Effects of 5-hydroxymethyl-2′-deoxyuridine and 3-aminobenzamide on chromosome aberrations in cultured human lymphocytes

Effects of 5-hydroxymethyl-2′-deoxyuridine (HmdUrd, a thymidine analog) and 3-aminobenzamide (3AB) on chromosome aberrations in cultured human lymphocytes were studied. The results show that HmdUrd is an effective clastogen in human peripheral lymphocytes. When cells were treated with HmdUrd and 3AB, a synergistic effect on chromatid gaps, breaks and exchanges was found. These findings support the hypotheses that 5-hydroxymethyluracil (HmuRa) residues in DNA are formed and then removed by the action of 5-HmUra-DNA glycosylase (Teeber et al., 1984) and that 3AB interferes with the completion of DNA repair following HmUra excision.     

Increased estrogen-16α-hydroxylase activity in women with breast and endometrial cancer

We have measured the three principal oxidative transformations of estradiol by means of a radiometric procedure in women with breast or endometrial cancer and in age matched controls. No difference between the 17β-o1 oxidation or 2-hydroxylation of the hormone was observed between the study groups. In contrast, 16α-hydroxylation was strikingly elevated in the women with breast and endometrial cancer relative to the age matched controls. Evidence is presented that this increased activity precedes the clinical evidence of the disease and that it represents a significant risk factor for these estrogen dependent tumors. This risk may be mediated by one of the products of 16α-hydroxylation, 16α-hydroxyestrone, which exhibits unique biological properties.     

Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.     

Structural characterization of human cholesterol 7α-hydroxylase

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B′ helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.     

Distribution and regulation of the 25-hydroxyvitamin D3 1α-hydroxylase in human parathyroid glands

Parathyroid glands express the 25-hydroxyvitamin D(3) 1α-hydroxylase (1αOHase). 1,25-dihydroxyvitamin D(3) (calcitriol) synthesized by extrarenal tissues generally does not enter the circulation, but plays an autocrine/paracrine role specific to the cell type, and is regulated by the needs of that particular cell. While the role of calcitriol produced in the parathyroid glands presumably is to suppress PTH and cell growth, its regulation in this cell type has not been defined. In the present study, we found that regulation of the human parathyroid 1αOHase differs from the renal enzyme in that it is induced by FGF-23 and extracellular calcium. Hyperplastic parathyroid glands from patients with chronic kidney failure normally display a heterogeneous cellularity. We found that the 1αOHase is expressed at much higher levels in oxyphil cells than in chief cells in these patients. Recent findings indicate that oxyphil cell content is increased by treatment with calcium receptor activators (calcimimetics). Here, we demonstrate that the calcimimetic cinacalcet increases the expression of 1αOHase in human parathyroid cultures. Additionally, we found that the 1αOHase in human parathyroid cultures is functionally active, as evidenced by the ability of the enzyme to 1-hydroxylate 25(OH)D(3) in parathyroid monolayers. Calcium, as well as cinacalcet, also induced expression of the degradation enzyme 24-hydroxylase, indicating the presence of a negative feedback system in the parathyroid cells. Therefore, local production of 1αOHase suggests an autocrine/paracrine role in regulating parathyroid function and may mediate, in part, the suppression of PTH by calcium and FGF-23.     

Ultrastructural evaluation of 8–16 cell human embryos cultured in vitro

The fine structure of eight human embryos cleaving in culture are described. Preovulatory human oocytes aspirated at diagnostic laparoscopies were fertilized and developed in vitro by methods which produced normal pregnancies. Three 8-cell and five 10–16 cell embryos showing delayed cleavage were fixed, serially sectioned and examined to determine whether the embryos were developing normally or otherwise.The organization of two 8-cell embryos was apparently normal while the other embryos showed varying aspects of abnormal development. Most blastomeres contained organelles normally present in human ova and their fine structure closely resembled those of comparable mammalian embryos. Nearly all the cellular components encountered in early mammalian embryos were observed. Certain morphogenetic changes were also noted during early cleavage involving nuclei, smooth endoplasmic reticulum and mitochondria.Both normal and abnormal features of the embryos are reported. Multi-nucleated blastomeres and partial fragmentation were commonly seen in abnormal embryos. The importance of ultrastructural evaluation of embryos in an in vitro programme is revealed in this investigation.     

Simultaneous evaluation of mRNAs of dopamine β-hydroxylase,tyrosine hydroxylase and proenkephalin a from three human pheochromocytomas

Using the rabbit reticulocyte cell-free translation system, the relative proportions of in vitro translatable mRNAs of three proteins in three human pheochromocytomas: tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and proenkephalin A have been compared.TH expression appeared rather constant in the three tumors. In contrast, those of DBH and proenkephalin A were more variable. Though the actual level of each mRNA was not determined, the identical value of DBH/proenkephalin A mRNAs ratio in the three tumors could suggest a coordination in the expression of these two proteins.     

Synthesis of 16α-3H androgen and estrogen substrates for 16α-hydroxylase

The synthesis of 16α-³H androgens and estrogens is described. 1-(³H)-Acetic acid in the presence of zinc dust reacts with 16α-bromo-17-ketosteroids to produce 16α-³H-17-ketosteroids. This chemical reaction was used to prepare 16α-³H-dehydroepiandrosterone (I) and 16α-³H-estrone acetate (XI) from 16α-bromo-dehydroepiandrosterone (X) and from 16α-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16α-³H-androstenedione (II) and 16α-³H-estradiol-17β (VII). 16α-³H-Estrone (VI) was obtained by the chemical hydrolysis of 16α-³H-estrone acetate. The label distribution as determined by microbiological 16α-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16α position. These substrates can be used for measuring the 16α hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).     

Purification and properties of human serum dopamine-β -hydroxylase

1. Two different molecular forms of dopamine-beta-hydroxylase were isolated from human serum; a major component (Peak I enzyme) with a molecular weight of 368000 and with a higher specific activity and a minor component (Peak II enzyme) with a molecular weight of 188000 and with a lower specific activity. 2. Both forms require ascorbic acid for the activity, and are stimulated by fumarate. Addition of N-ethylmaleimide or copper also increased the activity. The optimal pH of both forms in the presence of 20mM tyramine as substrate is 5.0. 3. Km values toward tyramine of Peak I enzyme and Peak II enzyme were 1.67 mM and 14.2 mM respectively. 4. Both Peak I enzyme and Peak II enzyme are glycoprotein.     

Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450k in rat kidney cortex: I. Characteristics of the hydroxylase system

Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450_K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450_K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450_K follow the same pattern of inactivation with increasing temperature. The apparent K_m for the induced hydroxylase was 7.7 μm and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450_K-dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10^?5m), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450_K-dependent aryl hydroxylase activity which differs from that present in the control rat.     

Immunofluorescent and biochemical studies on tyrosine hydroxylase and dopamine-β-hydroxylase of the bullfrog sciatic nerves

Summary Immunofluorescence specific for tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH) was accentuated in both proximal and distal segments of the sciatic nerve after ligation. Estimations of the enzyme activities confirmed the above results. Mean axoplasmic flow rates of TH and DBH in bullfrog sciatic nerve were found to be 8 and 123 mm/day, respectively. They were decreased by colchicine or by cold temperatures (4° C).     

β-Glucuronidase activity in human T and B lymphocytes and the Tμ and Tγ subpopulations

Summary The -glucuronidase staining characteristics of isolated T cell populations and the T and T enriched fractions derived of them were studied. T lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The rosette-forming cells in the pellet were referred to as T lymphocytes, whereas the lymphocytes in the interface were referred to as T depleted or T lymphocytes. B cells were studied on rosetted mononuclear cells with either mouse erythrocytes or with Staphylococcus Aureus (Cowan I) bacteria, after a preceeding polyvalent anti-human Ig treatment of the cells. While B cells showed mostly no reactivity, T and T cells were respectively characterised by a dot-like and granular pattern of reactivity. These findings are in agreement with those observed by others after -naphthyl-acetate esterase or acid phosphatase staining. Within the T lymphocyte fraction, the T non-, non lymphocytes seemed to have a granular pattern of reactivity. The same staining pattern was found in non-B, non-T lymphocytes.     

Phytanic acid α-oxidation in human cultured skin fibroblasts

We studied the oxidation of [1-¹⁴C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and ¹⁴CO₂ in human skin fibroblasts. The specific activity for α-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid α-oxidation enzyme system in peroxisomes.     

Testicular in vitro conversion of progesterone to testosterone and androstenedione in 17α-hydroxylase deficiency

Incubations of [³H]-progesterone with testicular tissue obtained from a new case of male with 17α-hydroxylase deficiency were performed. The per cent conversion to androstenedione and testosterone was virtually absent when compared to that obtained from an identical incubation performed using testicular tissue from a normal male with cryptochordism. The findings provide an in vitro evidence in support of the existence of 17α-hydroxylase testicular defect in this disorder.     

Genetic diversity of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) genes in cattle breeds

DNA from four cattle breeds was used to re-sequence all of the exons and 56% of the introns of the bovine tyrosine hydroxylase (TH) gene and 97% and 13% of the bovine dopamine β-hydroxylase (DBH) coding and non-coding sequences, respectively. Two novel single nucleotide polymorphisms (SNPs) and a microsatellite motif were found in the TH sequences. The DBH sequences contained 62 nucleotide changes, including eight non-synonymous SNPs (nsSNPs) that are of particular interest because they may alter protein function and therefore affect the phenotype. These DBH nsSNPs resulted in amino acid substitutions that were predicted to destabilize the protein structure. Six SNPs (one from TH and five from DBH non-synonymous SNPs) were genotyped in 140 animals; all of them were polymorphic and had a minor allele frequency of > 9%. There were significant differences in the intra- and inter-population haplotype distributions. The haplotype differences between Brahman cattle and the three B. t. taurus breeds (Charolais, Holstein and Lidia) were interesting from a behavioural point of view because of the differences in temperament between these breeds.     

Age-related decline in osteoblastogenesis and 1α-hydroxylase/CYP27B1 in human mesenchymal stem cells: stimulation by parathyroid hormone

With aging, there is a decline in bone mass and in osteoblast differentiation of human mesenchymal stem cells (hMSCs) in vitro. Osteoblastogenesis can be stimulated with 1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and, in some hMSCs, by the precursor 25‐hydroxyvitamin D₃ (25OHD₃). CYP27B1/1α‐hydroxylase activates 25OHD₃ and, to a variable degree, hMSCs express CYP27B1. In this study, we tested the hypotheses (i) that age affects responsiveness to 25OHD₃ and expression/activity of CYP27B1 in hMSCs and (ii) that parathyroid hormone (PTH) upregulates CYP27B1 in hMSCs, as it does in renal cells. There were age‐related declines in osteoblastogenesis (n = 8, P = 0.0286) and in CYP27B1 gene expression (n = 27, r = ?0.498; P = 0.008) in hMSCs. Unlike hMSCs from young subjects (≤50 years), hMSCs from older subjects (≥55 years) were resistant to 25OHD₃ stimulation of osteoblastogenesis. PTH1‐34 (100 nm ) provided hMSCs with responsiveness to 25OHD₃ (P = 0.0313, Wilcoxon matched pairs test) and with two episodes of increased 1,25(OH)₂D₃ synthesis, of cAMP response element binding protein (CREB) activation, and of CYP27B1 upregulation. Both increases in CYP27B1 expression by PTH were obliterated by CREB‐siRNA or KG‐501 (which specifically inhibits the downstream binding of activated CREB). Only the second period of CREB signaling was diminished by AG1024, an inhibitor of insulin‐like growth factor‐I receptor kinase. Thus, PTH stimulated hMSCs from elders with responsiveness to 25OHD₃ by upregulating expression/activity of CYP27B1 and did so through CREB and IGF‐I pathways.