Achim Trebst,my senior,and our joint research

In this tribute, I offer my best wishes to Achim Trebst on his 80th birthday on June 9, 2009. At the invitation of Govindjee, I present here a perspective of some of our joint research during 1970s through 2000s.     

Genetic diversity within and among populations of shortleaf pine (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.)

Shortleaf pine (n = 93) and loblolly pine (n = 112) trees representing 22 seed sources or 16 physiographic populations were sampled from Southwide Southern Pine Seed Source Study plantings located in Oklahoma, Arkansas, and Mississippi. The sampled trees were grown from shortleaf pine and loblolly pine seeds formed in 1951 and 1952, prior to the start of intensive forest management across their native ranges. Amplification fragment length polymorphism (AFLP) markers were developed and used to study genetic diversity and its structure in these pine species. After screening 48 primer pairs, 17 and 21 pairs were selected that produced 794 and 647 AFLPs in shortleaf pine and loblolly pine, respectively. High-AFLP-based genetic diversity exists within shortleaf pine and loblolly pine, and most (84.73% in shortleaf pine; 87.69% in loblolly pine) of this diversity is maintained within physiographic populations. The high value of unbiased measures of genetic identity and low value of genetic distance for all pairwise comparisons indicates that the populations have similar genetic structures. For shortleaf pine, there was no significant correlation between geographic distance and genetic distance (r = 0.28), while for loblolly pine there was a weak but significant correlation (r = 0.51).     

Porous bone morphogenetic protein-2 microspheres: Polymer binding and in vitro release

This research compared the binding and release of recombinant human bone morphogenetic protein 2 (rhBMP-2) with a series of hydrophobic and hydrophilic poly-lactide-co-glycolide (PLGA) copolymers. Porous microspheres were produced via a double emulsion process. Binding and incorporation of protein were achieved by soaking microspheres in buffered protein solutions, filtering, and comparing protein concentration remaining to nonmicrosphere-containing samples. Protein release was determined by soaking bound microspheres in a physiological buffer and measuring protein concentration (by reversed-phase high-performance liquid chromatography) in solution over time. Normalized for specific surface area and paired by polymer molecular weight. microspheres made from hydrophilic 50∶50 or 75∶25 PLGA bound significantly more protein than microspheres made from the corresponding hydrophobic PLGA. Increased binding capacity correlated with higher polymer acid values. With certain polymers, rhBMP-2 adsorption was decreased or inhibited at high protein concentration, but protein loading could be enhanced by increasing the protein solution:PLGA (volume:mass) ratio or by repetitive soaking. Microspheres of various PLGAs released unbound protein in 3 days, whereas the subsequent bound protein release corresponded to mass loss. RhBMP-2 binding to PLGA was controlled by the acid value, protein concentration, and adsorption technique. The protein released in 2 phases: the first occurred over 3 days regardless of PLGA used and emanated from unbound, incorporated protein, while the second was controlled by mass loss and therefore was dependent on the polymer molecular weight. Overall, control of rhBMP-2 delivery is achievable by selection of PLGA microsphere carriers. Published: October, 7, 2001.     

Website for avian flu information and bioinformatics

Highly pathogenic influenza A virus H5N1 has spread out worldwide and raised the public concerns. This increased the output of influenza virus sequence data as well as the research publication and other reports. In order to fight against H5N1 avian flu in a comprehensive way, we designed and started to set up the Website for Avian Flu Information () from 2004. Other than the influenza virus database available, the website is aiming to integrate diversified information for both researchers and the public. From 2004 to 2009, we collected information from all aspects, i.e. reports of outbreaks, scientific publications and editorials, policies for prevention, medicines and vaccines, clinic and diagnosis. Except for publications, all information is in Chinese. Till April 15, 2009, the cumulative news entries had been over 2000 and research papers were approaching 5000. By using the curated data from Influenza Virus Resource, we have set up an influenza virus sequence database and a bioinformatic platform, providing the basic functions for the sequence analysis of influenza virus. We will focus on the collection of experimental data and results as well as the integration of the data from the geological information system and avian influenza epidemiology. Contributed equally to this work Special Project of Informatization of Chinese Academy of Sciences in “the Eleventh Five-Year Plan”, E-Science Application of Research on Resources, Disease Monitoring and Risk Assessment of Important Wild Birds in Qinghai Lake Region (Grant No. INFO-115-D02), China International Science and Technology Cooperation of the Ministry of Science and Technology, China (MOST) (Grant Nos. 2006DFB32010 and 2007DFC30240), MOST Grant 2005CB523001 (Program 973), National Key Technologies Research & Development Program (Grant 2006BAD06A01), and a grant from NIH (Grant No. 3 U19 AI051915-05S1), the China Post-doctor fellowship (Grant No. 20070420542).     

Expression pattern of <Emphasis Type="Italic">serine protease inhibitor kazal type 3 (Spink3)</Emphasis> during mouse embryonic development

Recent evidence shows that the serine protease inhibitor Kazal type 3 (Spink3) has more diverse functions than expected. To gain insight into its function, we analyzed the spatiotemporal expression profile of Spink3, using in situ hybridization (ISH) and a Spink3 ( +/lacZ ) knock-in mouse, in which lacZ was inserted into the Spink3 locus. Spink3 ( lacZ ) expression was first observed in the foregut, midgut, hindgut and the forebrain/midbrain junction region at 9.5 days post coitus (dpc). In the pancreas, Spink3 mRNA was detected at 11.5 dpc, before formation of the typical shape of the exocrine structure of the pancreas. Acinar cell expression was clearly identified by 13.5 dpc. After differentiation of the intestinal tract, Spink3 ( lacZ ) expression was observed in the large intestine at 11.5 dpc, followed by expression in the small intestine at 13.5 dpc, before appearance of intestinal digestive enzymes. Spink3 mRNA and Spink3 ( lacZ ) activity were also detected in other tissues, including the mesonephric tubules and the urogenital ridge at 11.5 dpc, the genital swelling at 13.5 dpc, the ductus epididymis at 17.5 dpc, and the seminal vesicle at 8 weeks. These data suggest that Spink3 may play important roles in proliferation and/or differentiation of various cell types during development.     

Regeneration of grape (Vitis labruscana cv. Kyoho) by shoot-tip culture

We investigated the optimal levels of growth regulators, culture media, and pH on callus growth and organogenesis of in-vitro cultured ‘Kyoho’ grapes. Calli were induced by culturing leaf blades on an MS basal medium supplemented with 1 mg/IL BA and 0.01 mg/L 2,4-D. In addition, calli originating from the exocarp and mesocarp of grape fruits devel-oped on MS media supplemented with 0.1 mg/L IAA, NAA, or 2,4-D, or with 0.2 mg/L BA. In testing the potential for plant regeneration from shoot tips on various media, we found that the Nitsch medium, with I mg/L BA, was optimal for caulogenesis. The type of shoot development depended on the pH of the medium, with vigorous multiple-shoot devel-opment occurring at pH 6.0, and single shoots forming at pH 5.0. Finally, we were able to obtain rooted seedlings from the regenerated shoots that had been cultured on 1/4-strength Nitsch medium supplemented with 0.03 mg/L NAA.     

Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines

Pancreatic β-cell apoptosis is known to participate in the β-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB).     

T cell integrin overexpression as a model of murine autoimmunity

Integrin adhesion molecules have important adhesion and signaling functions. They also play a central role in the pathogenesis of many autoimmune diseases. Over the past few years we have described a T cell adoptive transfer model to investigate the role of T cell integrin adhesion molecules in the development of autoimmunity. This report summarizes the methods we used in establishing this murine model. By treating murine CD4+ T cells with DNA hypomethylating agents and by transfection we were able to test thein vitro effects of integrin overexpression on T cell autoreactive proliferation, cytotoxicity, adhesion and trafficking. Furthermore, we showed that the ability to inducein vivo autoimmunity may be unique to the integrin lymphocyte function associated antigen-1 (LFA-1). Published: October 24, 2003     

Response

The offer of the fast food company gives rise to suspicion. This seems to be based on unfounded stereotypes, however. This paper argues that we need to preserve choices in taking particular courses of action. There is nothing inherently wrong in fast food consumption so long as consumers are made aware of the importance of weight management and proper nutrition.     

A provisional model to predict blood pressure response to biofeedback-assisted relaxation

Blood pressure (BP) response to biofeedback-assisted relaxation is not uniform among hypertensive individuals. The purpose of this exploratory study was to determine if selected psychophysiological variables could be used to identify individuals able to lower blood pressure using biofeedback-assisted relaxation. Responders were defined using a preset criterion of 5 mm Hg or greater decrease in mean arterial pressure. A logistic regression model derived from five variables (heart rate, finger temperature, forehead muscle tension, plasma renin response to furosemide, and mean arterial pressure response to furosemide) provided significant predictive power for BP response, exhibiting a sensitivity of 84.6% and a specificity of 80.0%. With future validation, the proposed model may provide useful information to identify patients likely to benefit from biofeedback-assisted relaxation.     

Ecomorphology of the giant bear-dogs Amphicyon and Ischyrocyon

Giant bear-dogs of the genera Amphicyon and Ischyrocyon (Carnivora, Amphicyonidae, Amphicyoninae) were the largest carnivorans in North America during middle and late Miocene (17.5–8.8 Mya) with a dental and skeletal morphology that combined features found in living Ursidae, Canidae, and Felidae. This study tests previously proposed models of diet and hunting behaviour of these extinct carnivorans. Relative grinding area (RGA) of lower molars and wear pattern on upper molars suggest that bear-dogs were carnivorous. Amphicyon and Ischyrocyon possessed skeletal features of both ambush (short distal limb segments) and pursuit (caudally bent olecranon process of ulna) living predators. Therefore, bear-dogs probably pursued their prey (mediportal ungulates) for a longer distance but at a slower speed than do living ambush predators. Upon catching up to its prey a bear-dog probably seized it with powerfully muscled forelimbs and killed it by tearing into its ribcage or neck with canines set in a narrow rostrum.     

Organotypic cocultures as skin equivalents: A complex and sophisticated <Emphasis Type="Italic">in vitro</Emphasis> system

To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and thus the precise analysis of growth regulatory mechanisms. Published: April 12, 2004     

Polyamine metabolism in hants : Arginine and omithine decarboxylase activity in ripeningTrlticum durum seeds

The pattern of the activity of arginine decarboxylase (ADC) and omithine decarboxylase (ODC) involved in polyamine synthesis in ripening wheat seeds was examined. The aim was to study the polyamines and the activity of the two enzymes in correlation with the growth processes occurring in the developing wheat seeds. The results obtained showed a very different pattern of polyamine content in the two organs of caryopsis, and that the two enzymes in the embryos have a higher activity than in the endosperms. Moreover, while in the embryos the ADC exhibits higher activity than the ODC, in the endosperms the activity of ODC is about similar to that of ADC. This pattern is discussed in relation to the different histological characteristics of embryo and endosperm tissues during seed development.     

Molecular characterization of a cDNA for a cysteine-rich antifungal protein from<Emphasis Type="Italic">Capsicum annuum</Emphasis>

We have isolated a cDNA clone for the antifungal protein,CaAFP, from hot pepper,Capsicum annuum L. Its open reading frame encodes 85 amino acids, including 8 cysteine residues. CaAFP consists of three domains: a signal peptide, a chitin-binding domain, and a C-terminal peptide domain. The deduced amino acid sequence of the chitin-binding domain shows 92% and 85% similarity to the same domain from PnAMPs and hevein, respectively. Southern blot analysis indicated that CaAFP is present as a single copy, while the northern blots revealed that the clone is highly expressed in the leaves and flower buds, but not in the roots. However, wounding treatments and chemicals generally known to induce PR proteins did not stimulate its expression.In situ hybridization also showed that CaAFP is expressed in the parenchyma cells of the floral sepals. As seen in our functional analysis, this clone was expressed inEscherichia coli, and the fusion protein was purified using nickel-affinity column chromatography. This purified AFP fusion protein inhibited spore germination and appressoria formation in several plant pathogenic fungi, includingFusarium oxisporum andColletotrichum gloeosporioides. Our results suggest CaAFP is an antifungal protein that defends developing seeds against pathogen invasion while also having a specific biological role during floral development.     

Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts

Live rabbits have previously been generated through nuclear transfer using adult cells as nuclear donors. We demonstrated in this study that transfected adult rabbit fibroblasts are also capable of supporting full-term development. The fibroblasts were transfected with a pEGFP-C1 plasmid using lipofectamine^™ 2000, and the transgenic cells were derived from conditioned medium. The transgenic fibroblasts were cultured until confluent and then serum-starved prior to be used as nuclear donors. After nuclear transfer and activation, 22% (12/55) of the transgenic cloned embryos developed to the blastocyst stage. A total of 114 embryos at the 4- to 8-cell stage were transferred to the oviducts of 8 pseudo-pregnant mothers; 5 of these animals became pregnant, and 3 of the 5 mother rabbits carried the pregnancy to term. Caesarean section was performed on the 3 pregnant mothers, yielding 4 kits, one of which has survived for more than 9 months. Green fluorescence could be detected in the toenails of the living cloned rabbit and the offspring from the living cloned rabbit under ultraviolet light. DNA analyses confirmed that all 4 cloned rabbits were genetically identical to the transgenic donor cells, and that they all carried the EGFP gene. The present study demonstrated that transgenic rabbits can be generated through nuclear transfer. These results may facilitate future developments in the genetic engineering of rabbits.