A continuous spectrophotometric assay and nonlinear kinetic analysis of methionine γ-lyase catalysis

In this article, we present a new, easy-to-implement assay for methionine γ-lyase (MGL)-catalyzed γ-elimination reactions of l-methionine and its analogues that produce α-ketobutyrate (α-KB) as product. The assay employs ultraviolet–visible (UV–Vis) spectrophotometry to continuously monitor the rate of formation of α-KB by its absorbance at 315 nm. We also employ a nonlinear data analysis method that obviates the need for an “initial slope” determination, which can introduce errors when the progress curves are nonlinear. The spectrophotometric assay is validated through product analysis by ¹H NMR (nuclear magnetic resonance), which showed that under the conditions of study l-methionine (l-met) and l-methionine sulfone (l-met sulfone) substrates were converted to α-KB product with greater than 99% yield. Using this assay method, we determined for the first time the Michaelis–Menten parameters for a recombinant form of MGL from Porphyromonas gingivalis, obtaining respective k_cat and K_m values of 328 ± 8 min⁻¹ and 1.2 ± 0.1 mM for l-met γ-elimination and 2048 ± 59 min⁻¹ and 38 ± 2 mM for l-met sulfone γ-elimination reactions. We envisage that this assay method will be useful for determining the activity of MGL γ-elimination reactions that produce α-KB as the end product.     

DESI-MSI and METASPACE indicates lipid abnormalities and altered mitochondrial membrane components in diabetic renal proximal tubules

Metabolomics - Food and dietary ingredients have significant effects on metabolism and health. To evaluate whether and how different diets affected the serum lipidomic profile of dogs. Sixteen...     

Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

We showed earlier that 15 deoxy Δ^12,14 prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion [1]. However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.     

Influence of growth regulators on plant regeneration from epicotyl and hypocotyl cultures of two groundnut (Arachis hypogaea L.) cultivars

This study was designed to evaluate the effect of phytohormones on plant regeneration from epicotyl and hypocotyl explants of two groundnut (Arachis hypogaea) cultivars. Explants cultured on media with auxins and in combination with cytokinin produced high frequency of callus. After four weeks, callus from these cultures was transferred to medium with cytokinin and reduced auxin, shoot buds regenerated from the cultures. A high rate of shoot bud regeneration was observed on medium supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA. Among the different auxins tested, NAA was found to be most effective, producing the highest frequency of shoot buds per responding cultures. Of the two explants tested, epicotyl was found to be best for high frequency shoot bud regeneration. Multiple shoots arose on MS medium supplemented with BAP or kinetin (1.0–5.0 mg/L) plus IBA (1.0 mg/L), with maximum production occurring at 5.0 mg/L. The elongated shoots developed rootsin vitro upon transfer to MS medium supplemented with NAA or IBA (0.5–2.0 mg/L) and kinetin (0.5 mg/L) for 15 days.In vitro produced plantlets, were transferred to soil and placed in a glasshouse developed successfully, matured, and set seeds.     

Genetic Identification of all Four Mangrove Mud Crab Species (genus Scylla) Using Multiple Molecular Markers

Biochemical Genetics - Misleading identification and subsequent publications on biological, molecular, and aquaculture data of mangrove mud crab (genus Scylla de Hann 1833) is a major concern in...     

MODEL DEVELOPMENT AND PROCESS OPTIMIZATION FOR SOLVENT EXTRACTION OF POLYPHENOLS FROM RED GRAPES USING BOX–BEHNKEN DESIGN

The objective of the present study is to find out the optimum extraction conditions for extraction of polyphenols from red grapes using Box–Behnken design. Red grapes polyphenols were extracted using acid–ethanol solvent at various extraction temperature (40–60°C), extraction time (20–100 min) and different solid–liquid ratio (1:5–1:15 g:ml). The effect (main and interactive) of extraction conditions on total anthocyanin, phenolic and flavonoid content were studied using Box–Behnken design (three factors at three levels). The results showed that the contribution of the quadratic model was significant for all the responses. Second-order mathematical regression models were developed and were found to fit well with observed data. Derringer's desirability function methodology was performed to find out the optimal conditions based on both individual and combinations of all responses (extraction temperature: 57°C, time: 61 min, and solid–liquid ratio: 1:8.7 g:ml) were established. At this optimal condition, the anthocyanin yield, total phenolic and flavonoid content were 73.92 mg/100 g, 221.4 mg GAE/100 g, and 79.08 mg CE/100 g, respectively. A desirability value of 0.902 was achieved at this point.     

Manumycin A inhibits triple-negative breast cancer growth through LC3-mediated cytoplasmic vacuolation death

Therapy resistance can be attributed to acquisition of anti-apoptotic mechanisms by the cancer cells. Therefore, developing approaches that trigger non-apoptotic cell death in cancer cells to compensate for apoptosis resistance will help to treat cancer effectively. Triple-negative breast cancers (TNBC) are among the most aggressive and therapy resistant to breast tumors. Here we report that manumycin A (Man A), an inhibitor of farnesyl protein transferase, reduces cancer cell viability through induction of non-apoptotic, non-autophagic cytoplasmic vacuolation death in TNBC cells. Man A persistently induced cytoplasmic vacuolation and cell death through the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins along with endoplasmic reticulum (ER) stress markers, Bip and CHOP, and accumulation of ubiquitinated proteins. As inhibitors of apoptosis and autophagy failed to block cytoplasmic vacuolation and its associated protein expression or cell death, it appears that these processes are not involved in the death induced by Man A. Ability of thiol antioxidant, NAC in blocking Man A-induced vacuolation, death and its related protein expression suggests that sulfhydryl homeostasis may be the target of Man A. Surprisingly, normal human mammary epithelial cells failed to undergo cytoplasmic vacuolation and cell death, and grew normally in presence of Man A. In conjunction with its in vitro effects, Man A also reduced tumor burden in vivo in xenograft models that showed extensive cytoplasmic vacuoles and condensed nuclei with remarkable increase in the vacuolation-associated protein expression together with increase of p21, p27, PTEN and decrease of pAkt. Interestingly, Man A-mediated upregulation of p21, p27 and PTEN and downregulation of pAkt and tumor growth suppression were also mimicked by LC3 knockdown in MDA-MB-231 cells. Overall, these results suggest novel therapeutic actions by Man A through the induction of non-apoptotic and non-autophagic cytoplasmic vacuolation death by probably affecting ER stress, LC3 and p62 pathways in TNBC but not in normal mammary epithelial cells.     

PTEN loss defines a TGF-β-induced tubule phenotype of failed differentiation and JNK signaling during renal fibrosis

We investigated the signaling basis for tubule pathology during fibrosis after renal injury. Numerous signaling pathways are activated physiologically to direct tubule regeneration after acute kidney injury (AKI) but several persist pathologically after repair. Among these, transforming growth factor (TGF)-β is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-β signaling after AKI is accompanied by PTEN loss from proximal tubules (PT). With time, subpopulations of regenerating PT with persistent loss of PTEN (phosphate and tension homolog) failed to differentiate, became growth arrested, expressed vimentin, displayed profibrotic JNK activation, and produced PDGF-B. These tubules were surrounded by fibrosis. In contrast, PTEN recovery was associated with epithelial differentiation, normal tubule repair, and less fibrosis. This beneficial outcome was promoted by TGF-β antagonism. Tubule-specific induction of TGF-β led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-β depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-β antagonism increased PTEN, promoted differentiation, and decreased JNK activity. Cre-Lox PTEN deletion suppressed differentiation, induced growth arrest, and activated JNK. The low-PTEN state with JNK signaling and fibrosis was ameliorated by contralateral nephrectomy done 2 wk after unilateral ischemia, suggesting reversibility of the low-PTEN dysfunctional tubule phenotype. Vimentin-expressing tubules with low-PTEN and JNK activation were associated with fibrosis also after tubule-selective AKI, and with human chronic kidney diseases of diverse etiology. By preventing tubule differentiation, the low-PTEN state may provide a platform for signals initiated physiologically to persist pathologically and cause fibrosis after injury.