Use of two calcium concentrations in hemodialysis--report of a 20-year clinical experience.

Over the past almost 50 years several calcium concentrations in the dialysate (CaD) have been used to balance calcium in hemodialysis (HD) patients but a consensus as to which is most appropriate has not been established. Moreover, since the late 1980s, further confusion has been caused following the use of calcium salts as intestinal phosphate binders. This paper reports results of 387 chronic HD patients with respect to secondary hyperparathyroidism (sHPT) and renal osteodystrophy (ROD) of a single center over 20 years. The most important therapeutic measures applied were use of only 2 CaD, 1.5 and 1.75 mmol/l, with very few exceptions, administration of either calcium-containing or calcium-magnesium-containing and/or calcium-free phosphate binders, no dietary restrictions and continuous compensation of uremic acidosis via dialysate and oral supplements of bicarbonate. Using one of the two CaD and selective administration of different phosphate binders for fine adjustment of serum calcium through this combination, we were able to maintain in the long term almost physiological conditions. With exception of the phosphate metabolism, most physiological functions with regard to sHPT and ROD returned close to normal. As a result, the incidence of hypercalcemia, hypocalcemia, extraosseous, extravascular calcification, bone pain and spontaneous bone fractures was extremely low. We conclude that the clinical advantages of the therapeutic measures, above all precise balance of calcium homeostasis, in our investigation were demonstrated by high survival rates (92% after the first year on HD, 82% after 2, and 55% after 5 years), low incidence of cardiovascular fatalities (about 25%), and very low incidence of sHPT (mostly normal parathyroid hormone levels, 1 parathyrdoidectomy within 20 years).

Phytoestrogens and carcinogenesis-differential effects of genistein in experimental models of normal and malignant rat endometrium.

The phytoestrogen genistein was studied in normal and malignant experimental uterine models in vivo. The action of genistein on the uterus and vagina of ovariectomized DA/Han rats after 3 day oral administration (25, 50 or 100 mg/kg/BW/d) was compared to ethinyl oestradiol (0.1 mg/kg/BW/d). Effects on uterine and vaginal morphology, uterine growth and uterine gene expression were studied. A dose dependent increase of the uterine wet weight and the uterine and vaginal epithelial height, a dose dependent up-regulation of complement C3, down-regulation of clusterin mRNA expression and a stimulation of the vaginal cornification was observed after administration of genistein. Uterine gene expression and vaginal epithelium respond to genistein at doses where no significant effects on uterine wet weight were detectable. In general the vagina was more sensitive to genistein than the uterus. To analyse the action of genistein in malignant uterine tissue, the impact of a 28 d treatment with 50 mg/kg/d of genistein on the in-vivo tumour growth of RUCA I endometrial adenocarcinoma cells, following subcutaneous inoculation into syngeneic DA/Han rats, was assessed. In contrast to ethinyl oestradiol (0.1 mg/kg/BW/d), a dose of 50 mg/kg/BW/d of genistein did not affect tumour growth. Nevertheless C3 and TRPM2 mRNA expression in the tumour were both significantly stimulated by ethinyl oestradiol and genistein. In comparison to ovariectomized animals genistein up-regulated uterine wet weight and uterine dependent gene expression in tumour bearing animals. In conclusion, four independent uterine and vaginal parameters indicate genistein is a weak oestrogen receptor agonist in the uterus and vagina of female DA/Han rats, and evidence is provided for a selective oestrogen receptor modulator (SERM)-like action of genistein in normal and malignant uterine tissue.

Construction of 700 human/mouse A9 monochromosomal hybrids and analysis of imprinted genes on human chromosome 6.

As an in vitro assay system for the identification of human imprinted genes, a library of human/mouse A9 monochromosomal hybrids containing a single, intact bsr-tagged human chromosome of known parental origin, derived from normal human fibroblasts, has been previously generated by microcell-mediated chromosome transfer (MMCT). To supplement this assay system, we constructed additional 700 A9 monochromosomal hybrids, using a pSTneo or pPGKneo selection marker. To validate the A9 hybrids, we screened them with chromosome-specific polymorphic markers, and identified the hybrids containing either human chromosome 6, 7, 14, 18, or 21 of known parental origin. Matching paternal and maternal chromosome pairs of A9 hybrids were identified for chromosomes 6, 7, 14, and 18. The paternal-specific expression of ZAC (zinc finger protein, which regulates apoptosis and cell cycle arrest) and HYMAI (hydatidiform mole-associated and imprinted transcript), and the maternal-specific methylation of a CpG island within an imprinted domain on human chromosome 6q24, were maintained in A9 hybrids. For an example, we profiled the expression of expressed sequence tags (ESTs) and the methylation of CpG islands in the 300-kb imprinted domain around 6q24, which may be associated with cancers and transient neonatal diabetes mellitus (TNDM). Thus, the 700 A9 hybrids should be useful for various aspects of imprinting studies.

Mutagenicity of diesel exhaust particles from two fossil and two plant oil fuels.

Particulate matter of diesel engine exhaust from four different fuels was studied for content of polynuclear aromatic compounds and mutagenic effects. Two so-called biodiesel fuels, rapeseed oil methylesters (RME) and soybean oil methylesters (SME), were compared directly with two fossil diesel fuels with the normal (DF) and a low sulfur content (LS-DF). Diesel exhaust particles were sampled on filters from the diluted and cooled exhaust of a test engine at five different speeds and loads. Filters were weighed for total particulate matter, Soxhlet extracted with dichloromethane and the content of insoluble material determined. The soluble organic fraction was analysed for polynuclear aromatic compounds. Mutagenicity was determined using the Salmonella typhimurium/mammalian microsome assay with strains TA98 and TA100. Compared with DF, the exhaust particles of LS-DF, RME and SME contained less insoluble material, which consisted mainly of the carbon cores of diesel exhaust particles. The concentrations of individual polynuclear aromatic compounds varied widely among the different exhaust extracts, but total concentrations of the compounds were approximately double for DF and SME compared with LS-DF and RME. In TA98 significant increases in mutation rates were obtained for the soluble organic fractions of all fuels for engines running at full speed (load modes A and D), but for DF revertants were 2- to 10-fold more frequent as compared with LS-DF, RME and SME. Revertant frequencies for DF and partly for LS-DF were also elevated in TA100, while RME and SME gave no significant increase in mutations. The results indicate that diesel exhaust particles from RME, SME and LS-DF contain less black carbon and total polynuclear aromatic compounds and are significantly less mutagenic in comparison with DF. A high sulfur content of the fuel and high engine speeds (rated power) and loads are associated with an increase in mutagenicity of diesel exhaust particles.

Ability of xeno- and phytoestrogens to modulate expression of estrogen-sensitive genes in rat uterus: estrogenicity profiles and uterotropic activity.

The function of the uterus is regulated by female sex steroids and it is, therefore, used as the classical target organ to detect estrogenic action. Uterine response to estrogens involves the activation of a large pattern of estrogen-sensitive genes. This fact offers the opportunity to analyze the estrogenic activity of xeno- and phytoestrogens, and the mechanisms of their molecular action by a correlation of the uterotropic activity and their ability to modulate the expression of estrogen-sensitive genes. We have analyzed the expression of androgen receptor (AR), progesterone receptor (PR), estrogen receptor (ER), clusterin (CLU), complement C3 (C3), and GAPDH mRNA in the rat uterus following oral administration of ethinylestradiol (EE), bisphenol A (BPA), o,p'-DDT (DDT), p-tert-octylphenol (OCT) and daidzein (DAI). A significant stimulation of the uterine wet weight could be observed after administration of all the substances. The activity of all analyzed compounds to stimulate uterine weight was low in comparison to EE. DDT has the highest activity to stimulate uterine weight whereas BPA and DAI turned out to be less potent. The analysis of gene expression revealed a very specific profile of molecular action in response to the different compounds which cannot be detected by judging the uterotropic response alone. A dose dependent analysis revealed that C3 mRNA is already modulated at doses where no uterotropic response was detectable. Although DAI and BPA were very weak stimulators of uterine growth, these substances were able to alter the expression of AR, ER and C3 very strongly. Based on these investigations the analyzed compounds can be subdivided into distinct classes: First, compounds which exhibit a similar gene expression fingerprint as EE (e.g. OCT); second, compounds exhibiting a significant uterotropic activity, but inducing a pattern of gene expression different from EE (e.g. DDT); and third, compounds like BPA and especially DAI which exhibit a very low uterotropic activity, but nevertheless modulate the expression of estrogen-sensitive genes. These findings strongly suggest that the fingerprint of uterine gene expression is a very sensitive tool to investigate estrogenicity of natural and synthetic compounds and offers the possibility to get information in regard to the molecular mechanisms involved in the action of the respective compounds.

The antiovulatory potential of progesterone antagonists correlates with a down-regulation of progesterone receptors in the hypothalamus, pituitary and ovaries.

These studies analyze the regulation of progesterone receptors (PRs) in central and peripheral tissues with the aim of further understanding mechanistically the inhibition of ovulation by progesterone antagonists (PA). Therefore, it was of interest to investigate the influence of the progesterone receptor antagonist, Onapristone (ON), on PRs in the ovary, pituitary (PT), and hypothalamus (HYP), since ON effectively inhibits ovulation in rats. For this study PMSG/hCG-primed immature and adult female rats were treated with ON. Immunohistochemistry was used for the detection of PRs. Progesterone (P4) and estradiol (E2) levels were determined by RIA. PR expression in the ovaries of immature rats was not detectable until after hCG administration. In these animals, ON caused a reduction in the staining intensity of PR in the tertiary follicles at the time when the preovulatory P4-surge was inhibited (6 h post hCG). Adult rats treated for 15 days with ON showed a decreased PR expression in PT and HYP. At this time (proestrus, 7 p.m.) the P4 and E2 levels are significantly lowered. These results suggest that after treatment with ON the expression of PR is reduced in the ovary, PT and HYP. The regulation of PR in the ovary seems to be less dependent on estrogens than on LH. Thus, it is conceivable that the reduced PR expression after ON treatment may be a result of decreased LH sensitivity in the ovary. In the pituitary and hypothalamus, PR expression is stimulated by estrogens and progesterone, and therefore the fall in the P4 and E2 levels in ON-treated animals may be responsible for the reduced PR expression in PT and HYP, and may contribute to the antiovulatory effect of ON. We therefore conclude that the mechanism of the antiovulatory potency of progesterone antagonists is based on a reduced preovulatory P4-production and PR expression in the ovary and also on the down-regulation of PR in the anterior pituitary and hypothalamus.

Splenic abscess caused by Salmonella braenderup, treated with percutaneous drainage and antibiotics.

Splenic abscess is a rare condition and its optimal treatment is still debated. We report on a 17-year-old immunocompetent female patient, hospitalized with Salmonella braenderup gastroenteritis and splenic abscess, who was treated with ciprofloxacin, percutaneous catheter drainage and despite remaining drainage of 50 ml/24 h, the catheter was removed and the antibiotic treatment was stopped when the fluid was clear. Following removal a transient increase in the size of the splenic cavity was observed, but without any clinical symptoms or deterioration of laboratory parameters. At the 1-year follow-up, ultrasound examination of the spleen disclosed only a 8 mm scar.

An unusual arrangement of 13 zinc fingers in the vertebrate gene Z13.

The zinc finger is a protein domain that imparts specific nucleic acid-binding activity on a wide range of functionally important proteins. In this paper we report the molecular cloning and characterization of a novel murine zinc-finger gene, mZ13. Analysis of mZ13 cDNAs revealed that the gene expresses a 794-amino-acid protein encoded by a 2.7 kb transcript. The protein has an unusual arrangement of 13 zinc fingers into a 'hand' of 12 tandem fingers and a single isolated finger near the C-terminus. This structural organization is conserved with the probable chicken homologue, cZ13. mZ13 also contained an additional domain at the N-terminus which has previously been implicated in the regulation of zinc-finger transcription factor DNA-binding, via protein-protein interactions. mZ13 expression was detected in a wide range of murine embryonic and adult tissues. The structural organization of mZ13 and its expression profile suggest that it may function as a housekeeping DNA-binding protein that regulates the expression of specific genes.

Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P-450 mRNA.

A cloned complementary DNA sequence has been isolated from a human placental cDNA library in the bacteriophage expression vector lambda gt11 after screening with polyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-450Arom). A single recombinant clone, lambda hAROM1, was characterized by its ability to generate a beta-galactosidase fusion protein that reacted independently with polyclonal antibodies raised against beta-galactosidase and cytochrome P-450Arom and with monoclonal antibodies specific for cytochrome P-450Arom. The cDNA insert, which was found to be 1.8 kilobases in length, was radiolabeled and used to analyze poly(A)+ RNA isolated from human placenta and total RNA isolated from human adipose stromal cells cultured in the absence or presence of regulatory factors. The radiolabeled cDNA hybridized to several size species of mRNA in both placental and adipose stromal cell RNA fractions. Changes in the levels of adipose stromal cell RNA that hybridized to the cDNA insert were associated with comparable changes in the levels of translatable cytochrome P-450Arom mRNA and aromatase system activity. These findings are indicative that lambda hAROM1 contains DNA sequences complementary to human cytochrome P-450Arom mRNA and are suggestive that regulatory factors affect aromatase activity by altering the transcriptional activity of the cytochrome P-450Arom gene.

Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H.

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.

Iodination with Iodo-gen and radioimmunoassay of cholecystokinin (CCK) in acidified plasma, CCK release, and molecular CCK components in man.

The preparation of a stable fully immunoreactive 125I-labeled CCK39 using a modified Iodo-gen method with high specific radioactivity; the production of an avid and specific cholecystokinin (CCK) antiserum, and a sensitive, precise and specific radioimmunoassay method allowing measurements of fasting plasma CCK in the low picomole per liter range together with the significant rises in plasma CCK following a test meal and duodenal infusion of fat are described. Apparent immunoreactive fasting plasma CCK was eluted from a Sephadex G-50 Fine column in one peak probably representing plasma CCK bound to plasma proteins and nonspecific plasma effects. Apparent immunoreactive postprandial plasma CCK was eluted from a Sephadex G-50 Fine column in four peaks. The first peak probably represents plasma CCK bound to plasma proteins and nonspecific plasma effects; the second peak probably represents component I with a molecular weight between some 5,000 and 30,000; the third peak probably represents component II or CCK33, and the fourth peak probably represents component IV or CCK8.