The development of mouse oocyte cortical reaction competence is accompanied by major changes in cortical vesicles and not cortical granule depth

The basis for the incompetence of the cortical reaction in germinal vesicle stage (GV) mouse oocytes was studied by evaluating cortical granules (CGs) and vesicles in GV and mature oocyte cortices. Dark and light CGs had a similar mean distance of 0.4-0.6 micron from the plasma membrane for GV and mature cortices. The cortex of mature oocytes had a large population of membrane-bounded, 0.1-1.0 micron (diameter) vesicles. More than three times as many vesicles were observed in the CG domains of mature oocytes as were observed in GV oocytes. This lack of cortical vesicles (with their potential to store calcium) and not CG depth may account for cortical reaction incompetence in GV oocytes.     

Spontaneous allelic variant in deafness–blindness gene Ush1g resulting in an expanded phenotype

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1g^bw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1g^bw/bw mice had no substantial retinal degeneration compared with Ush1g^bw/+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.     

Occurrence of a Lysogenic Streptomyces sp. on the Nodule Surface of Black Gram (Vigna mungo (L.) Hepper)

A lysogenic Streptomyces sp., strain NS.A4, which was isolated from the nodule surface of black gram (Vigna mungo (L.) Hepper), was found to inhibit rhizobia of fast-and slow-growing strains of cowpeas and soybeans. It exhibited plaques when there was a change in cultural conditions. Repeated culturing of the organism in nutrient agar and broth confirmed the infection of Streptomyces sp. strain NS.A4 by an actinophage. Addition of the culture filtrate of Streptomyces sp. strain NS.A4 to shaken broth cultures of three other Streptomyces spp. resulted in phage infection.     

Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida

Fertilization results in cortical granule exocytosis, which is thought to be involved in modifications of the zona pellucida that constitute the zona pellucida block to polyspermy. A previous report demonstrated that a decrease in the number of Lens culinaris agglutinin-staining granules, which are likely to be cortical granules, occurred during in vivo mouse oocyte maturation with arrest at metaphase II, as well as the formation of a cortical granule-free domain in the area of the metaphase II spindle (T. Ducibella, E. Anderson, D.F. Albertini, J. Aalberg, and S. Rangarajan, 1988, Dev. Biol. 130, 184-197). We extend these observations by reporting here that germinal vesicle-intact oocytes matured in vitro to metaphase II in either the absence or the presence of serum develop a cortical granule-free domain and have reduced numbers of cortical granules when compared to germinal vesicle-intact oocytes; these changes are similar to those of oocytes matured in vivo. The reduction in the number of cortical granules requires germinal vesicle breakdown, since it is prevented by dibutyryl cAMP, which inhibits germinal vesicle breakdown in vitro. The ability of oocytes to respond to the calcium ionophore A23187 with a reduction in the number of cortical granules is also associated with meiotic maturation and develops between 7 and 12 hr after initiation of maturation. The maturation-associated reduction in the number of cortical granules is likely to represent cortical granule exocytosis, since this reduction is accompanied by the formation of a cortical granule-free domain and a conversion of ZP2 to ZP2f when the oocytes are matured in vitro in serum-free medium; this zona pellucida modification occurs following fertilization and is thought to be due to cortical granule exocytosis. In contrast, the loss of cortical granules and development of the cortical granule-free domain of oocytes matured in vitro in the presence of serum is not accompanied by the modification of ZP2. The inhibitory effect of serum on the ZP2 modification may afford in vivo a physiological mechanism to prevent a precocious modification of the zona pellucida that could result in a premature block to polyspermy and hence inhibit fertilization.     

Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog

Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive ³H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the non-radioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive ³H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive ³H-2-deoxy-D-glucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed.     

A new generation of predictive models: The added value of hybrid models for manufacturing processes of therapeutic proteins

Due to the lack of complete understanding of metabolic networks and reaction pathways, establishing a universal mechanistic model for mammalian cell culture processes remains a challenge. Contrarily, data-driven approaches for modeling these processes lack extrapolation capabilities. Hybrid modeling is a technique that exploits the synergy between the two modeling methods. Although mammalian cell cultures are among the most relevant processes in biotechnology and indeed looks ideal for hybrid modeling, their application has only been proposed but never developed in the literature. This study provides a quantitative assessment of the improvement brought by hybrid models with respect to the state-of-the-art statistical predictive models in the context of therapeutic protein production. This is illustrated using a dataset obtained from a 3.5 L fed-batch experiment. With the goal to robustly define the process design space, hybrid models reveal a superior capability to predict the time evolution of different process variables using only the initial and process conditions in comparison to the statistical models. Hybrid models not only feature more accurate prediction results but also demonstrate better robustness and extrapolation capabilities. For the future application, this study highlights the added value of hybrid modeling for model-based process optimization and design of experiments.     

Genetic diversity of Bradyrhizobium strains isolated from Arachis hypogaea

Rhizobia are used exclusively in agricultural systems for enhancing the ability of legumes to fix atmospheric nitrogen. Knowledge about the indigenous population is necessary for the selection and application of inoculant strains. In this study, we have assessed the genetic diversity of Bradyrhizobium strains isolated from the host plant, Arachis hypogaea along the coastline of Tamil Nadu. Different populations collected from varying environmental conditions were analysed for salt and pH tolerance. Genetic diversity among the strains was studied using RAPD markers and PCR-RFLP of 16S rDNA and nifD genes. The approaches used in this study yielded consistent results, which revealed a high degree of heterogeneity among strains and detection of two distinct genetic groups.     

The netrin receptor frazzled is required in the target for establishment of retinal projections in the Drosophila visual system

Retinal axons in Drosophila make precise topographic connections with their target cells in the optic lobe. Here we investigate the role of the Netrins and their receptor Frazzled in the establishment of retinal projections. We find that the Netrins, although expressed in the target, are not required for retinal projections. Surprisingly, Frazzled, found on both retinal fibers and target cells, is required in the target for attracting retinal fibers, while playing at best a redundant role in the retinal fibers themselves; this finding demonstrates that target attraction is necessary for topographic map formation. Finally, we show that Frazzled is not required for the differentiation of cells in the target. Our data suggest that Frazzled does not function as a Netrin receptor in attracting retinal fibers to the target; nor does it seem to act as a homotypic cell adhesion molecule. We favor the possibility that Frazzled in the target interacts with a component on the surface of retinal fibers, possibly another Netrin receptor.     

Mutation detection using Surveyor nuclease

We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations. This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3' side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucleotides. Surveyor nuclease technology involves four steps: (i) PCR to amplify target DNA from both mutant and wild-type reference DNA; (ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA; (iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes; and (iv) analysis of digested DNA products using the detection/separation platform of choice. The technology is highly sensitive, detecting rare mutants present at as low as 1 in 32 copies. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis. The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures.