Fecal coliform elevated-temperature test: a physiological basis.
The physiological basis of the Eijkman elevated-temperature test for differentiating fecal from nonfecal coliforms was investigated. Manometric studies indicated that the inhibitory effect upon growth and metabolism in a nonfecal coliform at 44.5 degrees C involved cellular components common to both aerobic and fermentative metabolism of lactose. Radioactive substrate incorporation experiments implicated cell membrane function as a principal focus for temperature sensitivity at 44.5 degrees C. A temperature increase from 35 to 44.5 degrees C drastically reduced the rates of [14C]glucose uptake in nonfecal coliforms, whereas those of fecal coliforms were essentially unchanged. In addition, relatively low levels of nonfecal coliform beta-galactosidase activity coupled with thermal inactivation of this enzyme at a comparatively low temperature may also inhibit growth and metabolism of nonfecal coliforms at the elevated temperature. (+info)
The effects of digestive enzymes on characteristics of placental insulin receptor. Comparison of particulate and soluble receptor preparations.
The role of the surrounding membrane structure on the binding characteristics of the insulin receptor was studied by using several digestive enzymes. The effects observed with particulate membrane preparations are compared with those from soluble receptor preparations. beta-Galactosidase and neuraminidase had no effect on insulin binding to either particulate or soluble receptors from human placentae. Exposure to 2 units of phospholipase C/ml increased insulin binding to particulate membranes, but was without effect on the soluble receptor preparation. The increase in binding to particulate membranes was shown to be due to an increase in apparent receptor number. After 5 min exposure to 500 microgram of trypsin/ml there was an increase in insulin binding to the particulate membrane fraction, owing to an increase in receptor affinity. After 15 min exposure to this amount of trypsin, binding decreased, owing to a progressive decrease in receptor availability. In contrast, this concentration of trypsin had no effect on the solubilized receptor preparation. Because of the differential effects of phospholipase C and trypsin on the particulate compared with the solubilized receptor preparations, it is concluded that the effects of these enzymes were due to an effect on the surrounding membrane structure. Changes in receptor configuration due to alterations within the adjoining membrane provide a potential mechanism for mediating short-term alterations in receptor function. (+info)
Astrocyte-specific expression of tyrosine hydroxylase after intracerebral gene transfer induces behavioral recovery in experimental parkinsonism.
Parkinson's disease is a neurodegenerative disorder characterized by the depletion of dopamine in the caudate putamen. Dopamine replacement with levodopa, a precursor of the neurotransmitter, is presently the most common treatment for this disease. However, in an effort to obtain better therapeutic results, tissue or cells that synthesize catecholamines have been grafted into experimental animals and human patients. In this paper, we present a novel technique to express tyrosine hydroxylase (TH) in the host's own astrocytes. This procedure uses a transgene in which the expression of a TH cDNA is under the control of a glial fibrillary acidic protein (GFAP) promoter, which confers astrocyte-specific expression and also increases its activity in response to brain injury. The method was tested in a rat model of Parkinson's disease produced by lesioning the striatum with 6-hydroxydopamine. Following microinjection of the transgene into the denervated striatum as a DNA-liposome complex, expression of the transgene was detected by RT-PCR and TH protein was observed specifically in astrocytes by using double-labeling immunofluorescence for GFAP and TH coupled with laser confocal microscopy. Efficacy was demonstrated by significant behavioral recovery, as assessed by a decrease in the pharmacologically induced turning behavior generated by the unilateral denervation of the rat striatum. These results suggest this is a valuable technique to express molecules of therapeutic interest in the brain. (+info)
Thyroid hormone effects on Krox-24 transcription in the post-natal mouse brain are developmentally regulated but are not correlated with mitosis.
Krox-24 (NGFI-A, Egr-1) is an immediate-early gene encoding a zinc finger transcription factor. As Krox-24 is expressed in brain areas showing post-natal neurogenesis during a thyroid hormone (T3)-sensitive period, we followed T3 effects on Krox-24 expression in newborn mice. We analysed whether regulation was associated with changes in mitotic activity in the subventricular zone and the cerebellum. In vivo T3-dependent Krox-24 transcription was studied by polyethylenimine-based gene transfer. T3 increased transcription from the Krox-24 promoter in both areas studied at post-natal day 2, but was without effect at day 6. An intact thyroid hormone response element (TRE) in the Krox-24 promoter was necessary for these inductions. These stage-dependent effects were also seen in endogenous Krox-24 mRNA levels: activation at day 2 and no effect at day 6. Moreover, similar results were obtained by examining beta-galactosidase expression in heterozygous mice in which one allele of the Krox-24 gene was disrupted with an inframe Lac-Z insertion. However, bromodeoxyuridine incorporation showed mitosis to continue through to day 6. We conclude first, that T3 activates Krox-24 transcription during early post-natal mitosis but that this effect is extinguished as development proceeds and second, loss of T3-dependent Krox-24 expression is not correlated with loss of mitotic activity. (+info)
Pathogenicity island 2 mutants of Salmonella typhimurium are efficient carriers for heterologous antigens and enable modulation of immune responses.
The potential use as vaccine delivery system of Salmonella typhimurium strains harboring defined mutations in the sseC (HH104) and sseD (MvP101) genes, which encode putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2, was evaluated and compared with that of the well-characterized aroA mutant strain SL7207 by using beta-galactosidase (beta-Gal) as a model antigen. When orally administered to immune-competent or gamma interferon-deficient (IFN-gamma-/-) BALB/c mice, both mutants were found to be highly attenuated (50% lethal dose, >10(9) bacteria). Both strains were also able to efficiently colonize and persist in Peyer's patches. Immunization with HH104 and MvP101 triggered beta-Gal-specific serum and mucosal antibody responses equivalent to or stronger than those observed in SL7207-immunized mice. Although immunoglobulin G2 (IgG2) serum antibodies were dominant in all groups, IgG1 was also significantly increased in mice vaccinated with MvP101 and SL7207. Comparable beta-Gal-specific IgA and IgG antibodies were detected in intestinal lavages from mice immunized with the different strains. Antigen-specific CD4(+) T-helper cells were generated after vaccination with all vaccine prototypes; however, responses were significantly more efficient when HH104 and MvP101 were used (P < 0.05). Significantly higher levels of IFN-gamma were produced by restimulated spleen cells from mice immunized with HH104 than from those vaccinated with the MvP101 or SL7207 derivatives (P +info)
Adenoviral gene transfer of the human V2 vasopressin receptor improves contractile force of rat cardiomyocytes.
BACKGROUND: In congestive heart failure, high systemic levels of the hormone arginine vasopressin (AVP) result in vasoconstriction and reduced cardiac contractility. These effects are mediated by the V1 vasopressin receptor (V1R) coupled to phospholipase C beta-isoforms. The V2 vasopressin receptor (V2R), which promotes activation of the Gs/adenylyl cyclase system, is physiologically expressed in the kidney but not in the myocardium. Expression of a recombinant V2R (rV2R) in the myocardium could result in a positive inotropic effect via the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: A recombinant adenovirus encoding the human V2R (Ad-V2R) was tested for its ability to modulate the cardiac Gs/adenylyl cyclase system and to potentiate contractile force in rat ventricular cardiomyocytes and in H9c2 cardiomyoblasts. Ad-V2R infection resulted in a virus concentration-dependent expression of the transgene and led to a marked increase in cAMP formation in rV2R-expressing cardiomyocytes after exposure to AVP. Single-cell shortening measurements showed a significant agonist-induced contraction amplitude enhancement, which was blocked by the V2R antagonist, SR 121463A. Pretreatment of Ad-V2R-infected cardiomyocytes with AVP led to desensitization of the rV2R after short-term agonist exposure but did not lead to further loss of receptor function or density after long-term agonist incubation, thus demonstrating resistance of the rV2R to downregulation. CONCLUSIONS: Adenoviral gene transfer of the V2R in cardiomyocytes can modulate the endogenous adenylyl cyclase-signal transduction cascade and can potentiate contraction amplitude in cardiomyocytes. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in congestive heart failure by bypassing the desensitized beta-adrenergic receptor signaling. (+info)
Multiple cis-acting regulatory regions are required for restricted spatio-temporal Hoxa5 gene expression.
Genetic analyses have revealed the essential role of the murine Hoxa5 gene for the correct specification of the cervical and upper thoracic region of the skeleton, and for the normal organogenesis and function of the respiratory tract, both structures expressing Hoxa5 during embryogenesis. To understand how the expression domains of the Hoxa5 gene are established during development, we have analyzed the cis-acting control regions mediating Hoxa5 gene expression using a transgenic approach. Four transcripts are derived from the Hoxa5 locus. The shortest and most abundant one displays a specific spatio-temporal profile of expression at earlier stages and in more anterior structures along the embryonic axis than the larger forms. We established that an 11.1 kilobase pair (kb) genomic fragment, extending from position -3.8 kb to +7.3 kb relative to Hoxa5 transcription initiation site, was sufficient to reproduce the temporal expression and substantially reconstitute the spatial pattern of the major Hoxa5 transcript. By deletion analyses, we identified a 2.1 kb fragment located downstream of the Hoxa5 gene that possesses mesodermal enhancer activity. Overall, the findings demonstrate that cis-acting regulatory elements essential for the correct expression of the major Hoxa5 transcript are located both upstream and downstream of the Hoxa5 coding sequences. (+info)
Ectopic expression of the transforming growth factor beta type II receptor disrupts mesoderm organisation during mouse gastrulation.
Transforming growth factor beta (TGFbeta) regulates the cell cycle and extracellular matrix (ECM) deposition of many cells in vitro. We have analysed chimaeric mouse embryos generated from embryonic stem cells with abnormal receptor expression to study the effect of TGFbeta on these processes in vivo and the consequences for normal development. The binding receptor for TGFbeta, TbetaRII, is first detected in the embryo proper around day 8.5 in the heart. Ectopic expression of TbetaRII from the blastocyst stage onward resulted in an embryonic lethal around 9.5 dpc. Analysis of earlier stages revealed that the primitive streak of TbetaRII chimaeras failed to elongate. Furthermore, although cells passed through the streak and initially formed mesoderm, they tended to accumulate within the streak. These defects temporally and spatially paralleled the expression of the TGFbeta type I receptor, which is first expressed in the node and primitive streak. We present evidence that classical TGFbeta-induced growth inhibition was probably the cause of insufficient mesoderm being available for paraxial and axial structures. The results demonstrate that (1) TGFbeta mRNA and protein detected previously in early postimplantation embryos is present as a biologically active ligand; and (2) assuming that ectopic expression of TbetaRII results in no other changes in ES cells, the absence of TbetaRII is the principle reason why the embryo proper is unresponsive to TGFbeta ligand until after gastrulation. (+info)