Reconstitution of the Anti-Apoptotic Bcl-2 Protein into Lipid Membranes and Biophysical Evidence for Its Detergent-Driven Association with the Pro-Apoptotic Bax Protein

The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2) protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax), are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM) and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD) spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23)-lauryl-ether (Brij-35) detergent at a level below its critical micelle concentration (CMC). Additional surface plasmon resonance (SPR) measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A₂ (a known inhibitory ligand of Bcl-2) to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC). Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.     

The investigation of therapeutic potential of oxytocin and liraglutide on vincristine‐induced neuropathy in rats

The aim of this study was to assess the therapeutic potential of oxytocin and liraglutide (LIR), a GLP‐1 analogue, in a rat model of vincristine‐induced neuropathy. Rats were injected with vincristine (VCR) at a dose of 4 mg/kg twice a week for 5 weeks. The VCR‐administered rats were divided into three groups and received saline, oxytocin, or liraglutide simultaneously with VCR. After the treatment period, electrophysiological, biochemical, histological, and immunohistochemical investigations were performed. Electromyography (EMG) recordings demonstrated significant alterations in the VCR + saline group (p < .001). Also, motor performance was decreased in the VCR + saline group (p < .05). Histologically, the axonal diameter was decreased in all groups. VCR + saline group showed significantly increased lipid peroxidation and decreased nerve growth factor (NGF) expression. However, the administration of oxytocin and liraglutide significantly prevented the EMG alterations, lipid peroxidation, and reduction in neuronal NGF expression. On the basis of these findings, oxytocin and liraglutide may be considered as potential agents for the prevention of VCR‐induced neuropathy.     

An Efficient Plant Regeneration System of <i>Hydrangea bretschneideri</i> Dipp via Stem Segments as Explants

Hydrangea bretschneideri Dipp is a highly popular ornamental plant for garden decoration. Genetic engineering technology has been successfully used in many plant species, but it is limited in Hydrangea. Here we established an efficient regeneration system by using stem segments as explants for the first time. In our study, the plant growth regulators (PGRs) were evaluated at the different regeneration processes, including axillary shoots regeneration and root induction. We found that the optimal concentration for axillary buds’ induction was 2.0 mgL⁻¹ 6-BA and 0.5 mgL⁻¹ 1 IAA, its highest induction rate was 70%. Moreover, the highest axillary shoots proliferation coefficient was 10.7 on the Murashige and Skoog (MS) medium with 2.0 mgL⁻¹ 6-benzyladenine (BA), 0.2 mgL⁻¹ indole-3-butyric acid (IBA), and 1.0 mgL⁻¹ gibberellin A3 (GA3). The highest frequency of root induction was 80.0 ± 0.06% by culturing the elongated shoots in 1/2 MS medium containing 0.1 mgL⁻¹ IBA. In summary, our study will provide an effective technology for large-scale propagation and important pathway for promoting the popularization and application of Hydrangea bretschneideri Dipp.     

内生Bacillus svelezensis HBB5菌株发酵盾叶薯蓣产薯蓣皂苷元的研究

目的探讨内生Bacillus svelezensis HBB5菌株发酵宿主植物盾叶薯蓣产薯蓣皂苷元的能力。方法接种内生B.svelezensis HBB5及B.subtilis ATCC 6633菌株(0.35×10~8 CFU/mL)至含盾叶薯蓣地下茎组织的液体培养基,32℃、165~185 r/min连续发酵108 h,检测发酵液细菌、pH、淀粉、麦芽糖、葡萄糖、淀粉酶(α-amylase)及薯蓣皂苷元溶出率等指标。结果内生B.svelezensis HBB5菌株有较强的酸、碱耐受力[pH(4.8±0.2)~(8.4±0.2)],相比B.subtilis ATCC 6633[pH(5.2±0.2)~(8.7±0.2)]差异不明显;前者达峰值生长量(60×10~(8±2) CFU/mL)明显高于后者(32×10~(8±2) CFU/mL)。发酵36~60 h时,B.svelezensis HBB5、B.subtilis ATCC 6633菌株发酵液的淀粉、麦芽糖、葡萄糖浓度达峰值,分别为(37.41±3.12)、(27.83±2.14)ng/mL,(21.06±1.25)、(16.54±1.08)ng/mL,(54.33±3.12)、(36.65±2.10)ng/mL,前者均高于后者。同时,B.svelezensis HBB5菌株维持高的α-amylase酶活性及薯蓣皂苷元溶出率。结论内生B.svelezensis HBB5菌株拥有较强的耐酸碱、降解淀粉、提高薯蓣皂苷元溶出的能力,为工业生产薯蓣皂苷元提供了一个新的方法。     

Influence of DGAT1 K232A Polymorphism on Milk Fat Percentage and Fatty Acid Profiles in Romanian Holstein Cattle

Milk and dairy products are considered the main sources of saturated fatty acids, which are a valuable source of nutrients in the human diet. Fat composition can be adjusted through guided nutrition of dairy animals but also through selective breeding. Recently, a dinucleotide substitution located in the exon 8 of the gene coding for acyl CoA: diacylglycerol acyltransferase 1 (DGAT1), that alters the amino acid sequence from a lysine to an alanine (p.Lys232Ala) in the mature protein, was shown to have a strong effect on milk fat content in some cattle breeds. Therefore, the objectives of this work were to study the occurrence of the DGAT1 p.Lys232Ala polymorphism in Romanian Holstein cattle and Romanian Buffalo breeds and to further investigate its possible influence on fat percentage and fatty acid profiles. The results obtained in this study show that in Romanian Holstein cattle the K allele is associated with increased fat percentage and higher levels of C16:0 and C18:0 fatty acids. The ratio of saturated fatty acids versus unsaturated fatty acids (SFA/UFA) was also higher in KK homozygous individuals, whereas the fractions of C14:0, unsaturated C18 decreased. The DGAT1 p.Lys232Ala polymorphism revealed a high genetic variance for fat percentage, unsaturated C18, C16:0, and SFA/UFA. Although the effect of this polymorphism was not so evident for short chain fatty acids such as C4:0–C8:0, it was significant for C14:0 fatty acids. We concluded that selective breeding of carriers of the A allele in Romanian Holsteins can contribute to improvement in unsaturated fatty acids content of milk. However, in buffalo, the lack of the A allele makes selection inapplicable because only the K allele, associated with higher saturated fatty acids contents in milk, was identified.     

Agricultural intensification in Brazil and its effects on land‐use patterns: an analysis of the 1975–2006 period

Does agricultural intensification reduce the area used for agricultural production in Brazil? Census and other data for time periods 1975–1996 and 1996–2006 were processed and analyzed using Geographic Information System and statistical tools to investigate whether and if so, how, changes in yield and stocking rate coincide with changes in cropland and pasture area. Complementary medium‐resolution data on total farmland area changes were used in a spatially explicit assessment of the land‐use transitions that occurred in Brazil during 1960–2006. The analyses show that in agriculturally consolidated areas (mainly southern and southeastern Brazil), land‐use intensification (both on cropland and pastures) coincided with either contraction of both cropland and pasture areas, or cropland expansion at the expense of pastures, both cases resulting in farmland stability or contraction. In contrast, in agricultural frontier areas (i.e., the deforestation zones in central and northern Brazil), land‐use intensification coincided with expansion of agricultural lands. These observations provide support for the thesis that (i) technological improvements create incentives for expansion in agricultural frontier areas; and (ii) farmers are likely to reduce their managed acreage only if land becomes a scarce resource. The spatially explicit examination of land‐use transitions since 1960 reveals an expansion and gradual movement of the agricultural frontier toward the interior (center‐western Cerrado) of Brazil. It also indicates a possible initiation of a reversed trend in line with the forest transition theory, i.e., agricultural contraction and recurring forests in marginally suitable areas in southeastern Brazil, mainly within the Atlantic Forest biome. The significant reduction in deforestation that has taken place in recent years, despite rising food commodity prices, indicates that policies put in place to curb conversion of native vegetation to agriculture land might be effective. This can improve the prospects for protecting native vegetation by investing in agricultural intensification.     

Cell cycle regulators cyclin D1 and CDK4/6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity

Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER−ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER−ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER−ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6.   In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.     

High-Fat Diet Reduces the Formation of Butyrate,but Increases Succinate,Inflammation, Liver Fat and Cholesterol in Rats,while Dietary Fibre Counteracts These Effects

Introduction

Obesity is linked to type 2 diabetes and risk factors associated to the metabolic syndrome. Consumption of dietary fibres has been shown to have positive metabolic health effects, such as by increasing satiety, lowering blood glucose and cholesterol levels. These effects may be associated with short-chain fatty acids (SCFAs), particularly propionic and butyric acids, formed by microbial degradation of dietary fibres in colon, and by their capacity to reduce low-grade inflammation.

Objective

To investigate whether dietary fibres, giving rise to different SCFAs, would affect metabolic risk markers in low-fat and high-fat diets using a model with conventional rats for 2, 4 and 6 weeks.

Material and Methods

Conventional rats were administered low-fat or high-fat diets, for 2, 4 or 6 weeks, supplemented with fermentable dietary fibres, giving rise to different SCFA patterns (pectin – acetic acid; guar gum – propionic acid; or a mixture – butyric acid). At the end of each experimental period, liver fat, cholesterol and triglycerides, serum and caecal SCFAs, plasma cholesterol, and inflammatory cytokines were analysed. The caecal microbiota was analysed after 6 weeks.

Results and Discussion

Fermentable dietary fibre decreased weight gain, liver fat, cholesterol and triglyceride content, and changed the formation of SCFAs. The high-fat diet primarily reduced formation of SCFAs but, after a longer experimental period, the formation of propionic and acetic acids recovered. The concentration of succinic acid in the rats increased in high-fat diets with time, indicating harmful effect of high-fat consumption. The dietary fibre partly counteracted these harmful effects and reduced inflammation. Furthermore, the number of Bacteroides was higher with guar gum, while noticeably that of Akkermansia was highest with the fibre-free diet.     

Psychosocial Working Conditions and Cognitive Complaints among Swedish Employees

Background

Cognitive complaints involving problems with concentration, memory, decision-making and thinking are relatively common in the work force. The sensitivity of both subjective and objective cognitive functioning to common psychiatric conditions, stress levels and to cognitive load makes it plausible that psychosocial working conditions play a role in cognitive complaints. Thus, this study aimed to test the associations between psychosocial work factors and cognitive complaints in nationally representative samples of the Swedish work force. Cross-sectional (n = 9751) and prospective (n = 3644; two time points two years apart) sequential multiple regression analyses were run, adjusting for general confounders, depressive- and sleeping problems. Additional prospective analyses were run adjusting for baseline cognitive complaints.

Cross-sectional results

High quantitative demands, information and communication technology (ICT) demands, underqualification and conflicts were positively associated with cognitive complaints, while social support, good resources at work and overqualification were negatively associated with cognitive complaints in all models. Skill discretion and decision authority were weakly associated with cognitive complaints. Conflicts were more strongly associated with cognitive complaints in women than in men, after adjustment for general confounders.

Prospective results

Quantitative job demands, ICT demands and underqualification were positively associated with future cognitive complaints in all models, including when adjusted for baseline cognitive complaints. Decision authority was weakly positively associated with future cognitive complaints, only after adjustment for depressive- and sleeping problems respectively. Social support was negatively associated with future cognitive complaints after adjustment for general confounders and baseline cognitive complaints. Skill discretion and resources were negatively associated with future cognitive complaints after adjustment for general confounders. The associations between quantitative demands and future cognitive complaints were stronger in women.

Discussion/Conclusions

The findings indicate that psychosocial working conditions should be taken into account when considering cognitive complaints among employees.