• Journal of Veterinary Clinics
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• v.22 no.4
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• pp.386-391
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• 2005

This study was done to produce a mutated protein inactivated cytotoxicity of Shigatoxin 2e (Stx2e) of E.coli O139 isolates by deletional mutagenesis of Stx2e A subunit gene encoding active-site cleft of enzymatic domain in ST2e holotoxin. Cytotoxicity of the toxoid expressed from the mutant Stx2e gene was compared with wild type Stx2e for development of vaccine candidate. A recombinant plasmid pED18 containing Stx2e gene ot E.coli O139 isolates was used to generate mutation plasmid. Deletion mutagenesis was conducted for Stx2e A subunit gene encoding enzymatically active domain by polymerase chain reaction (PCR) using ot designed primer to induce deletional mutation. DNA sequence analysis was confirmed that the pentamer (Typ 202- Ser 206) that lies within the proposed active-site cleft in the second region was completely deleted. A DNA fragment of 1.1 kb that encode the new mutant Stx2eA gene was inserted into plasmid pRSET vector digested with EcoRV-Hind III and named pEDSET The PEDSET was transformed in E. coli for expression of mutant protein and the protein was confirmed by SDS-PACE and Western-blotting. The protein expressed by the mutant was tested to confirm the reduction of cytotoxic activities on Vero cell using microcytotoxicity assay compared with wild type Stx2e, the cytotoxicity of deletional mutant protein was at least reduced by 3,000-fold on Vero cell.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.10
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• pp.1475-1481
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• 2003

The aim of this paper was to use the Cornell Net Carbohydrate and Protein System (CNCPS), that reports diet energy and protein value and animal requirements, as net energy for lactation ($NE_1$) and metabolizable protein (MP) respectively, to evaluate some rations for lactating Italian Mediterranean buffaloes. The investigation was carried out on six farms in the province of Caserta (southern Italy), where the milk production was controlled four times monthly on 10 animals (changing every time) chosen at different lactation days (5 categories): <2 months (A), 2-4 months (B), 4-6 months (C), 6-8 months (D), >8 months (E). Milk fat and protein were determined. Diet $NE_1$ and MP were estimated with the CPM-Dairy program (1998) using diet component chemical characteristics; then energy and protein intakes were estimated. $NE_1$ and MP requirements were estimated with two methods: 1) using CPM-Dairy that considers produced milk, fat and protein content, lactation phase and body condition score as main factors; 2) by applying the theory that to produce 1 kg of energy corrected milk, the buffalo needs 3.56 MJ of $NE_1$ and the efficiency to convert the absorbed aminoacids into milk protein is lower than cow (CNCPS). As regards energy, with method 1 the requirements were satisfactory starting from category A (4 out of 6 farms) and category B (5/6 farms); however, a surplus resulted for category E (5/6 farms). With method 2 a deficit in category A (5/6 farms) and B (3/6 farms) was observed, while the energy requirements were satisfied for all categories except E, where on only one buffalo farm had a surplus of energy intake. As regards protein, with method 1 the requirements were substantially satisfied for all the categories except E (3/6 farms); with method 2 the MP trend was much less favourable than with method 1. Indeed, a protein deficit was observed for all animals in categories A and B (5/6 farms). Moreover, on one farm the protein intake never satisfied animal requirements. In our experimental conditions, the use of the CNCPS to characterise diets for lactating buffalo and to calculate their requirements led to satisfactory results. By contrast, we cannot say the same for method 2, which applies a lower use efficiency of NE and MP for lactation in buffalo compared to cow.

• BMB Reports
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• v.41 no.12
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• pp.852-857
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• 2008

Little attention has been paid to the specificity between E2 and the target protein during ubiquitination, although RING-E3 induces a potential intra-molecular reaction by mediating the direct transfer of ubiquitin from E2 to the target protein. We have constructed artificial E2 fusion proteins in which a target protein (p27) is tethered to one of six E2s via a flexible linker. Interestingly, only three E2s (UbcH5b, hHR6b, and Cdc34) are able to ubiquitinate p27 via an intra-molecular reaction in this system. Although the first ubiquitination of p27 (p27-Ub) by Cdc34 is less efficient than that of UbcH5b and hHR6b, the additional ubiquitin attachment to p27-Ub by Cdc34 is highly efficient. The E2 core of Cdc34 provides specificity to p27, and the residues 184-196 are required for possessive ubiquitination by Cdc34. We demonstrate direct E2 specificity for p27 and also show that differential ubiquitin linkages can be dependent on E2 alone.

• Journal of Korean Biological Nursing Science
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• v.13 no.1
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• pp.1-7
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• 2011

Purpose: The purpose of this study was to compare the hypertrophic effects of low-intensity exercise on weight, myofibrillar protein content and Type I, II fiber cross-sectional area of hindlimb muscles of rats between every other day exercise and every day exercise. Methods: Adult male Sprague-Dawley rats were assigned to 1 of 3 groups: control group (C, n=6), experimental group 1 (E1, n=7) and experimental group 2 (E2, n=7). Rats in E1 group had 7 sessions (every other day) and those in E2 group had 14 sessions (every day) of exercise in which they ran on a treadmill for 30 min/day at 10 m/min. Results: Muscle weight, cross-sectional area of type I fiber and myofibrillar protein content of soleus and myofibrillar protein content of plantaris in E1 group, and myofibrillar protein content of soleus and cross-sectional area of type I fiber of plantaris in E2 group were greater than those in C group. Cross-sectional area of type I fiber of soleus of E1 group was higher than E2 group while cross-sectional area of type I fiber of plantaris of E2 group was higher than E1 group. Conclusion: Hypertrophy of hindlimb muscles occurs from every other day exercise similar to every day exercise.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.4 no.2
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• pp.238-242
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• 2001

Food allergy is a disease caused by an abnormal immunological reaction to specific food proteins. Whole milk and soy beans are the most frequent causes of food allergy, some studies show that 2.2~2.8% of children aged between 1 and 2 year are allergic to milk. It can be classified to acute (urticaria, asthma, anaphylaxis) or chronic (diarrhea, atopic dermatitis) allergy according to clinical symptoms, or to IgE related or non IgE related allergy by an immunological aspect. Generally, allergies invading only the GI tract are mostly due to a non IgE related reaction. These hypersensitive, immunologic reactions of the GI tract, not related to specific IgE for food, present themselves in many ways such as food protein-induced enteropathy, food protein-induced enterocolitis syndrome (FPIES), celiac disease, food induced protocolitis, or allergic eosinophillic gastroenteritis. FPIES is one kind of non IgE related allergic reaction and is manifested as severe vomiting and diarrhea in infants between 1 week and 3 months. We report a case of FPIES in a 40-day old male infant presenting with 3 times of repeated events of watery diarrhea after cow's milk feeding.

• Microbiology and Biotechnology Letters
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• v.47 no.2
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• pp.278-288
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• 2019

In the present investigation, we attempted to design a protocol to develop a hybrid protein with better bioremediation capacity. Using in silico approaches, a Hybrid Open Reading Frame (Hybrid ORF) is developed targeting the genes of microorganisms known for degradation of organophosphates. Out of 21 genes identified through BLAST search, 8 structurally similar genes (opdA, opd, opaA, pte RO, pdeA, parC, mpd and phnE) involved in biodegradation were screened. Gene conservational analysis categorizes these organophosphates degrading 8 genes into 4 super families i.e., Metallo-dependent hydrolases, Lactamase B, MPP and TM_PBP2 superfamily. Hybrid protein structure was modeled using multi-template homology modeling (3S07_A; 99%, 1P9E_A; 98%, 2ZO9_B; 33%, 2DXL_A; 33%) by $Schr{\ddot{o}}dinger$ software suit version 10.4.018. Structural verification of protein models was done using Ramachandran plot, it was showing 96.0% residue in the favored region, which was verified using RAMPAGE. The phosphotriesterase protein was showing the highest structural similarity with hybrid protein having raw score 984. The 5 binding sites of hybrid protein were identified through binding site prediction. The docking study shows that hybrid protein potentially interacts with 10 different organophosphates. The study results indicate that the hybrid protein designed has the capability of degrading a wide range of organophosphate compounds.

• Journal of Life Science
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• v.23 no.7
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• pp.941-946
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• 2013

Local protein synthesis at subsynaptic sites plays a key role in the regulation of the protein composition in local domains. In this study, we carried out immunocytochemistry of cultured rat hippocampal neurons in various developmental stages to investigate the expression of eIF4E and its binding protein, eIF4EBP1. Both proteins were distributed in dendrites. In addition, eIF4EBP1 was highly expressed in the nucleus throughout the development, whereas eIF4E was not expressed in the nucleus. Punctate expression of eIF4E and eIF4EBP1 was evident in DIV 3. The colocalization rates of eIF4E or eIF4EBP1 puncta with PSD95 were higher in the dendrogenic than in the mature stages. In contrast, the colocalization rates of eIF4E and eIF4EBP1 puncta were higher in the mature than in the dendrogenic stages. As eIF4E is inactive when it is bound to eIF4EBP1, these data indicate that most dendritic eIF4E's are active during development but that they are mostly under inhibition in mature neurons.

• Development and Reproduction
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• v.22 no.1
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• pp.95-104
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• 2018

CREBZF (cAMP-response element binding protein zhangfei) is a member of ATF/CREB family, and which regulates various cellular functions by suppressing major factors with direct interaction. In this study, we have examined the expression of CREBZF on mouse endometrium during uterus estrous cycles and estrogen (E2) treatment. In uterus, CREBZF mRNA expression was higher than other organs and mRNA and protein of CREBZF was increased in proestrus phase and decreased in estrus phase. The expression of CREBZF in 3-weeks old mouse uterus was reduced by E2 injection in endometrium. In addition, the expression of progesterone receptor, a marker of E2 in ovariectomized mice was found to be strongly expressed in stroma, while CREBZF was only expressed in epithelium. Also, we conformed that E2-suppressed CREBZF was restored by co-injection of ICI 182,780, an estrogen receptor antagonist. Overall, these results suggest that CREBZF is regulated by estrogen and involved in ER signaling pathway in mouse uterus.

• BMB Reports
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• v.40 no.1
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• pp.107-112
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• 2007

To study the functioning of HSP70 in Escherichia coli, we selected NtHSP70-2 (AY372070) from among three genomic clones isolated in Nicotiana tabacum. Recombinant NtHSP70-2, containing a hexahistidine tag at the amino-terminus, was constructed, expressed in E. coli, and purified by $Ni^{2+}$ affinity chromatography and Q Sepharose Fast Flow anion exchange chromatography. The expressed fusion protein, $H_6NtHSP70$-2 (hexahistidine-tagged Nicotiana tabacum heat shock protein 70-2), maintained the stability of E. coli proteins up to 90$^{\circ}C$. Measuring the light scattering of luciferase (luc) revealed that NtHSP70-2 prevents the aggregation of luc without ATP during high-temperature stress. In a functional bioassay (1 h at 50$^{\circ}C$) for recombinant $H_6NtHSP70$-2, E. coli cells overexpressing $H_6NtHSP70$-2 survived about seven times longer than those lacking $H_6NtHSP70$-2. After 2 h at 50$^{\circ}C$, only the E. coli overexpressing $H_6NtHSP70$-2 survived under such conditions. Our NtHSP70-2 bioassays, as well as in vitro studies, strongly suggest that HSP70 confers thermo-tolerance to E. coli.

• Archives of Pharmacal Research
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• v.23 no.4
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• pp.267-282
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• 2000

Cytochrome P45O (CYP) 2E1 catalyzes the metabolism of a wide variety of therapeutic agents, procarcinogens, and low molecular weight solvents. CYP2E1-catalyzed metabolism may cause toxicity or DNA damage through the production of toxic metabolites, oxygen radicals, and lipid peroxidation. CYP2E1 also plays a role in the metabolism of endogenous compounds including fatty acids and ketone bodies. The regulation of CYP2E1 expression is complex, and involves transcriptional, post-transcriptional, translational, and post-translational mechanisms. CYP2E1 is transcriptionally activated in the first few hours after birth. Xenobiotic inducers elevate CYP2E1 protein levels through both increased translational efficiency and stabilization of the protein from degradation, which appears to occur primarily through ubiquitination and proteasomal degradation. CYP2E1 mRNA and protein levels are altered in response to pathophysiologic conditions by hormones including insulin, glucagon, growth hormone, and leptin, and growth factors including epidermal growth factor and hepatocyte growth factor, providing evidence that CYP2E1 expression is under tight homeostatic control.