Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals

Human interferon-alpha 2b (IFN-α2b) was cloned and expressed in Pichia pastoris under the control of alcohol oxidase promoter (AOX1) using three different secretion signals. Native secretion signal of IFN-α2b, Saccharomyces cerevisiae MF-α factor prepro sequence and a mutated α prepro sequence without the Glu-Ala (EAEA) repeats were used separately for directing the secretion of IFN-α2b into the culture medium of P. pastoris. The native secretion signal of IFN-α2b did not secrete protein into the culture medium of P. pastoris. The α prepro sequence without the EAEA repeats directed the secretion of maximum amount of IFN-α2b (200 mg/l) into the culture medium, with the same amino acid sequence as that of the native IFN-α2b secreted by human lymphocytes. The full α prepro sequence, having both the protease cleavage sites for KEX2 and STE13 gene products, also secreted an equivalent amount of IFN-α2b into the culture medium. However, two interferon bands with similar molecular masses were observed, when full α prepro sequence was used for the secretion of IFN-α2b. The difference in the molecular masses of the two bands was found to arise due to the difference in the molecular masses of the N-terminal fragment, and the inefficient processing of secretion signal.     

Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I

The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin-myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin-myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin-myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.     

High glucose stimulates synthesis of fibronectin via a novel protein kinase C,Rap1b,and B-Raf signaling pathway

The molecular mechanism(s) by which high glucose induces fibronectin expression via G-protein activation in the kidney are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and activation, and the relevance of protein kinase C (PKC) and Raf pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a dose-dependent increase in Rap1b expression in glomeruli of diabetic rat kidneys. Similarly, in vitro exposure of MCs to HG led to an up-regulation of Rap1b with concomitant increase in fibronectin (FN) mRNA and protein expression. The up-regulation of Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and bisindolymaleimide, while also reducing HG- induced FN expression in non-transfected MCs. Overexpression of Rap1b by transfection with pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis; however, the PKC inhibitors had no significant effect in reducing FN expression in Rap1b-transfected MCs. Transfection of Rap1b mutants S17N (Ser --> Asn) or T61R (Thr --> Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf and Raf-1 expression was investigated to assess whether Rap1b effects are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was increased in MCs transfected with Rap1b. HG also caused activation of Rap1b, which was largely unaffected by anti-platelet-derived growth factor (PDGF) antibodies. HG-induced activation of Rap1b was specific, since Rap2b activation and expression of Rap2a and Rap2b were unaffected by HG. These findings indicate that hyperglycemia and HG cause an activation and up-regulation of Rap1b in renal glomeruli and in cultured MCs, which then stimulates FN synthesis. This effect appears to be PKC-dependent and PDGF-independent, but involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix synthesis, a hallmark of diabetic nephropathy.     

Convolutional neural networks for reconstruction of undersampled optical projection tomography data applied to in vivo imaging of zebrafish

Optical projection tomography (OPT) is a 3D mesoscopic imaging modality that can utilize absorption or fluorescence contrast. 3D images can be rapidly reconstructed from tomographic data sets sampled with sufficient numbers of projection angles using the Radon transform, as is typically implemented with optically cleared samples of the mm‐to‐cm scale. For in vivo imaging, considerations of phototoxicity and the need to maintain animals under anesthesia typically preclude the acquisition of OPT data at a sufficient number of angles to avoid artifacts in the reconstructed images. For sparse samples, this can be addressed with iterative algorithms to reconstruct 3D images from undersampled OPT data, but the data processing times present a significant challenge for studies imaging multiple animals. We show here that convolutional neural networks (CNN) can be used in place of iterative algorithms to remove artifacts—reducing processing time for an undersampled in vivo zebrafish dataset from 77 to 15 minutes. We also show that using CNN produces reconstructions of equivalent quality to compressed sensing with 40% fewer projections. We further show that diverse training data classes, for example, ex vivo mouse tissue data, can be used for CNN‐based reconstructions of OPT data of other species including live zebrafish.      

The yolk nucleus in the freshwater teleost Garra gotyla gotyla (Gray)

The structure and functional significance of the yolk nucleus in Garra are described. The study shows that the yolk nucleus appears in the cytoplasm of the early perinucleolus stage of the oocyte. It is present as a deeply stained structure enclosed in a vesicle. It disappears in the yolk vesicle stage and vitellogenesis begins soon afterwards. This probably indicates that it plays a role in the initiation of vitellogenesis. When yolk formation starts, the yolk nucleus disappears; this also suggests that it is of functional significance in yolk synthesis.     

Effect of second-leaf removal or kinetin treatment on the nucleic acid metabolism of senescing first seedling leaf of barley

1. Changes in nucleic acid metabolism in first seedling leaves of barley plants during aging (from 7 to 27 days) were followed, and the effect of continual removal of the second leaf and basal meristem or of treating the first leaf with 20p.p.m. kinetin on these changes was examined. During aging of the first seedling leaves the ribosomal RNA, DNA and soluble RNA declined, with ribosomal RNA showing the most rapid fall. This was, however, accompanied by increased incorporation of ³²P into RNA, which reached its peak on the fifteenth day. 2. Second-leaf removal partially suppressed first-leaf senescence as judged by retarded chlorophyll and nucleic acid decline and by a decreased extent of RNA labelling. Treatment with kinetin, however, did not prove effective. 3. No significant differences in the sucrose-gradient pattern of ³²P-labelled nucleic acids or in the ³²P-labelled nucleotide composition of RNA fractions during aging or during the two treatments were noted, except for a decrease in CMP content of soluble RNA during aging. 4. The results demonstrate that important changes in RNA metabolism are associated with leaf senescence.     

Terminal deoxynucleotidyl transferase activity in a cell line (molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia

A terminal deoxynucleotidyl transferase having a sedimentation coefficient of 3–4S has been found associated with the chromatin from a cell line (Molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia.     

Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett-Burman design (PBD). Only Zn(2+), Mn(2+) and MoO(4)(2-) were the most significant components (p<0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn(2+) (ZnSO(4)·7H(2)O), Mn(2+) (MnCl(2)·4H(2)O), and MoO(4)(2-) (Na(2)MoO(4)·2H(2)O) were 83, 42 and 135μM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25μg DAPG/g carrier) when stored at 28°C for 6 months. The structure of the purified DAPG was confirmed using (1)H NMR and mass spectrometry.