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A novel ATP-dependent nuclear DNA unwinding enzyme from pea has been purified to apparent homogeneity and characterized. This enzyme is present at extremely low abundance and has the highest specific activity among plant helicases. It is a heterodimer of 54 and 66 kDa polypeptides as determined by SDS/PAGE. On gel filtration chromatography and glycerol gradient centrifugation it gives a native molecular mass of 120 kDa and is named as pea DNA helicase 120 (PDH120). The enzyme can unwind 17-bp partial duplex substrates with equal efficiency whether or not they contain a fork. It translocates unidirectionally along the bound strand in the 3′→5′ direction. The enzyme also exhibits intrinsic single-stranded DNA- and Mg2+ -dependent ATPase activity. ATP is the most favoured cofactor but other NTPs and dNTPs can also support the helicase activity with lower efficiency (ATP > GTP = dCTP > UTP > dTTP > CTP > dATP > dGTP) for which divalent cation (Mg2+ > Mn2+ ) is required. The DNA intercalating agents actinomycin C1 , ethidium bromide, daunorubicin and nogalamycin inhibit the DNA unwinding activity of PDH120 with K i values of 5.6, 5.2, 4.0 and 0.71 µMs, respectively. This inhibition might be due to the intercalation of the inhibitors into duplex DNA, which results in the formation of DNA–inhibitor complexes that impede the translocation of PDH120. Isolation of this new DNA helicase should make an important contribution to our better understanding of DNA transaction in plants. [ABSTRACT FROM AUTHOR]

A full-length cDNA of 1951 bp encoding a calnexin (CNX) protein was cloned from a Pisum sativum expression library. The open reading frame (ORF) within this cDNA encodes a 551-amino acid protein with a calculated molecular mass of 62.47 kDa that exhibits extensive homology with the CNX proteins from soybean (80%), Arabidopsis thaliana (70%), maize (70%), and dog (39%). The characteristic CNX signature motifs, KPEDWDE and GXW, generally found in molecular chaperones, are present in pea CNX (PsCNX), along with putative sites for Ca2[sup +] binding and phosphorylation. In PsCNX, a signal sequence and a single transmembrane domain are also present at the N- and C-terminal ends, respectively. The PsCNX protein is expressed constitutively at the RNA level in vegetative and flowering tissues, as was evident from Northern analysis. Expression of PsCNX was light independent. In vitro translation of PsCNX cDNA yielded a 75-kDa precursor, which, in the presence of canine microsomal membranes, was cotranslationally processed into a 72.5-kDa product and was imported and localized to the endoplasmic reticulum. Trypsin treatment of the in vitro translated PsCNX in the presence of canine microsomes generated a further processed 67-kDa intraluminal form. The results with PsCNX also showed that the plant protein is a phosphoprotein containing phosphoserine residues, as evidenced by immunoprecipitation of PsCNX with anti-phosphoserine antibody. The PsCNX protein was also phosphorylated by endogenous kinases of pea microsomes. [ABSTRACT FROM AUTHOR]

An ATP-dependent DNA helicase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA ligase or nuclease activities. The enzyme requires Mg2+ or Mn2+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine. [ABSTRACT FROM AUTHOR]

In this study, the expression of four reference genes (β-actin, EF1-α, GAPDH, and SubF0), two prophenoloxidase genes (proPO1 and proPO2), and the distribution of phenoloxidase (PO) activity in eight tissues (carapace, cuticle, hemolymph, hepatopancreas, muscle, pereiopods, pleopods, and uropods) of black tiger shrimp (Penaeus monodon) were evaluated. According to the geNorm and NormFinder algorithms, the most stable reference gene or the best combinations of reference genes for the evaluation of proPO1 and proPO2 expression are SubF0 for cuticle, muscle, and uropods, EF1-α for hemolymph and pereiopods, EF1-α + SubF0 for carapace, β-actin + SubF0 for hepatopancreas, and β-actin + GAPDH for pleopods. In the hemolymph and uropods, proPO1 expression was significantly stronger than that of proPO2 (433 and 5 times higher, respectively). However, in hepatopancreas, proPO2 expression was significantly stronger than proPO1 (36 times higher). Trypsin cleaves the proPO zymogen into active PO in most of the studied tissues, except hepatopancreas. PO activity (units/mg protein) following trypsin activation varied among the tissues in the following order: uropods > hemolymph > pleopods > pereiopods > hepatopancreas > carapace > cuticle > muscle. The results indicate that proPO1 is dominant in hemolymph, whereas proPO2 is dominant in hepatopancreas. Altogether, these results contribute to the elucidation of the phenoloxidase function in P. monodon. [ABSTRACT FROM AUTHOR]

The expression stability of four potential reference genes (EF1‐α, β‐actin, GAPDH and SubF0) was evaluated to normalize the expression of six immune‐related genes (proPO1, proPO2, MnSOD, PAP, Rab7 and NOS) in different tissues of black tiger shrimp (Penaeus monodon) following melanin injection and using the geNorm and NormFinder algorithms. The stability rankings of the reference genes were as follows: EF1‐α > β‐actin > SubF0 > GAPDH in the muscle, GAPDH > EF1‐α > SubF0 > β‐actin in the carapace and EF1‐α > GAPDH > β‐actin > SubF0 in the haemolymph. To normalize the expression of six immune‐related genes of P. monodon in different tissues under the effect of melanin injection, EF1‐α and GAPDH alone were the most suitable reference genes for the muscle and carapace, respectively, whereas the combination of EF1‐α + β‐actin was the most suitable for the haemolymph. The expression of these six immune‐related genes in P. monodon was upregulated in the haemolymph, downregulated in the carapace and nearly unchanged in the muscle of the melanin‐injected group compared to that in the control group. Our results provide reference genes for reverse transcription quantitative polymerase chain reaction to analyse the protective effects of melanin in black tiger shrimp. [ABSTRACT FROM AUTHOR]

This study aimed to characterize and determine the distribution of proteases in various anatomical parts of Penaeus monodon using mainly electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels impregnated with casein (casein-SDS-PAGE) and specific protease inhibitors. Twelve caseinolytic bands designated A-L, with estimated molecular masses of 11.4-49.6 kDa were detected by SDS-PAGE, and were most abundant in the hepatopancreas. Among the 12 bands, E, J, K and L were predominant, A, G and H were identified as chymotrypsin-like proteases, D, E, I, J, K and L were identified as trypsin-like proteases, and B, C and F were identified as serine proteases. The proteases were most active at 50°C and pH 7.0. The proteolytic and trypsin-like activities of Penaeus monodon were most abundant in hepatopancreas extracts and least abundant in extracts from the haemolymph. Trypsin-like proteases in the hepatopancreas extract were found to activate prophenoloxidase (proPO) to form phenoloxidase (PO). Résumé: Cette étude vise à caractériser et à déterminer la distribution des protéases dans diverses parties anatomiques de Penaeus monodon , en utilisant principalement la technique de l'électrophorèse en gel de polyacrylamide contenant du dodécylsulfate de sodium (SDS) imprégné de caséine (casein SDS-PAGE) et des inhibiteurs spécifiques de protéase. La technique de l'électrophorèse SDS-PAGE révèle l'existence de douze bandes d'activité caséinolytique, désignées A-L et de poids moléculaire variant de 11,4 à 49,6 KDa. Ces bandes sont les plus abondantes dans l'hépatopancréas. Sur les douze bandes révélées, quatre ont été trouvées prédominantes à savoir E, J, K et L. Les bandes A, G et H ont été identifiées comme des protéases de type chymotrypsine, les bandes D, E, I, J, K et L comme des protéases de type trypsine et les bandes B, C et F des comme des protéases à sérine. La meilleure activité protéasique a été observée à 50°C et à pH 7,0. Les activités protéolytiques et celles de type trypsine de Penaeus monodon ont été trouvées plus abondantes dans l'hépatopancréas et moins abondantes dans l'hémolymphe. La formation de la phénoloxydase (PO), dans l'hépatopancréas, est due à l'activation de la prophénoloxydase (proPO) par des protéases de type trypsine. [ABSTRACT FROM AUTHOR]

Plants respond to the adverse environment by activating a series of stress-inducible genes, including genes encoding transcription factors. The expression of these genes is regulated by a core DNA sequence contained in their promoter region called cis-acting element. The promoter region of several stress-responsive genes contains several stress-regulated cis-acting elements such as dehydration-responsive element, Crepeat, low-temperature-responsive element, NAC recognition sequence and ZFHD recognition sequence. In this study we isolated a cDNA for a transcription factor named nuclear LIM interactor-interacting factor from rice cDNA library by yeast one-hybrid screening using two target sequences of 50 nucleotides derived from two stress-inducible promoters, JRC0528 and JRC0332, of cold-inducible genes OsZF1 and OsNAC6 respectively, as baits. The NLI-IF protein showed both DNA-binding and transcriptional activities in yeast experiments. Expression of OsNLI-IF was found to be induced by cold, heat, salt and drought stresses. The OsNLI-IF gene overexpressing transgenic tobacco plants showed improvement in drought tolerance. The present study emphasizes that OsNLI-IF could be useful for development of drought-tolerant transgenic crop plants. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) is the causative pathogen of coronavirus disease-19 (COVID-19). The COVID-19 pandemic has resulted in millions of deaths and widespread socio-economic damage worldwide. Therefore, numerous studies have been conducted to identify effective measures to control the spreading of the virus. Among various potential targets, the 3 chymotrypsin-like protease (3CLpro), also known as Mpro, stands out as the key protease of SARS-CoV-2, playing an essential role in virus replication and assembly, is the most prospective. In this study, we modified the commercial vector, pETM33- Nsp5-Mpro (plasmid # 156475, Addgene, USA), by inserting an autocleavage site (AVLQ) of 3CLpro and 6 × His-tag encoding sequences before and after the Nsp5-Mpro sequence, respectively. This modification enabled the expression of 3CLpro as an authentic N terminal protease (au3CLpro), which was purified to electrophoretic homogeneity by a single-step chromatography using two tandem Glutathione- and Ni-Sepharose columns. The enzyme au3CLpro demonstrated significantly higher activity (3169 RFU/min/μg protein) and catalytic efficiency (K cat /K m of 0.007 μM−1.s-1) than that of the 3CLpro (com3CLpro) expressed from the commercial vector (pETM33- Nsp5-Mpro) with specific activity 889 RFU/min/μg and K cat /K m of 0.0015 μM−1.s-1, respectively. Optimal conditions for au3CLpro activity included a 50 mM Tris-HCl buffer at pH 7, containing 150 mM NaCl and 0.1 mg/ml BSA at 37 °C. [ABSTRACT FROM AUTHOR]

Bacillus aquimaris SH6 spores produce carotenoids that are beneficial to white-leg shrimp (Litopenaeus vannamei) health. However, the optimal dose and mechanisms behind these effects are not well understood. We investigated the fate of SH6 spores in the gut of L. vannamei. Shrimp were divided into six groups administrated with either feed only (negative control) or SH6 spores at 5 × 106 CFU/g pellet (high dose, SH6 spore-H group), 1 × 106 CFU/g pellet (medium dose, SH6 spore-M group), 2 × 105 CFU/g pellet (low dose, SH6 spore-L group), astaxanthin at 0.5 mg/g pellet (Carophyll group), or carotenoids from SH6 vegetative cells at 5 μg/g pellet (SH6 carotenoid group). The growth rate was highest in SH6 spore-H (3.38%/day), followed by SH6 spore-M (2.84%/day) and SH6 spore-L (2.25%/day), which was significantly higher than the control (1.45%/day), Carophyll (1.53%/day) or SH6 carotenoid (1.57%/day) groups. The astaxanthin levels (1.9–2.0 μg/g shrimp) and red-colour scores (21–22) in SH6 spore-H/M were higher than the control (astaxanthin: 1.2 μg/g shrimp; red score: 20) or SH6 spore-L, but lower than the Carophyll and SH6 carotenoids. Feeding with medium and high doses of SH6 spores after 28 days resulted in respective 1.3-2-fold increases in phenol oxidase activity and 8–9 fold increases in Rho mRNA expression compared to the control and low dose group. The live-counts of SH6 in the gut gradually increased during the 28-day feeding period with SH6 spores at different concentrations, starting from 4.1, 8.2, and 5.4 × 104 CFU/g gut at day 1 and reaching 5.3, 5.1, and 4.4 × 105 CFU/g gut in the SH6-H/M/L groups, respectively, at day 28. Gut microbiota became more diversified, resulting in a 2-8-fold increase in total bacterial live-counts compared to the controls. SH6 spore germination was detected by measuring the mRNA expression of a specific sequence coding for SH6 amylase at 4 h, reaching saturation at 24 h. Our results confirm that SH6 spores colonize and germinate in the gut to improve the microbial diversity and boost the immune system of shrimp, exhibiting beneficial effects at >1 × 106 CFU/g pellet. [ABSTRACT FROM AUTHOR]

Leigh syndrome is a rare inherited, heterogeneous and progressive neurometabolic disorder that is mainly caused by specific mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). The present study reported a case of childhood Leigh syndrome with a point mutation at bp 8,993 in the mitochondrial ATPase6 gene. A 21‑month‑old male child had developed epilepsy, muscular weakness and vomiting, which was accompanied by high fever. Magnetic resonance imaging indicated typical characteristics of Leigh syndrome, including a symmetric abnormal signal in the dorsal medulla oblongata and Sylvian fissure enlargement in association with an abnormal signal in the periventricular white matter and in the putamina and caudate heads. The diagnosis was further supported with genetic tests including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), sequencing, and quantitative PCR. The patient was found to carry a mitochondrial T8993C (m.T8993C) mutation in peripheral blood with 94.00±1.34% heteroplasmy. Eight of his relatives were also subjected to quantification of the m.T8993C mutation. The percentages of heteroplasmy in samples taken from the grandmother, mother, aunt, cousin 1, and cousin 2 were 16.33±1.67, 66.81±0.85, 71.66±3.22, 87.00±1.79, and 91.24±2.50%, respectively. The mutation was not found in samples taken from the father, the husband of the aunt, or the grandfather of the patient. The obtained data showed that the mutation was maternally inherited and accumulated through generations. Even though the heteroplasmy levels of his mother, aunt, cousin 1, and cousin 2 were relatively high (66.81‑91.24%), they remained asymptomatic, indicating that the threshold at which this mutation shows effects is high. To the best of our knowledge, this is the first report of a case of Leigh syndrome in a Vietnamese individual harboring a mtDNA mutation at the 8,993 bp site, and showing a correlation between the heteroplasmy and clinical phenotype. These findings may be useful in helping to improve the clinical diagnosis and treatment of Leigh syndrome. [ABSTRACT FROM AUTHOR]

Vietnamese patients (106) tentatively diagnosed with encephalomyopathy were screened for the presence of 15 common point mutations in mitochondria using PCR-RFLP. The screened mutations include A3243G, T3271C and T3291C for Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like episodes (MELAS); A8344G and T8356C for Myoclonus Epilepsy and Rag-Red Fibers (MERRF); G11778A, G3460A and T14484C for Leber's Hereditary Optic Neuropathy (LHON); T8993G/C and T9176G for Leigh syndrome; A1555G for deafness syndrome; G4298A, T10010C, T14728C and T14709C for neuromuscular syndrome. As a result, 6 cases of A3243G (5.7%) and 2 cases of T14727C (3.9%) were found. The 6 cases of A3243G mutation were heteroplasmic at different levels (4.23–80.85%). The T14727C change was discovered for the first time in the MTTE gene encoding for tRNAGluand showed homoplasmy. The T14727C change was probably a mutation because it was further confirmed as vertically inherited from the mother and not the result of isolated polymorphism. [ABSTRACT FROM AUTHOR]

Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS–PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min −1 mg −1 at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. [ABSTRACT FROM AUTHOR]

The A3243G mitochondrial mutation is the major cause of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). The severity of the disease is correlated with the heteroplasmy level of the mutation. Here we describe for the first time the validation of a real-time polymerase chain reaction (PCR) assay with Taqman locked nucleic acid (LNA) fluorescent (FAM for mutant, HEX for wild type) probes for quantification of heteroplasmy levels in a total of 18 family members from 5 Vietnamese MELAS patients carrying A3243G. Almost no background of FAM signals was detected in normal samples, indicating that the probes were allele-specific. Standard curves indicate sensitive detection at 0.1% mutants and high reliability with R > 0.985. The correlation line between measured % mutant and expected % mutant was highly reliable, with a slope of 0.993 and R of 0.998. All positive A3243G mutant samples pre-screened by PCR-restriction fragment length polymorphism (RFLP) were confirmed, and their heteroplasmy levels quantified to be from 3.68 to 80.85%. The heteroplasmy levels in patients were higher than in their family members and generally correlated well with the severity of their clinical symptoms. Overall, this work is the first demonstration of the application of LNA probes for sensitive and highly reliable quantification of heteroplasmy levels in human mitochondria. [ABSTRACT FROM AUTHOR]

The envelope protein VP28 of white spot syndrome virus ( WSSV) is considered a candidate antigen for use in a potential vaccine to this important shrimp pathogen (the cause of white spot syndrome, WSS). Here, we used spores of Bacillus subtilis to display VP28 on the spore surface. Trials were conducted to evaluate their ability to protect shrimps against WSSV infection. The gene cotB-vp28 was integrated into the chromosome of the laboratory strain B. subtilis PY79, and expression of Cot B- VP28 was detected by Western blotting and immunofluorescence. Expression of Cot B- VP28 was equivalent to 1000 molecules per spore. PY79 and Cot B- VP28 spores were mixed with pellets for feeding of whiteleg shrimps ( Litopenaeus vannamei), followed by WSSV challenge. Superoxidase dismutase ( SOD), phenoloxidase activities and mortality rates of the two shrimp groups were evaluated. Groups fed with PY79 and Cot B- VP28 spores at day 7 had increased SOD activities of 29% and increased phenoloxidase activities of 15% and 33%, respectively, compared to those of the control group. Fourteen days postchallenge, 35% of vaccinated shrimps had died compared to 49% of those fed naked spores ( PY79) and 66% untreated, unchallenged animals. These data suggest that spores expressing VP28 have potential as a prophylactic treatment of WSS. [ABSTRACT FROM AUTHOR]

Carriers of drugs in cancer therapy are required to reduce side-effects of the drugs to normal cells. Here we constructed killed recombinant Bacillus subtilis spores (SA1) that expressed streptavidin as a chimeric fusion to the spore coat protein CotB and used the spores as bioparticle carrier. When bound with biotinylated cetuximab these spores could specifically target to the epidermal growth factor receptor on HT 29 colon cancer cells, thereby delivered paclitaxel to the cells with 4-fold higher efficiency, as indicated by fluorescent intensity of paclitaxel Oregon Green 488 bound to HT29 cells. Based on real-time monitoring of cell index, the IC50 of growth of HT29 cells by paclitaxel-SA1-cetuximab was estimated to be 2.9 nM approximately 5-fold lower than water-soluble paclitaxel (14.5 nM). Instability of DNA content was observed when cells were treated with 16 nM paclitaxel-SA1-cetuximab, resulting in a 2-fold enhancement in polyploidy cells. Thus, by targeting the release of paclitaxel to HT29 cells, spore-associated cetuximab augmented the inhibitory effect of paclitaxel on cell division and proliferation. The SA1 could be used as a 'universal' drug carrier to target specific biomarkers on cancer cells by conjugating with suitable biotinylated antibodies. [ABSTRACT FROM AUTHOR]

Magnetic Fe3O4 nanoparticles were prepared by coprecipitation and then coated with silica. These Fe3O4/SiO2 nanoparticles consisted of a 10-15 nm magnetic core and a silica shell of 2-5 nm thickness. The superparamagnetic property of the Fe3O4/SiO2 particles with the magnetization of 42.5 emu/g was confirmed by vibrating sample magnetometer (VSM). We further optimized buffers with these Fe3O4/SiO2 nanoparticles to isolate genomic DNA of hepatitis virus type B (HBV) and of Epstein-Barr virus (EBV) for detection of the viruses based on polymerase chain reaction (PCR) amplification of a 434 bp fragment of S gene specific for HBV and 250 bp fragment of nuclear antigen encoding gene specific for EBV. The purification efficiency of DNA of both HBV and EBV using obtained Fe3O4/SiO2 nanoparticles was superior to that obtained with commercialized Fe3O4/SiO2 microparticles, as indicated by (i) brighter PCR-amplified bands for both HBV and EBV and (ii) higher sensitivity in PCR-based detection of EBV load (copies/mL). The time required for DNA isolation using Fe3O4/SiO2 nanoparticles was significantly reduced as the particles were attracted to magnets more quickly (15-20 s) than the commercialized microparticles (2-3 min). [ABSTRACT FROM AUTHOR]