Localization of the receptor binding site of IFN-alpha 2b

The receptor binding site of IFN-alpha is not precisely known. To further characterize this site, mAb against IFN-alpha 2b were selected that block the binding of radiolabeled IFN-alpha 2b to its cell surface receptor. These antibodies also neutralized the anti-viral and anti-proliferative properties of IFN-alpha 2b. A subset of these antibodies (group 1) do not recognize IFN-alpha 2a, either in solid-phase immunoassays or functional assays, whereas a second subset (group 2), with no cross-reactivity with group 1, recognizes both IFN-alpha subtypes. Because IFN-alpha 2b and IFN-alpha 2a differ by only alpha Arg23-Lys23 substitution, group 1 antibodies must recognize an epitope within the receptor binding region of IFN-alpha 2b that includes Arg23. Group 2 antibodies recognize a separate and distinct epitope within the binding site that does not include Arg23.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion (Allium cepa) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 microgram of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E2.     

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39 degrees C) during the early part of the virus cycle (between 0- to 20-h postinfection) greatly inhibited virus production. This effect was not observed in revertant or wild-type cells. Molecular studies by the Southern transfer procedure of the unintegrated viral DNA synthesized in these cells at a permissive (33 degrees C) or nonpermissive temperature revealed that the levels of linear double-stranded viral DNA (8.8 kilobase pairs) were nearly identical in mutant or revertant cells incubated at 33 or 39 degrees C. However, the levels of two species of supercoiled viral DNA (with one or two long terminal repeats) were significantly lower in mutant cells incubated at 39 degrees C than in mutant cells incubated at 33 degrees C or in revertant cells incubated at 39 degrees C. Pulse-chase experiments showed that linear viral DNA made at 39 degrees C could not be converted into supercoiled viral DNA in mutant cells after a shift down to 33 degrees C. In contrast, such conversion was observed in revertant cells. Restriction endonuclease analysis did not detect differences in the structure of linear viral DNA made at 39 degrees C in mutant cells as compared to linear viral DNA isolated from the same cells at 33 degrees C. However, linear viral DNA made at 39 degrees C in mutant cells was poorly infectious in transfection assays. Taken together, these results strongly suggest that this X-linked gene, affecting mouse cell DNA synthesis, is operating in the early phase of murine leukemia virus replication. It seems to affect the level of production of unintegrated linear viral DNA only slightly while greatly reducing the infectivity of these molecules. In contrast, the accumulation of supercoiled viral DNA and subsequent progeny virus production are greatly reduced. Our pulse-chase experiments suggest that the apparent, but not yet identified, defect in linear viral DNA molecules might be responsible for their subsequent impaired circularization.     

Ordered replication of DNA sequences: synthesis of mouse satellite and adjacent main band sequences.

The replication of mouse satellite DNA was delayed when synchronized 3T3 cells were exposed to low concentrations of hydroxyurea during S phase, It appears that the onset of satellite replication is not a time dependent event, but instead requires that a certain amount of main band DNA be synthesized first. Using hydroxyapatite chromatography and S1 nuclease digestion, a procedure was developed to quantitate the synthesis of both satellite and neighboring main band sequences. The replication kinetics of satellite determined by this method agree with previous estimates. Main band sequences adjacent to satellite appear to replicate in concert with satellite DNA. The results are discussed and related to the limitations of the techniques utilized.     

Study of rust resistance genes in wheat germplasm with DNA markers

Wheat is a vital dietary component for human health and widely consumed in the world. Wheat rusts are dangerous pathogens and contribute serious threat to its production. In present study, PCR-Based DNA Markers were employed to check the rust resistance genes among 20 wheat genotypes and 22 markers were amplified. NTSYS-pc 2.2 was used to calculate genetic diversity and Nei and Li''s coefficients ranged from 0.55 to 0.95. Cluster analysis was obtained using UPGMA (Unweighted Pair Group Method of Arithmetic Average) algorithm. Maximum no. of genes (23) was amplified from TW-760010 genotype whereas minimum no of genes (14) were amplified from TW-76005 genotype. The data gained from present study open up new ways to produce new varieties by breeding rust resistant germplasm to avoid the economic and food loss and varieties with improved characteristics.     

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of −150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.     

Purification and Characterisation of Rat Kidney Glutathione Reductase

Glutathione reductase [GR, E.C.1.8.1.7] catalyses NADPH dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH). Thus, it is the crucial enzyme to maintain high [GSH]/[GSSG] ratio and physiological redox status in cells. Kidney and liver tissues were considered as a rich source of GR. In this study, rat kidney GR was purified and some of its properties were investigated. The enzyme was purified 2,356 fold with a yield of 16% by using heat-denaturation and Sephadex G25 gel filtration, 2′,5′-ADP Agarose 4B, PBE94 column chromatographies. The purified enzyme had a specific activity (Vm) of 250 U/mg protein and the ratio of absorbances at wavelengths of A ₂₇₃/A _463, A ₂₈₀/A ₄₆₀, A ₃₆₅/A ₄₆₀, and A ₃₇₉/A ₄₆₃, were 7.1, 6.8, 1.2 and 1.0, respectively. Each mol of GR subunit bound 0.97 mol of FAD. NADH was used as a coenzyme by rat kidney GR but with a lower efficiency (32.7%) than NADPH. Its subunit molecular weight was estimated as 53 kDa. An optimum pH of 6.5 and optimum temperature of 65 °C were found for rat kidney GR. Its activation energy (Ea) and temperature coefficient (Q₁₀) were calculated as 7.02 kcal/mol and 1.42, respectively. The Km_(NADPH) and kcat/Km _(NADPH) values were found to be 15.3 ± 1.4 μM and 1.68 × 10⁷ M⁻¹ s⁻¹ for the concentration range of 10-200 μM NADPH and when GSSG is the variable substrate, the Km_(GSSG) and the kcat/Km_(GSSG) values of 53.1 ± 3.4 μM and 4.85 × 10⁶ M⁻¹ s⁻¹ were calculated for the concentration range of 20–1,200 μM GSSG.     

Ki-67 immunostaining and stereologic estimation of nuclear volume in melanocytic skin tumors

OBJECTIVE: To investigate the proliferative activity and mean nuclear volume (MNV) of melanocytic skin tumors. STUDY DESIGN: Proliferative activity, assessed by immunostaining for the Ki-67 monoclonal antibody (reactive with all actively cycling cells), and MNV, estimated by means of a stereologic method, were determined in 60 cutaneous melanocytic tumors, including 28 primary malignant melanomas (PMM), 13 compound nevi (CN), 11 dysplastic nevi and 8 metastatic malignant melanomas. RESULTS: Both MNV and Ki-67 expression differed significantly between CN and other melanocytic tumors and showed a good correlation with Clark's level (a well-established prognostic parameter in PMM). CONCLUSION: The association of proliferative activity and quantitative nuclear features may be helpful in the interpretation of the degree of malignancy in melanocytic skin tumors.     

Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre- and postimmortalization, should be useful in defining molecular changes associated with immortalization.