Glycemic Control Promotes Pancreatic Beta-Cell Regeneration in Streptozotocin-Induced Diabetic Mice

Background

Pancreatic beta-cells proliferate following administration of the beta-cell toxin streptozotocin. Defining the conditions that promote beta-cell proliferation could benefit patients with diabetes. We have investigated the effect of insulin treatment on pancreatic beta-cell regeneration in streptozotocin-induced diabetic mice, and, in addition, report on a new approach to quantify beta-cell regeneration in vivo.

Methodology/Principal Findings

Streptozotocin-induced diabetic were treated with either syngeneic islets transplanted under the kidney capsule or subcutaneous insulin implants. After either 60 or 120 days of insulin treatment, the islet transplant or insulin implant were removed and blood glucose levels monitored for 30 days. The results showed that both islet transplants and insulin implants restored normoglycemia in the 60 and 120 day treated animals. However, only the 120-day islet and insulin implant groups maintained euglycemia (<200 mg/dl) following discontinuation of insulin treatment. The beta-cell was significantly increased in all the 120 day insulin-treated groups (insulin implant, 0.69±0.23 mg; and islet transplant, 0.91±0.23 mg) compared non-diabetic control mice (1.54±0.25 mg). We also show that we can use bioluminescent imaging to monitor beta-cell regeneration in living MIP-luc transgenic mice.

Conclusions/Significance

The results show that insulin treatment can promote beta-cell regeneration. Moreover, the extent of restoration of beta-cell function and mass depend on the length of treatment period and overall level of glycemic control with better control being associated with improved recovery. Finally, real-time bioluminescent imaging can be used to monitor beta-cell recovery in living MIP-luc transgenic mice.     

Towards a global database of weed risk assessments: a test of transferability for the tropics

Worldwide spread and establishment of alien plant species continues to accelerate and damage ecological and agricultural systems. Early warning and prevention of high-risk introductions is the most cost-effective approach to minimise losses while maximising benefits, and the Australian Weed Risk Assessment (A-WRA) system has been the most well-developed and successful predictive scheme. However, any system would be limited if the results or scores were confined to the locality of assessment. We compiled A-WRA scores conducted in four tropical to sub-tropical regions and tested the accuracy of these scores for predicting naturalisations for a separate well-documented, equatorial, exotic flora where weed risk assessments have never been conducted. Receiver Operating Characteristic (ROC) curves reflect high accuracies of predictions, comparable to those in other studies. No significant differences in accuracy were found between each regional subset and the compiled set of scores. Our results show that A-WRA scores assessed at one locality can be used for others of similar climate, increasing the utility of every species’ assessment. A global database of A-WRA scores would enable rapid local decision-making in border controls on imported plant species. A growing record of species assessments would also facilitate monitoring evolutionary and ecological aspects of invasive species.     

Tea epigallocatechin-3-gallate increases 8-isoprostane level and induces caudal regression in developing rat embryos

Tea is the most common beverage after water. Concerns have been raised about the safety of tea during pregnancy, especially for embryo development. We aimed at studying the effects of active tea components on developing embryos by in vitro rat embryo culture. Rat embryos during early organogenesis were cultivated in serum supplemented with one of the tea catechins. Developmental hallmarks and malformations (Mal) in the developing embryos were compared and evaluated by a standard morphological scoring system. The embryotoxicity of each tea catechin was classified according to the European Center for the Validation of Alternative Methods. Cell viability was assessed by supervital dye staining, apoptosis by TUNEL assay, and peroxidation by the 8-isoprostane EIA method. We found that (+)-catechin had the least effect on developing embryos (Mal(50)=715.1 mg/L; IC50(Mal)=435 mg/L), whereas (-)-epigallocatechin gallate had the most adverse effect (Mal(50)=54.2 mg/L; IC50(Mal)=45.8 mg/L). The major malformation in affected embryos included caudal retardation with abnormal axial flexion and delayed hind-limb formation. All catechins were classified as nonembryotoxic except (-)-epigallocatechin gallate, which was classified as weakly embryotoxic. With (-)-epigallocatechin gallate, increased numbers of nonviable and apoptotic cells in the malformed embryos were associated with increased embryo 8-isoprostane.     

Testis-specific expression and genomic multiplicity of the rat Rtdpoz genes that encode bipartite TRAF- and POZ/BTB-domain proteins

Based on bioinformatics analysis, we previously hypothesized the existence of a bipartite TDPOZ protein family members of which carry the TRAF domain (TD) and POZ/BTB [Huang, C.-J., Chen, C.-Y., Chen, H.-H., Tsai, S.-F., Choo, K.-B., 2004. TDPOZ, a family of bipartite animal and plant proteins that contain the TRAF (TD) and POZ/BTB domains. Gene 324, 117-127.]. Conservation in animals and plants suggests important biological functions for the putative TDPOZ proteins. In this work, we report testis-specific expression of two new Tdpoz members, Rtdpoz-T1 and -T2, of the rat genome; the result clearly indicates that members of the hypothetical gene family are, indeed, expressed. T1 and T2 cDNA sequences were derived by rapid amplification of cDNA ends (RACE). The exons of the genes were determined by queries of the rat genome sequence draft and selectively confirmed in splicing assays. The results indicate that T1 and T2 share a common leader exon indicative of alternative splicing, and that the genes are uninterrupted by introns in their respective coding sequences. Database interrogations also reveal a combined 297 hits of Rtdpoz-like sequences on 7 chromosomes; however, the bulk of the hits (264) and 26 putative TDPOZ-encoding genes, including T1 and T2, are found in a approximately 2.5 Mb cluster in the Rn2_2148 supercontig on chromosome 2. Our data signify retrotransposition in the generation and expansion of the Rtdpoz repertoire in the rat genome. We also anticipate spatio-temporal-specific expression of many more TDPOZ members in the rat or other animals and plants.     

RNAi knockdown of Oryza sativa root meander curling gene led to altered root development and coiling which were mediated by jasmonic acid signalling in rice

Jasmonic acid (JA) is a well-known defence hormone, but its biological function and mechanism in rice root development are less understood. Here, we describe a JA-induced putative receptor-like protein (OsRLK, AAL87185) functioning in root development in rice. RNA in situ hybridization revealed that the gene was expressed largely in roots, and a fusion protein showed its localization on the plasma membrane. The primary roots in RNAi transgenic rice plants meandered and curled more easily than wild-type (WT) roots under JA treatment. Thus, this gene was renamed Oryza sativa root meander curling (OsRMC). The transgenic primary roots were shorter, the number of adventitious roots increased and the number of lateral roots decreased as compared to the WT. As well, the second sheath was reduced in length. Growth of both primary roots and second sheaths was sensitive to JA treatment. No significant change of JA level appeared in the roots between the transgenic rice line and WT. Expression of RSOsPR10, involved in the JA signalling pathway, was induced in transgenic rice. Western blotting revealed OsRMC induced by JA. Our results suggest that OsRMC of the DUF26 subfamily involved in JA signal transduction mediates root development and negatively regulates root curling in rice.     

Profiling of Substrate Specificity of SARS-CoV 3CLpro

Background

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection.

Methodology/Principal Findings

To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3'' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1'' position prefers small residues, while P2'' and P3'' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3'' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence.

Conclusions/Significance

Our results demonstrated a strong structure-activity relationship between the 3CL^pro and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors.     

OsAGAP, an ARF-GAP from rice, regulates root development mediated by auxin in Arabidopsis

Arf (ADP-ribosylation factor) proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the action of GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) for their function. In the present study the OsAGAP gene in rice, which encoded a protein with predicted structure similar to ArfGAP, was identified. The purified OsAGAP-GST fusion protein was able to stimulate the GTPase activity of rice Arf. Furthermore, OsAGAP can rescue the defect of vesicular transport in the yeast gcs1 delta glo3 delta double-mutant cells. Transgenic Arabidopsis with OsAGAP constitutively expression showed reduced apical dominance, shorter primary roots, increasing number of longer adventitious roots. Many of the phenotypes can be phenocopied by treatment of exogenous indoleacetic acid level (IAA) in wild-type plants. Determination of whole-plant IAA level showed that there is a sharp increase of free IAA in OsAGAP transgenic Arabidopsis seedlings. In addition, removal of the 4-day-old shoot apex could inhibit the adventitious root formation in the transgenic seedlings. These results suggest OsAGAP, an ARF-GAP of rice, maybe involved in the mediation of plant root development by regulating auxin level.     

Determination of the enantiomeric composition of (R*,S*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate, the male-produced aggregation pheromone of Sitophilus granarius

The enantiomeric composition of sitophilate, the granary weevil [Sitophilus granarius (L.)] male-produced aggregation pheromone [(R^*,S^*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate)], was determined by three methods: (1) bioassaying the synthetic (2S,3R) and (2R,3S) enantiomers of the active (R^*,S^*) diastereomer; (2) ¹H NMR spectroscopy of Mosher ester derivatives of the natural pheromone and synthetic (2S,3R)-and (2R,3S)-sitophilate; and (3) capillary GLC comparisons of the retention times of derivatized natural pheromone and the two synthetic enantiomers. The combined methods confirmed the (2S,3R) enantiomer as the active form of sitophilate. Male granary weevils were shown to produce >96% (2S,3R)-sitophilate. No significant attraction of S. granarius by the (2R,3S) enantiomer was observed. Rice and maize weevils [S. oryzae (L.) and S. zeamais Motschulsky] were not attracted by (2S,3R)-sitophilate. S. granarius L. est un déprédateur important des grains stockés. Le (R^*,S^*)-1-éthylpropyl 2-méthyl-3-hydroxypentanoate a été identifié en 1987 comme le principal composé du sitophilate, la phéromone mâle d'agrégation de S. granarius. La composition énantiométrique du sitophilate a été déterminée par 3 méthodes:

蛋白酶抗性人睫状神经营养因子突变体基因的构建及其在巴斯德毕赤酵母中的表达

AX15是一种比天然睫状神经营养因子具有更高的生物学活性、更好的稳定性和可溶性的hCNTF突变体。在巴斯德毕赤酵母中表达时AX15易发生降解。氨基酸序列分析表明降解位于由12和13位氨基酸残基组成的双碱性氨基酸之后。根据KEX2蛋白酶的底物专一性把双碱性氨基酸从RR变为RK,构建了KEX2抗性的AX15突变体AX15（R13K）。AX15（R13K）的稳定性得到了显著的提高,在诱导100 h后也未发生降解。利用超滤浓缩和凝胶过滤得到了纯度>90%的AX15（R13K）。TF-1细胞存活实验表明AX15（R13K）具有与AX15相同的生物学活性。蛋白酶抗性人睫状神经营养因子突变体可能具有更好的体内稳定性,在临床应用上具有潜在的优势。