Germ cell transplantation in goats

Transplantation of spermatogonial stem cells provides a unique approach for the study of spermatogenesis and manipulation of the male germ line. This technique may also offer an alternative to the currently inefficient methods of producing transgenic domestic animals. We have recently established the technique of spermatogonial transplantation, originally developed in laboratory rodents, in pigs, and this study was aimed to extend the technique to the goat. Isolated donor testis cells were infused into the seminiferous tubules of anesthetized recipient goats through an ultrasonographically-guided catheter inserted into the rete testis. Donor cells were obtained by enzymatic digestion of freshly collected testes from immature goats (either from the recipients' contralateral testis or from unrelated donors). Prior to transplantation, testis cells were labeled with a fluorescent marker to allow identification after transplantation. Recipient testes were examined for the presence and localization of labeled donor cells at 3-week intervals up to 12 weeks after transplantation. Labeled donor cells were found in the seminiferous tubules of all testes, comprising 10-35% of the examined tubules. Histological examination of the recipient testes did not reveal evident tissue damage, except for limited fibrotic changes at the site of needle insertion. Likewise there were no detectable local or systemic signs of immunologic reactions to the transplantations. These results indicate that germ cell transplantation is technically feasible in immature male goats and that donor-derived cells are retained in the recipient testis for at least three months and through puberty. This study represents the first report of germ cell transplantation in goats.     

Activation of Wnt/β-Catenin Pathway in Monocytes Derived from Chronic Kidney Disease Patients

Patients with chronic kidney disease (CKD) have significantly increased morbidity and mortality resulting from infections and cardiovascular diseases. Since monocytes play an essential role in host immunity, this study was directed to explore the gene expression profile in order to identify differences in activated pathways in monocytes relevant to the pathophysiology of atherosclerosis and increased susceptibility to infections. Monocytes from CKD patients (stages 4 and 5, estimated GFR <20 ml/min/1.73 m²) and healthy donors were collected from peripheral blood. Microarray gene expression profile was performed and data were interpreted by GeneSpring software and by PANTHER tool. Western blot was done to validate the pathway members. The results demonstrated that 600 and 272 genes were differentially up- and down regulated respectively in the patient group. Pathways involved in the inflammatory response were highly expressed and the Wnt/β-catenin signaling pathway was the most significant pathway expressed in the patient group. Since this pathway has been attributed to a variety of inflammatory manifestations, the current findings may contribute to dysfunctional monocytes in CKD patients. Strategies to interfere with this pathway may improve host immunity and prevent cardiovascular complications in CKD patients.     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> bacteriocin Bac 14B with a broad inhibitory spectrum: Purification,amino acid sequence analysis,and physicochemical characterization

Bacillus subtilis strain 14B was used to produce a novel antimicrobial peptide (bacteriocin) called Bac 14B. Pure bacteriocin was obtained after heat and acidic treatments (80°C and pH 4), precipitation by ammonium sulfate, and chromatography on Sephadex G-50 and Mono Q Sepharose columns. Based on MALDI-TOF mass spectrometry analysis, purified Bac 14B is a monomer protein with a molecular mass of 20110.13 Da. N-terminal sequencing allowed for the straightforward identification of its first 12 residues, which were of a pure bacteriocin. It also revealed that this bacteriocin contained a unique sequence, namely M-L-K-A-N-L-Q-N-P-L-N-A, suggesting the identification of a novel compound. Bac 14B was stable for 1 h at temperatures up to 80°C and pH of 4 ∼ 8. It also proved sensitive to various proteases, which demonstrated its protein nature. Bac 14B displayed a bacteriolytical mode of action and a broad range of inhibitory spectra toward Gram-positive and -negative pathogens. Interestingly, based on conventional agronomic seed vigor parameters, the application of Bac 14B (500 activity units/mL) to various crops revealed that this bacteriocin was a potent exogenous enhancer of growth that stimulated the seedling vigor of tomatoes and muskmelons. Compared to those of the control, the germination percentage, shoot weight, shoot height, and root length were all significantly enhanced in Bac 14B-treated plant seeds. Bac 14B also exhibited effective disinfectant properties against a wide range of seedborne diseases and significant effects on the control of damping off diseases, particularly at the pregermination stage. It also proved to be effective against root rot diseases caused by Alternaria solani and other bacterial seedborne pathogens such as wilt diseases. The findings indicate that Bac 14B is the first B. subtilis-produced bacteriocin ever reported to exhibit such promising biological properties.     

Studies on new,centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimer's disease, and other memory disorders.     

Octanoylation of the lipoyl domains of the pyruvate dehydrogenase complex in a lipoyl-deficient strain of Escherichia coli

The overexpression of a subgene encoding a hybrid lipoyl domain of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli has previously been shown to result in the formation of lipoylated and unlipoylated products. Overexpression of the same subgene in a lipoic acid biosynthesis mutant growing under lipoate-deficient conditions has now been shown to produce domains modified by octanoylation as well as unmodified domains. It was concluded from the mass of a lipoyl-binding-site peptide that the modification involves N6-octanoylation of the lysine residue (Lys244) that is normally lipoylated, and this was confirmed by the trypsin-insensitivity of the corresponding Lys244-Ala-245 bond, and the absence of modification in a mutant domain in which Lys244 is replaced by Gln. This novel protein modification raises interesting questions concerning the pathway of lipoic acid biosynthesis and the mechanism of enzyme lipoylation.     

Proteomics studies in inner ear disorders: pathophysiology and biomarkers

Although proteomics has been exploited in a wide range of diseases for identification of biomarkers and pathophysiological mechanisms, there are still biomedical disciplines such as otology where proteomics platforms are underused due to technical challenges and/or complex features of the disease. Thus, in the past few years, healthcare and scientific agencies have advocated the development and adoption of proteomic technologies in otological research. However, few studies have been conducted and limited literature is available in this area. Here, we present the state of the art of proteomics in otology, discussing the substantial evidence from recent experimental models and clinical studies in inner-ear conditions. We also delineate a series of critical issues including minute size of the inner ear, delicacy and poor accessibility of tissue that researchers face while undertaking otology proteomics research. Furthermore, we provide perspective to enhance the impact and lead to the clinical implementation of these proteomics-based strategies.     

Tetanus toxin selectively impairs anti-tumoral but not anti-microbial macrophage-mediated effector functions

Abstract The present study was designed to establish the susceptibility of macrophage-mediated effector functions to tetanus toxin (TT). Using the murine macrophage cell line, GG2EE, generated in vitro by v- raf /v- myc oncogenes, we have previously provided evidence that TT selectively inhibits interferon gamma (IFN-γ), but not basal, lysozyme activity. Here we show that while neither phagocytic nor candidacidal activities are affected by TT treatment, antitumoral activity is significantly impaired after exposure to TT. This phenomenon, which is dose-dependent, is fully ascribed to the holotoxin, as heat inactivated TT, C or A-B fragments result ineffective. Furthermore, C but not A-B fragment competes with TT in abrogating its inhibitory effects. Overall, these data indicate that TT is not a broad-spectrum, down-regulating signal on macrophage-mediated functions, thus implying that its toxic action is exerted on specific molecular targets.     

Thermal Unfolding Pathway of PHD2 Catalytic Domain in Three Different PHD2 Species: Computational Approaches

Prolyl hydroxylase domain 2 containing protein (PHD2) is a key protein in regulation of angiogenesis and metastasis. In normoxic condition, PHD2 triggers the degradation of hypoxia-inducible factor 1 (HIF-1α) that induces the expression of hypoxia response genes. Therefore the correct function of PHD2 would inhibit angiogenesis and consequent metastasis of tumor cells in normoxic condition. PHD2 mutations were reported in some common cancers. However, high levels of HIF-1α protein were observed even in normoxic metastatic tumors with normal expression of wild type PHD2. PHD2 malfunctions due to protein misfolding may be the underlying reason of metastasis and invasion in such cases. In this study, we scrutinize the unfolding pathways of the PHD2 catalytic domain’s possible species and demonstrate the properties of their unfolding states by computational approaches. Our study introduces the possibility of aggregation disaster for the prominent species of PHD2 during its partial unfolding. This may justify PHD2 inability to regulate HIF-1α level in some normoxic tumor types.     

Comparative analysis of the methods used for finding surface energy to investigate protein interaction behavior on chromatographic supports

Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.