Genetic manipulation of antibiotic-producing Streptomyces

The genetic manipulation of Streptomyces species has been facilitated by the development of versatile cloning vectors, robust gene transfer systems and transposable elements. These molecular genetic tools have been used to construct antibiotic-producing strains with improved properties and recombinants for the production of hybrid glycopeptide and macrolide antibiotics, novel anthelminthic agents and novel cytotoxic anthracyclines.     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 microns during maturation and increased to 106 microns after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination.     

Real-time patch-cram detection of intracellular cGMP reveals long-term suppression of responses to NO and muscarinic agonists

Cyclic GMP (cGMP) is a crucial intracellular messenger in neuronal, muscle, and endocrine cells. The intracellular concentration of cGMP is regulated by various neurotransmitters, including acetylcholine (ACh) and nitric oxide (NO). While much is known about the biochemical steps leading to cGMP synthesis, little is known about cGMP kinetics in intact cells. Here, we use "patch-cramming," in which an excised, inside-out membrane patch containing cyclic nucleotide-gated ion channels is used as a biosensor, to obtain the first real-time measurements of cGMP in intact cells. Patch-cramming experiments on neuroblastoma cells show that both muscarinic agonists and NO rapidly elevate cGMP. NO elicits cGMP responses repeatedly without decrement, whereas responses to muscarinic agonists exhibit a profound and prolonged desensitization. Remarkably, muscarinic agonists also cause long-term (>30 min) suppression (LTS) of cGMP responses elicited by NO. Biochemical measurements reveal that rat sympathetic neurons also exhibit LTS of cGMP, suggesting that LTS is a widespread mechanism that may contribute to synaptic plasticity.     

Binding and lysing of blood clots using MRX-408

RATIONALE AND OBJECTIVES: A thrombus-specific ultrasound contrast agent, MRX-408, has been developed recently. This agent consists of phospholipid-coated microbubbles with a ligand capable of targeting the GPIIb/IIIa receptor, thereby allowing the microbubbles to bind with thrombi rich in activated platelets. In vitro and in vivo animal experiments have been conducted to examine imaging enhancement and sonothrombolysis using this agent compared with a nontargeted agent. METHODS: For clot binding, blood-smeared slides were incubated with microbubbles and examined under a light microscope. Change in backscatter signals from the blood clots after binding was examined by both an ultrasound scanner and two single-element transducers arranged in a transmitter-receiver pair. For clot lysis, either 1-MHz or 20-KHz ultrasound was used to enhance the lysing effects of MRX-408 with or without urokinase. RESULTS: Evidence of binding was demonstrated under a microscope. In vitro experiments showed that the "acoustic signature", or properties, of blood clots changed after binding. Clots became more echogenic and nonlinear. In vivo fundamental ultrasound imaging confirmed that as a result of binding, blood clots were more visible, the area of detection was improved, and shadowing behind clots was more noticeable. Under 1-MHz ultrasound and 30 minutes of treatment, lysis efficiency reached 34% with MRX-408, whereas there was no visible clot lysis with saline. CONCLUSION: The results of these preliminary studies show that as a contrast agent, MRX-408 enhanced clots under ultrasound imaging and facilitated sonothrombolysis with or without thrombolytic drugs.     

Collection and transfusion of blood and blood components in the United States, 1989

A poor response to L-DOPA in addition to parkinsonian, cerebellar, and autonomic signs is commonly regarded as indicative of clinical multiple system atrophy (MSA). We compared the motor response to a single oral administration of 250 mg L-DOPA/25 mg carbidopa in eight MSA patients and eight Parkinson's disease (PD) patients with the "on-off" phenomenon, evaluating L-DOPA peripheral pharmacokinetics. Motor response was consistently good in all PD patients, but only four MSA patients had a (moderate) response. Pharmacokinetic parameters did not differ between the groups. The varying extent of putaminal damage could be responsible for the differing motor response to L-DOPA in MSA patients.     

Age incidence of meningococcal infection England and Wales, 1984-1991

Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. This suggests that retroviral-mediated gene transfer might permit targeting of gene integration into malignant cells in organs composed mainly of quiescent nonproliferating cells, such as in the brain. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs ("suicide" genes) into proliferating brain tumors may be used to treat this cancer. We investigated the efficacy and dynamics of in vivo transduction of growing brain tumors with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir. Ganciclovir is phosphorylated by thymidine kinase to toxic triphosphates that interfere with DNA synthesis, resulting in the preferential death of the transduced tumor cells. Rats inoculated with 4 x 10(4) 9L gliosarcoma cells into the frontal lobe were treated 7 days later with an intratumoral stereotaxic injection of murine fibroblasts (NIH 3T3 cells) that were producing a retroviral vector containing the herpes simplex-thymidine kinase gene. Controls received vector producer and nonproducer NIH 3T3 cell lines containing the Escherichia coli lacZ (beta-galactosidase) gene as well as nonproducer NIH 3T3 cells containing the thymidine kinase gene. The animals were rested for 7 days to allow time for in situ transduction of the proliferating tumor cells with the herpes-thymidine kinase retroviral vector. The animals were then treated with ganciclovir, 15 mg/kg i.p. twice a day for 14 days. Gliomas receiving an injection of 3-5 x 10(6) thymidine kinase producer cells regressed completely in 23 of 30 rats given ganciclovir therapy, while 25 of 26 control rats developed large tumors. Intratumoral injection of a lower concentration of thymidine kinase vector producer cells (1.8 x 10(6)) resulted in a lower frequency of tumor regression (5 of 13 rats). To estimate the efficiency of in vivo gene transfer, 9L brain tumors were given injections of 5 x 10(6) beta-galactosidase vector producer cells. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranaside staining revealed maximal staining of beta-galactosidase within the tumor 7-14 days after injection of the vector producer cells. In vivo transduction rates in harvested tumors ranged from 10 to 70%. There was no evidence of transduction of the surrounding normal neural tissue. Occasional blood vessel endothelial cells within or adjacent to the tumor were observed to be 5-bromo-4- chloro-3-indolyl-beta-D-galactopyranaside positive.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of the mouse heart adenosine 5',5"'-P1-P4-tetraphosphate receptor

We have previously demonstrated that mouse brain membrane fractions have a specific, saturable receptor for diadenylated nucleotides. Binding is specific for two adenosines, and the length of the phosphate bridge is critical, with four phosphates being optimal [Hilderman et al. (1991) J. Biol. Chem. 266, 6915-6918]. In this report, we demonstrate that adenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding to its receptor is dependent upon an activation step that requires divalent cations and a serine protease. Monoclonal antibodies (Mabs) are identified that inhibit Ap4A binding to its membrane receptor. These antibodies recognize a 212-kDa membrane protein. However, SDS-PAGE analysis of Ap4A cross-linked to membrane fractions reveals that Ap4A is not attached to the 212-kDa peptide but to a 30-kDa polypeptide. Appearance of the 30-kDa polypeptide is dependent on the activation step, and one of the inhibitory antibodies blocks its appearance. We suggest that the protease-dependent processing step involves cleavage of the 212-kDa component with the appearance of an active 30-kDa receptor.     

Multifrequency tympanometry in chinchillas

Multifrequency tympanometry (MFT), using probe frequencies ranging from 226-2,000 Hz, was performed on normal chinchillas to obtain normative data against which to compare results from animals with middle ear pathology. A series of validating experiments was conducted to determine the effects of anatomical alterations of the middle ear on MFT. These included artificially extending the ear canal, opening the bulla, injecting saline into the middle ear, and disrupting the ossicular chain. The results indicate that MFT characteristics of chinchilla ears are qualitatively similar to those observed in normal humans and patients with middle ear disease, and MFT provides information that is not available from the 226-Hz tympanogram.     

Spontaneous and ligand-induced trafficking of CXC-chemokine receptor 4

A chimeric protein consisting of CXC-chemokine receptor 4 (CXCR4) and the green fluorescent protein (GFP) was used for studying receptor localization and trafficking in real time in stably transduced HeLa, U-937, CEM, and NIH/3T3 cells. CXCR4-GFP was fully active as a co-receptor in mediating human immunodeficiency virus (HIV) entry. Both CXCR4 and CXCR4-GFP were found to undergo significant spontaneous endocytosis. Only 51.5 +/- 7.8% of receptor molecules were found on the plasma membrane in CD4-positive cells, 43.9 +/- 8.5% were found in CD4-negative HeLa cells, 75.6 +/- 9.7% were found in U-937 cells, 72.5 +/- 7.9 were found in CEM cells, and almost none were found in in NIH/3T3 cells. Stromal cell-derived factor-1alpha induced rapid endocytosis of cell surface receptor molecules. A significant part of CXCR4 was targeted to lysosomes upon binding of the ligands, and recycling of internalized CXCR4 was not efficient. Only about 30% of receptor molecules recycled back to the cell surface in HeLa cells, 5% recycled in U937, and 10% recycled in CEM cells, suggesting that the protective effect of chemokines against HIV infection can be attributed not only to competition for binding but also to depletion of the co-receptor molecules from the cell surface. Envelope glycoprotein gp120 of syncytia-inducing/lymphocyte tropic HIV-1 strains induced rapid internalization of CXCR4 in both CD4-negative and CD4-positive cells, suggesting that gp120 is a high affinity ligand of CXCR4.     

Role of active site tyrosine residues in catalysis by human glutathione reductase

Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.