Genomic organization of the CYP19b genes in the rainbow trout (Oncorhynchus mykiss Walbaum).

We report the occurrence of two CYP19b genes, namely CYP19b-I and CYP19b-II, encoding forms I and II of cytochrome P450aromB, the prevalently cerebral variant of aromatase in fish, in the nuclear genome of the rainbow trout. The CYP19b-I gene is 7.6 kbp-long, more than double the size of the known fish CYP19a and b genes, owing to the presence of three introns (1, 4 and 5) that enclose repeated sequences and are longer than 1 kbp. Unlike the CYP19a genes, but similarly to the CYP19b gene of the Nile tilapia, it contains 10, and not 9, exons, including an untranslated exon 1 (83 bp), as found also in the 5' non-coding region of mammalian CYP19 genes. The 5'-UTR is composed by exon 1 and the first 41 bp of exon 2 (150 bp), whose coding region covers the first 36 amino acid residues that incorporate the transmembrane domain. The CYP19b-II gene is only 2.5 kbp-long, because it contains only one intron, corresponding to the third intron of CYP19b-I, and lacks also its first two exons. Thus, it encodes for a presumably soluble protein. Apart from this difference, the rest of the coding region is virtually the same as that of the CYP19b-I gene. The 5'-UTR corresponds in part to the 3'-end (132 bp) of the second intron of the CYP19b-I gene, while the remaining portion (208 bp) bears no homology. CYP19b-II could be regarded as a pseudogene of the CYP19b-I gene, though it is unclear whether it is a processed or a duplicated pseudogene. Moreover, since it is transcriptionally active, it may retain a functional role for the overall brain aromatase activity in the rainbow trout.

[The hemodiafiltration with infusion of acetate-free dialysis fluid can modify the inflammatory response in patients "high responders" to inflammatory stimuli?]. / Il trattamento in emodiafiltrazione con infusione di liquido di dialisi (PHF acetate free) può modificare la risposta infiammatoria in pazienti già identificati come "high responders" a stimoli infiammatori?

PURPOSE: This study aimed to verify the effects of paired hemodiafiltration on-line (PHF-AF) on inflammation in patients who were "high responders" to inflammatory stimuli: elevated C-reactive protein (CRP), genetic polymorphisms influencing a low transcription for interleukin-10 (IL-10) and a high transcription for IFN-gamma. METHODS: Ten patients selected as high responders for IFN-gamma and low responders for IL-10 were included in a crossover study to compare PHF-AF and standard bicarbonate hemodialysis (BHD). At study entry and before the start of each dialysis session the following examinations were performed: CRP, albumin, fibrinogen, ferritin, transferrin, prealbumin and serum levels of IL-6, IL-10, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha). After the 1st and 3rd week of the study, the blood samples were also collected after the dialysis session. RESULTS: . There was a significant reduction in albumin and prealbumin in PHF-AF patients during the study; none of the other parameters were changed in both patient groups. CRP tended to be elevated after dialysis in both PHF-AF and BHD. While IL-6, IL-10 and IFN-gamma were unchanged during the dialysis session, there was a significant variation in TNF-alpha levels, which were increased in BHD (from 10.9 +/- 3.1 to 14.7 +/- 4.1 pg/mL; p=0.004) and reduced in PHF-AF (from 11.9 +/-2.8 to 6.3 +/- 2.2 pg/mL; p=0.0004). CONCLUSION: Although the cytokine levels were unchanged during the study in both BHD and PHF-AF, the modification of TNF-alpha during the dialysis session was considered as inflammatory significance.

Myostatin expression during development and chronic stress in zebrafish (Danio rerio).

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle mass in mammals. We have studied myostatin expression during embryonic and post-hatching development in zebrafish by semiquantitative RT-PCR. The transcript is present in just-fertilized eggs and declines at 8 h post-fertilization (hpf), suggesting a maternal origin. A secondary rise occurs at 16 hpf, indicating the onset of embryonic transcription at the time of muscle cell differentiation. The level of myostatin mRNA decreases slightly at 24 hpf, when somitogenesis is almost concluded, and rises again at and after hatching, during the period of limited muscle hyperplastic growth that is typical of slow-growing, small fish. In the adult muscle, we found the highest expression of myostatin mRNA and protein, which were detectable by Northern and Western blot analyses respectively. Although only the precursor protein form was revealed in the adult lateral muscle, we demonstrated that zebrafish myostatin is proteolytically processed and secreted in cultured cells, as is its mammalian counterpart. These results suggest that myostatin may play an important regulatory role during myogenesis and muscle growth in fish, as it does in mammals. In chronically stressed fish, grown from 16 days post-fertilization to adulthood in an overcrowded environment, we observed both depression of body growth and a diminished level of myostatin mRNA in the adult muscle, as compared with controls. We propose that chronic stunting in fish brings about a general depression of muscle protein synthesis which does not spare myostatin.

[Thermal energy balance during hemodialysis: the role of the filter membrane]. / Studio del bilancio termico durante la dialisi: ruolo svolto dalla membrana dei filtri.

BACKGROUND: Body temperature tends to increase during conventional haemodialysis; this might interfere with normal cardiovascular response to dialytic ultrafiltration, thus facilitating the occurrence of symptomatic hypotension. Putative factors responsible for changes in thermal balance during haemodialysis include heat load from the dialysis bath, reduction in convective heat loss secondary to skin vessel vasoconstriction, heat overproduction due to central stimulation by bioincompatibility reactions to the filter membranes. The aim of the present study was twofold: to define thermal energy balance (ET) during dialysis and to investigate the effect of membrane bioincompatibility on energy balance METHODS: We measured ET in 12 patients (9M, 3F) during two identical dialysis sessions, differing only in the membrane composition of the filters used: cuprophane 1.3- 1.6 mq and LF polysulphone 1.3- 1.6 mq. Thermal energy balance studies were performed by the Blood Temperature Monitor (Fresenius Medical Care) under conditions in which the core temperature of the patients was maintained unchanged from the start to the end of the dialysis procedure. RESULTS: Arterial blood temperatures were constant, while dialysate and venous blood temperatures progressively decreased (from 36.9 to 35.4 C and from 36.5 to 35.1 C for cuprophane; from 36.9 to 35.2 and from 36.9 to 35.1 for polysulphone membrane). Mean thermal energy transfer was negative (removal of energy from the patients to the extracorporeal circuit) with both filters, equal to 146 KJ with cuprophane and to 163 KJ with polysulphone. When a stepwise multiregression analysis was applied, hourly energy transfer (ET) was significantly and independently correlated with both ultrafiltration rate (UF=% b.w.) and heart rate changes (HR) according to the equation: ET= -92.03+41.29 UF+1.04 HR (p<0.0003). Conclusions. In this study we present experimental evidence that increased body temperature during dialysis is not caused by heat load from the dialysis bath, nor by heat over production secondary to bioincompatibility reactions. Consequently, haemodynamic responses to dialytic ultrafiltration may be regarded as the main regulatory factor of thermal balance.

Hormonal steroidogenesis in liver and small intestine of the green frog, Rana esculenta L.

We have investigated the potential of autonomous hormonal steroidogenesis in liver and small intestine of male and female frogs, Rana esculenta, during the recovery phase. After incubation of mitochondrial fractions with [4-14C]cholesterol, female liver and intestine formed pregnenolone at a rate of 0.63 and 2.3 pmol/mg protein/h, respectively, whereas conversion by male organs was only c. 0.03 pmol/mg protein/h. Minced tissues preparations transformed [4-14C]pregnenolone into progesterone and 17alpha-hydroxypregnenolone, the former prevailing in the liver, the latter in the intestine. Moreover, both tissues produced 20alpha-dihydropregnenolone, 20alpha-dihydroprogesterone and dehydroepiandrosterone. From incubates with [4-14C]dehydroepiandrosterone, androstenedione and androst-5-ene-3beta, 17beta-diol were identified, the former being more abundant in the liver, the latter in the intestine. These results indicate that both liver and intestine in frog can be independent sources of hormonally active steroids in both sexes.

Validation of a simple method for assessing sodium intake in dialysis patients.

It has been reported that sodium intake can be estimated in dialysis patients by the increment in the body sodium pool from the end of a dialysis session to the beginning of the following one. To verify the reliability of this method we compared the sodium intake, estimated by the interdialytic changes in plasma sodium concentration (C) and body water volume (V), to sodium removal during three consecutive sessions. For this purpose we investigated 9 nondiabetic patients, 5 females and 4 males, under chronic hemofiltration treatment. Sodium intake was estimated by the formula (C(pre) V(pre)) - (C(post) V(post)) using a flame photometer and electrical bioimpedance to determine the plasma sodium concentration and total body water, respectively. Sodium removal was calculated by the difference between sodium loss into the ultrafiltrate and sodium gain with the reinfusion fluid. The mean values of sodium intake calculated during the three intervals corresponded with the sodium losses measured during the three hemofiltration sessions in each patient (338+/-55 vs. 329+/-67 mEq; r = 0.92, p<0.0001). A direct relationship was also observed between sodium intake and both interdialytic body weight increase (r = 0.89, p< 0.0001) and fluid loss during the sessions (r = 0.88, p<0.0001). This data demonstrates that sodium intake can be properly estimated by the interdialytic changes in body water and plasma sodium concentrations. They also suggest that fluid intake may be influenced by sodium consumption and that sodium intake monitoring could be useful for the control of excessive interdialytic fluid gain.

Regulatory regions in the promoter and third intron of the growth hormone gene in rainbow trout, Oncorhynchus mykiss walbaum.

The mechanisms involved in the transcriptional regulation of the rainbow trout (Oncorhynchus mykiss) growth hormone (tGH) gene have been investigated. Transient transfection assays, using deletion mutants of the tGH promoter, demonstrated that the -226/+24 5'-flanking region, bearing three binding sites for the pituitary-specific transcription factor GHF1/Pit1 and a cAMP-response element, is necessary and sufficient to confer strong tissue-specific and cAMP-stimulated expression to a luciferase reporter gene. This region is also upregulated by the synthetic glucocorticoid dexamethasone (DEX), the combined effects of cAMP, and DEX being synergistic. Footprinting and gel shift assays revealed that GHF1 binds to a recognition element in the third intron of the tGH gene, suggesting that GHF1 can affect the expression of this gene by interacting with response elements in the transcription unit. These results may be exploited to design tGH gene constructs for the production of autotransgenic fish, in which the expression of the isospecific transgene driven by a constitutive proximal promoter is specifically targeted to the pituitary and physiologically controlled.

Potential for estrogen synthesis and action in human normal and neoplastic thyroid tissues.

To investigate the potential of intracrine or paracrine estrogen synthesis and action in the human thyroid gland and thyroid tumors, the presence of the messenger ribonucleic acids (mRNAs) of both cytochrome P450 aromatase (P450arom) and estrogen receptor (ER) was investigated by RT-PCR with primers designed on the respective coding regions and Southern hybridization analysis with specific probes in neoplastic (n = 42), hyperplastic (n = 7), and adjacent histologically normal thyroid tissues (n = 33) obtained from 43 female and 7 male patients. Most thyroid tissues were positive for both mRNAs, but 2 normal and 3 neoplastic tissues were negative for P450arom mRNA only, 3 normal and 1 hyperplastic tissues were negative for ER mRNA only, and 2 normal tissues were double negative. In some patients, P450arom mRNA was absent in either the neoplastic tissue or the normal one. Single and double negative samples were relatively more frequent in men (n = 4) than in women (n = 7). All negative samples were positive for beta-actin mRNA. RT-PCR amplification and Southern blotting of promoter-specific untranslated 5'-termini revealed that the human thyroid gland and tumors mainly use the ovarian-type promoter, promoter II, for CYP19 expression. Transcripts with either exon I.4 or I.1 were present only in some samples and in very low copy number. When 18 neoplastic samples with their surrounding normal tissues were analyzed immunohistochemically, 57% of those that were positive for P450arom mRNA also had a positive immunoresponse for the corresponding protein. In the case of ER, the percentage was 58%. Immunostaining for P450arom was often particularly intense in neoplastic samples. When 3 adenomata and 1 papillary cancer were incubated with [1,2,6,7-3H]testosterone, 17beta-estradiol could be radiochemically identified with a maximal yield of 10.5 fmol/mg x h. In conclusion, the human thyroid gland appears to have the potential for both estrogen synthesis and intracrine or paracrine estrogen responsiveness, which seem to be greater in women than men and may become enhanced with the process of tumorigenesis.

Immunodetection of cell adhesion molecules in rat Sertoli cell cultures.

PROBLEM: The presence of cell adhesion molecules (CAMs) in Sertoli cells has not been explored extensively. The expression of CAMs involved in cell-matrix and cell-to-cell interactions in Sertoli cell cultures was examined. METHOD OF STUDY: Immunohistochemical and Western blot techniques were applied to rat Sertoli cell cultures using specific antibodies to alpha 3, alpha 5, and alpha 6 integrin subunits; NCAM; and cadherins. RESULTS: Expression of alpha 3 and alpha 6 integrin subunits (mainly laminin receptors) and lack of expression of alpha 5 integrin subunit (fibronectin receptor) was observed in Sertoli cells by immunohistochemistry. These cells also expressed neural CAM (NCAM) and N-cadherin. By Western blot analysis, Sertoli cell extracts reacted with antibodies to alpha 3 integrin subunit revealed a band approximately 130 kDa, whereas no expression of alpha 5 integrin subunit was detected. Cell extracts incubated with antibodies to pan cadherin exhibited a band approximately 120 kDa, whereas bands of 180, 140, and 120 kDa were observed with antibodies to NCAM. CONCLUSION: New data about the expression of receptors for extracellular matrix proteins (alpha 3 and alpha 6 integrin subunits) as well as cell-to-cell adhesion molecules (NCAM and cadherins) are reported in rat Sertoli cell cultures.

Developmentally regulated expression and activity of 17alpha-hydroxylase/C-17,20-lyase cytochrome P450 in rat liver.

We have investigated the developmental pattern of expression and activity of 17alpha-hydroxylase/C-17,20-lyase cytochrome P450 (cytochrome P450c17) in the liver, stomach, duodenum, and testis of rats from day 18 of pregnancy to adulthood. In the male liver, the enzyme became detectable at birth (135 pmol/mg protein x min) at a level comparable to that in the testis (188 pmol/mg protein x min). The activity then increased dramatically, reaching a peak at 8 days (691 pmol/mg protein x min), which was more than 4-fold the testicular levels in rats of the same age or in adults. Thereafter it declined steadily, becoming undetectable from puberty onward. The hepatic peak followed a depression in testicular activity (58 pmol/mg protein x min) on day 6. Northern and immunoblot analyses showed a good temporal correlation between enzyme activity and the occurrence of P450c17 messenger RNA (mRNA) and protein. The same patterns of mRNA and protein occurrence were observed in female rat liver, indicating that the hepatic CYP17 expression is not sexually dimorphic. Sequencing confirmed a complete identity in the coding region between hepatic and gonadal mRNAs. Hepatic P450c17 mRNA, however, was 150-200 bases longer than the gonadal counterparts. No significant expression of mRNAs encoding P450scc and P450arom was observed in liver of either sex at any age. In stomach and duodenum, enzyme activity was much lower (maxima at 25 and 14 pmol/mg protein x min, respectively) than that in liver, but persisted from the time of weaning onward. It is suggested that the hepatic peak in P450c17 activity may serve to convert circulating progestogens into androgens for gonadal aromatization during Sertoli and granulosa cell proliferation.

Immunodetection of cell adhesion molecules and extracellular matrix proteins in rat Leydig cell cultures.

Cell adhesion molecules (CAMs) as well as extracellular matrix (ECM) proteins were identified in Leydig cell cultures using immunohistochemical and Western blot analysis. Leydig cells were isolated from 60-day-old rats and cultured for 4 days. For immunofluorescence and immunoperoxidase techniques, Leydig cells were incubated with antisera to ECM proteins (antibodies to laminin, type IV collagen and fibronectin); antisera to integrins (antibodies to beta 1, alpha 3, alpha 5 and alpha 6 integrin subunits) and antisera to cell-to-cell adhesion molecules (antibodies to N-CAM and N-cadherin). Results of the two immunohistochemical techniques were similar. Laminin and type IV collagen were detected in the perinuclear area of Leydig cell cytoplasm and cell processes as bright granular immunofluorescence or as a brown reaction product using the immunoperoxidase technique. Leydig cells expressed alpha 3 and alpha 6 integrin subunits (mainly laminin receptors), while no reaction was detected with antibodies to the alpha 5 integrin subunit (fibronectin receptor). Leydig cells also expressed cell-to-cell adhesion molecules such as N-CAM and N-cadherin. Using Western blot analysis, Leydig cell extracts incubated with antibodies to laminin revealed two bands of around 200 kDa, which is characteristic of laminin 1 light chains. A band with electrophoretic mobility similar to that of the alpha 2 (IV) collagen chain from EHS sarcoma and a band of around 230 kDa similar to fibronectin were also detected in Leydig cell extracts using specific antisera. Leydig cells incubated with antibodies to the alpha 3 integrin subunit revealed two bands below 120 kDa. Finally, Western blot results showed that Leydig cells expressed N-CAM as two faint bands of around 140 kDa and N-cadherin as a 120 kDa band. The present data suggest that Leydig cells in culture are able to synthesize ECM proteins and express ECM receptors (integrins), as well as cell-to-cell adhesion molecules such as N-CAM and N-cadherin.

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat.

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17alpha-hydroxyprogesterone, androstenedione and testosterone. The transition from C21 to C19 steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17alpha-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-3H]progesterone and [1,2-3H]17alpha-hydroxyprogesterone as substrates. In the testis and male and female duodena, the Km values for progesterone 17alpha-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the Vmax values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the Km values for exogenous 17alpha-hydroxyprogesterone were 525, 675 and 637 nM, whereas the Vmax values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the Km values were calculated with respect to intermediate 17alpha-hydroxyprogesterone formed from progesterone, they were similar to the Km values for 17alpha-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the Vmax values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of Km values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17alpha-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17beta inhibited 17alpha-hydroxylation but not bond scission on intermediate 17alpha-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.

The transcriptional regulation of the growth hormone gene is conserved in vertebrate evolution.

Growth hormone (GH) gene expression in mammals is regulated by the interaction of the transcription factor Pit-1 with two binding sites within the proximal promoter. Four sequences, homologous to the mammalian Pit-1 motif occur in the rainbow trout (Oncorhynchus mykiss) GHII (rtGH) gene promoter, two of which partly overlap. The three regions containing these putative Pit-1 binding sequences were protected from deoxyribonuclease I digestion by nuclear extracts of GC cells, a rat pituitary tumor cell line producing Pit-1. In gel shift assays, nuclear proteins from GC cells and from trout pituitaries were found to interact specifically with one of these protected sites. Transfection experiments showed that the rtGH promoter is transcriptionally active in GC cells, the response being strongly enhanced in the presence of a cAMP analogue. The results demonstrate that rat Pit-1 binds to and activates the rtGH promoter, indicating that the basic mechanisms regulating GH gene transcription have been conserved between fish and mammals.

The influence of reproductive and hormonal factors on thyroid cancer in women.

We conducted a study on 165 women with thyroid cancer and 214 hospital controls in order to investigate the role of reproductive and hormonal factors in the aetiology of thyroid cancer. Late age at menarche (Relative risk (RR) for menarche at greater than or equal to 14 vs less than or equal to 11 = 2.8), menstrual irregularity (RR = 1.7), late age at first birth (RR for first birth at greater than or equal to 28 vs less than or equal to 21 = 2.4) and at last birth (RR for last birth greater than or equal to 30 vs less than 30 = 2.2) significantly increased the risk of the development of thyroid cancer both in premenopausal and postmenopausal women. Parity was, in the present study rather inconsistently related to disease status whereas voluntary abortions and miscarriages were completely unrelated. A non significant increasing risk was observed with age at menopause older than 50, and with the use of oral contraceptives in premenopausal women. The mechanism of action of female hormones on the thyroid gland remains largely obscure, but the observation that age at first and, perhaps, subsequent pregnancies may be relevant points to an interplay of reproductive factors (and, possibly, their hormonal correlates) more complex than previously suggested.

Effect of intravenous supplementation of a new essential amino acid formulation in hemodialysis patients.

Protein energy undernutrition (PEU) and abnormalities of amino acid (AA) metabolism are common in maintenance hemodialysis patients (MHP). A new EAA formulation (BS695), enriched with valine and threonine, containing some histidine, and low in phenylalanine and methionine was recently developed. We randomly supplemented 11 MHP with this solution (treated group, TG) and 10 MHP with a standard AA solution containing both essential and non-essential AA (control group, CG). Both groups received 3.65 g of nitrogen, i.v. three times per week during hemodialysis for six months. During treatment, dietary intake remained stable in both groups. Before treatment, after three and six months of treatment, and six months after the end of treatment, we determined routine blood chemistries, anthropometry, serum protein levels (albumin, transferrin), delayed cutaneous sensitivity (Multi-test), protein catabolic rate (PCR), plasma AA content and motor nerve conduction velocity (MNCV). Before treatment PEU, predominantly of marasmic type, was common. After treatment anthropometry and immune response were unchanged in both groups; PCR increased more in CG than in TG; serum albumin levels decreased significantly only in CG; MNCV improved in TG and worsened in CG. These preliminary results suggest that this new EAA formulation may have beneficial effects on some nutrition related abnormalities of MHP. Better results might occur with long-term AA supplementation, particularly if it is associated with a higher energy intake.

Risk factors for thyroid cancer in northern Italy.

We conducted a hospital based case-control study of 245 cases of thyroid cancer (62 males and 183 females) and 411 controls in three areas of Northern Italy. Subjects with thyroid cancer more often had a history of benign thyroid nodules (18 cases versus 0 controls, lower 95% confidence limit = 7.84), goitre (RR = 5.61, 95% confidence interval (CI): 2.13-14.77) and residence in endemic goitre areas (RR for residence longer than 20 years = 2.29, 95% CI: 1.23-4.29). Heavy (greater than 1000 rads) irradiation of the neck was reported only by seven cases (lower 95% confidence limit: 2.45). Among 31 food items considered, a few showed direct association, including starchy foods and various sources of animal fats or proteins, whereas frequent consumption of other foods, including major sources of dietary iodine (such as fish, green vegetables and fruit) gave significant protection. When analysis was restricted to various subgroups of patients (ie, different histological types, sexes and major groups of hospital controls), it yielded similar results.

Evaluation of use patterns of psychotropic drugs in an Italian geriatric hospital.

This study is a survey of psychotropic drug use patterns among 934 elderly patients admitted to the Padua Geriatric Hospital. 34% of the patients took psychotropic drugs during their stay in hospital. The drugs most commonly used are anxiolytics and hypnotics of the benzodiazepine class. Women subjects living alone, and to a lesser extent those suffering from particular disorders, as of the central nervous system and the musculoskeletal apparatus, seem to be at higher risk in the use of psychotropic drugs. Moreover, the results show that psychotropic drugs are prescribed during the stay in hospital and even on discharge to a high percentage of subjects who did not use such drugs before admission to the hospital.