Activation and induction of glycine N-methyltransferase by retinoids are tissue- and gender-specific

Betaine-homocysteine S-methyltransferase (BHMT; EC2.1.1.5) is a zinc metalloenzyme that catalyzes the transfer of a methyl group from betaine to homocysteine to produce dimethylglycine and Met, respectively. This enzyme is a member of a family of zinc-dependent methyltransferases that use thiols or selenols as methyl acceptors and which contain the following motif: G[ILV]NCX(20, 100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I. We recently reported that the three cysteine residues within this motif function as ligands to zinc in BHMT because changing any of them to alanine abolished zinc-binding and enzyme activity (A. P. Breksa, III, and T. A. Garrow, 1999, Biochemistry 38, 13991-13998). To determine if other amino acid residues in this motif were critical for enzyme function, the two regions defined by the motif in human BHMT, GVNCH(218) and VRYIGGCCGFEPYHI(307), were subjected to semirandom and random site-directed mutagenesis. Mutant enzymes were classified as either active or inactive based on their ability to complement the Met auxotrophy of Escherichia coli strain J5-3. The Gly residue at position 214 was found to be absolutely essential for complementation. The positions occupied by Gly297, Gly298, and Gly301 favored substitutions of small amino acids like Ala and Ser. We hypothesize that these Gly residues provide the necessary flexibility to the Zn-binding region to permit coordination of the metal.     

125I[Sar(1)Ile(8)] angiotensin II has a different affinity for AT(1) and AT(2) receptor subtypes in ovine tissues

Iodinated angiotensin II (Ang II) and its analogues are often assumed to have equal affinities for AT(1) and AT(2) receptor subtypes. However, using saturation and competition binding assays in several tissues from pregnant, nonpregnant, and fetal sheep, we found the affinity of 125I[Sar(1)Ile(8)] Ang II for Ang II receptors was different (P<0.05) between tissue types. The dissociation constants (Kd) and half maximal displacements of [Sar(1)Ile(8)] Ang II (Sar IC(50)) were directly related (P<0.05) to proportions of AT(1) receptors, and inversely related (P<0.05) to proportions of AT(2) receptors in tissues from all groups combined, in tissues from individual groups (pregnant, nonpregnant or fetal), and in some individual tissues (uterine arteries and aortae). This suggests that 125I[Sar(1)Ile(8)] Ang II has a different affinity for AT(1) and AT(2) receptors in ovine tissues. The Kds of 125I[Sar(1)Ile(8)] Ang II for "pure" populations of AT(1) and AT(2) receptors were 1.2 and 0.3 nM, respectively, i.e. affinity was four-fold higher for AT(2) receptors. We corrected the measured proportions of the receptor subtypes using their fractional occupancies. In tissues which contained at least 10% of each receptor subtype, the corrected proportions were significantly altered (P<0.05), even in some tissues, to the extent of being reversed.     

The effect of captivity and diet on KLH isoform ratios in Megathura crenulata

Aquaculture of the giant keyhole limpet, Megathura crenulata, may provide a reliable long-term supply of keyhole limpet hemocyanin (KLH) for many promising biomedical applications. However, previous studies have reported a complete loss of the KLH1 isoform under certain cultivation conditions. We examined whether captivity per se and diet caused a significant change in the isoform profile of M. crenulata. Although there was a trend toward a decreasing percentage of KLH1 in some animals, in general isoform profiles were not significantly affected by captivity or dietary limitations. Further, the percentage of KLH1 significantly increased for limpets with previously low levels of KLH1 when fed a supplemental mixed diet. Our results indicate that normal isoform profiles can be maintained in limpets held in captivity even when fed insufficient diets, and that these conditions do not cause a complete loss of either KLH isoform. Notably, the enhancement of abnormally low levels of KLH1 suggests that variability in isoform profiles could potentially be minimized through diet. While there is a need for further research on the factors responsible for the variability of KLH, overall, these results support the premise that culture of M. crenulata may provide a sustainable source of this biomedically important product.     

Chronic ethanol exposure increases the binding of HuR to the TNFalpha 3'-untranslated region in macrophages

Tumor necrosis factor alpha (TNFalpha) expression is a key mediator of ethanol-induced liver disease. Increased lipopolysaccharide (LPS)-stimulated TNFalpha expression in macrophages after chronic ethanol feeding is associated with a stabilization of TNFalpha mRNA (Kishore, R., McMullen, M. R., and Nagy, L. E. (2001) J. Biol. Chem. 276, 41930-41937). Here we show that the 3'-UTR of murine TNFalpha mRNA was sufficient to mediate increased LPS-stimulated expression of a luciferase reporter in RAW 264.7 macrophages after chronic ethanol exposure. Further, we show that HuR, a nuclear/cytoplasmic shuttling protein, which binds to TNFalpha mRNA, is required for increased expression of TNFalpha after chronic ethanol. In Kupffer cells, HuR was primarily localized to the nucleus and then translocated to the cytosol in response to LPS in both pair- and ethanol-fed rats. After chronic ethanol feeding, HuR quantity in the cytosol was greater, both at baseline and in response to LPS, compared with pair-fed controls. Using RNA gel shift assays, we found that LPS treatment increased HuR binding to the 65-nucleotide A + U-rich element of the TNFalpha 3'-UTR by 2-fold over baseline in Kupffer cells from pair-fed rats. After chronic ethanol feeding, HuR binding to the TNFalpha A + U-rich element was increased by more than 5-fold at baseline and in response to LPS, compared with pair-fed controls. Down-regulation of HuR expression by RNA interference prevented the chronic ethanol-induced increase in expression of luciferase reporters containing the TNFalpha 3'-UTR. Taken together, these data demonstrate that increased binding of HuR to the TNFalpha 3'-UTR contributes to increased LPS-stimulated TNFalpha expression in macrophages after chronic ethanol exposure.     

Development of an integrated laboratory information management system for the maize mapping project

MOTIVATION: The development of an integrated genetic and physical map for the maize genome involves the generation of an enormous amount of data. Managing this data requires a system to aid in genotype scoring for different types of markers coming from both local and remote users. In addition, researchers need an efficient way to interact with genetic mapping software and with data files from automated DNA sequencing. They also need ways to manage primer data for mapping and sequencing and provide views of the integrated physical and genetic map and views of genetic map comparisons. RESULTS: The MMP-LIMS system has been used successfully in a high-throughput mapping environment. The genotypes from 957 SSR, 1023 RFLP, 189 SNP, and 177 InDel markers have been entered and verified via MMP-LIMS. The system is flexible, and can be easily modified to manage data for other species. The software is freely available. AVAILABILITY: To receive a copy of the iMap or cMap software, please fill out the form on our website. The other MMP-LIMS software is freely available at http://www.maizemap.org/bioinformatics.htm.     

Donor cell transplantation for myocardial disease: does it complement current pharmacological therapies?

Heart failure secondary to ischemic heart disease, hypertension, and myocardial infarction is a common cause of death in developed countries. Although pharmacological therapies are very effective, poor prognosis and shorter life expectancy of heart disease patients clearly indicate the need for alternative interventions to complement the present therapies. Since the progression of heart disease is associated with the loss of myocardial cells, the concept of donor cell transplantation into host myocardium is emerging as an attractive strategy to repopulate the damaged tissue. To this end, a number of donor cell types have been tested for their ability to increase the systolic function of diseased hearts in both experimental and clinical settings. Although initial clinical trials with bone marrow stem cells are encouraging, long-term consequences of such interventions are yet to be rigorously examined. While additional laboratory studies are required to address several issues in this field, there is also a clear need for further characterization of drug interactions with donor cells in these interventions. Here, we provide a brief summary of current pharmacological and cell-based therapies for heart disease. Further, we discuss the potential of various donor cell types in myocardial repair, mechanisms underlying functional improvement in cell-based therapies, as well as potential interactions between pharmacological and cell-based therapies.     

Determination of a clinical value for the repair half-time (T1/2) of the trigeminal nerve based on outcome data from gamma knife radiosurgery for facial pain

Stereotactic radiosurgery (GKRS) using the Leksell Gamma Knife is a treatment option for patients with trigeminal pain. We analyzed a database of 326 GKRS procedures performed over 4.6 years at three discrete dose levels commonly described in the published literature. Logistic regression was used to model the logit of response as a function of treatment time. The resulting coefficient was converted to an estimated probability of response for the shortest and longest treatment times in clinical practice. The two estimated probabilities were then compared to yield the estimated difference in the biologically effective dose (BED) between the two doses, using a modified linear-quadratic model for stereotactic radiosurgery. This difference was used to back-calculate a clinical value for T(1/2), resulting in a range of 1.28-1.77 h for T(1/2). The biological model appeared to accurately predict that, given the doses and treatment times used in general clinical practice, there would be no significant difference in clinical outcome.     

Hepatocyte expressed chemerin-156 does not protect from experimental non-alcoholic steatohepatitis

Molecular and Cellular Biochemistry - Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed...