In vitro Induction of Germinal Vesicle Breakdown in Xenopus laevis Oocytes by Melittin

The bee venom, melittin, is an amphipathic polypeptide comprising 26 amino acids with known sequence. It consists of a hydrophobic and a basic hydrophilic segment, possesses lipolytic activity, and stimulates Na⁺-K⁺ pump activity. At 1.5 μM melittin induces 98% germinal vesicle breakdown (GVBD) in stage VI (Dumont) oocytes and 96% in stage IV oocytes. Progesterone (30 μM) induced 100% GVBD in stage VI oocytes and none in stage IV oocytes. GVBD occurs earlier with melittin than with progesterone, i.e., 3 h compared to 5 h. An unusual morphologic change observed with melittin is the occurrence of mottling of the animal pole. The inner boundary of the melanin layer appears irregular with projections extending into the cytoplasm.
When stage VI oocytes were microinjected with 60 nl of 3 mM melittin only 48% showed GVBD indicating that the effectiveness of melittin was dependent upon the route of administration. On the other hand, 60% of stage VI oocytes underwent GVBD when microinjected with the cytosol fraction obtained from melittin-treated oocytes. Dissolution of isolated germinal vesicles did not occur when they were incubated in modified Earth's medium containing 3 mM melittin. The present results suggest that melittin induces GVBD by promoting the production of maturation promoting factor.     

Ontogenic Development of Corticotrophs in Fetal Buffalo (Bubalus Bubalis) Pituitary Gland

To evaluate the subpopulation of corticotrophs in developing buffalo (Bubalus bubalis) fetus, pituitary glands were recovered (n=6 per group) from late first, second and third gestational female buffalo dams. The corticotrophs were identified by using specific antibodies against proopiomelanocortin (POMC) and adrenocorticotrophic hormone (ACTH) through immunohistochemistry. There was a significant (P≤0.05) increase of immunoreactive (ir) ir-ACTH cells during late 2^nd trimester while, ir-POMC cells were more (P≤0.05) at late 3^rd trimester of gestation as compared to other age groups. The quantity of co-localized cells for POMC and ACTH was significantly (P≤0.05) greater at the end of 1^st gestation rather than 2^nd and 3^rd gestational fetal adenohypophyseal cells. This study is the first to demonstrate co-localization of POMC+ACTH and the affect of gestational age on the expression of these cells in buffalo fetus adenohypophysis.Key words: buffalo, corticotrophs, fetus, pituitary gland, immunohistochemistry     

水牛皮肤成纤维细胞的分离与体外培养

探讨水牛成纤维细胞的分离与传代培养方法。组织块培养法培养的成纤维细胞原代生长较慢,需12天左右方可汇合形成单层,而酶消化法培养的成纤维细胞原代生长相对生长快,仅需8天便可汇合形成单层。两种方法传代细胞的生长速度相似,仅需4-5天就可汇合形成单层。通过体细胞的核型分析发现,成纤维细胞在传代培养过程中的核型变化不大,66．67％～81．67％的细胞具有正常的二倍体核型,各代之间无显著差异。结果表明,水牛成纤维细胞均能稳定地进行传代培养。     

Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Molecular Cloning,Identification, and Expression Patterns of Myostatin Gene in Water Buffalo (Bubalus Bubalis)

Myostatin (MSTN), also named growth differentiation factor 8 (GDF8), is a transforming growth factor-β (TGF-β) family member with a key role in the negative regulation of skeletal muscle growth. However, its role in ovarian folliculogenesis remains unclear. To provide us with a basis for understanding this role, we cloned MSTN and examined its expression patterns in water buffalo (Bubalus bubalis). The complete ORF of the water buffalo MSTN gene is 1,128 nucleotides, which encode a 375 amino acid protein and sharing 99% identity at the deducted amino acid level with that of Bos taurus. Protein sequence analysis showed that MSTN is a weakly acerbic extracellular protein, consisting of signal peptides at 18-19 sites, a TGF-β propeptide, and a TGF-β domain. RT-PCR analyses demonstrated that water buffalo MSTN was expressed in multiple tissues but not limited to muscle. Immunohistochemistry staining confirmed the presence of MSTN in oocytes and granulosal cells. To our knowledge, this is the first study to confirm the expression of MSTN in the water buffalo ovary, suggesting an additional role of MSTN in water buffalo folliculogenesis, along with its role in skeletal muscle growth regulation. Further study of the regulatory mechanism of MSTN in water buffalo reproduction is warranted.

Abbreviations: MSTN, myostatin; ORF, open reading frame.     

Amnion Epithelial Cells of Buffalo (Bubalus bubalis) Term Placenta Expressed Embryonic Stem Cells Markers and Differentiated into Cells of Neurogenic Lineage In Vitro

Aim of the present study was the isolation, culture, and characterization of amniotic membrane-derived epithelial cells (AE) from term placenta collected postpartum in buffalo. We found that cultured cells were of polygonal in shape, resistance to trypsin digestion and expressed cytokeratin-18 indicating that they were of epithelial origin. These cells have negative expression of mesenchymal stem cell markers (CD29, CD44, and CD105) and positive for pluripotency marker (OCT4) genes indicated that cultured cells were not contaminated with mesenchymal stem cells. Immunofluorescence staining with pluripotent stem cell surface markers, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 indicated that these cells may retain pluripotent stem cell characteristics even after long period of differentiation. Differentiation potential of these cells was determined by their potential to differentiate into cells of neurogenic lineages using retinoic acid. In conclusion, we demonstrate that AE cells expressed pluripotent stem cell markers and have propensity to differentiate into cells of neurogenic lineage upon directed differentiation in vitro.     

Evaluation of The Probiotic Potential of Lactobacillus Delbrueckii Ssp. indicus WDS-7 Isolated from Chinese Traditional Fermented Buffalo Milk In Vitro

The present study aimed to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from Chinese traditional fermented buffalo milk. Out of 22 isolates, 11 were putatively identified as LAB preliminarily. A total of six LAB strains displayed strong adhesion to HT-29 cells and all these strains showed preferable tolerance to artificially simulated gastrointestinal juices. WDS-4, WDS-7, and WDS-18 exhibited excellent antioxidant capacities, including DPPH radical, ABTS⁺ radical, and superoxide anion scavenging activities. Compared with the other two LAB strains, WDS-7 had a stronger inhibition effect on four pathogens. Based on the 16S rRNA gene sequencing and phylogenetic analysis, WDS-7 was identified as Lactobacillus delbrueckii ssp. indicus and selected to assess the potential and safety of probiotics further. The results revealed that WDS-7 strain had a strong capacity for acid production and good thermal stability. WDS-7 strain also possessed bile salt hydrolase (BSH) activity. Compared to LGG, WDS-7 was a greater biofilm producer on the plastic surface and exhibited a better EPS production ability (1.94 mg/ml as a glucose equivalent). WDS-7 was proved to be sensitive in the majority of tested antibiotics and absence of hemolytic activity. Moreover, no production of biogenic amines and β-glucuronidase was observed in WDS-7. The findings of this work indicated that L. delbrueckii ssp. indicus WDS-7 fulfilled the probiotic criteria in vitro and could be exploited for further evaluation in vivo.     

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence,Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.     

Purification of glucose 6-phosphate dehydrogenase from Buffalo (Bubalus bubalis) erythrocytes and investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from buffalo (Bubalus bubalis) erythrocytes and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: hemolysate preparation and 2('),5(')-ADP-Sepharose 4B affinity gel chromatography. Thanks to the two consecutive procedures, the enzyme, having a specific activity of 69.7EU/mg proteins, was purified 650-fold with a yield of 31%. Optimal pH, stable pH, optimal temperature, molecular weight, and K(M) and V(max) values for NADP(+) and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. In addition, K(i) values and the type of inhibition were determined by means of Lineweaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADPH, and NADH.     

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes,Followed by Intracytoplasmic Sperm Injection,to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.     

Comparative In Vitro Development of Precocious and Normal Strains of Eimeria tenella (Coccidia)

SYNOPSIS. Eimeria tenella strain Wis-F is known to develop in chickens with a significantly shortened prepatent period and its pathogenicity is virtually completely attenuated. In vitro development of this strain paralleled development of the control (Wisconsin) strain through the first asexual generation. Instead of entering 2nd generation schizogony, however, most of the Wis-F merozoites developed into microgamonts or macrogamonts. Wall-forming bodies were prominent in developing macrogametes at 80–88 hr and began coalescing into the oocyst wall by 88 hr. Microgamete development paralleled that of macrogametes, with the appearance of multinucleate, immature forms at 72–80 hr and with recognizable, spermlike microgametes being prominent at 88–96 hr. Pathogenicity attenuation and reduction of the length of the prepatent period clearly resulted from omission of a portion of the life cycle (2nd generation schizogony).     

Ketoconazole Inhibits the Growth and Development of Ichthyophonus sp. (Mesomycetozoa) In Vitro

ABSTRACT. We determined the in vitro effect of the azol-derivative antifungal ketoconazole (KZ) on the morphology, growth, and development of teleost fish parasite Ichthyophonus sp. The KZ was delivered to culture medium using liposomes (L) or a lipid emulsion (E) at five different doses (i.e. 5, 50, 100, 200, and 400 μg/ml) for both L and E formulations. Controls consisted of Eagle's minimum essential medium (MEM) supplemented with 10% foetal bovine serum (MEM-10) alone (C-MEM) or containing amounts of L or E equivalent to those used in the KZ100 and KZ400 treatments (i.e. 100L, 400L, 100E, and 400E, respectively). Morphological alterations, such as a decrease in the number of dividing spores and nuclei, and condensation or even destruction of the cytoplasm, were observed using light and electron microscopy in the MEM-cultured organisms receiving KZ formulations, especially with KZ400L preparations, at both 7- and 14-d postinoculation. The KZ treatments also demonstrated a statistically significant inhibition of Ichthyophonus growth in MEM. These treatments also had an inhibitory effect on subsequent Ichthyophonus germination in Earle's fish saline agar (EFSA) medium, which was more evident for L formulations when the organism was treated for 7 d and for E formulations at 14 d. Our results endorse the potential use of KZ for the treatment for ichthyophonosis and provide support to proceed to in vivo assays.     

Development and Evaluation of Ethyl Cellulose-Based Transdermal Films of Furosemide for Improved In Vitro Skin Permeation

Transdermal films of the furosemide were developed employing ethyl cellulose and hydroxypropyl methylcellulose as film formers. The effect of binary mixture of polymers and penetration enhancers on physicochemical parameters including thickness, moisture content, moisture uptake, drug content, drug–polymer interaction, and in vitro permeation was evaluated. In vitro permeation study was conducted using human cadaver skin as penetration barrier in modified Keshary–Chein diffusion cell. In vitro skin permeation study showed that binary mixture, ethyl cellulose (EC)/hydroxypropyl methylcellulose (HPMC), at 8.5:1.5 ratio provided highest flux and also penetration enhancers further enhanced the permeation of drug, while propylene glycol showing higher enhancing effect compared to dimethyl sulfoxide and isopropyl myristate. Different kinetic models, used to interpret the release kinetics and mechanism, indicated that release from all formulations followed apparent zero-order kinetics and non-Fickian diffusion transport except formulation without HPMC which followed Fickian diffusion transport. Stability studies conducted as per International Conference on Harmonization guidelines did not show any degradation of drug. Based on the above observations, it can be reasonably concluded that blend of EC–HPMC polymers and propylene glycol are better suited for the development of transdermal delivery system of furosemide.     

BRCA-1 Gene Expression and Comparative Proteomic Profile of Primordial Follicles from Young and Adult Buffalo (Bubalus bubalis) Ovaries

In our previous study, we demonstrated that the repair efficiency of DNA double-strand breaks declines with increasing age in rat primordial follicles. In the present study, we extended our studies to buffalo (Bubalus bubalis) wherein we studied the expression of BRCA-1 related DNA repair genes in primordial follicles of young (12 months-22 months) and adult (72–96 months) buffaloes. The relative expression of selected genes, as determined by RT-PCR, revealed a significant (p?相似文献   

Evaluation of the Probiotic Potential of Saccharomyces cerevisiae Strain (C41) Isolated from Tibicos by In Vitro Studies

Probiotics and Antimicrobial Proteins - Artisanal fermented beverages have been associated with beneficial effects for a long time. In Mexico, there are a wide variety of artisanal fermented...     

In Vitro Studies for the Evaluation of Insecticidal Potential of the Venom of Endoparasitic Wasp Aenasius arizonensis (Girault) (Hymenoptera,Encyrtidae)

International Journal of Peptide Research and Therapeutics - Aenasius arizonensis (Hymenoptera: Encyrtidae) a nymphal, solitary endoparasitoid is a potent biocontrol agent of cotton mealybug,...     

Bacterial Reduction of Cr(VI) and Fe(III) in In Vitro Conditions

ABSTRACT Chemical reduction of Cr(VI) can be a strategy to detoxify toxic metals in oxidized states, whereas reduction of Fe(III) could enhance the availability of Fe in the form of Fe(II) to boost plant growth. However, it creates another problem of chemical sludge disposal. Hence, microbial conversion of Cr(VI) to Cr(III) and Fe(III) to Fe(II) is preferred over the chemical method. Out of 11 bacterial strains isolated from the rhizospheric zone of Typha latifolia growing on fly ash dump sites, four isolates were selected for the reduction of Cr(VI) and Fe(III) and were identified as Micrococcus roseus NBRFT2 (MTCC 9018), Bacillus endophyticus NBRFT4 (MTCC 9021), Paenibacillus macerans NBRFT5 (MTCC 8912), and Bacillus pumilus NBRFT9 (MTCC 8913). These strains were individually tested for survival at different concentrations of Cr(VI) and Fe(III), pH, and temperature, and then, their ability for reduction of both metals was evaluated at optimum pH 8.0 and temperature 35°C. The results indicated that NBRFT5 was able to reduce the maximum amount, 99% Cr(VI) and 98% Fe(III). Other strains also reduced these metals to different levels, but less than NBRFT5. Hence, these strains may be used for decontamination of metal-contaminated sites, particularly with Cr(VI) and Fe(III) through the reduction process.     

In Vitro Membrane Penetration of Modified Peptide Nucleic Acid (PNA)

Abstract

Efficient cellular uptake is crucial for the success of any drug directed towards targets inside cells. Peptide nucleic acid (PNA), a DNA analog with a promising potential as a gene-directed drug, has been shown to display slow membrane penetration in cell cultures. We here used liposomes as an in vitro model of cell membranes to investigate the effect on penetration of a PNA molecule colvalently modified with a lipophilic group, an adamantyl moiety. The adamantyl attachment was found to increase the membrane-penetration rate of PNA three-fold, as compared to corresponding unmodified PNA. From the penetration behaviour of a number of small and large molecules we could conclude that passive diffusion is the mechanism for liposome-membrane passage. Flow linear dichroism (LD) of the modified PNA in presence of rod-shaped micelles, together with octanol-water distribution experiments, showed that the adamantyl-modified PNA is amphiphilic; the driving force behind the observed increased membrane-penetration rate appears to be an accumulation of the PNA in the lipid double layer.