Mechanism and Aquaculture Application of Teleost Enzymes Adapted at Low Temperature.

Environment temperature highly influences the physiological condition of poikilothermic teleost. There are different physiological and biochemical responses between fish in different habitats. In order to take profit of fish adapted to different temperatures, some important enzymes have been isolated, assayed, and analyzed. Enzyme expression patterns and properties were evaluated in lactate dehydrogenase, creatine kinase, and stearoyl-CoA desaturase studies. In this chapter, we try to identify the mechanisms of enzyme activity at low temperature by comparing different studies on enzyme kinetics and regulation. The protein-protein interaction between monomers, protein-solvent interaction, and protein substrate correlation are discussed. Studying fish enzymes could accumulate the understanding of marine organism's enzyme function during adaptation in different temperature zones. Based on these mechanisms, the application of cold-adapted enzymes in aquaculture system is illustrated. Furthermore, this information may create a possible explanation of fish physiological and biochemical evolution route and construct an appropriate strategy to overcome the climate change.

Genetic polymorphisms of Chinese patients with ischemic stroke and concurrent stenoses of extracranial and intracranial vessels.

The etiology of concurrent stenoses of extracranial and intracranial vessels in patients with ischemic stroke is poorly understood, but hereditary factors are believed to be important. We aimed to determine whether genetic polymorphisms affecting homocysteine and lipid metabolism are associated with concurrent stenoses. The genotypes of 191 Han Chinese patients with acute ischemic stroke, of whom 47 (25%) had concurrent stenoses, and 167 healthy control patients in Hong Kong were examined for the following polymorphisms: paraoxonase 1 (PON1) Q192R, methylenetetrahydrofolate reductase (MTHFR) A222V, glutamate-cysteine ligase catalytic-subunit (GCLC)-129C>T, and oxidized low-density lipoprotein receptor (OLR) 3' untranslated region C>T (rs1050283). The genotype distributions of PON1 Q192R and MTHFR A222V, which affect lipid and homocysteine metabolism, differed significantly between patients with stroke and healthy controls. The presence of at least one R allele in PON1 Q192R and a TT allele in OLR rs1050283 were associated with concurrent stenoses. We also identified a possible association between the presence of at least one V allele in MTHFR A222V and concurrent stenoses. This study shows that genetic polymorphisms affecting homocysteine and lipid metabolism are possible risk factors for stroke and concurrent stenoses.

Long-term outcomes of ischemic stroke patients with concurrent intracranial and extracranial stenoses and ischemic heart disease.

BACKGROUND AND PURPOSE: Coexisting ischemic heart disease (IHD) and concurrent atherosclerosis of intracranial and extracranial vessels is common in Asians. This study aims to investigate the long-term outcomes of ischemic stroke patients with concurrent stenoses and IHD. METHODS: This was a prospective cohort study in Hong Kong. Consecutive Chinese patients with acute ischemic stroke underwent MRI, magnetic resonance angiography and carotid duplex sonography. RESULTS: A total of 428 patients were included. The mean follow-up period was 65 months (up to 87 months). Ninety-three patients (22%) died of any cause and 104 patients (22%) suffered from nonfatal vascular events. Fifty-four patients (13%) had IHD. Among them, 27 patients (50%) had concurrent stenoses. In patients with concurrent stenoses and IHD, only 3 (11%) were free of death and recurrent vascular events. Eight (30%) had recurrent nonfatal stroke, 7 (26%) had nonfatal myocardial infarct (MI) and 11 (41%) died, 6 (22%) of them due to fatal MI. The overall 5-year cumulative rates of mortality, recurrent vascular events and combined poor outcomes were 21, 23 and 43%, respectively. In patients with concurrent stenoses and IHD, these rates were 40, 50 and 83%, respectively. More deaths (log rank: 6.56; p = 0.01), recurrent vascular events (log rank: 25.24; p < 0.001) and poor outcomes (log rank: 27.50; p < 0.001) were found among patients with concurrent stenoses and IHD. CONCLUSIONS: Ischemic stroke patients with concurrent stenoses and IHD had high risks of death and recurrent vascular events. Future studies on aggressive medical therapy and early cardiac interventions in this high-risk group of stroke patients are warranted.

Use of magnetic resonance angiography to predict long-term outcomes of ischemic stroke patients with concurrent stenoses in Hong Kong.

PURPOSE: To determine the long-term outcome of ischemic stroke patients with concurrent intracranial and extracranial atherosclerosis using magnetic resonance angiography. METHODS: A prospective cohort of patients in Hong Kong with acute ischemic stroke was studied with magnetic resonance angiography of the brain and carotid duplex. All patients were followed up regularly for the development of recurrent stroke, cardiac events, or death. RESULTS: Totally 343 patients with acute ischemic stroke were included, of whom 104 (30%) had concurrent intracranial and extracranial lesions. The follow-up period was up to 76 months (mean 44.5 months). Overall, 55 patients (15.5%) died of any cause and 91 patients (26.5%) suffered a further nonfatal vascular event. The overall 5-year cumulative rates of mortality, restroke and poor outcomes (combined death and further vascular events) were 18, 27 and 37%, respectively. In patients with concurrent lesions, these rates were 31, 41 and 51%, respectively. The corresponding rates were 13, 22 and 31% in patients without concurrent lesions. The risks were highest in the first year after stroke. More deaths (log rank, 16.3; p = 0.0001), restrokes (log rank, 9.71; p = 0.002) and poor outcomes (log rank, 13.87; p = 0.0001) were found among patients with concurrent lesions. The presence of concurrent vascular lesions, advanced age, smoking, hyperlipidemia and previous history of stroke were independent predictors of poor outcomes. CONCLUSIONS: The long-term prognosis of ischemic stroke patients with concurrent atherosclerosis of intracranial and extracranial vessels is poor. They are at high risk of further vascular events or death.

A double casting technique to minimize distortion when constructing fixed partial dentures on implants.

The casting of extensive implant superstructures that have a good passive fit may be technically demanding. A simple, 2-stage casting technique is presented that avoids common problems associated with the casting of large superstructures. The fabrication of a 7-unit metal-ceramic screw-retained fixed partial denture supported by 5 implants is used to illustrate this technique.

Development of DNA delivery system using Pseudomonas exotoxin A and a DNA binding region of human DNA topoisomerase I.

Gene therapy is defined as the delivery of a functional gene for expression in somatic tissues with the intent to cure a disease. Thus, highly efficient gene transfer is essential for gene therapy. Receptor-mediated gene delivery can offer high efficiency in gene transfer, but several technical difficulties need to be solved. In this study, we first examined the DNA binding regions of the human DNA topoisomerase I (Topo I), using agarose gel mobility shift assay, in order to identify sites of noncovalent binding of human DNA Topo I to plasmid DNA. We identified four DNA binding regions in human DNA Topo I. They resided in aa 51-200, 271-375, 422-596, and 651-696 of the human DNA Topo I. We then used one of the four regions as a DNA binding protein fragment in the construction of a DNA delivery vehicle. Based on the known functional property of each Pseudomonas exotoxin A (PE) domain and human DNA Topo I, we fused the receptor binding and membrane translocation domains of PE with a highly positively charged DNA binding region of the N-terminal 198 amino acid residues of human DNA Topo I. The resulting recombinant protein was examined for DNA binding in vitro and transfer efficiency in cultured cells. The results show that this DNA delivery protein is a general DNA delivery vehicle without DNA sequence, topology, and cell-type specificity. The DNA delivery protein could be used to target genes of interest into cells for genetic and biochemical studies. Therefore, this technique can potentially be applied to cancer gene therapy.

Recombinant protein composed of Pseudomonas exotoxin A, outer membrane proteins I and F as vaccine against P. aeruginosa infection.

We have constructed a chimeric protein composed of the receptor binding and membrane translocation domains of Pseudomonas exotoxin A (PE) with the outer membrane proteins I and F, together designated as PEIF. The potential of PEIF as a vaccine against Pseudomonas infection was evaluated in BALB/c mice and New Zealand white rabbits. We examined titers of anti-PE and anti-OprF antibodies, and the ability both to neutralize PE cytotoxicity and to increase opsonophagocytic uptake of Pseudomonas aeruginosa strain PAO1, serogroups 2 and 6. The results showed that PEIF can induce antibodies not only to neutralize the PE cytotoxicity but also to promote the uptake of various strains of P. aeruginosa by murine peritoneal macrophages. In a burned mouse model, PEIF afforded significant protection against infection by the homologous P. aeruginosa strain PAO1, heterologous serogroup 2, and the PE hyperproducing strain PA103. These observations thus indicate that PEIF may be used as a novel vaccine against P. aeruginosa infection.

A nontoxic Pseudomonas exotoxin A induces active immunity and passive protective antibody against Pseudomonas exotoxin A intoxication.

Pseudomonas exotoxin A (PE) is one of the most potent cytotoxic agents produced by Pseudomonas aeruginosa. In this study, we examined the possibility of using PE with a deletion of 38 carboxyl-terminal amino acid residues, designated PE(Delta576-613), for active immunization against PE-mediated disease. We first examined the toxic effects of PE and PE(Delta576-613) on 5- and 9-week-old ICR mice. The results show that the subcutaneous administration of PE(Delta576-613) at a dose of 250 microg was still nontoxic to 5- and 9-week-old ICR mice, while native PE was lethal at a dose of 0.5 and 1 microg, respectively. PE(Delta576-613) was then used to immunize ICR mice. The minimum dose of PE(Delta576-613) that could effectively induce anti-PE antibodies in 5- and 9-week-old ICR mice was found to be 250 ng. However, immunization with 250 ng PE(Delta576-613) failed to protect the immunized mice from a lethal dose of PE. The effective immunization dose of PE(Delta576-613) that could protect mice against a 2 microg PE challenge was found to be 15 microg. In addition, sera obtained from PE(Delta576-613)-immunized ICR mice were able to neutralize PE intoxication and effectively protect mice from PE. Thus, PE(Delta576-613) may be used as an alternative route to new PE vaccine development.

Cloning, characterization, and expression in Escherichia coli of three creatine kinase muscle isoenzyme cDNAs from carp (Cyprinus carpio) striated muscle.

In vertebrates, the creatine kinase isoenzyme family consists of four types of isoforms: cytosolic muscle type (M-CK), cytosolic brain type (B-CK), mitochondrial ubiquitous, acidic type (Miu-CK), and mitochondrial sarcomeric, basic type (Mis-CK). Until recently, the existence of more than one subisoform of CK isoenzyme has been demonstrated only in fishes by starch gel electrophoresis. We report herein the isolation of three full-length cDNAs that correspond to three closely related creatine kinase M-CK genes from common carp (Cyprinus carpio), designated the M1-CK, M2-CK, and M3-CK genes. Using oligonucleotide probes that correspond to the same region but with the most variable sequences, different restricted genomic hybridization patterns have been obtained. These Southern blot results indicate that the three cDNAs come from different genes. Northern blot analysis using probes that correspond to the 3'-untranslated regions further show that all three subisoforms are expressed specifically in carp muscle. The deduced amino acid sequences of these three subisoforms of carp M-CK show about 85% identity to mammalian M-CK isoenzyme. Finally, the three cDNAs have been expressed in Escherichia coli with a molecular mass of approximately 43,000 Da, and these recombinant proteins exhibit creatine kinase activity. All of these data suggest that the M-CK isoenzymes have at least three subisoforms in carp.

Involvement of serine proteinase in infectious pancreatic necrosis virus capsid protein maturation and NS proteinase cleavage in CHSE-214 cells.

An investigation of virus-specific protein maturation in infectious pancreatic necrosis virus (IPNV) infected Chinook salmon embryo cells (CHSE-214) was undertaken. The precursor protein (pVP2-1) of the major mature capsid protein (VP2) was processed sequentially from pVP2-1 to pVP2-2 and VP2. Experiments using serine proteinase inhibitors showed that the maturation of the VP2 was blocked in the pVP2-1 post-translational cleavage steps. A protinin, a potent proteinase inhibitor, at 800 µg ml(-1) blocked pVP2-2 to VP2 and the cleavage of VP4 (28 kDa) to VP4-1 (25 kDa). Therefore, our data showed that the maturation of the capsid protein (VP2) and cleavage of VP4 (NS proteinase) can be blocked by serine proteinase inhibitors.

Variation in mitochondrial DNA and population structure of the Taipei treefrog Rhacophorus taipeianus in Taiwan.

Mitochondrial DNA (mtDNA) from 40 samples of the Taipei treefrog Rhacophorus taipeianus collected from seven populations in Taiwan were sequenced to document the DNA sequence variation in anuran mtDNA and to elucidate the phylogeographic population structure in the Taipei treefrog. Sequences of 722-764 bases in length, including a 108-bp segment of the cytochrome b gene and a 614-656-bp D-loop segment, were obtained by direct sequencing using polymerase chain reaction (PCR). The variation in length was due to a 40-bp region that tandemly repeated four to five times in the D-loop region. The first repeat is the most conserved one among the five repeats because there are no variable sites in this repeat. Besides the 40-bp length variation, 28 positions in the 764-bp sequences are variable and distributed evenly in the cytochrome b gene fragment and D-loop region. Variation in the D-loop of the Taipei treefrog is comparable to those of other vertebrates. Two well-differentiated lineages (northern and central) differing by mean sequence divergence of 1.7% are identified and concordant with their geographic distributions. The two lineages are inferred to have split from a common ancestral population in the early Pleistocene. However, the interpopulation divergence of the northern lineage (< 0.33%) is apparently lower than that of the central lineage (1.11%), implying that the two lineages evolved independently and had different demographic histories after divergence. This study reveals that anuran D-loop has potential as a genetic marker in phylogenetic and population genetic analyses of anurans.

Population structure of the Japanese eel, Anguilla japonica.

Mitochondrial DNA (mtDNA) sequences that include (a) a part of the cytochrome b gene, (b) two tRNA genes, and (c) a part of the noncoding D-loop region of 31 Anguilla japonica (Japanese eel) and 1 A. marmorata collected from Taiwan, Japan, and mainland China were determined to evaluate the population structure of Japanese eel. Among 30 genotypes identified from the 31 Japanese eel mtDNAs sequenced, there are 58 variable sites, predominantly clustered at the D-loop region. The phylogenetic tree constructed by the unweighted pair-group method with arithmetic mean shows neither significant genealogical branches nor geographic clusters. Furthermore, the sequence-statistics test reveals little, if any, significant genetic differentiation. These results indicate that the 31 Japanese eels might come from a single population. Analysis of sequence variation in mtDNA by using the relationship between the number of segregating sites and the average number of nucleotide differences under the neutral mutation hypothesis reveals that neutral mutation acts as a major factor influencing the evolutionary divergence of the Japanese eel mitochondrial genome sequenced, especially in the noncoding region.

Analysis of the Drosophila gene for the laminin B1 chain.

We have isolated and sequenced a Drosophila genomic DNA that encodes the entire coding region of the laminin B1 chain. The genomic DNA sequenced spans 11,787 bp, including a 1.1-kb 5'-flanking region, 5 exons, 4 introns, and a 1.4-kb 3'-flanking region. The open reading frame is within the two largest exons, the exons 3 and 4, while the first two and the last exons are much smaller and are untranslated. The structure of the Drosophila laminin B1 gene is similar to the Drosophila laminin B2 gene. Their exon-intron lengths and Eco RI, Pst I restriction maps are quite conserved. Both of their open reading frames are very compact, and their first introns are much larger than all of the rest of the introns. These results are consistent with the suggestion that the B1 and B2 genes could be derived from an ancestral gene. The similarity of the proximal 5'-flanking regions of the Drosophila B1 and B2 genes is 46.6%. Also, similar sequences of transcriptional regulatory elements, even though not site conserved, are found in both proximal 5'-flanking regions of the B1 and B2 genes. When transfected into Drosophila SL-2 cells, pCAT plasmid containing 1,048 bp of 5'-flanking region shows a strong expression of chloramphenicol acetyltransferase (CAT) activity. The deletion clones that contain sequences between nucleotides -462 to +150, and -282 to +150 all show strong CAT activity. These results suggest that this 5'-flanking promoter region may contain DNA sequences that can promote the expression of the laminin B1 gene.

The complete nucleotide sequence of the Crossostoma lacustre mitochondrial genome: conservation and variations among vertebrates.

The complete mitochondrial (mt) genome of Crossostoma lacustre, a freshwater loach from mountain stream of Taiwan, has been cloned and sequenced. This fish mt genome, consisting of 16558 base-pairs, encodes genes for 13 proteins, two rRNAs, and 22 tRNAs, in addition to a regulatory sequence for replication and transcription (D-loop), is similar to those of the other vertebrates in both the order and orientation of these genes. The protein-coding and ribosomal RNA genes are highly homologous both in size and composition, to their counterparts in mammals, birds, amphibians, and invertebrates, and using essentially the same set of codons, including both the initiation and termination signals, and the tRNAs. Differences do exist, however, in the lengths and sequences of the D-loop regions, and in space between genes, which account for the variations in total lengths of the genomes. Our observations provide evidence for the first time for the conservation of genetic information in the fish mitochondrial genome, especially among the vertebrates.

Structure of the Drosophila gene for the laminin B2 chain.

We isolated and sequenced a Drosophila genomic DNA sequence that encodes the entire coding region of the laminin B2 chain. The 11,464-bp genomic sequence contains a 2.1-kb of 5'-flanking DNA, ten exons, nine introns, and the 3'-flanking region. The first exon encodes a 5' untranslated region; the ATG translational start codon is in exon 2. The entire translated region is within a 8.3-kb Eco RI fragment. The Drosophila laminin B2 gene differs substantially in size and exon pattern from those of the human B1 and B2 genes. However, as in the case of the human B1 gene, the overall exon pattern of the Drosophila B2 gene does not correlate well with the highly conserved structural domains and internal repeats of the B2 polypeptide chain. Unlike the human and mouse B1 and B2 genes, the 2.1-kb 5'-flanking region of the Drosophila B2 gene contains a TATA box and two CAAT boxes. Other potential transcriptional regulatory sequences include two reverse complementary cAMP response element sequences; two sequences that are homologous to the retinoic acid response element motifs of the mouse B1 gene; and sequences homologous to the binding sites for transcription factors dFRA and dJRA, zeste, and possibly GAGA. When transfected into Drosophila SL-2 cells, pCAT plasmid containing 2,090 bp of 5'-flanking region shows a 3.0- to 3.5-fold increase in chloramphenicol acetyltransferase activity after induction with retinoic acid and/or 8-bromo-cAMP. These results suggest that this 5'-flanking promoter region may contain DNA sequences that can regulate the expression of the laminin B2 gene.

Primary structure of the Drosophila laminin B2 chain and comparison with human, mouse, and Drosophila laminin B1 and B2 chains.

Laminin, a major component of basement membranes, is a large glycoprotein consisting of three disulfide-bonded subunits, A, B1, and B2. We have isolated and sequenced a Drosophila laminin B2 chain cDNA clone that spans 5737 nucleotides. The deduced amino acid sequence predicts that the mature and nonglycosylated polypeptide has a chain length of 1606 residues (Mr = 178,665). This B2 chain contains 100 half-cystine residues, most of which are located in two cysteine-rich domains, and 11 N-X-S or N-X-T sequences which are potential sites of N-linked glycosylation. The predicted secondary structure reveals the presence of six structurally distinct domains, of which two are mainly alpha-helical, two are cysteine-rich with homologous repeats, and two are globular regions. The Drosophila B2 chain is 40.3 and 41.1% identical to the human and mouse B2 chains, respectively, and 29.6, 30.0, and 29.4% identical to the Drosophila, human, and mouse B1 chains, respectively.

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 4. Influence of DNA topology.

Pulsed-field gel electrophoresis is a powerful technique for the fractionation of linear DNA molecules with sizes above 50 kilobase pairs (kb). Here it is demonstrated that this technique is also effective for separating smaller DNAs including linear, circular, and supercoiled species. The mobilities of linear DNAs larger than 8 kb can be modulated by pulse times between 0.1 and 100 s. The mobility of supercoiled DNA molecules up to 16 kb is generally unaffected by these pulse times except that 10-s pulse times cause a small but distinct increase in the mobility. The general insensitivity of small supercoiled DNAs to pulse time presumably occurs because these species reorient so rapidly that they spend most of their time undergoing conventional electrophoresis. However, the mobilities of larger supercoiled DNAs are affected by pulse times of less than 1 s, and at 0.1 s the molecules are better resolved by pulsed electrophoresis than by ordinary electrophoresis. The mobility of 3-19 kb nicked and relaxed circular DNA molecules is also affected by pulse time but in a complex way.