Gender-specific proteomic responses in zebrafish liver following exposure to a selected mixture of brominated flame retardants.

Proteomic effect screening in zebrafish liver was performed to generate hypotheses following exposure (21 days) to a structurally diverse mixture of brominated flame retardants (BFRs). Fish were exposed to two doses (10 and 100 nmol/g feed). Two-dimensional gel-electrophoresis, image analysis and MALDI-TOF mass-spectrometry revealed 13 and 19 significant responses in males and females, respectively. Effects on proteins related to cellular maintenance and stress were observed in both genders. Regulated proteins were gender-specific, but functionally indicated common protective responses (peroxiredoxin 6 and Zgc:92891 in males and transketolase in females) suggesting oxidative stress. Betaine homocysteine methyltransferase (BHMT) was induced in both genders. In addition a female-specific downregulation of ironhomeostatic proteins (iron-regulatory protein 1 and transferrin) were observed. Our proteomic approach revealed novel responses that suggest important gender-specific sensitivity to BFRs that should be considered when interpreting adverse effects of BFRs.

Elevated zinc protoporphyrin/heme ratios in umbilical cord blood after diabetic pregnancy.

OBJECTIVE: Offspring of diabetes patients may suffer from tissue iron deficiency. Erythrocyte zinc protoporphyrin/heme (ZnPP/H) ratios measure impaired iron status. The aim of the study was to examine whether cord ZnPP/H ratios were associated with pregnancy glycemic control. METHODS: ZnPP/H was measured in cord blood from 31 pregnancies with insulin-treated diabetes (diabetes group) and compared to population normal values. Maternal glycemic control was assessed by daily glucose log, glycosylated hemoglobin and birth weight. RESULTS: Median cord ZnPP/H was higher in the diabetes group than the population normal values (106 (65.2 to 146.8) microM/M vs 68.2 (37.6 to 98.8) micro/M, P < 0.0001). Ratios were directly correlated to surrogates of control (glycosylated hemoglobin, P = 0.05, and birth weight, P < 0.04). Cord ZnPP/H ratios from pregnancies with pre-existing and gestational diabetes were similar. CONCLUSION: Because cord ZnPP/H was higher in large offspring of diabetic pregnancy, it might identify greater iron utilization for fetal erythropoiesis.

Maternal pregnancy weight gain and cord blood iron status are associated with eosinophilia in infancy.

OBJECTIVE: Allergic disease is multifactorial in origin. Because iron nutrition affects immune responses and maternal pregnancy weight gain impairs fetal iron delivery while increasing fetal demands for growth, the study examined maternal pregnancy weight gain, newborn iron status and an index of atopic disease, infant eosinophilia. STUDY DESIGN: Within a larger prospective study of healthy newborns at risk for developing iron deficiency anemia, umbilical cord iron indicators were compared to infant eosinophil counts. RESULT: Infants who developed eosinophilia exhibited higher cord reticulocyte-enriched zinc protoporphyrin/heme ratio, P<0.05 and fewer cord ferritin values in the highest (best) quartile, P<0.05. If cord ferritin was in the upper three quartiles, the negative predictive value for infant eosinophilia was 90%. High maternal pregnancy weight gain predicted infant eosinophil counts, P<0.04, and contributed to cord ferritin predicting eosinophilia, P<0.003. CONCLUSION: Poor fetal iron status may be an additional risk factor for infant eosinophilia.

Involvement of differential metallothionein expression in free radical sensitivity of RTG-2 and CHSE-214 cells.

The role of metallothionein (MT) in free radical regulation and scavenging was investigated using two fish cell lines, the rainbow trout gonadal (RTG-2) cell line and the chinook salmon embryonic (CHSE-214) cell line. Exposure of RTG-2 cells to H(2)O(2) resulted in upregulation of both MT mRNA and MT protein and was also demonstrated by immunocytochemistry, confirming that MT was regulated by free radicals. We then compared the H(2)O(2) resistance in RTG-2 and CHSE-214 cells following metal treatment with Zn or Cd to induce MT. Comparison of survival of control cells and metal-exposed cells showed that metal treatment, which induced MT, significantly raised the H(2)O(2) tolerance in a dose-dependent manner in RTG-2 cells, while no increased H(2)O(2) resistance was observed in CHSE-214 cells. Transient over-expression of MT in CHSE-214: 59 cells also resulted in a dose-dependent increase in resistance to H(2)O(2) exposure. The raised resistance against H(2)O(2) in metal treated RTG-2 cells as well as transfected CHSE-214: 59 cells strongly demonstrate that MT is involved in the protection against H(2)O(2) and suggest a physiologically important function for MT when cells or whole organisms are exposed to oxidative stress.

In vitro and in vivo evaluation of dihydropyrimidinone C-5 amides as potent and selective alpha(1A) receptor antagonists for the treatment of benign prostatic hyperplasia.

alpha(1) Adrenergic receptors mediate both vascular and lower urinary tract tone, and alpha(1) receptor antagonists such as terazosin (1b) are used to treat both hypertension and benign prostatic hyperplasia (BPH). Recently, three different subtypes of this receptor have been identified, with the alpha(1A) receptor being most prevalent in lower urinary tract tissue. This paper explores 4-aryldihydropyrimidinones attached to an aminopropyl-4-arylpiperidine via a C-5 amide as selective alpha(1A) receptor subtype antagonists. In receptor binding assays, these types of compounds generally display K(i) values for the alpha(1a) receptor subtype <1 nM while being greater than 100-fold selective versus the alpha(1b) and alpha(1d) receptor subtypes. Many of these compounds were also evaluated in vivo and found to be more potent than terazosin in both a rat model of prostate tone and a dog model of intra-urethral pressure without significantly affecting blood pressure. While many of the compounds tested displayed poor pharmacokinetics, compound 48 was found to have adequate bioavailability (>20%) and half-life (>6 h) in both rats and dogs. Due to its selectivity for the alpha(1a) over the alpha(1b) and alpha(1d) receptors as well as its favorable pharmacokinetic profile, 48 has the potential to relieve the symptoms of BPH without eliciting effects on the cardiovascular system.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 1. Structure-activity relationship in dihydropyrimidinones.

Dihydropyrimidinones such as compound 12 exhibited high binding affinity and subtype selectivity for the cloned human alpha(1a) receptor. Systematic modifications of 12 led to identification of highly potent and subtype-selective compounds such as (+)-30 and (+)-103, with high binding affinity (K(i) = 0.2 nM) for alpha(1a) receptor and greater than 1500-fold selectivity over alpha(1b) and alpha(1d) adrenoceptors. The compounds were found to be functional antagonists in human, rat, and dog prostate tissues. Compound (+)-103 exhibited excellent selectively to inhibit intraurethral pressure (IUP) as compared to lowering diastolic blood pressure (DBP) in mongrel dogs (K(b)(DBP)/K(b)(IUP) = 40) suggesting uroselectivity for alpha(1a)-selective compounds.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 4. Structure-activity relationship in the dihydropyrimidine series.

We have previously disclosed dihydropyridines such as 1a,b as selective alpha(1a) antagonists as a potential treatment for benign prostatic hyperplasia (BPH). The propensity of dihydropyridines toward an oxidation led us to find suitable replacements of the core unit. The accompanying papers describe the structure-activity relationship (SAR) of dihydropyrimidinones 2a,b as selective alpha(1a) antagonists. We report herein the SAR of dihydropyrimidines such as 4 and highlight the similarities and differences between the dihydropyrimidine and dihydropyrimidinone series of compounds.

Cerebral metabolism of L-[2-18F]fluorotyrosine, a new PET tracer of protein synthesis.

L-[2-18F]fluorotyrosine (2-18FTyr) was evaluated as a tracer of cerebral protein synthesis for positron emission tomography (PET). Its metabolism in murine cerebrum was studied. The uptake in brain reaches a value of approximately 2% of the injected dose per gram tissue after 60 min. The incorporation of the tracer into tissue proteins was proven by discontinuous SDS gel electrophoresis. The protein bound fraction of tissue activity increased to 84% and 89% after 60 and 120 min p.i., respectively. High performance liquid chromatography analysis showed a concomitant decrease of free 2-18FTyr in tissue with time. The sum of free 2-18FTyr, tRNA-and protein-bound 2-18FTyr in cerebral tissue gave an almost quantitative activity balance of 96 +/- 4% at all times examined. A significant formation of fluorodopa or fluorodopamine must therefore be excluded. This shows that L-[2-18F]fluorotyrosine is a promising tracer for quantitation of protein synthesis rates with PET based on a three-compartment model.

Increased amino acid transport into brain tumors measured by PET of L-(2-18F)fluorotyrosine.

The uptake of L-(2-18F)fluorotyrosine (F-Tyr), a newly synthetized amino acid tracer, was studied in 15 patients with various brain tumors by dynamic PET. The higher F-Tyr accumulation in tumors (mean 27% above contralateral tissue) was associated with two-fold transport rates into tumors, while the rate constants describing irreversible incorporation were decreased. The increased F-Tyr transport was not correlated to 68Ga-EDTA accumulation and cannot be explained by disruption of the blood-brain barrier. Kinetic analysis of 2-(18F)-fluoro-deoxy-glucose accumulation in the same patients demonstrated that increased metabolic rates in tumors are mainly caused by altered phosphorylation rates while transport of glucose is less affected. Since F-Tyr transport rates clearly separated tumors from normal tissue and since F-Tyr accumulation was related to tumor grade, PET studies of F-Tyr uptake are of clinical value for diagnosis and classification of brain tumors.

SPECT studies of brain tumors with L-3-[123I] iodo-alpha-methyl tyrosine: comparison with PET, 124IMT and first clinical results.

L-3-[123I]iodo-alpha-methyltyrosine (123IMT) like tyrosine has been reported previously to have a high affinity for a transport system in the blood-brain-barrier (BBB). We examined the kinetic behavior of 124IMT in brain and plasma in two patients with glioblastoma using dynamic positron emission tomography (PET). 124IMT accumulated in brain and tumor tissue, reaching a maximum after 15 min, with a washout of 20% to 35% at 60 min postinjection. Animal experiments confirmed the accumulation of the intact tracer in murine brain, but there was no incorporation into proteins. SPECT studies with 123IMT in patients with different types of brain tumors showed increased uptake in 26 of 32 tumors. Although nonspecific uptake in tumors must be considered, the accumulation of IMT in normal brain and in some tumors with intact BBB suggests a specific uptake of IMT. As transport is the main determinant of initial amino acid uptake, 123IMT appears to be a suitable SPECT tracer of amino acid uptake although it is not incorporated into protein.

The effect of halofenate or halofenate free acid on human, rat and guinea pig platelet aggregation.

Halofenate free acid (HFA), the major metabolite of the hypolipemic agent halofenate, blocked the secondary phase of human platelet aggregation induced by ADP, epinephrine, or thrombin; higher concentrations of clofibrate free acid (CFA) were required to produce similar inhibitory effects on platelet aggregation. HFA and CFA inhibited collagen-induced aggregation of human, rat, or guinea pig platelets. Halofenate orally administered to rats caused inhibition of collagen-induced aggregation when plasma levels of HFA exceeded 300 mug/ml, a clinically achievable human plasma concentration. The platelet inhibitory effects of clofibrate administration were less than those observed with halofenate administration.

A 7-year old white-male boy with progressive neurological deterioration.

A 9-month-old boy presented with rapid deterioration of psychomotor development. He developed seizures at 2 months, and shortly thereafter lost motor skills and developed feeding difficulties, increased startle response, red maculas, and decreased vision. His measurements, including head circumference, were greater than the 95th centile. No organomegaly was found. Serum determination of the hemoxsaminidases confirmed the diagnosis of Sandhoff disease.

Prognostic value of malignant cells in pleural lavage at thoracotomy for bronchial carcinoma.

Despite seemingly radical surgery many patients operated on for bronchial carcinoma will die from their disease. Some patients might benefit from postoperative treatment and a prognostic factor that could identify those with an increased risk for tumor relapse would be of great clinical importance. One possible such factor is the occurrence of malignant cells in pleural lavage performed at operation. To test this hypothesis 224 consecutive patients who had been operated on due to verified or strongly suspected bronchial carcinoma, preoperatively staged as stage I or II, were investigated. After opening the thorax and before manipulation or palpation of the lungs, 300 ml of physiological saline solution was installed into the pleura. After excluding patients who were not radically operated, there remained 138 patients with histologically confirmed lung cancer (carcinoids excluded) and 12.3% showed tumour cells in the washings. Two of 18 patients with metastatic lung disease ( 11%) and one of ten patients with carcinoid tumor also showed malignant cells in the lavage. The patients with lung cancer have been followed for 3 years or until death. After three years 60.2% of those without malignant cells in the pleural lavage were still alive, while this figure was 41.2% in the other group. The difference was not statistically significant. Other factors, such as spread to local lymph nodes, size of tumor, etc. were related to the occurrence of malignant cells in the pleura, and these factors were also better prognostic ones. We conclude that the clinical use of pleural lavage cytology is limited.

Score for neonatal acute physiology and phlebotomy blood loss predict erythrocyte transfusions in premature infants.

OBJECTIVE: To test the hypothesis that utilization of a previously described measure of acuity (ie, the score for neonatal acute physiology [SNAP]) during the first 7 postnatal days predicts which infants with a birth weight of 1500 g or less received erythrocyte transfusion during the initial hospitalization. DESIGN: Retrospective chart review. SETTING: A regional tertiary care newborn intensive care unit at the Arizona Health Sciences Center, University Medical Center, Tucson. MATERIALS: Medical records of premature infants (birth weight, < or = 1500 g) who were admitted from October 1993 to January 1995. MAIN OUTCOME MEASURES: Occurrence or nonoccurrence of erythrocyte transfusion was determined in 47 infants who were compared for demographic information, phlebotomy blood loss, diagnoses, medications, and the SNAP at 0, 1, 2, and 7 days of life. RESULTS: Infants with a birth weight of 1500 g or less received a mean +/- SD of 1.9 +/- 2.9 transfusions with 22 (47%) of the infants given transfusions Infants who were given transfusions vs those who were not given transfusions were of a lower mean +/- SD birth weight (971 +/- 238 g vs 1272 +/- 144 g; P < .001) and a lower gestational age (27.7 +/- 1.6 weeks vs 30.7 +/- 2.8 weeks; P < .001), and they had a greater mean phlebotomy blood loss (3.3 +/- 1.6 mL/kg per day vs 1.4 +/- 0.5 mL/kg per day; P < .001) during the first postnatal week. The SNAP indexes in those who received transfusions were higher at 1, 2, and 7 days of life (P = .03, P = .001, and P < .001, respectively). Using stepwise logistic regression, phlebotomy blood loss and the SNAP at 7 days of life were significant predictors of the number of transfusions. The logistic model predicted which infants had been administered transfusions with 86% sensitivity and 88% specificity. CONCLUSIONS: The efficacy and cost-effectiveness of recombinant human erythropoietin therapy in premature infants remain under study. As earlier treatment with recombinant human erythropoietin may be more efficacious, early identification of which infants currently undergo transfusion may identify those who will receive the greatest benefit from recombinant human erythropoietin therapy. The SNAP distinguished those infants who were given transfusions from those who did not receive transfusions, even after adjusting for phlebotomy blood loss.

Oral prophylaxis with neomycin and erythromycin in colorectal surgery. More proof for efficacy than failure.

In an open, prospective, and randomized investigation on the prophylactic efficacy of peroral neomycin sulfate-erythromycin base vs intravenous ceftriaxone-metronidazole preparation in colorectal surgery, no significantly diverging results between regimens were recorded (1/27 [3.7%] and 2/27 [7.4%] wound infections, respectively). Commentary is made about the diverging results from earlier studies on antimicrobial prophylaxis and on the multifactorial causality of surgical infection. We believe that variables such as physical condition of the patients, virulence and local resistance patterns of bacteria, and technical skill of the surgeons are far more important in regard to the postoperative outcome concerning septic complications than is the choice of proper antibiotics. Thus, to determine the efficacy of antimicrobial prophylaxis, we call for larger investigations in the future, preferably double-blind, where it is possible to better control and diminish the influence of determinants other than the antibiotics being compared.

High and low (Gpp(NH)p-sensitive) affinity sites for beta 2-adrenergic blockers as antagonists of isoproterenol in the field-stimulated rat vas deferens.

Schild plots for (-)-propranolol, timolol and IPS 339, but not penbutolol or carazolol, as antagonists of isoproterenol in the isolated, field-stimulated rat vas deferens exhibited biphasic curves indicating the presence of both high and low affinity sites for these agents in this preparation. Schild plots determined for (-)-propranolol in the presence of prazosin (0.3 micron) or using the prostatic end of the rat vas deferens yielded two similar affinity sites suggesting the lack of involvement of postsynaptic alpha 1-adrenergic mechanisms in the observed response. In the presence of Gpp(NH)p(4 micron), (-)-propranolol, timolol and IPS 339 exhibited only a single affinity site which corresponded to their high affinity site in antagonizing isoproterenol in the absence of Gpp(NH)p. Similarly, only a single high affinity site was observed for (-)-propranolol when salbutamol, rather than isoproterenol, was used as the agonist. The data suggest the two affinity sites observed with some beta-adrenergic blockers in this preparation reflects a possible action of isoproterenol and the antagonists upon two subpopulations of beta 2-adrenergic receptors, one of which is sensitive to Gpp(NH)p.

In vitro pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors.

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl) [1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5-b]pyridine) inhibited specific 125I-[Sar1, Ile8]angiotensin II binding to angiotensin AT1 receptor (Ki = 0.11-0.20 nM) in rabbit aorta, rat adrenal and human angiotensin AT1 receptor in CHO (Chinese hamster ovary transformed) cells and to AT2 receptor (Ki = 0.14-0.23 nM) in rat adrenal and brain receptors. L-163,017 also had a high affinity in the presence of bovine serum albumin (2 mg/ml), for angiotensin AT1 and AT2 receptors on human adrenal (Ki 3.9 and 4.3 nM), aorta (Ki 0.45 and 0.96 nM) and kidney (Ki 3.6 and 2.3 nM). The much higher Ki values in human tissues were likely due to the presence of bovine serum albumin in the binding assay buffer since L-163,017 had Ki values of 0.13 +/- 0.04 and 2.0 +/- 0.04 nM in the absence and presence of bovine serum albumin, respectively, in inhibiting 125I-[Sar1,Ile8]angiotensin II binding to angiotensin AT1 receptor in rat adrenal membranes. Scatchard analysis of 125I-[Sar1,Ile8]angiotensin II binding in the presence of bovine serum albumin (2 mg/ml) in rabbit aorta and bovine cerebellum indicated a competitive interaction of L-163,017 with angiotensin AT1 and AT2 receptors (Ki values 2.5 and 2.1 nM respectively). L-163,017 inhibited angiotensin II-induced aldosterone release in rat adrenal demonstrating that L-163,017 acted as a competitive antagonist (pA2 = 9.9) and lacked agonist activity. L-163,017 also inhibited angiotensin II responses in rat vascular tissues. The specificity of L-163,017 was shown by its lack of activity on the above functional responses produced by other agonists and in several binding assays.

Thyrotropin-releasing hormone receptors in gut tissues resemble pituitary receptors.

The relative order of activity of thyrotropin-releasing hormone (TRH) and various analogs in contracting the isolated guinea pig antrum and duodenum correlated with their potencies in activating thyroid-stimulating hormone (TSH) release. The action of TRH in both tissues was selectively antagonized by the putative pituitary TRH receptor antagonist, chlordiazepoxide (10 microM). The data indicate that the contractions produced by TRH in these gut tissues are mediated by TRH receptors with similar characteristics as the pituitary TRH receptors responsible for TSH release.

A new simple mouse model for the in vivo evaluation of cholecystokinin (CCK) antagonists: comparative potencies and durations of action of nonpeptide antagonists.

A new simple mouse assay for the in vivo evaluation of CCK antagonists which is based upon visual determination of the gastric emptying of a charcoal meal is described. CCK-8 (24 micrograms/kg s.c.) but not various other peptide and nonpeptide agents effectively inhibited gastric emptying in this test system. The effect of CCK-8 was antagonized by established peripheral CCK antagonists but not representative agents of various other pharmacological classes. The rank order of potency of the CCK antagonists were: L-364,718 (ED50 = 0.01 mg/kg, i.v.; 0.04 mg/kg, p.o.) greater than Compound 16 (ED50 = 1.5 mg/kg, i.v.; 2.0 mg/kg p.o.) greater than asperlicin (ED50 = 14.8 mg/kg i.v.) greater than proglumide (ED50 = 184 mg/kg i.v.; 890 mg/kg, p.o.). Duration of action studies based upon ED50 values determined at various time intervals after oral administration showed that L-364,718 and proglumide are considerably longer acting than Compound 16. Asperlicin (ED50 greater than 300 mg/kg, p.o.) was ineffective as a CCK antagonist when administered orally. These data provide the first direct comparisons of the in vivo potencies of current CCK antagonists and demonstrate the utility of a new simple mouse assay for the in vivo characterization of peripheral CCK antagonists.

Neuropeptide Y (NPY) binding sites in rat brain labeled with 125I-Bolton-Hunter NPY: comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens.

The binding of biologically active 125I-Bolton-Hunter (BH)-NPY to rat brain membranes was saturable and reversible and regulated by inorganic cations and guanyl nucleotides consistent with other neurotransmitter receptor systems. The concentration of specific 125I-NPY binding differed in various brain regions, being highest in the hippocampus and lowest in the cerebellum. Scatchard analysis of 125I-NPY binding showed a single class of receptor sites with a Kd = 0.1 nM and Bmax of 3 pmole/g tissue in hippocampus. Peptide YY, porcine and human NPY inhibited the specific 125I-BH-NPY binding with IC50 values of 50-120 pM. In contrast, human NPY free acid and pancreatic polypeptides from human (HPP), rat (RPP) and avian (APP) sources were much weaker (IC50 greater than or equal to 300 nM). The rank order of potencies for NPY analogs and the inactivity of APP and HPP fragment (31-36) on brain binding appeared to correlate with their relative activities in inhibiting contractions of the field-stimulated rat vas deferens. However, PYY, HPP and RPP exhibited activity in the field-stimulated rat vas deferens indicative of a possible action upon sites distinct from the brain NPY binding site.