Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

The common severe Z mutation (E342K) of α₁-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular ‘snapshots’, the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α₁-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P₁₄P₁₂ and P₄, but not P₁₀P₈ or P₂P_1′, resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P₁₄P₁₂ region self-inserts early during activation, while the interaction between P₆P₄ and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P₆P₄, but not P₁₄P₁₂, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol⁻¹.     

Methoprene modulates the effect of diet on male melon fly,Bactrocera cucurbitae,performance at mating aggregations

The effect of access to dietary protein (P) (hydrolyzed yeast) and/or treatment with a juvenile hormone analogue, methoprene (M), (in addition to sugar and water) on male aggregation (lekking) behaviour and mating success was studied in a laboratory strain of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Six‐day‐old males were treated with (1) protein and methoprene (M+P+), (2) only protein (M?P+), or (3) only methoprene (M+P?), and compared with 14‐day‐old sexually mature untreated males (M?P?). The lekking behaviour of the four groups of males when competing for virgin sexually mature females (14 –16 days old) was observed in field cages. The following parameters were measured at male aggregations: lek initiation, lek participation, males calling, male–male interaction, female acceptance index, and mating success. For all these parameters, the M+P+ males significantly outperformed the other males. Moreover, for all parameters, there was a similar trend with M+P+ > M?P+ > M?P? > M+P?. More M+P+ males called and initiated and participated in lek activities than all other types of male, which resulted in higher mating success. They had also fewer unsuccessful copulation attempts than their counterparts. Whereas treatment with methoprene alone had a negative effect in young males with only access to sugar, access to dietary protein alone significantly improved young male sexual performance; moreover, the provision of methoprene together with protein had a synergistic effect, improving further male performance at leks. The results are of great relevance for enhancing the application of the sterile insect technique (SIT) against this pest species. The fact that access to dietary protein and treatment of sterile males with methoprene improves mating success means that SIT cost‐effectiveness is increased, as more released males survive to sexual maturity.     

Prospect of phytoaccumulation of arsenic by Brassica juncea (L.) in Bangladesh

The phytoaccummulation of arsenic by Brassica juncea (L.) was investigated for varying concentrations selected within the range that is evident in Bangladeshi soil. B. juncea (Rai and BARI-11) was grown in the hydroponic media under greenhouse condition with different concentrations (0.5, 1.0, 15, 30, 50 and 100 ppm) of sodium arsenite. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used to analyze the data. Mapping of potential area of phytoaccumulation of arsenic by B. juncea was done using Geographic information system (GIS). Arsenic was detected at lower concentrations (0.5 and 1.0 ppm) only at root system of the plant. For higher concentrations (15, 30, and 50 ppm) arsenic was detected both in the root and shoot systems. The results suggested that at 15 and 50 ppm uptake was higher compared to 30 ppm. For 100 ppm of arsenic no plant growth was observed. In Bangladesh, where concentration of arsenic is at lower level and present only at rooting zone, B. juncea may be used for phytoaccumulation of arsenic keeping usual agronomic practices. However, for higher concentrations, B. juncea can be regarded as a good accumulator of arsenic where uptake of arsenic was up to 1% of total biomass of the plant.     

Elevated Soluble CD163 in Gestational Diabetes Mellitus: Secretion from Human Placenta and Adipose Tissue

Recently soluble CD163 (sCD163), a cleaved form of the macrophage receptor CD163, was identified as a macrophage-specific risk-predictor for developing Type 2 Diabetes. Here, we investigate circulating levels of sCD163 in gestational diabetes mellitus (GDM). Furthermore, given the role of the placenta in the pathogenesis of GDM, we assessed placental contribution to sCD163 secretion. Paired maternal (venous) and umbilical vein blood samples from GDM (n = 18) and Body Mass Index (BMI) matched control women (n = 20) delivered by caesarean section at 39–40 week gestation were assessed for circulating levels of sCD163, Tumour necrosis factor alpha (TNF-α) and Interleukin 6 (IL-6). Media from explant culture of maternal subcutaneous fat and corresponding placental tissues were assayed for these same molecules. CD163 positive cell numbers were determined in placental and adipose tissues of GDM and control women. We found significantly elevated circulating sCD163 levels in GDM mothers (688.4±46.9 ng/ml vs. 505.6±38.6 ng/ml) and their offspring (418.2±26.6 ng/ml vs. 336.3±24.4 ng/ml [p<0.05 for both]) as compared to controls, together with elevated circulating TNF-α and IL-6 levels. Moreover, both GDM placentae (268.1±10.8 ng/ml/mg vs. 187.6±20.6 ng/ml/mg) and adipose explants (41.1±2.7 ng/ml/mg vs. 26.6±2.4 ng/ml/mg) released significantly more sCD163 than controls. Lastly, significantly more CD163 positive cells were observed in GDM placentae (25.7±1.1 vs. 22.1±1.2) and adipose tissue (19.1±1.1 vs 12.7±0.9) compared to controls. We describe elevated sCD163 levels in GDM and identify human placenta as a novel source of sCD163 suggesting that placental tissues might contribute to the increased levels of circulating sCD163 in GDM pregnancies.     

Adaptation of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> to Thermal Stress Yields a Thermotolerant Variant Which Also Exhibits Improved Survival at pH 2

Loss in probiotic viability upon exposure to stressful storage and transport conditions has plagued the probiotic market worldwide. Lactobacillus acidophilus is an important probiotic that is added to various functional foods. It is known to be fairly labile and susceptible to temperature variations that it encounters during processing and storage which increases production cost. It has been repeatedly demonstrated that pre-exposure to sub-lethal doses of stress, particularly, temperature and pH, leads to improved survival of various probiotics when they subsequently encounter the same stress of a much greater magnitude. Attempts to adapt L. acidophilus to temperatures as high as 65 °C to arrive at a thermotolerant variant have not been reported previously. To improve viability at elevated temperatures, we gradually adapted the L. acidophilus NCFM strain to survival at 65 °C for 40 min. Following adaptation, the variant showed a 2-log greater survival compared to wild-type at 65 °C. Interestingly, this thermotolerant variant also demonstrated a 2-log greater stability compared to wild-type at pH 2.0. The improved pH and temperature stress tolerance exhibited by this variant remained unaltered even when the strain was lyophilized. Moreover, the thermotolerant variant demonstrated improved stability compared to wild-type when stored for up to a week at 37 and 42 °C. Probiotic properties of the variant such as adherence to epithelial cells and antibacterial activity remained unaltered. This strain can potentially help address the issue of significant loss in viable cell counts of L. acidophilus which is typically encountered during probiotic manufacture and storage.     

Rutin–Nickel Complex: Synthesis,Characterization, Antioxidant,DNA Binding,and DNA Cleavage Activities

The rutin–nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV–visible spectroscopy, IR, mass spectrometry, ¹H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.     

Removal and recovery of Cu(II) and Zn(II) using immobilized Mentha arvensis distillation waste biomass

The potential use of the immobilized Mentha arvensis distillation waste (IMADW) biomass for removal and recovery of Cu(II) and Zn(II) from aqueous was evaluated in the present study. Biosorption capacity of Cu(II) and Zn(II) on IMADW increased with increase in pH reaching a maximum at 5 for Cu(II) and 6 for Zn(II). The equilibrium sorption data agreed well with Langmuir isotherm model and pseudo-second-order kinetic model in batch mode. Cu(II) and Zn(II) uptake by IMADW was best described by pseudo-first-order kinetic model in continuous mode. Maximum Cu(II) and Zn(II) uptake by IMADW was 104.48 and 107.75 mg/g, respectively. Fourier Transform Infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were also carried out to investigate functional groups and surface changes of biomass. The results showed that IMADW biomass is a potential biomaterial to remove Cu(II) and Zn(II) ions with a high biosorption capacity from aqueous solutions.     

Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration

Hemolin, a member of the immunoglobulin protein superfamily, functions in Lepidoptera as an opsonin in defence against potential pathogens and seems to play a role in tissue morphogenesis. We show that hemolin gene is expressed in several organs of Galleria mellonella larvae, including the nervous system and the silk glands. The expression in the silk glands of the wandering larvae and their isolated abdomens is enhanced within 6 h after an injection of bacteria, lipopolysaccharides, or peptidoglycans. The magnitude of silk gland response to bacterial challenge is similar to that seen in the fat body. A profound rise of hemolin expression without bacterial inoculation occurs in the silk glands of isolated abdomens when they are induced to pupate by a topical application of 20-hydroxyecdysone (20E). The induction of pupation is associated with silk gland programming for disintegration by apoptosis and phagocytosis. Administration of a juvenile hormone agonist prevents pupation and abolishes the stimulatory 20E effect on the hemolin expression. Hemolin protein can be immunodetected in the silk glands as well as in the spun-out cocoon silk. The results suggest that silk glands are a component of the insect immune system and that hemolin may mark the apoptic cells for the elimination by hemocytes.