Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice.

The FAM20 family of secreted proteins consists of three members (FAM20A, FAM20B, and FAM20C) recently linked to developmental disorders suggesting roles for FAM20 proteins in modulating biomineralization processes. The authors report here findings in knockout mice having null mutations affecting each of the three FAM20 proteins. Both Fam20a and Fam20c null mice survived to adulthood and showed biomineralization defects. Fam20b (-/-) embryos showed severe stunting and increased mortality at E13.5, although early lethality precluded detailed investigations. Physiologic calcification or biomineralization of extracellular matrices is a normal process in the development and functioning of various tissues (eg, bones and teeth). The lesions that developed in teeth, bones, or blood vessels after functional deletion of either Fam20a or Fam20c support a significant role for their encoded proteins in modulating biomineralization processes. Severe amelogenesis imperfecta (AI) was present in both Fam20a and Fam20c null mice. In addition, Fam20a (-/-) mice developed disseminated calcifications of muscular arteries and intrapulmonary calcifications, similar to those of fetuin-A deficient mice, although they were normocalcemic and normophosphatemic, with normal dentin and bone. Fam20a gene expression was detected in ameloblasts, odontoblasts, and the parathyroid gland, with local and systemic effects suggesting both local and/or systemic effects for FAM20A. In contrast, Fam20c (-/-) mice lacked ectopic calcifications but were severely hypophosphatemic and developed notable lesions in both dentin and bone to accompany the AI. The bone and dentin lesions, plus the marked hypophosphatemia and elevated serum alkaline phosphatase and FGF23 levels, are indicative of autosomal recessive hypophosphatemic rickets/osteomalacia in Fam20c (-/-) mice.

Hydraulic trade-offs and space filling enable better predictions of vascular structure and function in plants.

Plant vascular networks are central to botanical form, function, and diversity. Here, we develop a theory for plant network scaling that is based on optimal space filling by the vascular system along with trade-offs between hydraulic safety and efficiency. Including these evolutionary drivers leads to predictions for sap flow, the taper of the radii of xylem conduits from trunk to terminal twig, and how the frequency of xylem conduits varies with conduit radius. To test our predictions, we use comprehensive empirical measurements of maple, oak, and pine trees and complementary literature data that we obtained for a wide range of tree species. This robust intra- and interspecific assessment of our botanical network model indicates that the central tendency of observed scaling properties supports our predictions much better than the West, Brown, and Enquist (WBE) or pipe models. Consequently, our model is a more accurate description of vascular architecture than what is given by existing network models and should be used as a baseline to understand and to predict the scaling of individual plants to whole forests. In addition, our model is flexible enough to allow the quantification of species variation around rules for network design. These results suggest that the evolutionary drivers that we propose have been fundamental in determining how physiological processes scale within and across plant species.

Increased aortic atherosclerotic plaque development in female apolipoprotein E-null mice is associated with elevated thromboxane A2 and decreased prostacyclin production.

The production of thromboxane A(2) (TXA(2)) and prostacyclin (prostaglandin I(2), PGI(2)) is known to be increased in patients with atherosclerosis. In this study, we evaluated the influence of gender on TXA(2) and PGI(2) production, and their association with the progression of atherosclerosis in apolipoprotein E-null (ApoE(-/-)) mice maintained on a high fat diet for 3 months. En face analyses of aortas showed marked increases in plaque formation in female ApoE(-/-) mice. Quantification of the hematoxylin/eosin (H&E) stained cross sections of the aortic arch revealed 3 to 4-fold higher plaque thickness in female ApoE(-/-) mice. Analyses of 24-hours urine samples for 11-dehydro TXB(2) and 2, 3-dinor-6-keto PGF(1a) indicated that female ApoE(-/-) mice produce up to 15-fold more TXA(2) and 50% less PGI(2) than the age matched males. Interestingly, the serum cholesterol levels in ApoE(-/-) females were 20% lower than males on the high fat regimen. No gender-associated changes in the number of T lymphocytes, mast cells and macrophages were evident in the lesion areas of ApoE(-/-) mice. The results suggest that the markedly elevated TXA(2) production and reduced PGI(2) production are gender-related proatherogenic risk factors in female ApoE(-/-) mice.

Uterine effects of tamoxifen: a prospective study.

The purpose of the study was to evaluate tamoxifen-associated changes in the vagina and uterus in postmenopausal breast cancer patients. Between June 1994 and December 1998, 45 patients enrolled in a prospective study before commencing tamoxifen therapy. Patients with endometrial thickness >5 mm or neoplasia were excluded. Transvaginal ultrasonography, vaginal maturation indexes (VMI), and endometrial biopsy were performed at baseline and repeated at 6 months (n= 42), 1 year (n= 39), 2 years (n= 32), 3 years (n= 26), 4 years (n= 19), and 5 years (n= 15). For the 39 patients followed for 1 year, VMI (% parabasal/intermediate/superficial) was 21/71/8 at baseline compared with 1/90/9 at 1 year (P value = 0.0008/0.001/0.78). At baseline, mean endometrial thickness and uterine volume were 2.6 mm and 64 cm(3), respectively, compared with 5.8 mm and 84 cm(3) at 1 year (P= 0.0002, 0.002). At baseline, 80% of patients had atrophic endometrium and 9% proliferative endometrium compared with 61% and 26% at 1 year, respectively (P= 0.04). No cases of endometrial hyperplasia or adenocarcinoma were detected. Findings observed at 6 months persisted through 5 years of follow-up. Tamoxifen exerts a weak estrogenic effect on the vagina and uterus in highly prescreened postmenopausal women without preexisting endometrial pathology.

Solvent effects on coupling yields during rapid solid-phase synthesis of CGRP(8-37) employing in situ neutralization.

The success of solid-phase peptide synthesis is often dependent upon solvation of the resin and the growing resin-bound peptide chain. We investigated the relationship between solvent properties and solvation of the resin and peptide-resin in order to obtain satisfactory coupling yields for the rapid solid-phase peptide synthesis, using butyloxycarbonyl-(Boc)-amino acid derivatives, of human-alpha-calcitonin gene-related peptide(8-37) (CGRP(8-37)). Solvation of (p-methylbenzhydrylamine)copoly(styrene-1% divinylbenzene (DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8-37) (peptide-resin) was correlated to solvent Hildebrand solubility (delta) and hydrogen-bonding (delta(h)) parameters. Contour solvation plots of delta(h) vs. delta revealed maximum solvation regions of resin and peptide-resin. Maximum resin solvation occurred with N-methylpyrrolidinone (NMP), NMP : dimethylsulfoxide (DMSO) (8 : 2) and DMSO. Inefficient solvation of the peptide-resin occurred with these solvents and resulted in poor syntheses with average coupling yields of 78.1, 88.9 and 91.8%, respectively. Superior peptide-resin solvation was obtained using dimethylacetamide (DMA) and dimethylformamide (DMF), resulting in significantly higher average coupling yields of 98.0 and 99.5%, respectively. Thus, the region of maximum peptide-resin solvation shifts to solvents with higher delta(h) values. DMF provided the most effective peptide-resin solvation and was the only solvent from which CGRP(8-37) was obtained as a single major product in the crude cleaved material.

Immunological comparison of 124 isolates of Toxoplasma gondii.

We tested 124 isolates of Toxoplasma gondii, as determined morphologically and by their ability to elicit antibodies in the dye test with the RH strain of Toxoplasma in mice. They were compared for their capacity to immunize CF-1 mice against isolate T-1, and T-1 immune mice for their capacity to resist each of the 123 other isolates. Of the 125 isolates, 52 had been isolated in the continental USA, 33 in Central America, 15 in Europe, 9 in Hawaii, five in Japan, two in Taiwan, five in Australia, one in Indonesia, one in Tunisia, and one was of unknown origin. Complete cross-immunity was found. This suggests that only one immunotype of Toxoplasma is prevalent in the United States, and perhaps all over the earth. Vaccines are likely to immunize against most or all Toxoplasma isolates.

Determination of the genera of cyst-forming coccidia.

The following heteroxenous and cyst-forming coccidian genera, Besnoitia, Cystoisospora, Frenkelia, Hammondia, Neospora, Sarcocystis and Toxoplasma have been compared biologically, and a key to determine their tissue cysts is provided.

Controlling deamidation rates in a model peptide: effects of temperature, peptide concentration, and additives.

The rate of deamidation of the Asn residue in Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA), a model peptide, was determined at pH 9 (400 mM Tris buffer) as a function of temperature and peptide concentration. Over the temperature range 5-65 degrees C, deamidation followed Arrhenius behavior, with an apparent activation energy of 13.3 kcal/mol. Furthermore, increasing the peptide concentration slows the rate of deamidation. Self-stabilization with respect to deamidation has not been reported previously. The rate of deamidation was also determined in the presence of sucrose and poloxamer 407 (Pluronic F127). In both cases, the rate of deamidation was retarded by up to 40% at 35 degrees C. In aqueous solutions containing poloxamer 407, the degree of stabilization is independent of formation of a reversible thermosetting gel. With sucrose, maximum reduction in the deamidation rate was attained with as little as 5% (w/v). Addition of sucrose results in a greater conformational preference for a type II beta-turn structure, which presumably is less prone to intramolecular cyclization and subsequent deamidation.

Functional calcitonin gene-related peptide subtype 2 receptors in porcine coronary arteries are identified as calcitonin gene-related peptide subtype 1 receptors by radioligand binding and reverse transcription-polymerase chain reaction.

Calcitonin gene-related peptide (CGRP) receptors are classified into CGRP subtype 1 (CGRP(1)) and CGRP subtype 2 (CGRP(2)) based on the affinity of the antagonist, human alpha (halpha)-CGRP(8-37). halpha-CGRP(8-37) antagonizes CGRP(1) receptor-mediated responses with high affinity (K(B) < 100 nM) and antagonizes CGRP(2) receptor-mediated responses with low affinity (K(B) > 1 microM). CGRP(2) receptors have been previously reported to mediate relaxation of large porcine coronary arteries because this action is antagonized with low affinity by halpha-CGRP(8-37). In the present study, we used reverse transcription-polymerase chain reaction, radioligand binding, and values from our previously reported isolated tissue experiments to compare the CGRP receptor in porcine coronary arteries with the porcine CGRP(1) receptor stably expressed in human embryonic kidney (HEK) 293 cells. We identified calcitonin receptor-like receptor and receptor activity modifying protein 1 mRNA in coronary arteries. We also found that the ligand binding characteristics of the CGRP receptor in coronary arteries and the cloned CGRP(1) receptor were highly similar. K(I) values for halpha-CGRP(8-37) were 6.6 and 5.7 nM in porcine coronary arteries and the cloned CGRP(1) receptor, respectively. The affinities (K(B)) of halpha-CGRP(8-37) and five other antagonists were 22- to 707-fold lower in functional experiments measuring relaxation of coronary arteries than in radioligand binding experiments. Despite this difference in absolute affinity values, there was a high correlation of the rank order of affinity for the antagonists determined by the two methods. Thus halpha-CGRP(8-37) antagonizes CGRP-induced relaxation of porcine coronary arteries with low affinity at the CGRP(1) receptor. Taken together, these data do not support the existence of the CGRP(2) receptor.

Results of ambulatory electrocardiography in overtly healthy Doberman Pinschers with echocardiographic abnormalities.

OBJECTIVE: To identify, by means of 24-hour ambulatory electrocardiography, electrocardiographic abnormalities in overtly healthy Doberman Pinschers in which results of echocardiography were abnormal. DESIGN: Clinical case series. ANIMALS: 56 (35 male, 21 female) overtly healthy Doberman Pinschers with echocardiographic evidence of cardiomyopathy on initial examination that subsequently died of cardiomyopathy. PROCEDURE: Twenty-four-hour ambulatory electrocardiographic (Holter) recordings obtained at the time of initial examination were reviewed. For all dogs, scan quality was > 90%. RESULTS: Initial Holter recordings of all 56 dogs contained ventricular premature contractions (VPC). Thirty-six (65%) dogs had > 1,000 VPC/24 h, 17 (31%) had > 5,000 VPC/24 h, and 11 (19%) had > 10,000 VPC/24 h. Fifty-four (96%) dogs had couplets of VPC, 37 (66%) had triplets of VPC, and 36 (64%) had episodes of nonsustained (< 30 seconds) ventricular tachycardia. Number of VPC/24 h during the initial Holter recordings was positively correlated with numbers of couplets and triplets of VPC and number of ventricular escape beats and negatively correlated with left ventricular fractional shortening. Twenty-eight dogs died suddenly prior to the putative onset of congestive heart failure. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that along with echocardiography, 24-hour ambulatory electrocardiography can be used to help identify overtly healthy Doberman Pinschers with cardiomyopathy.

Isolation and characterization of insulin from the Brockmann body of Dissostichus mawsoni, an Antarctic teleost fish.

The Brockmann body of fish synthesizes and secretes insulin. The Brockmann body of Antarctic fish has been described anatomically and shown to contain insulin immunoreactive sites, however, the primary structure of an Antarctic fish insulin has yet to be reported. Insulin was isolated from the Brockmann bodies of the Antarctic perciform teleost, Dissostichus mawsoni. The peptide was purified to homogeneity by gel filtration and reversed-phase HPLC. Insulin-containing fractions were identified by radioimmunoassay using antisera raised against porcine insulin. Electrospray ionization-mass spectrometry determined the mass of the isolated product to be 5725.27 a.m.u. The amino acid composition and primary structure were determined for the pyridylethylated A- and B-chains. The amino acid sequences of the A chain and B chain were H-Gly-lle-Val-Glu-Gln-Cys-Cys-His-Gln-Pro10-Cys-Asn-Ile-Phe- Asp-Leu-Gln-Asn-Tyr-Cys20-Asn-OH and H-Ala-Pro-Gly-Pro-GIn-His-Leu-Cys-Gly-Ser10-His-Leu-Val-Asp-Ala-Le u-Tyr-Leu-Val-Cys20-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Asn-Pro-Lys30++ +-OH, respectively. The primary structure of insulin from Antarctic fish is compared with known structures of insulin from other vertebrates.

Association between results of ambulatory electrocardiography and development of cardiomyopathy during long-term follow-up of Doberman pinschers.

OBJECTIVE: To characterize ambulatory electrocardiographic results of overtly healthy Doberman Pinschers and determine associations between those results and development of dilated cardiomyopathy. DESIGN: Cohort study. ANIMALS: 114 (58 male, 56 female) overtly healthy Doberman Pinschers without echocardiographic evidence of cardiac disease on initial examination. PROCEDURE: Echocardiograms and 24-hour ambulatory electrocardiograms (Holter recordings) were obtained initially and at variable intervals. The status (live vs dead) of all dogs was known at least 2 years and as long as 10 years after initial examination (mean [+/- SD] follow-up time, 4.33 +/- 1.84 years). Associations between development of dilated cardiomyopathy and number of ventricular premature contractions (VPC), age, and sex were determined. RESULTS: 55 dogs (48%) did not have VPC on initial Holter recordings, and only 8 dogs had > 50 VPC/24 hours. The likelihood that a dog would have VPC was associated with increasing age and being male. At least 1 VPC/24 hours, and in particular, > 50 VPC/24 hours or > or = 1 couplet or triplet of VPC/24 hours, were predictive of subsequent development of dilated cardiomyopathy. Fifty-four dogs (47%) developed dilated cardiomyopathy; 12 were still alive at the end of the study, and 42 had died. Twenty-five of these 42 dogs died after the onset of congestive heart failure (CHF), 15 died suddenly before the onset of overt CHF, and 2 died of noncardiac causes. More males developed dilated cardiomyopathy than females, and dogs that died suddenly were approximately 1 year younger than those that developed CHF. CONCLUSIONS AND CLINICAL RELEVANCE: Results of high-quality Holter recordings may be used to identify overtly healthy Doberman Pinschers that are at a high risk for dilated cardiomyopathy.

Optical parametric generation in 2-mum-wavelength-pumped periodically poled LiNbO(3).

An optical parametric generator based on periodically poled LiNbO(3) and pumped by a 2.051-mum-wavelength laser has been demonstrated. Pump pulses of 50-ns duration of 50-Hz repetition frequency were converted into signal and idler pulses in the 3.4-5.2-mum wavelength range in a double-pass pump configuration by a 5-cm-long quasi-phase-matched crystal. An average pump power of 180 mW generated 30-mW average signal power at 3.64-mum wavelength, corresponding to 16.7% signal conversion efficiency.

Nursing interventions to decrease bleeding at the femoral access site after percutaneous coronary intervention. SANDBAG Nursing Coordinators. Standards of Angioplasty Nursing Techniques to Diminish Bleeding Around the Groin.

BACKGROUND: This trial is the first prospective, multicenter clinical nursing trial conducted to measure the effect of nursing interventions on bleeding at the femoral access site after percutaneous coronary intervention with or without a potent antiplatelet agent given along with heparin and aspirin. OBJECTIVE: To measure the relationship between nursing interventions and complications at the arterial access site in patients undergoing percutaneous coronary interventions and to recommend a standard of care to minimize bleeding complications. METHODS: In a descriptive, correlational 4010-patient study, nursing care interventions after coronary procedures were measured. Observed standards of care were assessed, and regression techniques were used to evaluate nursing interventions and the effect of the interventions on bleeding at the access site after percutaneous coronary procedures. RESULTS: Several significant correlations between nursing interventions and the occurrences of moderate to severe bleeding at the access site were found; however, most interventions had little effect. The most significant factors in decreasing complications at the access site were early removal of the arterial sheath, the type of pressure mechanism used to achieve arterial hemostasis, staffing allocation, and the person and method used to remove the sheath. CONCLUSION: Many nursing interventions after percutaneous coronary intervention have become routine in the absence of clinical outcome data. Most nursing interventions aimed at decreasing bleeding at the vascular access site increase nursing workload but do not significantly affect bleeding in the groin. These results underscore the importance of continued clinical research studies to validate nursing practice on the basis of patients' outcomes.

Predictive value of preclinical toxicology studies for platinum anticancer drugs.

Rodent and nonrodent toxicology studies are currently expected to support Phase I trials of antineoplastic drugs in the United States. To determine the predictive value of these studies, we initiated a project to compare preclinical and clinical toxicity data within various drug classes. The first class analyzed was the platinum anticancer drugs. Twelve platinum analogues that had both preclinical (mice, rats and/or dogs) and clinical data from matching drug administration schedules were identified. The rodent LD10 (the dose that causes lethality in 10% of treated animals) or dog toxic dose high (a dose that when doubled causes lethality in dogs) correlated well with the human maximally tolerated dose on a mg/m2 basis. For every platinum analogue investigated, one-third the rodent LD10 or one-third the dog toxic dose high in mg/m2 gave a starting dose and a first escalation dose that did not exceed the clinical maximally tolerated dose. The dose-limiting toxicities in patients were previously observed in 7 of 7, 7 of 8, and 9 of 11 mouse, rat, and dog studies, respectively. Our data indicate that mice, rats, and dogs all had value in predicting a safe starting dose and the qualitative toxicities in humans for platinum anticancer compounds. The efficiency of Phase 1 trials could have been improved without sacrificing patient safety by allowing higher starting doses for this drug class than conventionally permitted.

Limitations in using peptide drugs to characterize calcitonin gene-related peptide receptors.

Calcitonin gene-related peptide (CGRP) is an endogenous vasodilator peptide that produces its effects by activation of CGRP1 and CGRP2 receptor subtypes. These receptor subtypes are characterized in functional studies using the agonist Cys(Acm)2, 7-human-alpha-calcitonin gene-related peptide (Cys(ACM)2, 7-h-alpha-CGRP), which activates CGRP2 receptors, and the antagonist h-alphaCGRP(8-37) which has a high affinity for CGRP1 receptors and a low affinity for CGRP2 receptors. Our aim was to identify factors that may limit the use of these drugs to characterize CGRP receptor subtypes. We studied CGRP receptors using isolated ring segments of pig coronary and basilar arteries studied in vitro. The affinity of the antagonist h-alphaCGRP(8-37) for inhibiting h-alphaCGRP-induced relaxation of coronary arteries (log10 of the antagonist equilibrium dissociation constant = -5.33) was determined from Schild plots that had steep slopes. Therefore, we used capsaicin to investigate the role of endogenous CGRP in confounding affinity measurements for h-alphaCGRP(8-37). After capsaicin treatment, the slopes of the Schild plots were not different from one, and a higher affinity of h-CGRP(8-37) in blocking relaxation was obtained (log10 of the antagonist equilibrium dissociation constant = -6.01). We also investigated the agonist activity of the putative CGRP2 receptor selective agonist Cys(Acm)2,7-h-alphaCGRP. We found that maximal relaxation of coronary arteries caused by Cys(Acm)2,7-h-alphaCGRP was dependent upon the level of contractile tone induced by KCl. We also determined the KA for Cys(Acm)2,7-h-alphaCGRP and found that the KA (817 nM) was not significantly different from the EC50 (503 nM) for this drug in causing relaxation, indicating that Cys(Acm)2, 7-h-alphaCGRP is a partial agonist. Because experimental conditions affect the actions of h-CGRP(8-37) and Cys(Acm)2,7-h-alphaCGRP, the conditions must be carefully controlled to reliably identify CGRP receptor subtypes.