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.     

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.     

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.     

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli

Summary The effects of 25-fold overproduction ofEscherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from theBacillus subtilis chromosome and the mature part of TEM-β-lactamase, were studied inE. coli. One precursor (pre[A2d]-β-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[Al3i]-β-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature β-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-β-lactamase and pre(A2)-β-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export inE. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.     

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.     

Application of good laboratory practice (GLP) to culture collections of microbial and cell cultures

Although the principles and the necessity for good laboratory practice (GLP) guidelines to confirm the credibility, integrity, and quality of non-clinical laboratory studies have been known for more than a decade, culture collection activities are not subject to them. Because of recent advances in biotechnology, culture collections face increased demands not only for quality cultures but also current information. When applied in culture collections, GLP guidelines prove to be an excellent management tool as well as a cost-effective system of providing authentic and reliable microbial and cell cultures and associated data.     

Familial and iatrogenic cortisol receptor resistance.

Primary cortisol receptor resistance has been reported in 6 patients and 14 asymptomatic family members. We observed an additional 6 patients (2 males and 4 females). The male patients presented with hypertension. The female patients presented with acne, hirsutism and irregular menstruations. Dexamethasone therapy (1-1.5 mg/day) was of considerable clinical benefit. All 6 patients showed insufficient suppression of cortisol after 1 mg dexamethasone. The diurnal rhythm of ACTH and cortisol was intact, albeit at an elevated level. There was a normal increase of ACTH, cortisol, and GH to insulin-induced hypoglycemia, while cortisol production was (slightly) elevated. Adrenal androgen levels were increased in all patients. Glucocorticoid receptors measured in a whole cell dexamethasone binding assay in mononuclear leukocytes showed a lowered affinity in 1, and lowered numbers of receptors in 4 patients. In 1 patient no abnormalities were found. As a "bioassay" for glucocorticoid action dexamethasone suppressibility of mitogen-stimulated incorporation of [3H]thymidine in mononuclear leukocytes was measured. In this last patient dexamethasone suppressibility of [3H]thymidine incorporation was significantly lowered. Twelve months' treatment with 200 mg RU 486 per day in meningioma patients induced a similar biochemical picture as observed in primary cortisol receptor resistance. Partial cortisol receptor resistance might be less rare than previously thought. In the 6 patients presented at least 3 different forms can be recognized. Therapy with dexamethasone was successful in female patients with acne and hirsutism, as the secondary overproduction of adrenal androgens was effectively controlled. Chronic therapy with RU 486 causes a biochemical picture similar to primary cortisol receptor resistance.     

Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.