Biochemical and molecular medicine最新文献

Heme oxygenase the rate-limiting step in the degradation of heme to bilirubin, generates carbon monoxide. This gaseous molecule plays important roles in neuronal signaling and modulation of vascular tone. Additionally, carbon monoxide is involved in some pathological conditions (e.g., ischemia, endotoxic shock, excitotoxicity) as a protective or toxic factor. Bilirubin, another heme metabolite, exhibits intriguing biological activities as an antioxidant, an antimutagen, and an anti-complement agent. Vital functions and the dual nature displayed by these two heme metabolites are discussed.

A collaborative March of Dimes study was designed to examine the utility of dried blood spot (DBS) materials routinely collected from newborns as a source for monitoring cocaine exposure and to assess the prevalence of cocaine use among childbearing women in Georgia. We used a modified urinary radioimmunoassay (RIA) to anonymously detect the cocaine metabolite benzoylecgonine (BE) in DBSs. Extensive efforts were undertaken to assure absolute nonlinkage of BE data to any individual. The positive results found by RIA were confirmed by a mass spectrometry (MS) method specifically developed to detect BE in DBSs. BE was measured in 23,141 DBSs collected during 2 months of routine newborn screening in Georgia. A good correlation was observed for RIA results versus MS results (r²= 0.97). The estimated minimal statewide BE prevalence was 4.8 per 1000 childbearing women. We demonstrated that immunoassay testing for cocaine without confirmatory testing can yield falsely elevated prevalence rates. When proper confirmatory testing is done, DBSs are a valuable source for population-based monitoring of substance abuse among childbearing women.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cystic tubule enlargement and expansion of the interstitium associated with fibrosis. Our previous studies have analyzed the increased proliferation of cystic epithelial cells and this study examines the basis of increased proliferation of interstitial fibroblasts associated with ADPKD disease progression. ADPKD fibroblasts show phenotypic alterationsin vitro,have acquired the capacity to grow in soft agar, and show an increased mitogenic response to a variety of growth factors particularly acidic FGF (aFGF). ELISA, Western immunoblot analysis, and immunocytochemistry showed increased aFGF content in ADPKD tissues and fibroblasts in culture, and aFGF was secreted into the extracellular matrix and conditioned medium, respectively. No alterations in aFGF receptor number were found, but Scatchard analysis of¹²⁵I-aFGF binding suggested an increased affinity of binding to the low affinity receptor, and covalent cross-linking analysis suggested the presence of novel putative receptors (120 kDa) in ADPKD fibroblasts. Signaling abnormalities were found, since aFGF incubation resulted in the tyrosine phosphorylation of additional substrates, more rapidly and for a more sustained duration in ADPKD fibroblasts than in normal fibroblasts. These findings suggest an important role for acidic FGF in the hyperproliferation of interstitial fibroblasts associated with disease progression in human ADPKD.

Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver. The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolpyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes. The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase. Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia. Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions. These properties are consistent with the structure/function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver. Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species. Galactose oxidase-susceptible membrane-associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG. GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB³H₄. Serial t.l.c. revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver. These galactose–oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C fromBacillus thuringiensisand were resistant to glycosylphosphatidylinositol-specific phospholipase C fromTrypanosoma brucei.These data indicate that IPG molecules with insulin-like biological activities are present in human liver.

To understand the role of amylin, the novel pancreatic hormone, in fuel metabolism of neonatal mammals, the transcription of the amylin gene in newborn dogs was studied under different conditions, such as fasting, hyperinsulinemia, and hyper IGF-1. Our results showed (1) The amylin mRNA level decreased during a 24-h fasting period after birth, 59.1 ± 4.5% at 4 h, 80.1 ± 7.9% at 10 h, and 44.5 ± 3.0% at 24 h, compared to 0-h-fasted controls, respectively. In this period, the decreased mRNA level of the amylin gene and the increased mRNA levels of the gluconeogenic genes showed an inverse ratio relationship. (2) Euglycemic hyperinsulinemic clamp did not alter the amylin mRNA level, 39.6 ± 1.2% (hyperinsulinemia) vs 41.4 ± 3.1% (controls), in newborn dogs, but lowered the amylin mRNA by 35.3%, 64.7 ± 12.5% vs 100.0 ± 12.0%, in adult dogs. (3) Euglycemic hyper-IGF-1 clamp had no effect on the amylin mRNA levels of either newborn or adult dogs, 52.4 ± 9.1% (hyper IGF-1) vs 47.9 ± 4.3% (controls) in newborns and 95.2 ± 12.6% (hyper IGF-1) vs 100.0 ± 14.0% (controls) in adults. The data from the present study showed that amylin may be involved in carbohydrate homeostasis, but may not be able to stimulate gluconeogenesis in newborn dogs during a 24-h fasting period after birth. Whether amylin action may be another mechanism for neonatal hyperglycemia by inducing insulin resistance in peripheral tissues needs further investigation.

Oxidative stress has been suggested to play a crucial role in the pathogenesis of diabetic complications including nephropathy. However, the exact mechanism of diabetic nephropathy is still not clearly understood. Since oxidative stress is known to be a major component in the induction of apoptosis, we investigated the occurrence of apoptosis in diabetic rat kidney. The status of oxidative stress was determined as thiobarbituric acid reactive substances (TBARS). The TBARS in the control and diabetic rat kidney were 2.00 ± 0.963 and 3.83 ± 0.715 μmol/mg protein, respectively (P< 0.05). Apoptosis was determined by evaluating the DNA fragmentation using an enzyme-linked immunoassay andin situend labeling. DNA fragmentation increased approximately fourfold in diabetic rat kidney compared to the normal kidney (P< 0.05). Apoptagin situlabeling displayed negligible apoptosis in nondiabetic kidney while significant areas of apoptosis were observed in diabetic kidney. Our results suggest that increased oxidative stress in diabetic kidney could induce apoptosis, which may contribute to the development of diabetic nephropathy.

Experimental streptozotozin-induced diabetes resulted in important changes in body weight which were associated with abnormalities in water and food intake. In addition, diabetic rats showed a clear muscle atrophy involving a decrease in both skeletal muscle size and protein content. This was accompanied by a marked loss of total carcass nitrogen. These changes were related to important alterations in protein turnover in skeletal muscle. Thus, the diabetic animals showed changes in the fractional protein rates of both synthesis (decreased by 37%) and degradation (increased by 140%). The increased protein degradation observed in the muscle of the diabetic animals was associated with important changes in the concentration of both circulating and muscle amino acids. Interestingly, the diabetic animals did not show important changes in either liver or kidney protein turnover rates, in spite of having a clear increase (over 50%) in kidney mass. In addition, and although the total amino acid concentration was not affected by the diabetic state, the chemically induced diabetic animals showed important elevations of branched-chain amino acids (leucine, isoleucine, and valine) in both blood and skeletal muscle. Similarly, important decreases in the blood concentrations of glutamate + glutamine, alanine, glycine, proline, serine, and threonine were also observed. These observations reinforce the idea of the association between muscle protein wasting, increased protein turnover, and alterations in branched-chain amino acids previously proposed by our group.

Multiple exostoses is a polygenic disease of bone formation and development characterized by the presence of cartilage-capped osseous projections emanating from the end of the long bones. Two members of a recently defined multigene family of proteins (EXT1 and 2) were shown to be involved in this disease. To investigate the evolutionary relatedness of EXT genes across species we isolated the mouse EXT2 cDNA. As in the human counterpart, the mouse EXT2 cDNA contains an open reading frame of 2154 bp encoding a predicted protein of 718 amino acids. The nucleic acid sequence is 87% identical to the human EXT2 transcript, resulting in an amino acid sequence which is 95% identical to the human protein. The mouse EXT2 gene also shows significant sequence similarity to the mouse and human EXT1 gene. Northern blot analysis shows that this gene is expressed in early stages of embryonic development, andin situhybridizations suggest that EXT2 plays a role in limb development. The identification of the mouse EXT2 gene will allow functional analysis through insertional inactivation and reverse genetics in mice in order to better understand the formation of exostoses during bone formation.

Biotinidase deficiency is an autosomal recessive disorder that can result in neurologic and cutaneous symptoms if not treated with biotin supplementation. We have identified the most common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States. 1368A → C results in a substitution of histidine for glutamine 456 (Q456H) in exon D of the biotinidase gene. This mutation was found in at least one allele in 14 unrelated children from 27 different families or 15 of 54 alleles studied (28%). This mutation was not identified in 41 normal adults using SSCA, nor was it found in 296 normal newborns using allele-specific oligonucleotide analysis, suggesting that this change is not a polymorphism. In addition, biochemical data from a child homozygous for Q456H suggest that the aberrant enzyme has very low biotinyl-hydrolase activity, lacks biotinyl-transferase activity, and is not recognized by antibody prepared to purified, normal human biotinidase. The ethnic backgrounds of the parents contributing the Q456H allele are varied but are generally northern European.

The disposition of whole blood mono- to hexaglutamyl methylfolate and plasma homocysteine (HCY) was used to evaluate potential lesion sites in one-carbon metabolism which could be responsible for neural tube defect(NTD)-affected pregnancies. An isocratic high-performance liquid chromatographic system (HPLC) with photodiode array detection was used to quantify and speciate whole-blood methylfolate into mono-, di-, tri-, tetra-, penta-, and hexaglutamate forms. This technique was also used with off-line radioassay to identify nonmethyl whole-blood folates. Isocratic HPLC with fluorescence detection was used to quantify SBDF derivatized homocysteine in plasma. The study investigated blood from 11 women who had experienced a previous NTD- affected pregnancy and 11 controls of similar age and social class. No subjects were pregnant. HCY levels were significantly higher in NTD subjects (P= 0.0486, 95% CI −2.799,0.001 using the Mann–Whitney test), as was the ratio of known intracellular (tri- to hexaglutamyl) methylfolate compared to extracellular (mono- and diglutamyl) methylfolate (P= 0.0062 95% CI −0.543, 3.862 using the Mann–Whitney test). Vitamin B₁₂, red cell folate, circulating total methylfolate, and circulating mono- to hexaglutamyl methylfolates showed no difference between population groups. The disposition between individual and cumulative glutamate chain lengths of methylfolate showed significant trends which differed between population groups: (i) total blood methylfolate (Glu_1–6) appears to be utilized byN-5-methyltetrahydrofolate:homocysteine methyltransferase (MS) in control blood but not NTD blood, where it appears to accumulate following a 45-min incubation; (ii) whole-blood hexaglutamyl methylfolate (5CH₃-H₄PteGlu₆) becomes a larger proportion of the total blood methylfolate in NTD than in control populations; and (iii) the intermediate glutamate chains of methylfolate (Glu_1–5) remain relatively constant as 5CH₃-H₄PteGlu₆accumulates in NTD but appear to increase linearly with 5CH₃-H₄PteGlu₆in controls. The significant elevation of HCY in the NTD population is associated with the increasing proportion of 5CH₃-H₄PteGlu₆relative to the total methylfolate, since, when corrected for HCY level, the proportion of 5CH₃-H₄PteGlu₆to total methylfolate is similar in NTD and control populations. These trends are consistent with a defect at the level of vitamin B₁₂dependent MS which “traps” folate at the 5CH₃-H₄PteGlu₆level.