campylobacters

The heat resistance of Campylobacter jejuni strains AR6 and L51 and the heat resistance of Campylobacter coli strains DR4 and L6 were measured over the temperature range from 50 to 60°C by two methods. Isothermal measurements yielded D₅₅ values in the range from 4.6 to 6.6 min and z values in the range from 5.5 to 6.3°C. Dynamic measurements using differential scanning calorimetry (DSC) during heating at a rate of 10°C/min yielded D₅₅ values of 2.5 min and 3.4 min and z values of 6.3°C and 6.5°C for AR6 and DR4, respectively. Both dynamic and isothermal methods yielded mean D₅₅ values that were substantially greater than those reported previously (0.75 to 0.95 min). DSC analysis of each strain during heating at a rate of 10°C/min yielded a complex series of overlapping endothermic peaks, which were assigned to cell wall lipids, ribosomes, and DNA. Measurement of the decline in the numbers of CFU in calorimetric samples as they were heated showed that the maximum rate of cell death occurred at 56 to 57°C, which is close to the value predicted mathematically from the isothermal measurements of D and z (61°C). Both estimates were very close to the peak m₁ values, 60 to 62°C, which were tentatively identified with unfolding of the 30S ribosome subunit, showing that cell death in C. jejuni and C. coli coincided with unfolding of the most thermally labile regions of the ribosome. Other measurements indicated that several essential proteins, including the α and β subunits of RNA polymerase, might also unfold at the same time and contribute to cell death.     

Studies on clinical signs and haematological alterations in pneumonic aspergillosis due to Aspergillus flavus in Japanese quail

Intratracheal inoculation of 2-week old quail chicks with Aspergillus flavus spores resulted in the development of clinical signs within 24 h of infection. These were characterized by dullness, depression, anorexia, accelerated breathing, gasping and prostration leading to death. These signs continued up to 7 days followed by considerable decrease in the intensity of the symptoms as well as number of birds showing clinical signs. Mortality occurred primarily in the first week with a majority of the birds dying from 2–4 days after infection. The overall mortality during a 6-week observation period was 25%. The average body weight of the infected chicks was slightly lower than that of controls; the difference being significant at 2, 3 and 42 days post-infection. There was no appreciable difference in the mean values of haemoglobin, packed cell volume and total erythrocyte count between the infected and control chicks at any stage of infection, but total leucocyte count revealed a significant increase (p<0.05) from 3–7 days post-infection. This was due to increase in the percentage of heterophils and decrease in lymphocytes.     

Comparison of Effect on Tobacco Consumption and Carbon Monoxide Absorption of Changing to High and Low Nicotine Cigarettes

In 10 sedentary workers, smoking as they felt inclined over a five-hour period in the middle of a typical working day, changing to low nicotine cigarettes (<0·3 mg) caused an increase in the number and weight of cigarettes smoked, while changing to high nicotine cigarettes (3·2 mg) caused a decrease (P < 0·01). The average number and weight smoked in five hours for usual, low, and high nicotine brands were respectively 10·6 (6·00 g), 12·5 (6·52 g), and 6·7 (4·19 g). When smoking the usual brand the average blood carboxyhaemoglobin (COHb) increased 1·78% (from 6·38% to 8·16%). But on changing to either high or low nicotine cigarettes the COHb levels instead of increasing, tended to fall (P < 0·01). The average fall of 0·34% while smoking low nicotine cigarettes was due to the low carbon monoxide (CO) yield of these cigarettes, while the fall of 1·04% when smoking high nicotine cigarettes was attributable to reduced consumption. The findings support the view that smoking behaviour is modified to regulate nicotine intake. Besides having low tar and CO yields, the least harmful cigarettes for heavy smokers may be those with a high, rather than low, nicotine yield.     

Characteristics of apoptosis induction in human breast cancer cells treated with a ceramidase inhibitor

Cancer is a complex disease with high mortality rates. Breast cancer is one of the most fatal diseases both for men and woman. Despite the positive developments on cancer treatment, a successful treatment agent/method has not been developed, yet. Recently, cancer research has been involved in sphingolipid metabolism. The key molecule here is ceramide. Ceramides mediate growth suppress, apoptosis and aging regulation. Ceramidases metabolize ceramide and decrease its level in cells and cause escape the death. Inhibition of ceramidases as new targets for cancer treatment is shown in the literature. Herein, we found that d-erythro-MAPP and its nanoparticle formulation, reduce the viability of MCF-7 cells in a dose-dependent manner with IC₅₀ value of 4.4 µM, and 15.6 µM, respectively. Confocal and transmission electron microscopy results revealed apoptotic morphological and ultrastructural changes for both agents. Apoptosis and cell cycle arrest were supported by annexin-V, mitochondrial membrane potential changings and cell cycle analysis, respectively.     

Risk Factors for Death among Children Less than 5 Years Old Hospitalized with Diarrhea in Rural Western Kenya, 2005–2007: A Cohort Study

Background

Diarrhea is a leading cause of childhood morbidity and mortality in sub-Saharan Africa. Data on risk factors for mortality are limited. We conducted hospital-based surveillance to characterize the etiology of diarrhea and identify risk factors for death among children hospitalized with diarrhea in rural western Kenya.

Methods and Findings

We enrolled all children <5 years old, hospitalized with diarrhea (≥3 loose stools in 24 hours) at two district hospitals in Nyanza Province, western Kenya. Clinical and demographic information was collected. Stool specimens were tested for bacterial and viral pathogens. Bivariate and multivariable logistic regression analyses were carried out to identify risk factors for death. From May 23, 2005 to May 22, 2007, 1,146 children <5 years old were enrolled; 107 (9%) children died during hospitalization. Nontyphoidal Salmonella were identified in 10% (118), Campylobacter in 5% (57), and Shigella in 4% (42) of 1,137 stool samples; rotavirus was detected in 19% (196) of 1,021 stool samples. Among stools from children who died, nontyphoidal Salmonella were detected in 22%, Shigella in 11%, rotavirus in 9%, Campylobacter in 5%, and S. Typhi in <1%. In multivariable analysis, infants who died were more likely to have nontyphoidal Salmonella (adjusted odds ratio [aOR] = 6·8; 95% CI 3·1–14·9), and children <5 years to have Shigella (aOR = 5·5; 95% CI 2·2–14·0) identified than children who survived. Children who died were less likely to be infected with rotavirus (OR = 0·4; 95% CI 0·2–0·8). Further risk factors for death included being malnourished (aOR = 4·2; 95% CI 2·1–8·7); having oral thrush on physical exam (aOR = 2·3; 95% CI 1·4–3·8); having previously sought care at a hospital for the illness (aOR = 2·2; 95% CI 1·2–3·8); and being dehydrated as diagnosed at discharge/death (aOR = 2·5; 95% CI 1·5–4·1). A clinical diagnosis of malaria, and malaria parasites seen on blood smear, were not associated with increased risk of death. This study only captured in-hospital childhood deaths, and likely missed a substantial number of additional deaths that occurred at home.

Conclusion

Nontyphoidal Salmonella and Shigella are associated with mortality among rural Kenyan children with diarrhea who access a hospital. Improved prevention and treatment of diarrheal disease is necessary. Enhanced surveillance and simplified laboratory diagnostics in Africa may assist clinicians in appropriately treating potentially fatal diarrheal illness. Please see later in the article for the Editors'' Summary     

Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ⁴-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-¹⁴C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [¹⁴C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the ¹⁴C incorporated into this fraction during incubation with [U-¹⁴C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of ¹⁴C were incorporated into these lipid fractions from [1-¹⁴C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [¹⁴C]lactate and glucose 6-phosphate (C-1) derived from [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-glucose, and the ratio of [¹⁴C]carbon dioxide yields from [1-¹⁴C]glucose and [6-¹⁴C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of ¹⁴C from [1-¹⁴C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [³H]water, [¹⁴C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.     

Evaluation of Murine Norovirus, Feline Calicivirus, Poliovirus, and MS2 as Surrogates for Human Norovirus in a Model of Viral Persistence in Surface Water and Groundwater

Human noroviruses (NoVs) are a significant cause of nonbacterial gastroenteritis worldwide, with contaminated drinking water a potential transmission route. The absence of a cell culture infectivity model for NoV necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict NoV inactivation. The NoV surrogates murine NoV (MNV), feline calicivirus (FCV), poliovirus (PV), and male-specific coliphage MS2, in conjunction with Norwalk virus (NV), were spiked into surface water samples (n = 9) and groundwater samples (n = 6). Viral persistence was monitored at 25°C and 4°C by periodically analyzing virus infectivity (for all surrogate viruses) and nucleic acid (NA) for all tested viruses. FCV infectivity reduction rates were significantly higher than those of the other surrogate viruses. Infectivity reduction rates were significantly higher than NA reduction rates at 25°C (0.18 and 0.09 log₁₀/day for FCV, 0.13 and 0.10 log₁₀/day for PV, 0.12 and 0.06 log₁₀/day for MS2, and 0.09 and 0.05 log₁₀/day for MNV) but not significant at 4°C. According to a multiple linear regression model, the NV NA reduction rates (0.04 ± 0.01 log₁₀/day) were not significantly different from the NA reduction rates of MS2 (0.05 ± 0.03 log₁₀/day) and MNV (0.04 ± 0.03 log₁₀/day) and were significantly different from those of FCV (0.08 ± 0.03 log₁₀/day) and PV (0.09 ± 0.03 log₁₀/day) at 25°C. In conclusion, MNV shows great promise as a human NoV surrogate due to its genetic similarity and environmental stability. FCV was much less stable and thus questionable as an adequate surrogate for human NoVs in surface water and groundwater.     

Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix.     

Exocyclic amino groups of flanking guanines govern sequence-dependent adduct conformations and local structural distortions for minor groove-aligned benzo[a]pyrenyl-guanine lesions in a GG mutation hotspot context

The environmental carcinogen benzo[a]pyrene (BP) is metabolized to reactive diol epoxides that bind to cellular DNA by predominantly forming N²-guanine adducts (G*). Mutation hotspots for these adducts are frequently found in 5′-···GG··· dinucleotide sequences, but their origins are poorly understood. Here we used high resolution NMR and molecular dynamics simulations to investigate differences in G* adduct conformations in 5′-···CG*GC··· and 5′-···CGG*C··· sequence contexts in otherwise identical 12-mer duplexes. The BP rings are positioned 5′ along the modified strand in the minor groove in both cases. However, subtle orientational differences cause strong distinctions in structural distortions of the DNA duplexes, because the exocyclic amino groups of flanking guanines on both strands compete for space with the BP rings in the minor groove, acting as guideposts for placement of the BP. In the 5′-···CGG*C··· case, the 5′-flanking G · C base pair is severely untwisted, concomitant with a bend deduced from electrophoretic mobility. In the 5′-···CG*GC··· context, there is no untwisting, but there is significant destabilization of the 5′-flanking Watson–Crick base pair. The minor groove width opens near the lesion in both cases, but more for 5′-···CGG*C···. Differential sequence-dependent removal rates of this lesion result and may contribute to the mutation hotspot phenomenon.