Isolation of the vitellogenin-binding protein from locust ovaries

A rapid, efficient procedure for the isolation and purification of the vitellogenin binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl-β-D-glucoside and removal of the detergent, the binding protein is subjected to affinity chromatography on vitellogenin coupled covalently to Affi-Gel 15. The binding protein is eluted with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be identified as the vitellogenin binding protein. It retains its high-affinity binding properties. The specific binding of vitellogenin increases from 4.8 μg (intact ovarian membranes) to 170.9 μg (affinity purified binding protein) per mg membrane protein, which corresponds to a purification factor of 35.     

5-Hydroxyindoleacetic acid and homovanillic acid concentrations in cerebrospinal fluid in patients with Alzheimer's disease, depression and mild cognitive impairment

In this study we investigated the cerebrospinal fluid (CSF) concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) in Alzheimer (AD) patients (n=75), patients with mild cognitive impairment (MCI, n=9) and patients with depression (n=7). CSF HVA was significantly elevated in AD with depression (Geriatric Depression Scale, 15 point version GDS>5) in comparison to AD without depression (p<0.05, ANOVA) and CSF HVA showed a significant positive correlation with the GDS score of AD-patients (p=0.03, Spearman Rho: 0.38, Spearman Rank Correlation). In the group of AD patients CSF 5-HIAA was positively correlated with cerebrospinal fluid beta-amyloid 1-42 (Abeta42), p<0.05, Spearman Rho: 0.3, Spearman Rank Correlation, but not with CSF tau. Additionally, there was a significant positive correlation between cerebrospinal fluid 5-HIAA and HVA in the group of AD patients (p<0.0001, Rho: 0.47, Spearman Rank correlation). Neither 5-HIAA nor HVA in CSF could differentiate between mild cognitive impairment, depression and AD. The results of this study support the hypothesis that the serotonergic system plays a role in the course of AD. They further suggest an important role of dopamine metabolism in depression within AD patients.     

Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Specific activation of Gsalpha by an NDPK B.Gbetagamma complex in H10 cells

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.     

Plasma concentrations of 5-HT, 5-HIAA,norepinephrine, epinephrine and dopamine in ecstasy users

Recreational use of the synthetic methamphetamine derivative MDMA (3,4-methylenedioxymethamphetamine), the main constituent of the illegal drug "ecstasy", has increased dramatically in recent years. The reasons for ecstasy-associated cardiovascular complications like tachycardia, arrhythmias and hypertensive crises and psychiatric symptoms like psychotic episodes are not well understood. We have measured the plasma concentrations of 5-HIAA, 5-HT, norepinephrine, epinephrine and dopamine in 159 ecstasy users and controls. Ecstasy users showed elevated resting sympathetic activity, reflected in increased norepinephrine, epinephrine and dopamine levels. The levels of these catecholamines correlated positively with the cumulative dose and also with consumption during the last 30 days and 12 months. Although it is known that significant changes in 5-HT and 5-HIAA appear in the cerebrospinal fluid in ecstasy users, we could not detect alterations in serotonergic neurotransmitters in plasma in this large sample of subjects. Thus, in the drug-free interval, ecstasy users show lowered central serotonergic activity (lowered 5-HT and 5-HIAA concentrations in CSF) along with unchanged central noradrenergic and dopaminergic activity (HVA and MHPG unchanged in CSF) and elevated peripheral noradrenergic, dopaminergic and adrenergic activity along with unchanged peripheral serotonergic activity (plasma levels). We conclude, that the data presented here could argue for a noradrenergic hyperreactivity in the drug-free interval in ecstasy users resulting from previous ecstasy consumption. Also for an association with psychotic episodes and cardiovascular complications like tachycardia, arrhythmias.     

Prolonged blood glucose reduction in mrp-2 deficient rats (GY/TR(-)) by the glucose-6-phosphate translocase inhibitor S 3025

Chlorogenic acid derivatives are potent inhibitors of hepatic glucose production by inhibition of the glucose-6-phosphate translocase component of the hepatic glucose-6-phosphatase system. The pharmacological proof of concept was clearly demonstrated during i.v. infusion of potent derivatives (S 4048, S 3483) in rats. However, the blood glucose lowering effect of S 4048 after bolus i.v. injection lasted only 60-90 min. Plasma clearance of S 4048 was very high, and the parent compound was rapidly and efficiently excreted into the bile of Wistar and GY/TR(-) rats, indicating that mrp-2 was not involved in this hepatobiliary elimination process. About 72% of the total administered radioactivity appeared in the bile within 20 min after i.v. bolus injection of the radiolabeled analogue [(3)H]S 1743 in a Wistar rat. However, in GY/TR(-) rats the dicarboxylic analogue of S 4048, S 3025, was cleared from the plasma less rapidly than its parent compound and its biliary elimination was comparatively low. In contrast, S 3025 exhibited comparable pharmacokinetics and biliary elimination profile as S 4048 in Wistar rats, suggesting that biliary elimination of S 3025 is facilitated by mrp-2, functionally absent in GY/TR(-) rats. Targeting to mrp-2 resulted in a significantly prolonged reduction of blood glucose levels in GY/TR(-) rats after i.v. bolus administration of S 3025.     

Plasma beta carotene in Alzheimer's disease. Association with cerebrospinal fluid beta-amyloid 1-40, (Abeta40), beta-amyloid 1-42 (Abeta42) and total Tau

We studied the plasma beta carotene concentrations in 40 Alzheimer's disease patients and the association with cerebrospinal fluid beta-amyloid 1-40, (Abeta40), cerebrospinal fluid beta-amyloid 1-42 (Abeta42) and cerebrospinal fluid total Tau. We found that patients with plasma beta carotene levels below the 25th percentile had 55% reduced ratios of Abeta40/Tau and 51% reduced ratios of Abeta 40/Abeta 42 compared with patients in the highest quartile. Mean Tau concentrations in the lowest quartile of plasma beta-carotene levels were 74% higher compared with the highest quartile of plasma beta-carotene levels. Thus, we could demonstrate an statistically significant association between beta carotene levels in plasma and neurochemical markers in the cerebrospinal fluid of Alzheimer's disease patients.     

High energy phosphate transfer by NDPK B/Gβγcomplexes - an alternative signaling pathway involved in the regulation of basal cAMP production

The activation of heterotrimeric G proteins induced by G protein coupled receptors (GPCR) is generally believed to occur by a GDP/GTP exchange at the G protein α -subunit. Nevertheless, nucleoside diphosphate kinase (NDPK) and the β-subunit of G proteins (Gβ) participate in G protein activation by phosphate transfer reactions leading to the formation of GTP from GDP. Recent work elucidated the role of these reactions. Apparently, the NDPK isoform B (NDPK B) forms a complex with β; γ; dimers in which NDPK B acts as a histidine kinase phosphorylating G#x03B2; at His266. Out of this high energetic phosphoamidate bond the phosphate can be transferred specifically onto GDP. The formed GTP binds to the G protein α -subunit and thus activates the respective G protein. Evidence is presented, that this process occurs independent of the classical GPCR-induced GTP/GTP exchange und thus contributes, e.g. to the regulation of basal cAMP synthesis in cells.     

Vitellogenin Titres in Normal and Accelerated Maturation of Gregarious-Phase Schistocerca gregaria

Isolation of vitellogenin of the Schistocerca gregaria (Forskal) in its gregarious phase was achieved by a combination of gel permeation and anion exchange chromatography. Staining for carbohydrate and lipid moieties showed that the vitellogenin is a glycolipoprotein. The vitellogenin of S. gregaria has a native molecular weight of about 700 kDa. On SDS-PAGE, the protein showed nine apoproteins of about 124, 120, 105, 60, 59, 58, 57, 53 and 34 kD. Determination of the levels of vitellogenin by ELISA in the haemolymph of maturing females showed that those exposed to mature males from 1 to 2 days after ecdysis had increased levels of vitellogenin from day 10 (81.1 ± 4.5). In contrast, females exposed to immature males or kept alone showed an increase (107.3 ± 0.9 and 70.2 ± 2.7) not until day 16 or later, respectively. These results are consistent with the accelerating effect of pheromonal emissions from mature males on the maturation of female S. gregaria.     

Body Building on Diamonds

Whereas conservative therapies aim to stall the advance of disease,regenerative medicine strives to reverse it.The capacityof most tissues to regenerate derives from stem cells,but there are a number of barriers which have to be circumvented before itwill be possible to use stem-cell-based therapies.Such therapies,however,are expected to improve human health enormously,and knowledge gained from studying stem cells in culture and in model organisms is now laying the groundwork for a new eraof regenerative medicine.One of the most prominent methods to study stem cell differentiation is to let them to form embryoidbodies.Under favourable conditions any stem cell line will form embryoid bodies.However,the mechanism of the formation ofembryoid bodies is not very well understood,and to produce them in the laboratory is in no way trivial-an important technicalbarrier in stem cell research.Recently,the embryoid body cultivation step has been successfully circumvented for the derivationof osteogenic cultures of embryonic stem cells.Here we report on a simple and reusable system to cultivate embryoid bodies inextremely short times.The method is inspired by the principles that lead to the establishment of the biomimetic triangle.