Use of helper-free retroviral vector to direct a high expression of porcine growth hormone in mouse fibroblast cells.

A retroviral vector has been employed to express the cDNA coding for porcine growth hormone (pGH) in the mouse fibroblast cell NIH 3T3 in large quantity. In this study, a single gene vector which contained no selectable marker was used. We have coinfected NIH 3T3 cells with pGH retrovirus and Neo(r) retrovirus to obtain a stable, high-expression clone. Using a superinfection strategy, we further increased the copy number of proviral DNA in the host chromosome, thus increasing the pGH secretion from 22 to 55 micrograms/10(6) cells/24 h. The recombinant pGH produced from mouse fibroblast cells was heterogeneous at the N-terminus, which mimicked the situation with bovine growth hormone either from natural sources or from recombinant products derived from mouse fibroblasts. This technology is useful for many biologically important genes to be stably transduced by retroviral vector into mammalian cells and highly expressed.     

Protection of DNA damage by dietary restriction.

Dietary restriction is known to retard the aging processes and delay the onset of age-related neoplastic diseases. The mechanisms underlying these remarkable actions of nutritional intervention are not known in spite of recently intensified research efforts. However, the last couple of years' research on dietary restriction produced strong evidence indicating that its effective antiaging actions might be related to its ability to modulate free radical damage. In the present study, DNA damage and attenuation of the damage by dietary restriction were assessed by measuring 8-hydroxydeoxyguanosine 8-OH dG) in both nuclear DNA (nuDNA) and mitochondrial DNA (mitDNA) fractions. The data show that substantially more damage (approximately 15 times) occurred in mitDNA compared to nuDNA. More interestingly, the DNA damage was significantly attenuated in dietarily restricted rats.     

A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca(2+)-dependent K+ channels.

A 20 amino acid synthetic peptide, corresponding to the amino-terminal region of the Shaker B (ShB) K+ channel and responsible for its fast inactivation, can block large conductance Ca(2+)-dependent K+ channels from rat brain and muscle. The ShB inactivation peptide produces two kinetically distinct blocking events in these channels. At lower concentrations, it produces short blocks, and at higher concentrations long-lived blocks also appear. The L7E mutant peptide produces only infrequent short blocks (no long-lived blocks) at a much higher concentration. Internal tetraethylammonium competes with the peptide for the short block, which is also relieved by K+ influx. These results suggest that the peptide induces the short block by binding within the pore of Ca(2+)-dependent K+ channels. The long block is not affected by increased K+ influx, indicating that the binding site mediating this block may be different from that involved in the short block. The short block of Ca(2+)-dependent K+ channels and the inactivation of Shaker exhibit similar characteristics with respect to blocking affinity and open pore blockade. This suggests a conserved binding region for the peptide in the pore regions of these very different classes of K+ channel.     

Catalytic properties of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris nTR.

An X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5) was identified to be loosely bound on the inner cell membrane fraction of Lactococcus lactis subsp. cremoris nTR. The biosynthesis of X-PDAP was continuously increased before the late-log growth phase of the bacteria. Both Gly-Pro-pNA and Ala-Ala-pNA were hydrolyzed by X-PDAP; the kcat/Km value of the former was about 10-fold that of the latter. The Ki of X-Pro and Pro-X were more specific to X-PDAP than those of X-Ala. The enzyme splitting a dipeptide sequentially from beta-casomorphin as a model catalytic pattern was identified and some properties of the enzyme were further characterized.     

Epstein-Barr virus transmission via the donor organs in solid organ transplantation: polymerase chain reaction and restriction fragment length polymorphism analysis of IR2, IR3, and IR4.

Two organ transplant recipients who received organs from a common donor and were diagnosed as having an Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder were studied to determine the mode of EBV transmission. The results of restriction fragment length polymorphism, polymerase chain reaction, and minisatellite DNA analyses demonstrate that both patients had a common strain of EBV and that this strain was transmitted from the donor's organs to both recipients. Posttransplant lymphoproliferative disorder resulted from the proliferation of EBV-immortalized B lymphocytes of the recipient, not those of the donor.     

The relationship between ovarian steroids and uterine estrogen receptors during late pregnancy

Although a direct interdependence exists between the ovarian steroids, estrogen and progesterone, the exact role of these two hormones during pregnancy, especially late pregnancy, is not completely understood. Investigations have been conducted to determine whether the circulating levels of progesterone and estrogen or changes in the ratio of progesterone/estrogen in relation to the concentration of uterine estrogen receptors are associated with triggering parturition. Ninety-day old female rats were sacrificed at gestation days 14, 16, 18, 20 and two days post-partum. The plasma levels of estradiol and progesterone were measured by solid-phase radioimmunoassay. Uterine cytosol was subjected to a charcoal binding assay to determine the concentration of estrogen receptors. Our findings demonstrate that there is a significant (p less than 0.05) drop in both plasma progesterone and estradiol during late pregnancy. Also indicated is a significant (p less than 0.05) increase in uterine estrogen receptors throughout late pregnancy. Finally, during this period there is a direct correlation between the shift in the progesterone/estrogen ratio and the increase in the concentration of uterine estrogen receptors in late pregnancy.     

Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody.

A human monoclonal antibody designated 15e is reactive with the envelope glycoprotein (gp120) of multiple isolates of human immunodeficiency virus type 1 (HIV-1). Antibody 15e also neutralizes HIV-1 with broad specificity and blocks gp120 binding to CD4. Characterization of the 15e epitope shows that it is conformation dependent and is distinct from previously recognized functional domains of gp120, suggesting that this epitope represents a novel site important for HIV-1 neutralization and CD4 binding. These findings have implications for the development of a vaccine for AIDS.     

Simultaneous bilateral forearm revascularization

A successful simultaneous bilateral forearm revascularization was performed on a 17-year-old boy. Functional recovery of both forearms was evaluated 42 months after injury. The patient can use both hands for the activities of daily living. So far, he has been employed and has no significant psychological problems. Temporary intraluminal silicone shunts are extremely helpful for reducing ischemic damage to the injured limb. The sufficient skeletal shortening of the upper limb replantation is crucially important. The wounds must be managed by aggressive and repeated debridement. Accurate primary nerve repair is essential, and the early postoperative rehabilitation is also important to achieve a satisfactory functional return. The functional replanted or revascularized upper extremity is superior to an amputation or prosthesis, especially in the cases of bilateral upper extremity amputation or devascularization.     

Restoration of embryogenic competence in repeatedly subcultured carrot cell lines

Summary The production of somatic embryos from carrot suspension cultures invariably decreases through simple, repeated subculturing. Extracellular, concentrated compounds extracted from already established embryo culture not only recovered the embryogenic capability, but also accelerated the embryo production as much as two-fold (up to 1600 embryos/ml) compared with that of a control culture. Sugars, which were only a small portion of the total concentrate, were excluded as possible causative factors. It is likely that a protein fraction that is generated directly by competent, embryogenic cultures is important for the restoration of embryogenic potential.