Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage. Supported by National Natural Science Foundation of CHINA (30170060)     

Lead identification to generate isoquinolinedione inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.     

Effect of zinc on cellular levels of calmodulin and cyclic adenosine monophosphate in the adipocyte

A perturbation of zinc metabolism has been noted in subjects with obesity. Zinc may also participate in the intracellular signal cascade by affecting cellular calcium influx and a change in the calcium-calmodulin (CaM)-cyclic adenosine monophosphate (cAMP) pathway. The possible effects of zinc on cellular concentrations of CaM, a major cytosolic calcium-binding protein, in the adipocytes derived from obese (ob/ob) mice and their lean counterparts were studied. Adipocytes derived from both phenotypes of mice were treated either with 0.2 mM of zinc sulfate or without any additive for 1 h of incubation; the cellular levels of CaM and cAMP were then determined. The results showed that the obese mice had lower CaM and cAMP levels in their adipocytes compared to the lean mice. Zinc treatment reduced CaM and increased cAMP levels in all mice, although this effect was more pronounced in the lean mice. This study indicated that there was an inverse interaction between CaM and cAMP in their cellular levels in the mouse adipocytes and that might be affected by exogenous zinc addition.     

Using Next-Generation Sequencing and Bioinformatic Methods to Predict New Genes That May Be Regulated by CD47 in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world, and the incidence and death rate of OSCC in men is twice that of women. CD47 is a ubiquitous cell surface transmembrane protein, also known as integrin-related protein (IAP). Previous studies have pointed out that CD47 can inhibit the growth of OSCC, but the detailed mechanism is not clear. This study aimed to explore the effect of CD47 gene expression profiles in OSCC. The OSCC cell lines, OECM-1 and OC-2, overexpressed CD47, and the expression profiles of mRNAs were analyzed through next-generation sequencing (NGS) with a bioinformatic approach. A total of 14 differentially expressed genes (DEGs) were listed. In addition, ingenuity pathway analysis (IPA) was used to analyze the molecular function (MF), biological process (BP), and cellular component (CC) network signaling. The human protein atlas (HPA) database was used to analyze gene expression and the survivability of human cancer. The results found that HSPA5, HYOU1, and PDIA4 were involved in the IPA network and when highly expressed, mediated the survivability of cancer. In addition, HSPA5 was positively and significantly correlated with CD47 expression (p < 0.0001) and induced by CD47-overexpression in the OECM-1 and OC-2 OSCC cancer cell lines. These findings provide important insights into possible new diagnostic strategies, including unfolded protein for OSCC-targeting CD47.     

Comparison of secondary structures of insulin and proinsulin by FTIR

Although the structure of insulin is known in great detail, that of proinsulin has been little investigated, except for a few CD and NMR studies. The secondary structures of human proinsulin are now compared with those of insulin by Fourier Transformed Infrared (FTIR) studies. The deconvolved and second derivative spectra of proinsulin and insulin in the amide I' band region are closely similar with peaks corresponding to -helix, irregular helix, and 3₁₀ helix at nearly identical positions. For both proteins, the relative contents of the above structures as calculated from the peak areas are in good agreement with the values obtained from the known structure of crystalline porcine insulin. However, compared with insulin, proinsulin has markedly more unordered structures as indicated by the area under the peak at 1643.4 cm^–1. In addition, both peak positions and relative areas for turn structure of the prohormone are different from those for insulin. It appears from the above that the A-and B-chain segments of proinsulin and insulin are similar in their secondary structures, especially in helices. The C-chain segment is largely unordered except in a few -turns.     

Dopamine D1 Agonist SKF 38393 Increases the State of Phosphorylation of ARPP-21 in Substantia Nigra

Abstract: ARPP-21 is a cyclic AMP-regulated phosphoprotein (M_r= 21,000) that has a distribution in brain similar to that of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M_r= 32,000). It is enriched in the medium-sized spiny neurons in the striatum and in the striatonigral nerve terminals in the pars reticulata of the substantia nigra. The present study shows that dopamine D₁ agonist SKF 38393 increases the state of phosphorylation of ARPP-21 by 26% in nigral slices and that pretreatment of the slices with D₁ antagonist SCH 23390 blocks this effect. These results demonstrate that ARPP-21 is a dopamine-regulated phosphoprotein. Because D₁ receptors are localized on nerve terminals of striatonigral pathway, the phosphorylation of ARPP-21 is likely to mediate some of the intracellular effects of dopamine on these terminals.     

Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern.

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) caused excessive disease in infants upon subsequent natural infection with RSV. Recent studies with BALB/c mice have suggested that T cells are important contributors to lung immunopathology during RSV infection. In this study, we investigated vaccine-induced enhanced disease by immunizing BALB/c mice with live RSV intranasally or with FI-RSV intramuscularly. The mice were challenged with RSV 6 weeks later, and the pulmonary inflammatory response was studied by analyzing cells obtained by bronchoalveolar lavage 4 and 8 days after challenge. FI-RSV-immunized mice had an increased number of total cells, granulocytes, eosinophils, and CD4+ cells but a decreased number of CD8+ cells. The immunized mice also had a marked increase in the expression of mRNA for the Th2-type cytokines interleukin-5 (IL-5) and IL-13 as well as some increase in the expression of IL-10 (a Th2-type cytokine) mRNA and some decrease in the expression of IL-12 (a Th1-type cytokine) mRNA. The clear difference in the pulmonary inflammatory response to RSV between FI-RSV- and live-RSV-immunized mice suggests that this model can be used to evaluate the disease-enhancing potential of candidate RSV vaccines and better understand enhanced disease.     

Kinetics of trypsin inhibition by its specific inhibitors

The kinetics of inhibition of trypsin by its specific inhibitors, pancreatic trypsin inhibitor, ovomucoid trypsin inhibitor, and soybean trypsin inhibitor, has been studied by following the hydrolysis of benzoylarginine ethyl ester in the presence of the inhibitor, and the results have been analyzed with the method described previously [Tian & Tsou (1982) Biochemistry 21, 1028]. The results obtained are consistent with the following: (a) The enzyme binds with the pancreatic inhibitor irreversibly to form an inactive complex. (b) The binding with the ovomucoid inhibitor to form the inactive complex is reversible. (c) An intermediate is formed before the relatively stable inactive complex with the soybean inhibitor, and both steps are reversible. The respective microscopic rate constants are determined by suitable plots of the apparent rate constants under different substrate and inhibitor concentrations. The second-order rate constants for the initial binding step thus obtained are in accord with the apparent inactivation rate constants determined by measuring the activity remaining with a stopped-flow apparatus equipped with a multimixing system after the enzyme-inhibitor mixture has been incubated for different time intervals.