Cloning and functional expression of α-galactosidase cDNA from Penicillium janczewskii zaleski

Biologia ◽  
2011 ◽  
Vol 66 (2) ◽  
Author(s):  
Bo Zhang ◽  
Yiqun Chen ◽  
Zhimin Li ◽  
Wenqing Lu ◽  
Yunhe Cao
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Functional Expression ◽  
Recombinant Enzyme ◽  
Methanol Induction ◽  
Potential Applications ◽  
Prospective Application ◽  
Polymerase Chain ◽  
Penicillium Janczewskii ◽  
Rapid Amplification ◽  
Different Levels

AbstractIn recent years, α-galactosidase has been attracting more and more attention because of its potential applications in many aspects. Using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, a full-length cDNA sequence composed of 2,439 bp was cloned from Penicillium janczewskii zaleski and was subcloned into pPICZαA and transformed into Pichia pastoris strain X-33. In a 10-L fermentor, the recombinant yeast expressed α-galactosidase with a yield of 254 U/mL by methanol induction for 120 h. The recombinant enzyme showed the optimal activity at 40°C and pH 5.2. The K m values of the recombinant enzyme using p-nitrophenyl-α-D-galactopyranoside (pNPG), melibiose, raffinose and stachyose as substrates were 1, 16, 17.8 and 5.3 mM, respectively. V max values were 227.3, 116.7, 104.8, and 80.6 μM/min using pNPG, melibiose, raffinose and stachyose as substrates, respectively. The α-galactosidase exhibited no sensitivity to various metal ions and ethylenediaminetetraacetic acid, and hydrolyzed melibiose, raffinose and stachyose with different levels of galactose release. The biochemical characteristics of the α-galactosidase suggest that the enzyme may have a prospective application in feed industry as an additive.

scholarly journals Cloning, sequencing and expression of rat liver pyruvate carboxylase

1996 ◽  
Vol 316 (2) ◽  
pp. 631-637 ◽  
Author(s):  
Sarawut JITRAPAKDEE ◽  
Grant W. BOOKER ◽  
A. Ian CASSADY ◽  
John C. WALLACE
Keyword(s):  
Rat Liver ◽  
Pyruvate Carboxylase ◽  
Untranslated Region ◽  
Northern Analysis ◽  
Reading Frame ◽  
Liver Cdna Library ◽  
Lactating Mammary Gland ◽  
Polymerase Chain ◽  
Rapid Amplification ◽  
Sequencing And Expression

Overlapping clones encoding rat liver pyruvate carboxylase (PC) have been isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on total liver RNA. The sequence of rat PC cDNA contains an open reading frame of 3537 nucleotides encoding a polypeptide of 1178 amino acids with a calculated Mr of 129848. This is flanked by a 5′ untranslated region of 66 bp and a 3′ untranslated region of 421 bp including the poly(A) tail. The inferred protein sequence is 96.6% identical with mouse and 96.3% identical with human PCs, 68.4% identical with mosquito PC and 53.5% identical with yeast PC isoenzymes PC1 and PC2. On the basis of partial proteolysis and sequence homology with PC from other organisms (yeast, mosquito, mouse and human) and with other biotin enzymes, three functional domains, namely the biotin carboxylation domain, the transcarboxylation domain and the biotinyl domain, have been identified. Comparison with the known structure of the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase [Waldrop, Rayment and Holden (1994) Biochemistry 33, 10249–10256] highlights the functional importance of 11 highly conserved residues. Northern analysis revealed that PC mRNA is highly expressed in rat liver, kidney, adipose tissue and brain, moderately expressed in heart, adrenal gland and lactating mammary gland, and expressed at a low level in spleen and skeletal muscle.

scholarly journals Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris

2003 ◽  
Vol 376 (3) ◽  
pp. 781-787 ◽  
Author(s):  
Isabel SOARES-SILVA ◽  
Dorit SCHULLER ◽  
Raquel P. ANDRADE ◽  
Fátima BALTAZAR ◽  
Fernanda CÁSSIO ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Lactic Acid ◽  
Pichia Pastoris ◽  
Plasma Membranes ◽  
Membrane Vesicle ◽  
Dry Weight ◽  
Functional Expression ◽  
Acid Uptake ◽  
Methanol Induction

In Saccharomyces cerevisiae the activity for the lactate–proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a Vmax of 2.1 nmol·s−1·mg of dry weight−1. Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest Vmax (0.84 nmol·s−1·mg of dry weight−1) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels.

scholarly journals Genetic Diversity and Aggressiveness of Ralstonia solanacearum Strains Causing Bacterial Wilt of Potato in Uruguay

Plant Disease ◽  
2011 ◽  
Vol 95 (10) ◽  
pp. 1292-1301 ◽  
Author(s):  
M. I. Siri ◽  
A. Sanabria ◽  
M. J. Pianzzola
Keyword(s):  
Genetic Diversity ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Potato Tubers ◽  
Bacterial Strains ◽  
Potato Plants ◽  
Polymerase Chain ◽  
First Time ◽  
Different Levels

Bacterial wilt, caused by Ralstonia solanacearum, is a major disease affecting potato (Solanum tuberosum) production worldwide. Although local reports suggest that the disease is widespread in Uruguay, characterization of prevalent R. solanacearum strains in that country has not been done. In all, 28 strains of R. solanacearum isolated from major potato-growing areas in Uruguay were evaluated, including 26 strains isolated from potato tubers and 2 from soil samples. All strains belonged to phylotype IIB, sequevar 1 (race 3, biovar 2). Genetic diversity of strains was assessed by repetitive-sequence polymerase chain reaction, which showed that the Uruguayan strains constituted a homogeneous group. In contrast, inoculation of the strains on tomato and potato plants showed, for the first time, different levels of aggressiveness among R. solanacearum strains belonging to phylotype IIB, sequevar 1. Aggressiveness assays were also performed on accessions of S. commersonii, a wild species native to Uruguay that is a source of resistance for potato breeding. No significant interactions were found between bacterial strains and potato and S. commersonii genotypes, and differences in aggressiveness among R. solanacearum strains were consistent with previously identified groups based on tomato and potato inoculations. Moreover, variation in responses to R. solanacearum was observed among the S. commersonii accessions tested.

scholarly journals Cloning and characterization of novel human SLC4A8 gene products encoding Na+-driven Cl−/HCO3− exchanger variants NDCBE-A, -C, and -D

2008 ◽  
Vol 34 (3) ◽  
pp. 265-276 ◽  
Author(s):  
Mark D. Parker ◽  
Patrice Bouyer ◽  
Christopher M. Daly ◽  
Walter F. Boron
Keyword(s):  
In Silico Analysis ◽  
Functional Expression ◽  
Inhibitory Effect ◽  
Functional Difference ◽  
The Novel ◽  
Equivalent Position ◽  
Rapid Amplification ◽  
Novel Exon ◽  
Silico Analysis

The reported sequences of the human and mouse Na+-driven Cl−/HCO3− exchangers (NDCBEs) differ greatly in their extreme cytosolic COOH termini (Ct). In human NDCBE (NDCBE-B), a 17-amino acid (aa) sequence replaces 66 aa at the equivalent position in mouse NDCBE (NDCBE-A). We performed 5′- and 3′-rapid amplification of cDNA ends (RACE) on human brain cDNA, followed by PCR of full-length cDNAs to determine whether the human SLC4A8 gene was capable of producing the mouselike Ct sequence. Our study confirmed the presence in human cDNA of mouse NDCBE-like transcripts (human NDCBE-A) and also disclosed the existence of three further novel NDCBE transcripts that we have called NDCBE-C, NDCBE-D, and NDCBE-D′. The novel NDCBE-C/D/D′ transcripts initiate at a novel “exon 0” positioned ∼35 kb upstream of the first exon of NDCBE-A/B. NDCBE-C/D/D′ protein products are predicted to be truncated by 54 aa in the cytosolic NH2 terminus (Nt) compared with NDCBE-A/B. Our data, combined with a new in silico analysis of partial transcripts reported by others in the region of the human SLC4A8 gene, increase the known extent of the SLC4A8 gene by 49 kb, to 124 kb. A functional comparison of NDCBE-A/B/C/D expressed in Xenopus oocytes demonstrates that the Nt variation does not affect the basal functional expression of NDCBE, but those with the shorter Ct have a 25–50% reduced functional expression compared with those with the longer Ct. By comparison with an artificially truncated NDCBE that contains neither 17-aa nor 66-aa Ct cassette, we determined that the functional difference is unrelated to the 66-aa cassette of NDCBE-A/C, but is instead due to an inhibitory effect of the 17-aa cassette of NDCBE-B/D.

Isolation and Characterization of Mesenchymal Stem Cells from Chicken Liver

2020 ◽  
Vol 10 (1) ◽  
pp. 8-16
Author(s):  
Xibin Liu ◽  
Shuang Zhang ◽  
Weijun Guan ◽  
Dong Zheng
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Protein Markers ◽  
Multilineage Differentiation ◽  
Rt Pcr ◽  
Multipotent Stem Cells ◽  
Isolation And Characterization ◽  
Potential Applications ◽  
Polymerase Chain

Hepatic mesenchymal stem cells (HMSCs) are multipotent stem cells that is a vital part of the regeneration of hepatocytes after injury. In this study, HMSCs were isolated in embryonic livers from of 12-day-old chick embryo using collagenase, and the primary HMSCs were sub-cultured to passage. The protein markers of HMSCs, namely CD71, CD29 and CD44, were tested with immunofluorescence and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The proliferation of HMSCs in different passages was detected using growth curve, which shown a typically sigmoidal. And then, the pluripotent of HMSCs was analyzed, the results showed that HMSCs could directly induce to differentiate into neural-like cells, adipocytes, and osteoblasts. Our data illustrated that the chick HMSCs have same characteristics to those obtained from other species. The capacity of these cells for multilineage differentiation shows promise for many potential applications.

scholarly journals Prevalence of select vector-borne disease agents in owned dogs of Ghana

2014 ◽  
Vol 85 (1) ◽  
Author(s):  
Lorelei L. Clarke ◽  
Lora R. Ballweber ◽  
Kelly Allen ◽  
Susan E. Little ◽  
Michael R. Lappin
Keyword(s):  
Dirofilaria Immitis ◽  
Ethylenediaminetetraacetic Acid ◽  
Bartonella Henselae ◽  
Conventional Polymerase Chain Reaction ◽  
Ehrlichia Canis ◽  
Hepatozoon Canis ◽  
Veterinary Clinic ◽  
Vector Borne ◽  
Polymerase Chain ◽  
Edta Blood

Ticks, sera and ethylenediaminetetraacetic acid (EDTA) blood were collected from dogs evaluated at the Amakom Veterinary Clinic in Kumasi, Ghana. Sera were evaluated for Dirofilaria immitis antigen and antibodies against Borrelia burgdorferi, Anaplasma phagocytophilum and Ehrlichia canis. Conventional polymerase chain reaction assays designed to amplify the deoxyribonucleic acid (DNA) ofEhrlichia spp. or Anaplasma spp. or Neorickettsia spp. or Wolbachia spp., Babesia spp., Rickettsia spp., Hepatozoon spp., Bartonella spp. and the haemoplasmas were performed on DNA extracted from EDTA blood and all positive amplicons were sequenced. This small survey shows that the following vector-borne pathogens are present in urban Ghanian dogs: Ehrlichia canis, Hepatozoon canis,Dirofilaria immitis and Anaplasma platys. Bartonella henselae was isolated from ticks but not from the dogs.

scholarly journals OGG1 Involvement in High Glucose-Mediated Enhancement of Bupivacaine-Induced Oxidative DNA Damage in SH-SY5Y Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Zhong-Jie Liu ◽  
Wei Zhao ◽  
Qing-Guo Zhang ◽  
Le Li ◽  
Lu-Ying Lai ◽  
...  
Keyword(s):  
Dna Damage ◽  
High Glucose ◽  
Oxidative Dna Damage ◽  
Functional Expression ◽  
Culture Conditions ◽  
Control Culture ◽  
Ros Production ◽  
Dna Oxidative Damage ◽  
Repair Enzymes ◽  
Polymerase Chain

Hyperglycemia can inhibit expression of the 8-oxoG-DNA glycosylase (OGG1) which is one of the key repair enzymes for DNA oxidative damage. The effect of hyperglycemia on OGG1 expression in response to local anesthetics-induced DNA damage is unknown. This study was designed to determine whether high glucose inhibits OGG1 expression and aggravates bupivacaine-induced DNA damage via reactive oxygen species (ROS). SH-SY5Y cells were cultured with or without 50 mM glucose for 8 days before they were treated with 1.5 mM bupivacaine for 24 h. OGG1 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. ROS was estimated using the redox-sensitive fluorescent dye DCFH-DA. DNA damage was investigated with immunostaining for 8-oxodG and comet assays. OGG1 expression was inhibited in cells exposed to high glucose with concomitant increase in ROS production and more severe DNA damage as compared to control culture conditions, and these changes were further exacerbated by bupivacaine. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) prevented high glucose and bupivacaine mediated increase in ROS production and restored functional expression of OGG1, which lead to attenuated high glucose-mediated exacerbation of bupivacaine neurotoxicity. Our findings indicate that subjects with diabetes may experience more detrimental effects following bupivacaine use.

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