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.     

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.     

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.     

Persistent obstructive jaundice, cholangitis, and biliary cirrhosis due to common bile duct stenosis in chronic pancreatitis

Long strictures of the intrapancreatic portion of the common bile duct were found in 6 patients with chronic pancreatitis. These strictures were responsible for painless obstructive jaundice, recurrent cholangitis, secondary biliary cirrhosis, and chronic abdominal pain difficult to distinguish from that caused by pancreatitis. Endoscopic retrograde cholangiopancreatography and intraoperative cholangiography were invaluable in making the diagnosis and in planning surgical correction. Decompression of the biliary tree by anastomosis of the gallbladder or common duct to the small intestine completely relieved symptoms and allowed liver function to improve significantly. Common duct stricture as a complication of chronic pancreatitis should be considered in the differential diagnosis of extrahepatic biliary obstruction and whenever surgical treatment of chronic pancreatitis is contemplated.     

Bilateral pulmonary hilar lymphadenopathy. An unusual manifestation of metastatic renal cell carcinoma

The significance of glucagon for the alterations in carbohydrate and fat metabolism during swimming has been evaluated. Fed, male rats were used. Blood was drawn by cardiac puncture for glucose analysis and either rabbit-antiglucagonserum (A-rats) or normal rabbitserum (N-rats) injected. Twenty-nine rats were then forced to swim (S-rats) with a tail weight for 60 min, while 16 rats were resting controls (C-rats). Subsequently blood was drawn and samples of liver and muscle tissue collected. In SN-rats glucagon concentrations increased from 152 +/- 18 (S.E.) pg/ml (CN-rats) to 332 +/- 61 (P less than 0.05), while liver glycogen decreased (P less than 0.001) and blood glucose increased (P less than 0.05). In SA-rats, however, the changes in liver glycogen and blood glucose were halved indicating that increased glucagon secretion enhances hepatic glycogen depletion during prolonged exercise. NEFA rose in SA-rats (P less than 0.005) as well as in SN-rats (P less than 0.05). Glycerol concentrations, however, only increased in SA-rats (P less than 0.05) indicating a shift towards lipid combustion in antibody treated rats. Muscle glycogen and plasma insulin diminished and blood lactate increased uniformly in exercised rats.     

Immunoreactive growth hormone levels in pigs fed protein or energy restricted diets during the postweaning period

Developmental changes in growth hormone levels during protein or energy malnutrition was studied in weaned pigs. Three or 4 weeks old pigs from control dams were fed a control diet (18% protein), an energy-restricted diet (18% protein) or a low protein diet (6% protein) for 8 weeks. Energy restriction was achieved by feeding the control diet in amounts that allowed very little growth. After the restriction period, all pigs were fed the control diet ad libitum for another 8 weeks. Blood samples were collected at intervals throughout the experiment and the plasma was analyzed for growth hormone by radioimmunoassay. Post weaning protein deprivation resulted in higher growth hormone levels during the restriction period as compared to control pigs or pigs with a restricted energy intake.