Discovering orphans' sweet secret: NR4A receptors and hepatic glucose production

Aberrant hepatic gluconeogenesis contributes importantly to hyperglycemia in type II diabetic patients. A study by Pei et al. (2006b) identifies NR4A orphan nuclear receptors as a novel branch of cAMP-dependent regulators of hepatic glucose production under healthy and diabetic conditions.     

Comparison of ELISA and capillary electrophoresis with laser-induced fluorescence detection in the analysis of Ochratoxin A in low volumes of human blood serum

In this paper the determination of Ochratoxin A (OTA) in low volumes of human blood serum by enzyme-linked immunosorbent assay (ELISA) is compared with an appropriate capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) method. In order to use ELISA for high-throughput analysis in epidemiological studies no sample cleanup was performed. Both methods showed a limit of detection (LOD) of 0.5 ng/mL. Comparing the precisions of both methods, the data show that the quantified concentrations in ELISA are higher than the corresponding concentrations in the CE-LIF method. Using a matrix calibration curve instead of a standard calibration curve the reproducibilities of both methods are comparable. No additional matrix effect could be observed by adding phenylalanine as probable matrix compound to the serum.     

Strain-dependent regulation of neurotransmission and actin-remodelling proteins in the mouse hippocampus

Individual mouse strains differ significantly in terms of behaviour, cognitive function and long-term potentiation. Hippocampal gene expression profiling of eight different mouse strains points towards strain-specific regulation of genes involved in neuronal information storage. Protein expression with regard to strain- dependent expression of structures related to neuronal information storage has not been investigated yet. Herein, a proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) has been chosen to address this question by determining strain-dependent expression of proteins involved in neurotransmission and activity-induced actin remodelling in hippocampal tissue of five mouse strains. Of 31 spots representing 16 different gene products analysed and quantified, N-ethylmaleimide-sensitive fusion protein, N-ethylmaleimide-sensitive factor attachment protein-alpha, actin-like protein 3, profilin and cofilin were expressed in a strain-dependent manner. By treating protein expression as a phenotype, we have shown significant genetic variation in brain protein expression. Further experiments in this direction may provide an indication of the genetic elements that contribute to the phenotypic differences between the selected strains through the expressional level of the translated protein. In view of this, we propose that proteomic analysis enabling to concomitantly survey the expression of a large number of proteins could serve as a valuable tool for genetic and physiological studies of central nervous system function.     

Comparative proteomics of high light stress in the model alga Chlamydomonas reinhardtii

High light (HL) stress adversely affects growth, productivity and viability of photosynthetic organisms. The green alga Chlamydomonas reinhardtii is a model system to study photosynthesis and light stress. Comparative proteomics of wild-type and two very high light (VHL)-resistant mutants, VHL(R)-S4 and VHL(R)-S9, revealed complex alterations in response to excess light. A two-dimensional reference map of the soluble subproteome was constructed representing about 1500 proteins. A total of 83 proteins from various metabolic pathways were identified by peptide mass fingerprinting. Quantitative comparisons of 444 proteins showed 105 significantly changed proteins between wild type and mutants under different light conditions. Commonly, more proteins were decreased than increased, but different proteins were affected in each genotype. Proteins uniquely altered in either VHL(R) mutant may be involved in VHL resistance. Such candidate proteins similarly altered without light stress, thus possibly contributing to "pre-adaptation" of mutants to VHL, included decreased levels of a DEAD box RNA helicase (VHL(R)-S4) and NAB1 and RB38 proteins (VHL(R)-S9), and increased levels of an oxygen evolving enhancer 1 (OEE1) isoform and an unknown protein (VHL(R)-S4). Changes from increased levels in HL to decreased levels in excess light, included OEE1 (VHL(R)-S9) or the reverse change for NAB1, RB38, beta-carbonic anhydrase and an ABC transporter-like protein (VHL(R)-S4).     

Comparison of metabolic risk factors between severely and very severely obese patients

Objective: A prospective clinical intervention study was performed to estimate the metabolic risk factors in patients with very severe obesity (VSO) vs. severe obesity (SO). Research Methods and Procedures: Two hundred twenty‐eight VSO (BMI ≥ 50 kg/m²) and 221 SO patients (BMI = 40 to 49.9 kg/m²) participated in the study (367 women and 82 men). Metabolic measurements included plasma lipids, glucose and insulin, hemoglobin A_1c, leptin, and sex hormones, as well as hepatic steatosis in a subgroup of patients. Subgroups of patients with non–insulin‐dependent diabetes and hyperlipidemia (HLP) were examined. Results: The most unexpected result of our study was that VSO men showed significantly better lipid profiles than SO men. Furthermore, 18% of VSO men had no metabolic aberrations, whereas all SO men did. The advantageous metabolic status of VSO men was associated with sex hormone changes that favor gynoid fat distribution. The beneficial metabolic situation with VSO seems to be sex specific for men. Discussion: This study shows that the metabolic situation in VSO is not more severe than in the less obese cohort. These findings distinctly differ from the positive associations that have previously been reported between BMI, lipids, and other metabolic indices among individuals whose BMI is <40 kg/m².     

Inhibition of protein kinase A in murine enteric neurons causes lethal intestinal pseudo‐obstruction

A number of in vitro studies suggest that many important developmental and functional events in the enteric nervous system are regulated by the intracellular signaling enzyme cAMP protein kinase A (PKA). To evaluate the in vivo significance of these observations, a Cre‐inducible, dominant‐negative, mutant regulatory subunit (RIαB) of PKA was activated in enteric neurons by either a Proteolipid protein‐Cre transgene or a Hox11L1‐Cre “knock‐in” allele. In both models, RIαB activation resulted consistently in profound distension of the proximal small intestine within 2 weeks after birth. Intestinal transit of radio‐opaque tracers was severely retarded in the double‐transgenic animals, which died shortly after weaning. In the enteric nervous system, recombination was restricted to neurons as demonstrated by histochemical analysis and confocal microscopic colocalization of a Cre recombinase‐dependent reporter gene with the neuronal marker Hu(C/D), in contrast with the glial marker S100. Histochemical analysis of β‐galactosidase expression and acetylcholinesterase activity, as well as neuronal counts, demonstrated that intestinal dysmotility was not associated with obvious malformation of the myenteric plexus. However, inhibition of PKA activity in enteric neurons disrupted the major motor complexes of isolated intestinal segments in vitro. These results provide strong evidence that PKA activity plays a critical role in enteric neurotransmission in vivo, and highlight neuronal PKA or related signaling molecules as potential therapeutic targets in gastrointestinal motility disorders. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

Generation of a conditional null allele of jumonji

The jumonji (jmj) gene plays important roles in multiple organ development in mouse, including cardiovascular development. Since JMJ is expressed widely during mouse development, it is essential that conditional knockout approaches be employed to ablate JMJ in a tissue-specific manner to identify the cell lineage specific roles of JMJ. In this report, we describe the establishment of a jmj conditional null allele in mice by generating a loxP-flanked (floxed) jmj allele, which allows the in vivo ablation of jmj via Cre recombinase-mediated deletion. Gene targeting was used to introduce loxP sites flanking exon 3 of the jmj allele to mouse embryonic stem cells. Our results indicate that the jmj floxed allele converts to a null allele in a heart-specific manner when embryos homozygous for the floxed jmj allele and carrying the alpha-myosin heavy chain promoter-Cre transgene were analyzed by Southern and Northern blot analyses. Therefore, this mouse line harboring the conditional jmj null allele will provide a valuable tool for deciphering the tissue and cell lineage specific roles of JMJ.     

Should symptomatic menopausal women be offered hormone therapy?

Many physicians remain uncertain about prescribing hormone therapy for symptomatic women at the onset of menopause. The American Society for Reproductive Medicine (ASRM) convened a multidisciplinary group of healthcare providers to discuss the efficacy and risks of hormone therapy for symptomatic women, and to determine whether it would be appropriate to treat women at the onset of menopause who were complaining of menopausal symptoms. MAJOR FINDINGS: Numerous controlled clinical trials consistently demonstrate that hormone therapy, administered via oral, transdermal, or vaginal routes, is the most effective treatment for vasomotor symptoms. Topical vaginal formulations of hormone therapy should be preferred when prescribing solely for the treatment of symptoms of vulvar and vaginal atrophy. Data from the Women's Health Initiative indicate that the overall attributable risk of invasive breast cancer in women receiving estrogen plus progestin was 8 more cases per 10,000 women-years. No increased risk for invasive breast cancer was detected for women who never used hormone therapy in the past or for those receiving estrogen only. Hormone therapy is not effective for the treatment of cardiovascular disease and that the risk of cardiovascular disease with hormone therapy is principally in older women who are considerably postmenopause. CONCLUSIONS: Healthy symptomatic women should be offered the option of hormone therapy for menopausal symptoms. Symptom relief with hormone therapy for many younger women (at the onset of menopause) with menopausal symptoms outweighs the risks and may provide an overall improvement in quality of life. Hormone therapy should be individualized for symptomatic women. This involves tailoring the regimen and dose to individual needs.