Biochemical and molecular medicine最新文献

Based on multiple gel permeation chromatographic experiments, we report a Stokes radius of 59.7 Å forPseudomonas mevalonii3-hydroxy-3methylglutaryl coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.88) and its His381Asn, His381Gln, and His381Lys mutant enzymes. Comparison of this Stokes radius with the radius calculated from the crystal structure indicated that the active form ofP. mevaloniiHMG-CoA reductase was a hexamer and not a dimer as previously thought. The Stokes radius, anS_20,wof 11.0, and an estimated$\text{V}\text{̄}$of 0.723 were used in the Svedberg equation to calculate an anhydrous molecular mass of 270,084 Da forP. mevaloniiHMG-CoA reductase (monomer mass 45,538 Da), consistent with the enzyme being a hexamer in solution. The Stokes radii of all standard proteins examined correlated with the inverse error function complement of their partition coefficient,K_d.K_ddid not correlate with logarithm of the standard protein's molecular weight. Eight nonstandard proteins had Stokes radii that matched their crystallographic radii of longest axis. This indicated that the frozen conformation of a protein in its crystal form can dictate restraints on its shape in solution.

Free radical species associated with bilateral ureteral obstruction (BUO) are considered important in the pathogenesis of the glomerular and tubulointerstitial injury in BUO rats. We seek to test the hypothesis that the use of an easily administered antioxidant, vitamin E, at sufficient plasma concentrations, can decrease this release of free oxygen radicals in kidney tissue and ameliorate the increase of the fibrogenic cytokine, transforming growth factor β-1 (TGFβ-1). We used the unilateral ureteral obstruction (UUO) rat model, because the presence of the uninjured contralateral kidney provides a nonuremic internal milieu, in contrast to the uremic, acidotic, and hypercholesterolemic BUO model. Compared to sham controls, the UUO animals showed a dramatic increase in renal cortical TGFβ-1 mRNA, as quantitated by Northern blot analysis with cyclophilin internal standards. This increase in TGFβ-1 mRNA was reversed in UUO rats treated with vitamin E. The plasma malondialdehyde (MDA) concentration, an index of lipid peroxidation and an indirect index of free radical release, was significantly elevated in UUO animals compared to sham animals. The vitamin E-treated UUO animals showed a significant decrease in both plasma and renal cortical tissue MDA content. Taken together, these findings provide evidence of the important biological role of reactive free radical species in the tubulointerstitial injury of UUO and the novel role of vitamin E in modulating the mRNA of the fibrogenic TGFβ-1 in obstructive uropathy.

Serotonin (5-hydroxytryptamine) is believed to play a pathogenic role in skin damage and various skin abnormalities; however, its mechanism of action remains unknown. We show here that intradermal injection of serotonin in rats induced a marked reduction in the activities of the glycolytic enzymes, phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13), in both the cytoskeletal and cytosolic fractions from skin. Serotonin also decreased the levels of glucose 1,6-bisphosphate in skin, the powerful regulator of glucose metabolism. These serotonin-induced changes were accompanied by a marked decrease in ATP content in skin. All these pathological changes induced by serotonin were prevented by treatment with two structurally different calmodulin antagonists: thioridazine, an antipsychotic phenothiazine, or clotrimazole, from the group of the antifungal azole derivatives that were recently recognized as calmodulin antagonists. The present results suggest that calmodulin antagonists may be effective drugs in the treatment of skin damage under various pathological conditions and diseases in which serotonin levels are increased.

Methylprednisolone (MP) and related corticosteroids are a fundamental part of regimens used to treat lymphoma and leukemia. In many of these malignancies, oncogenic activation ofC-MYCandBCL2is seen. Abnormalities of the tumor suppressorp53,which exerts growth-suppressing and apoptosis-enhancing functions through the transcriptional regulation of downstream genes includingCDKN1, GADD45,andBCL2,are also often found. The goal was to determine the modulation of expression of the oncogenes (C-MYCandBCL2), thep53pathway described above, and the apoptosis markerTGF-β₁in the human Raji lymphoma following MP treatment. Raji xenografts were grown in nude mice and growth curves characterized by sequential measurement. Mice were treated daily for 8 days with MP. Tumors were harvested untreated, or at 1 or 8 days after cessation of MP treatment, and the RNA was extracted. RT–PCR was used to determine the level of mRNA expression of the genes. Tumor growth was greatly reduced in the MP-treated mice. Gene expression levels forC-MYCandBCL2were reduced at 1 day following MP and approached control levels 8 days after MP treatment. Expression levels ofp53, CDKN1,andGADD45were moderately and coordinately decreased at 1 day after cessation of MP treatment and remained repressed a week later.TGF-β₁exhibited no change in expression levels. These results suggest that decreased expression ofC-MYCandBCL2may play a role in the molecular events that initiate and are responsible for the growth inhibition of Raji lymphoma xenografts by MP.

The basic region of c-Myc and other basic helix–loop–helix (b-HLH)-containing proteins bind to the palindromic DNA sequences CANNTG. For the myogenic factor MyoD, a member of the b-HLH family, mutation of several basic region residues abrogates muscle differentiation, but not DNA binding. One of the amino acid positions displaying this behavior in MyoD aligns with a highly conserved asparagine in Myc. This conserved asparagine displays complete tolerance for alanine substitution as measured by DNA binding. To test the possibility of whether the basic region of Myc encodes a second biological function, the conserved asparagine in c-Myc (N360) was mutated to alanine and tested for the Myc-dependent functions of cellular transformation and apoptosis. In contrast to the deleterious effects of such mutations in MyoD, the alanine mutant functions normally for both Myc-dependent cellular transformation and apoptosis induction. Therefore, a basic region function distinct from DNA binding may not be a general feature of HLH transcription factors.

Leptin, the product of the ob gene, is a recently discovered hormone secreted by adipocytes. Serum leptin concentrations increase in correlation with the percentage of body fat, but besides that little is known about the physiological actions of leptin in humans. In order to understand the role of leptin in severe malnutrition, in the present work 10 patients recently diagnosed with anorexia nervosa were studied both before and 2 months later, after partial weight recovery, and were compared with 18 normal-weight women as controls. Leptin was measured by a newly developed radioimmunoassay and both IGF-I and IGFBP-3 were measured by commercial radioimmunoassays. The mean (±SE) serum leptin concentrations (in μg/liter) were 18.1 ± 2.0 in control women with BMI of 21.1 ± 0.3, significantly higher (P< 0.01) than that in the anorexia nervosa patients at diagnosis (2.2 ± 0.1, BMI 15.3 ± 0.6). These differences were also observed in IGF-I values (μg/liter) that were 228.0 ± 14.6 in controls and 157.4 ± 28.7 in anorexia nervosa patients (P< 0.02). No differences were observed in IGF-BP3. After treatment, patients with anorexia nervosa experienced an increase in BMI (17.1 ± 0.5,P< 0.0001 vs before) although they were still underweight. The partial recovery in weight led to a complete normalization of IGF-I levels (214.0 ± 21.0 μg/liter) and to an enhancement in leptin levels (3.3 ± 0.5 μg/liter;P< 0.03 vs before treatment), though still lower than those in normal-weight women (P < 0.05). Individually analyzed, a large dispersion was observed in control subjects, with leptin levels ranging from 5.5 to 38.7 μg/liter, while in all anorexia nervosa patients leptin levels were under 3 μg/liter. A treatment-induced increase in body weight led to an increase in leptin levels in 7 out of the 10 anorexia nervosa patients studied and the 3 patients with no increase in leptin were all initially under the 14.5 BMI. In conclusion, leptin levels are severely reduced in anorexia nervosa patients with severe malnutrition, and a significant rise occurred after partial weight recovery. There seems to be a level of BMI below which leptin levels do not drop further but also do not increase despite weight gain. While IGF-I reflects the energy intake of the previous few weeks, the serum leptin concentration reflects the true status of the adipose stores, a fact that has useful clinical implications.

The molecular techniques ofin situtranscription and antisense RNA amplification (IST/aRNA) have allowed for the monitoring of coordinate changes in the expression of multiple genes simultaneously. However, the analysis of their concurrent behavior during murine embryogenesis has been problematic. Studies involving the investigation of temporal and spatial gene expression during embryogenesis have focused solely on the analysis of isolated, single gene events. Such an approach has failed to provide an integrative picture of genetic control over the varied and complicated cellular processes governing embryogenesis. In order to interpret the enormous amount of gene expression data generated by these procedures, we have attempted to develop an analytical framework by employing the statistical concepts of principal components analysis (PCA). For the current study, we performed IST/aRNA on neural tubes dissected from the highly inbred LM/Bc murine strain collected during four gestational time periods. A subset of these genes, representing a partial signaling pathway in the developing neuroepithelium, was then subjected to PCA. Here, we report that PCA highlighted the transcriptional interplay among the genesp53, wee-1, Tgfβ-2,andbcl-2such that the combined reciprocal regulation of their gene products is suggestive of a predominant proliferative state for the developing neuroepithelium. The application of PCA to the gene expression data has elucidated previously unknown interrelationships among cell cycle genes, growth, and transcription factors on a transcriptional level during critical stages of neurulation. The information gleaned from this analysis, while not definitive, suggests distinct hypotheses to guide future research.

Myogenic regulatory factors (MRFs) promote differentiation of muscle cells from fibroblasts and are induced by insulin-like growth factor I (IGF-1). Prior studies have shown synthesis of new muscle protein and improved muscle morphology when maturedymice with muscular dystrophy are treated with IGF-1. We investigated whether these salutary effects of IGF-1 might be attributable to stimulation of MRFs. Maledy(129ReJ) mice and controls (129J) were assigned to IGF-1 treatment (10 μg twice daily) or nontreatment at about 5 weeks of life and sacrificed 6 weeks later. RNA was extracted from skeletal muscles, reverse transcribed, and amplified by polymerase chain reaction (PCR) using primers specific for each MRF. Competitive PCR was performed to quantifyMyoDexpression in response to IGF-1 treatment. Transcripts formyf-5, MRF4,and myogenin were detected in both control anddymouse muscles; no apparent differences were observed between treatment groups. Quantitative analysis of transcripts forMyoDindicated no significant basal differences between control anddymice. There was, however, significantly higherMyoDexpression in thedygroup, and a trend toward significance in the control group, following IGF-1 treatment. These data suggest that IGF-1 exerts itsin vivoeffects in postembryonal muscle by stimulating MRFs.

Our previous investigations demonstrated that unsuppressed gluconeogenesis under hyperinsulinemia in newborn dogs may be a mechanism of neonatal hyperglycemia. In the present study, the transcription of the gene for fructose-1,6-bisphosphatase (fru-1,6-P₂ase; E 3.1.3.11) of newborn dogs was studied under various metabolic perturbations (age, suckling, fasting, and hyperinsulinemia). Total RNAs isolated from livers and kidneys were hybridized with a rat fru-1,6-P₂ase cDNA probe. We observed that (i) fru-1,6-P₂ase mRNA was expressed in both kidney and liver at birth and was about 40 and 80% of those in kidney and liver of adult dog, respectively; (ii) suckling decreased the kidney fru-1,6-P₂ase mRNA level to 77.8 ± 1.7% (24 h) from 100.0 ± 8.0% (4 h), but increased liver mRNA to 158.6 ± 11.4% (24 h) from 100.0 ± 2.3% (4 h); (iii) during a 24-h period of fasting, the kidney fru-1,6-P₂ase mRNA level did not change in the first 10 h and then increased 18.5% at 24 h, whereas the liver fru-1,6-P₂ase mRNA increased ca. 20% during the first 10 h and then up to 161.1 ± 18.0% at 24 h compared to that at 100.0 ± 11.4% (0 h); (iv) euglycemic hyperinsulinemia did not change the renal fru-1,6-P₂ase mRNA level, but lowered the hepatic fru-1,6-P₂ase mRNA level to 56.0 ± 8.7 from 100.0 ± 11.8% (fasted controls) in newborn dogs, which was identical to that in adult dogs. These data suggest that the fru-1,6-P₂ase in liver may play a more important role in glucose homeostasis of newborn dogs than that in kidney during the first day of their lives and that the incomplete suppression of transcription of the hepatic fru-1,6-P₂ase gene by insulin in newborn dogs may not contribute to neonatal hyperglycemia due to insulin resistance.