A convenient method for saponin isolation in tumour therapy

Saponinum album (Merck), which is a crude mixture of saponins from Gypsophila paniculata L., was shown to improve the anti cancer therapy when used in vivo in combination with saporin-based targeted toxins. Unfortunately saponinum album cannot be used for further development since Merck has ceased its production in the 1990s. As pure saponins are mandatory for use in medical purposes we developed a convenient method for saponin isolation directly from the roots of Gypsophila paniculata L. The developed method is rapid, cheap and scaling up is also possible. By combining dialysis and HPLC three saponins were isolated in a one-step procedure. Chemical structures of the purified saponins were characterized by extensive one and two-dimensional NMR-spectroscopy and by using ESI-TOF-MS. The biological activities of the purified saponins were also investigated. The method presented herein enabled a rapid and cheap isolation of saponins for tumour therapy.     

Phosphoinositide 3-kinase γ has Multiple Phospholipid Binding Sites

Phosphoinositide 3-kinase γ is a multifunctional enzyme with lipid and protein kinase activities that also acts as a scaffold protein in many diverse signalling processes. The enzyme contains five different domains, but their individual contributions to membrane binding are not fully understood. Here, using in vitro liposome binding assays of individual domains and deletion constructs of human phosphoinositide 3-kinase γ, we show that each domain is capable of binding anionic phospholipids to varying degrees, depending on the charge of the anionic substrate. Moreover, with the exception of the C2-domain, deletion of any single protein domain results in a complete loss of kinase activity toward both lipids and proteins.     

Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, β-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.     

Characterization of rhesus macaque KIR genotypes and haplotypes

Certain combinations of the killer immunoglobulin-like receptors (KIR) and major histocompatibility complex class I ligands in humans predispose carriers to a variety of diseases, requiring sophisticated genotyping of the highly polymorphic and diverse KIR and HLA genes. Particularly, KIR genotyping is challenging due to polymorphisms (allelic substitutions), genomic diversity (presence/absence of genes), and frequent duplications. Rhesus macaques are often used as important animal models of human diseases such as, e.g. AIDS. However, typing of rhesus macaque KIR genes has not been described so far. In this study, we report the identification of additional novel rhesus macaque KIR cDNA sequences and a sequence-specific KIR genotyping assay. From a cohort of four rhesus macaque families with a total of 70 individuals, we identified 25 distinct KIR genotypes. Segregation analyses of KIR genes and of two polymorphic microsatellite markers allowed the identification of 21 distinct KIR haplotypes in these families, with five to 11 segregating KIR genes per haplotype. Our analyses confirmed and extended knowledge on differential gene KIR gene content in macaques and indicate that rhesus macaque and human KIR haplotypes show a comparable level of diversity and complexity.     

Homocysteine and vitamin therapy in stroke prevention and treatment: a review

Homocysteine (Hcy), a sulfur amino acid, is the only direct precursor for L-methionine synthesis through a reaction that requires vitamin B??, representing a connection with "one-carbon" units metabolism. Hcy catabolism requires vitamin B? and as a consequence, alteration in folic acid and B vitamins status impairs Hcy biotransformation. Numerous studies have indicated that Hcy is an independent risk factor for cardio- and cerebrovascular diseases. In the last decade, several clinical trials have investigated the possible correlation between the use of folic acid and vitamins B? and B?? for lowering Hcy plasma concentration and the reduced risk of stroke or its recurrence. This review is aimed to present some aspects of Hcy biochemistry, as well as the mechanisms through which it exerts the toxic effects on the vascular endothelium. We also discuss the results of some of the clinical trials developed to investigate the beneficial effects of vitamin therapy in the prevention and management of stroke.     

Deducing corticotropin-releasing hormone receptor type 1 signaling networks from gene expression data by usage of genetic algorithms and graphical Gaussian models

Background  

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a hallmark of complex and multifactorial psychiatric diseases such as anxiety and mood disorders. About 50-60% of patients with major depression show HPA axis dysfunction, i.e. hyperactivity and impaired negative feedback regulation. The neuropeptide corticotropin-releasing hormone (CRH) and its receptor type 1 (CRHR1) are key regulators of this neuroendocrine stress axis. Therefore, we analyzed CRH/CRHR1-dependent gene expression data obtained from the pituitary corticotrope cell line AtT-20, a well-established in vitro model for CRHR1-mediated signal transduction. To extract significantly regulated genes from a genome-wide microarray data set and to deduce underlying CRHR1-dependent signaling networks, we combined supervised and unsupervised algorithms.     

Effect of nitrate supply and mycorrhizal inoculation on characteristics of tobacco root plasma membrane vesicles

Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10–15 mM nitrate.     

Accelerated hand bone mineral density loss is associated with progressive joint damage in hands and feet in recent-onset rheumatoid arthritis

Introduction

To investigate whether accelerated hand bone mineral density (BMD) loss is associated with progressive joint damage in hands and feet in the first year of rheumatoid arthritis (RA) and whether it is an independent predictor of subsequent progressive total joint damage after 4 years.

Methods

In 256 recent-onset RA patients, baseline and 1-year hand BMD was measured in metacarpals 2-4 by digital X-ray radiogrammetry. Joint damage in hands and feet were scored in random order according to the Sharp-van der Heijde method at baseline and yearly up to 4 years.

Results

68% of the patients had accelerated hand BMD loss (>-0.003 g/cm²) in the first year of RA. Hand BMD loss was associated with progressive joint damage after 1 year both in hands and feet with odds ratios (OR) (95% confidence intervals [CI]) of 5.3 (1.3-20.9) and 3.1 (1.0-9.7). In univariate analysis, hand BMD loss in the first year was a predictor of subsequent progressive total joint damage after 4 years with an OR (95% CI) of 3.1 (1.3-7.6). Multivariate analysis showed that only progressive joint damage in the first year and anti-citrullinated protein antibody positivity were independent predictors of long-term progressive joint damage.

Conclusions

In the first year of RA, accelerated hand BMD loss is associated with progressive joint damage in both hands and feet. Hand BMD loss in the first year of recent-onset RA predicts subsequent progressive total joint damage, however not independent of progressive joint damage in the first year.     

Proteomic analysis of zymogen granules

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging and regulated apical secretion of digestive enzymes. ZG constituents play important roles in pancreatic injury and disease. The molecular mechanisms underlying these processes are still poorly defined. Thus, there is currently great interest in the identification and characterization of ZG components. Recent proteomic studies have greatly enhanced our knowledge regarding potential new 'players' in ZG biogenesis and regulated secretion. In this article, we present the latest advancements in and insights into the analysis of the ZG proteome by the combination of organelle isolation, protein separation, mass spectrometry and validation of protein identification. Recent developments in the analysis of ZG proteins from pancreatic juice and related proteins from saliva are also discussed.