Somatic embryogenesis and organogenesis inGuizotia Abyssinica

Summary Induction of somatic embryogenesis, shoot organogenesis, and subsequent plant regeneration in niger seem to be dependent on genotype, choice of explant, and composition of media growth regulators. Two distinct regeneration protocols have been developed for somatic embryogenesis and shoot organogenesis. Somatic embryogenesis was induced from epicotyls and cotyledonary explants (9 to 35%) (but not from hypocotyls and roots) in presence of 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxypropionic acid. These embryos matured in MS medium containing Kinetin plus naphthalene acetic acid (NAA), Kinetin plus Zeatin, and Kinetin plus abscisic acid (ABA). Matured embryos could be germinated on LS and MS basal media without hormones. Non-embryogenic callus initiated on Linsmaier and Skoog’s (LS) medium from cotyledons of six different genotypes produced shoots (9 to 32%) on Murashige and Skoog’s (MS) medium fortified with 6-benzylaminopurine (BAP, 0.5 mg · liter^−1), and BAP (1 mg · liter^−1) plus NAA (0.1 mg · liter^−1). These shoots were rooted with 100% frequency by using indole-3-acetic acid or NAA and transferred successfully to the soil.     

Analysis of a genomic DNA segment carrying the wheat high-molecular-weight (HMW) glutenin Bx17 subunit and its use as an RFLP marker

Summary A genomic fragment containing the Bx17 high-molecular-weight (HMW) glutenin gene was isolated from a wheat genomic library. The fragment contains a coding region of 2.82kb with 1.98-kb downstream and 12.8-kb upstream flanking regions. The fragment was sequenced and compared with previously published glutenin genes from chromosomes 1A, 1B and 1D using a computer alignment package. The Bx17 gene shows marked similarity to the Bx7 gene sequence. A phenetic tree derived from the alignments is presented. Also shown are restriction fragment length polymorphisms (RFLPs) at the glutenin loci in a set of Australian and international wheat varieties using different regions of the glutenin clone as probes. The RFLPs correlated well with the protein composition in all cultivars analysed.     

v-myb blocks granulocyte colony-stimulating factor-induced myeloid cell differentiation but not proliferation.

To understand the effects of v-myb expression on mammalian hematopoietic cell differentiation, we have constructed a retroviral vector which can efficiently express v-myb gene product in mammalian cells. Infection of interleukin-3-dependent murine progenitor cell line 32D Cl3, which undergoes terminal differentiation to mature granulocytes in the presence of granulocyte colony-stimulating factor (GCSF), with this recombinant retrovirus does not abrogate its requirement of interleukin-3 for growth. However, expression of v-myb in these cells blocks their ability to differentiate in response to GCSF. Instead, the v-myb-infected cells proliferate indefinitely in the presence of GCSF. 32D Cl3 cells infected with empty vector carrying only the neomycin resistance gene responded to the addition of GCSF in a manner identical to that of the uninfected cells and underwent terminal differentiation into granulocytes. These results suggest that oncogenic forms of myb gene bring about transformation by blocking the differentiation signal derived by cytokines while promoting the proliferative signal transduction pathways.     

Microcomputer BASIC programs for rapid determination of lethal dosages of biocides using logit transformations

The probit analysis model is generally used for the deterininationof lethal dosages in bioassay applications. However, the logitmodels, which use the logistic curve instead of the integratednonnal curve, could also be effectively used for the determinationof lethal dosages and regression coefficients. In this paper,two lypes of logit models, namely minimum logit chi square andmaximum likelihood, have been described in detail for the estimationof the parameters. The results of these two models are alsocompared with those of the probit model.     

Transgenic modifications of silkworms as a means to obtain therapeutic biomolecules and protein fibers with exceptional properties

Transgenic modification of Bombyx mori silkworms is a benign approach for the production of silk fibers with extraordinary properties and also to generate therapeutic proteins and other biomolecules for various applications. Silk fibers with fluorescence lasting more than a year, natural protein fibers with strength and toughness exceeding that of spider silk, proteins and therapeutic biomolecules with exceptional properties have been developed using transgenic technology. The transgenic modifications have been done primarily by modifying the silk sericin and fibroin genes and also the silk producing glands. Although the genetic modifications were typically performed using the sericin 1 and other genes, newer techniques such as CRISPR/Cas9 have enabled successful modifications of both the fibroin H-chain and L-chain. Such modifications have led to the production of therapeutic proteins and other biomolecules in reasonable quantities at affordable costs for tissue engineering and other medical applications. Transgenically modified silkworms also have distinct and long-lasting fluorescence useful for bioimaging applications. This review presents an overview of the transgenic techniques for modifications of B. mori silkworms and the properties obtained due to such modifications with particular focus on production of growth factors, fluorescent proteins, and high performance protein fibers.     

Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.     

Synthesis of methyl 2,4-di-O-methyl-3-O-(2-O-methyl-alpha-L-rhamnopyranosyl)-alpha-L- rhamnopyranoside and methyl 2,4-di-O-methyl-3-O-[2-O-methyl-3-O-(2-O-methyl-alpha-L-fucopyranosyl)- alpha-L-rhamnopyranosyl]-alpha-L-rhamnopyranoside: di- and tri-saccharide segments of a phenolic glycolipid of Mycobacterium kansasii.

Syntheses of the title glycosides are described. The critical O-glycosylations were carried out in the presence of boron trifluoride etherate with a high degree of alpha-selectivity.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.