Prolactin stimulates precursor cells in the adult mouse hippocampus

In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory.     

Multidrug transporters in prokaryotic and eukaryotic cells: physiological functions and transport mechanisms

Multidrug transporters mediate the extrusion of structurally unrelated drugs from prokaryotic and eukaryotic cells. As a result of this efflux activity, the cytoplasmic drug concentration in the cell is lowered to subtoxic levels and, hence, cells become multidrug resistant. The activity of multidrug transporters interferes with the drug-based control of tumours and infectious pathogenic microorganisms. There is an urgent need to understand the structure-function relationships in multidrug transporters that underlie their drug specificity and transport mechanism. Knowledge about the architecture of drug and modulator binding sites and the link between energy-generating and drug translocating functions of multidrug transporters may allow one to rationally design new drugs that can poison or circumvent the activity of these transport proteins. Furthermore, if one is to inhibit multidrug transporters in human cells, one should know more about their physiological substrates and functions. This review will summarize important new insights into the role that multidrug transporters in general, and P-glycoprotein and its bacterial homologue LmrA in particular, play in the physiology of the cell. In addition, the molecular basis of drug transport by these proteins will be discussed.     

Investigation of Ochratoxin A biosynthetic genes in<Emphasis Type="Italic">Penicillium verrucosum</Emphasis> by DDRT-PCR experiments: differential expression of OTA genes

A defined minimal medium has been developed which is either restrictive or permissive for the production of ochratoxin A byPenicillium verrucosum simply by changing the nitrogen and carbon source. The combination of ammonium/glycerin promoted, whereas the combination nitrate/glucose repressed ochratoxin production.In parallel mutants ofP verrucosum have been constructed by restriction endonuclease mediated integration, which were not able to produce ochratoxin. Different types of transformants, which either produce no detectable amounts of ochratoxin A but ochratoxin α, strains which produce only ochratoxin B and strains which produces none of these secondary metabolites, have been observed.     

Insulin and epidermal growth factor do not affect phosphoinositide metabolism in rat liver plasma membranes and hepatocytes

Recent studies with viral oncogene tyrosine kinases have suggested that these kinases may phosphorylate phosphoinositides and diacylglycerol. Since the receptors for insulin and epidermal growth factor (EGF) also possess tyrosine kinase activity, we have investigated possible effects of insulin and EGF on phosphoinositide metabolism in rat liver plasma membranes and rat hepatocytes. In plasma membranes prepared from rats injected 18 h prior with [3H]myo-inositol or incubated with [gamma-32P]ATP, phosphatidylinositol-4-P and phosphatidylinositol-4,5-P2 were formed, but there were no effects of either insulin or EGF although these agents stimulated protein tyrosine phosphorylation. In hepatocytes incubated with [3H]myo-inositol, label was incorporated into phosphatidylinositol, phosphatidylinositol-4-P, and phosphatidylinositol-4,5-P2, but there was no effect of insulin. Incubation of hepatocytes with [3H]myo-inositol plus insulin or EGF for 2 h also did not alter the formation of [3H]myo-inositol-1,4,5-P3 from [3H]phosphatidylinositol-4,5-P2 induced by vasopressin. These findings suggest that the tyrosine kinase activity of liver insulin and EGF receptors is not important in phosphoinositide formation.     

Characterization of responses of isolated rat hepatocytes to ATP and ADP

In isolated rat hepatocytes, ATP and ADP (10(-6) M) rapidly mobilize intracellular Ca2+ and increase the concentration of free cytosolic Ca2+ ([Ca2+]i) within 1-2 s. The increase in [Ca2+]i is maximal (2.5- to 3-fold) by about 10 s and is dose-dependent, with ATP and ADP being half-maximally effective at 8 X 10(-7) and 3 X 10(-7) M, respectively. At submaximal concentrations, the rise in [Ca2+]i is transient due to hydrolysis of the agonist. The increase in [Ca2+]i in response to ATP or ADP can be potentiated by low concentrations of glucagon (10(-9) M). In addition, the [Ca2+]i rise can be antagonized in a time- and dose-dependent manner by the tumor promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. Adenosine, at concentrations as high as 10(-4) M, does not alter [Ca2+]i. AMP is ineffective at 10(-5) M, but at 10(-4) M it increases [Ca2+]i approximately 1.5-fold after a 30-s lag and at a slow rate. Conversely, high concentrations (10(-4) M) of adenosine and AMP increases cell cAMP about 2- to 3-fold. ATP and ADP, at concentrations (10(-6) M) which near-maximally increase [Ca2+]i, do not affect hepatocyte cAMP. ATP and ADP increase the cellular level of myoinositol 1,4,5-trisphosphate (IP3), the putative second messenger for Ca2+ mobilization. The increase in IP3 is dose-dependent and precedes or is coincident with the [Ca2+]i rise. There is an approximate 20% increase in IP3 with concentrations of ATP or ADP which near-maximally induce other physiological responses. It is concluded that submicromolar concentrations of ATP and ADP mobilize intracellular Ca2+ and activate phosphorylase in hepatocytes due to generation of IP3. These effects may involve P2-purinergic receptors. In contrast adenosine and AMP interact with P1 (A2)-purinergic receptors to increase cAMP.     

The significance of sedoheptulose 1,7-bisphosphate in the metabolism and regulation of the pentose pathway in liver

Rat liver cytosolic enzyme preparation catalyses the formation of sedoheptulose 1,7-P2 (60% of total heptulose-P formed) from hexose 6-P and triose 3-P (reverse mode of pentose pathway operation). Smaller amounts of sedoheptulose 1,7-P2 are also formed from ribose 5-P during the non-oxidative synthesis of hexose 6-P (forward pentose pathway operation). The apparent absence of erythrose 4-P in biological systems may be explained by its contribution to carbons 4,5,6 and 7 of sedoheptulose 1,7-P2 as well as its pronounced ability to exist in dimeric form. Apart from the aldolase catalyzed formation of sedoheptulose 1,7-P2, 6-phosphofructokinase also catalyses its formation from sedoheptulose 7-P and fructose 1,6-bisphosphatase catalyses its dephosphorylation. These three enzymes may contribute to the regulation of carbon flux through the near equilibrium reactions of the non-oxidative pentose phosphate pathway in vivo. The phosphotransferase enzyme of the L-type pentose pathway is also able to catalyse the interconversion of sedoheptulose mono and bisphosphates via D-glycero D-ido octulose-P.     

Kinetic studies on the nitrite reductase of Wolinella succinogenes.

Calibration relationships were derived for cartilage proteoglycan subunit (PGS) that relate the inverse z-average hydrodynamic radius (Rs) and the weight-average Mr (Mw) to the partition coefficient (Kav.) for PGS when chromatographed on a Sepharose CL-2B column. PGS isolated from chick limb-bud chondrocyte cell cultures was fractionated chromatographically into eight pools, for which Mw and Rs were determined by total-intensity and dynamic light-scattering measurements. These data were found to be related to Kav. through the following empirical equations: log Mw = -(1.65 +/- 0.27)Kav. +(6.58 +/- 0.08); log Rs = -(0.69 +/- 0.04)Kav. +(2.75 +/- 0.01). Application of these relationships to the chromatographic data led to Mw = 1.48 X 10(6) and Rs = 38.7 nm (387 A) for the unfractionated specimens compared with values of Mw = 1.46 X 10(6) and Rs = 38.2 nm (382 A) determined by light-scattering. Our results were found to be consistent with previously proposed phenomenological models for the gel-filtration mechanism. Application of these calibration relationships to Kav. for several unfractionated specimens led to predicted values of Mw and Rs that are accurate to within 10%.     

Agonist-induced down-regulation of the angiotensin II receptor in primary cultures of rat hepatocytes

The ability of angiotensin II to down-regulate its receptor was tested on rat hepatocytes in primary culture for 4 h. Angiotensin II treatment decreased [3H]angiotensin II specific binding in a concentration- and time-dependent manner. The effect was maximum with 1 microM angiotensin II and after 2 h. There was a decrease in the maximum number of binding sites (56% of control) with no significant effect on the apparent dissociation constant. The down-regulation was blocked by the angiotensin II antagonist [Val4,Ile7]angiotensin III and was not induced by other hormones (e.g. vasopressin, norepinephrine, or glucagon) or by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate or A23187 ionophore. The decrease in angiotensin II receptors resulted in correlated decreases in the potency of angiotensin II to activate phosphorylase or lower glucagon-induced cAMP accumulation. However, high concentrations of the agonist were still able to elicit maximal responses in both parameters. Down-regulation of the receptor was not dependent upon active Gi, since it was still observed after ADP-ribosylation and inactivation of Gi by pertussis toxin. The above results indicate that the down-regulation of the hepatic angiotensin II receptor induced by its agonist is homologous and does not involve Gi, Ca2+, or protein kinase C. The correlation of receptor loss with decreases in the potency of angiotensin to activate phosphorylase and inhibit glucagon-induced cAMP accumulation is consistent with the idea that a single receptor population regulates two different messengers, i.e. calcium and cAMP.     

Characterization of the angiotensin II receptor in primary cultures of rat hepatocytes. Evidence that a single population is coupled to two different responses

The angiotensin II receptor of cultured rat hepatocytes was characterized using [3H]angiotensin II as radioligand. Binding at 23 degrees C was rapid (t1/2 = 0.65 min) with equilibrium being reached in 10-12 min. At this time, binding was completely reversible after 20 min (t1/2 = 3.5 min), indicating negligible internalization of the ligand. Analysis of the saturation binding curve showed one population of binding sites with an apparent KD of 8.6 nM and a Bmax of 35 fmol/mg of protein. The time courses of association and dissociation were also consistent with one class of binding sites with an apparent kinetically derived KD of 7.7 nM. The order of potency of different agonists and antagonists to increase cytosolic Ca2+ or phosphorylase a or inhibit the effects of angiotensin II on these parameters was the same as for their mimicry or reversal of angiotensin II inhibition of glucagon-induced cAMP accumulation, and was well correlated with their order of potency to inhibit angiotensin II specific binding. Treatment of cultured hepatocytes with dithiothreitol caused a time- and concentration-dependent inhibition of angiotensin II binding and corresponding alterations of angiotensin II effects on phosphorylase and cAMP. It also inhibited the actions of other hormones on phosphorylase. These results indicate that hepatocytes contain a homogeneous population of angiotensin II receptors that are coupled to two different biological effects apparently mediated by different G-proteins.