植物硫转运蛋白研究进展

硫转运蛋白在植物对硫酸盐的吸收和转运中起着重要的作用。已经在拟南芥、大麦和小麦等植物中分离到了40多种硫转运蛋白基因。这些基因序列与其他种类生物的硫转运蛋白基因序列有着高度的保守性。利用CLUSTAL程序建立的系统进化树将植物硫转运蛋白划分为5个亚群。使用多种拓扑预测程序推测出不同植物硫转运蛋白的共同结构特点是均含有12个跨膜域。在柱花草和大麦中,硫转运蛋白基因表达调控包括植物体内硫水平的负调控和O—乙酰丝氨酸的正调控两种方式。对硫转运蛋白的组织定位和功能研究表明,高亲和硫转运蛋白主要定位于根部,在根系硫酸盐吸收中起重要作用。     

Divergence of tRNA genes in chloroplast DNA of higher plants

The saturation hybridization between spinach chloroplast (ct) DNA and spinach ¹²⁵I-labelled chloroplast tRNA has shown that about 1.1% of the spinach ctDNA codes for tRNAs. The observed hybridization is a result of specific base-pairing as shown by competition hybridization experiments and thermal stability of the ctDNA-tRNA hybrids. The amount of hybridization shows that spinach ctDNA contains about 40 tRNA genes. Similar hybridization studies have shown that corn ctDNA contains about 28 tRNA genes. The cross-hybridizations between ctDNA and tRNAs of corn, spinach and pea have shown that tRNAs in chloroplasts of higher plants have undergone significant divergence. The pea and spinach tRNAs have been found to have 50% of the base sequences in common. The corn tRNAs have been found to have only about 30% of the base sequences in common with pea and spinach. These data have been confirmed by extensive heterologous competition experiments and thermal stability of the heterologous DNA-tRNA hybrids. The experiments have also shown that the base sequences of tRNAs common in all three plants are the same.     

Genome evolution of allopolyploids: a process of cytological and genetic diploidization

Allopolyploidy is a prominent mode of speciation in higher plants. Due to the coexistence of closely related genomes, a successful allopolyploid must have the ability to invoke and maintain diploid-like behavior, both cytologically and genetically. Recent studies on natural and synthetic allopolyploids have raised many discrepancies. Most species have displayed non-Mendelian behavior in the allopolyploids, but others have not. Some species have demonstrated rapid genome changes following allopolyploid formation, while others have conserved progenitor genomes. Some have displayed directed, non-random genome changes, whereas others have shown random changes. Some of the genomic changes have appeared in the F1 hybrids, which have been attributed to the union of gametes from different progenitors, while other changes have occurred during or after genome doubling. Although these observations provide significant novel insights into the evolution of allopolyploids, the overall mechanisms of the event are still elusive. It appears that both genetic and epigenetic operations are involved in the diploidization process of allopolyploids. Overall, genetic and epigenetic variations are often associated with the activities of repetitive sequences and transposon elements. Specifically, genomic sequence elimination and chromosome rearrangement are probably the major forces guiding cytological diploidization. Gene non-functionalization, sub-functionalization, neo-functionalization, as well as other kinds of epigenetic modifications, are likely the leading factors promoting genetic diploidization.     

Synthesis and partial characterization of hydrogels obtained via glutaraldehyde crosslinking of acetylated chitosan and of hyaluronan derivatives

Hydrophilic networks based on functionalized hyaluronic acid and on partially acetylated chitosan, respectively, have been obtained. In the case of hyaluronic acid (HA), primary amino functionalities have been introduced along the polysaccharide chains. The ensuing derivatives, i.e., HA-lysine (HA-K), HA-diamino pentane (HA-DAP), and HA-glycine-lysine (HA-GK), have been characterized by high field NMR spectroscopy. NMR 2D-DOSY experiments have allowed us to optimize the purification procedure. Chitosan was made soluble in water by partial acetylation. Cross-linking reactions have been performed using glutaraldehyde. The obtained networks have been qualitatively characterized by means of (13)C CP-MAS NMR technique. The hydrogels have been characterized also in terms of water uptake.     

Biomass estimation of orange roughy: a summary and evaluation of techniques for measuring stock size of a deep-water fish species in New Zealand

A number of techniques have been employed to measure biomass of orange roughy in New Zealand. Time series of stratified random trawl surveys in a number of areas over periods of 5–10 years have given relative abundance indices, which have been used in stock reduction analyses to estimate true biomass. This has given confident results for the Chatham Rise stock, and surveys have also shown strong trends in other regions. Acoustic surveys have been carried out on three grounds. They can give relative indices of orange roughy abundance in areas of fiat or medium sloping bottom, and have the potential for estimation of absolute biomass from a single survey. Egg production surveys have been employed on two grounds on the east coast of the North Island, where conditions of tight spawning aggregations and steep bottom topography have limited the success of other methods. Both daily fecundity reduction and annual egg production methods have been used. These have given estimates of true biomass from one-off surveys, although results are imprecise. Both unstandardized and standardized analyses of commercial catch-per-unit-effort data have given relative indices of abundance, which have formed an important part of stock assessment for several fisheries. No single technique used to measure the size of orange roughy stocks has proven ideal or appropriate in all New Zealand situations. All have advantages and disadvantages, depending on the characteristics of the fishing area and fish behaviour. For two areas, a combination of methods have been applied, which has given more confident results than those from a single technique.     

Host defense mechanisms of cephalopods

Humoral and cellular mechanisms of defense have been described for cephalopods, a relatively advanced group of mollusks. Typical of other mollusks, cephalopod agglutinins are the most documented component of humoral immunity. Lectins, which have agglutinating properties, have been described and characterized from octopuses. Agglutinins from cephalopod hemolymph have also been shown to agglutinate a variety of vertebrate red blood cells, as well as potential bacterial pathogens. Hemocytes are the primary component of cellular immunity. Although the hemocyte role in phagocytosis has been extensively studied in other mollusks, the mechanisms of phagocytosis have not been described extensively for cephalopods. Cephalopod hemocytes have phagocytic capabilities and may function in encapsulation and neutralization of foreign substances; however, the effects of environmental factors and the full extent of phagocytic capabilities of cephalopod hemocytes have not been reported. Hemocytes from cephalopods have a role in wound healing and inflammation which have been reported in detail by several investigators.     

Production of tetanolysin preparations and characteristics of their properties

As the result of the study of tetanolysin-producing Clostridium tetani strains, their populations have been found to be markedly heterogeneous with respect to the hemolytic activity of clone cultures. On the basis of normal and dialyzed cultures of selected variants with maximum activity the preparations of tetanolysin have been obtained, and their hemolytic activity and antigenic properties have been studied. Antihemolytic rabbit sera have also been obtained and characterized. Partially purified preparations of tetanolysin with high hemolytic activity have been obtained by the fractionation of C. tetani dialyzed cultures with ammonium sulfate.     

Neurobiology of disease

Advances in technology and basic sciences this past decade have transformed neurobiological research. Practitioners looking prospectively in 1990 could hardly have hoped for the diagnostics and rational therapeutics that have become part of regular practice today. Here, we discuss three areas that have had great impact: genetics, cell death, and stem cell/gene therapy research.     

Cryopreservation of islets of Langerhans

Development of techniques for cryopreservation of pancreatic islets of Langerhans could potentially allow for increased freedom from the time restrictions presently affecting viability in islet cell transplantation. While several investigators have attempted islet cell freezing and have obtained favorable in vitro results after thawing, there have been few reported in vivo successes with islets transplanted after freezing. We have developed a simple system for freezing islet cell pancreatic fragments to ?196 °C and have either stored them in liquid nitrogen for 24 hr or immediately thawed the islets prior to transplantation. In addition, antilymphoblast globulin has been used as graft pretreatment modality in order to modify islet cell immunogenicity. We found that ALG was effective in prolongation of graft survival after freezing as well as on fresh nonfrozen transplants. The use of freezing and ALG appears, therefore, to have a favorable effect on the immunogenicity of the pancreatic islet cell allograft.     

Plant systems biology comes of age

'Omics' research approaches have produced copious data for living systems, which have necessitated the development of systems biology to integrate multidimensional biological information into networks and models. Applications of systems biology to plant science have been rapid, and have increased our knowledge about circadian rhythms, multigenic traits, stress responses and plant defenses, and have advanced the virtual plant project.     

The place of metabolism in the origin of life

Metabolism and replication are generally considered essential features of biological life. Workers in the field of the origin of life are mostly split into two groups, depending on which of these two functions is postulated to have occurred first. Because of difficulties encountered by the replication-first (or genetics-first) approach, some workers have postulated that a highly developed metabolism must have originated before replication and the formation of a genetic apparatus. However, as supporters of a replication-first approach have pointed out, and as is discussed in this article, the alternative metabolism-first approach has fundamental problems that have not been sufficiently addressed.     

The construction of cosmid libraries of eukaryotic DNA using the Homer series of vectors

We have constructed and characterised a series of approved, disabled cosmid vectors which we call Homer cosmids and have examined the optimal conditions for the construction of libraries of eukaryotic DNA segments using these vectors. Analysis of these libraries shows that most of the sequences we have tested for are present at the expected frequency and that the libraries can be stably propagated. We have also directly tested the stability of cosmid clones carrying tandemly repeated inserts. This work shows that it should be possible to clone most eukaryotic genes using cosmid vectors and that such cloning systems have considerable advantages over those more commonly used.     

Identification of rat neurophysins: Complete amino acid sequences of MSEL- and VLDV-neurophysins

Two rat neurophysins have been purified by salt precipitation, molecular sieving and ion-exchange chromatography. The proteins, performic-acid oxidized or reduced-alkylated, have been split either by trypsin or by staphylococcal proteinase and fragments have been separated by peptide mapping. Amino acid sequences of tryptic peptides have been determined either directly or after cleaving the large fragments by subtilisin, chymotrypsin, elastase or staphylococcal proteinase and characterizing the subfragments. Tryptic peptides have been ordered through the fragments given by staphylococcal proteinase. The N-terminal sequences of both proteins have also been established by automated degradation.The two usual types of mammalian neurophysins have been identified. One neurophysin belongs to the MSEL-neurophysin family and shows 11 substitutions and a 2-residue C-terminal truncation when compared with bovine MSEL-neurophysin. The other belongs to the VLDV-neurophysin family and shows 8 substitutions when compared with bovine VLDV-neurophysin. There are 23 differences between the MSEL- and VLDV-neurophysins of the rat.     

Pharmacology of azoles

Azole antifungals have different pharmacokinetic characteristics: complete oral absorption for Voriconazole, and to a lesser extent for fluconazole. The absorption of posaconazole and itraconazole increases with food intake. All of them have high tissue distribution with low plasma concentrations, especially low in the case of posaconazole and itraconazole. Posaconazole and itraconazole have high plasmatic protein binding and consequently both have a very low free fraction. Elimination of azole antifungals is through a metabolic pathway with CYP450 isoenzymes, and has a non linear pharmacokinetics with a high risk of interation with other drugs since azoles have the ability of CYP450 isoenzymes inhibition. Possibly the parameter that defines more precisely their efficacy is AUIC with an optimum value near 20, although cut-off values must be defined since some azoles may have difficulty to reach this value.     

In vitro study of the biology of small cell carcinoma of the lung

We have developed three types of experimental systems for the study of SCCL: (1) serially heterotransplanted tumors in athymic nude mice; (2) continuous, clonable cell cultures; and (3) direct clonogenic assays for tumor specimens. These systems have their own individual advantages, applications, and limitations, but these are interrelated and complementary. The study of these systems has greatly aided our understanding of the biology of SCCL, and its relationship to other lung cancers and the APUD cell system. In addition, new markers for SCCL have been identified, such as a creatine kinase and its BB isoenzyme (CK-BB). These cellular markers may have clinical applications, as serum levels of CK-BB are an indicator of tumor burden. Assays for clonogenic tumor cells may permit selection of optimal drug combinations for the treatment of individual tumors. Variant cultures having the morphology of SCCL, but lacking some or all of the other features, have been identified. While our systems have been used primarily for biological studies, they have clinical applications for both diagnostic and therapeutic purposes.     

Role of innervation in the control of bone remodeling

During the last fifteen years, an increasing number of studies have examined the origin, the ontogeny, and the distribution of nerve fibers in bone. They have also investigated the nature of neuromediators conveyed by these skeletal nerve fibers. Experimental models of sensory and sympathetic denervation and clinical studies have shown that these two neuronal systems are involved in bone development, growth and remodeling. More recently, some new concepts regarding the role of nerve fibers in bone physiology have emerged with the demonstration of a leptin-dependent central control of bone formation via the sympathetic system. This new neural regulating pathway of bone cell functions could have enormous implications for human skeletal biology and treatment of bone pathologies.     

Analysis of the aliphatic 1H-NMR spectrum of plasminogen kringle 4. A comparative study of human, porcine, bovine and chicken homologs

The aliphatic 1H-NMR spectrum of the kringle 4 domain of human plasminogen has been studied via two-dimensional chemical shift correlated (COSY) and nuclear Overhauser correlated (NOESY) experiments at 300 MHz and 620 MHz. A number of aliphatic proton spin systems have been identified and several definite assignments have been made. This was mainly achieved by comparison of the human kringle 4 spectrum with spectra of the porcine, bovine and chicken homologs and also with that of the kringle 1 from human plasminogen on which we have reported previously. The three valyl and two leucyl residues of human kringle 4 have been assigned. The eleven threonyl spin systems have been identified via a RELAYED-COSY experiment and Thr17 has been assigned. The three alanyl spin systems have been identified and assigned. Six seryl spin systems have been identified and the signals from the seven glycyl residues of human kringle 4 have been located with Gly45 assigned. Furthermore, 24 AMX spin systems have been mapped in the COSY spectrum of human kringle 4 and H alpha-H beta,beta' spin systems of Tyr2, Tyr41, Tyr50, Tyr74, Trp25 and Trp62 have been assigned. From the spectrum of a deglycosylated chicken homolog, the epsilon-methyl singlets of Met28 and Met48 have been assigned. Finally, ligand effects on selected aliphatic resonances were observed which could be analyzed in terms of residues likely to neighbor the kringle lysine-binding site.     

Proteomic analysis of mitochondria from the Bos taurus heart. III. Identification of mitochondrial inner memrane proteins

We have conducted a research of mitochindrial internal membrane proteins. This fraction has been received in the form of submitochondrial particles (SMP). SMP have been processed by trypsinum, and the received peptides have been separated from so-called "smoothfaced vesicles". "Smoothfaced vesicles" were blasted, proteinse and peptides were processed by cyanogen bromide and trypsinum. We have received two groups of tryptic peptides and analyzed them separately with the help of proteomic methods, such as chromatography, mass spectrometry and protein identification in different databases. To identify more proteins and find minor components of mitochindrial proteome we have considered possible non-specific fragmentation of proteins. 298 proteins have been identified, we also have conducted the analisys of their functions and cell localization.