Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

The third disulfide loop (amino acids 33 to 42) of human epidermal growth factor (hEGF) encompasses the region of highest amino acid conservation among all of the EGF-like family of molecules. The importance of some of these highly conserved residues for the maintenance of biological activity, especially the aromatic amino acid tyrosine at position 37, has until now been considered essential on the basis of previous studies with the EGF-like molecule transforming growth factor alpha. Variants at the Tyr-37 position of hEGF were constructed by site-directed mutagenesis. The substituting amino acids were phenylalanine, histidine, serine, alanine, aspartic acid, arginine, and glycine. The variants were tested for their ability to competitively displace native [125I]hEGF from its receptor and to stimulate the protein-tyrosine kinase activity of the receptor; the order of efficacy of substituting amino acids was Phe greater than His greater than Ser greater than Ala greater than Asp greater than Arg greater than Gly in both assays. All were effective, with no or only moderate reduction in potency, in stimulating the incorporation of [3H]thymidine into acid-insoluble material of quiescent mouse A31 cells. Only Tyr-37----Ala, Tyr-37----Arg and Tyr-37----Gly were slightly less potent in the cell assay. Thus, neither tyrosine nor another aromatic amino acid at position 37 in hEGF is essential for full biological activity.

Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.

Effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on alpha-aminoisobutyrate uptake in friend Erythroleukemic cells.

In Friend erythroleukemic cells, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the rate of influx as well as the steady-state accumulation of the amino acid transport probe alpha-aminoisobutyric acid (AIB). This nonmetabolizable analog is concentrated in these cells by utilizing the energy of the Na+ electrochemical gradient. The effect of TPA is to increase the ouabain-sensitive component of uptake of AIB (and of the K+ analog 86Rb+). Transport changes are not observed until 30 min after application of the promoter and reach a maximum in about 6 hr. The effects are blocked by cycloheximide. The flux results are consistent with a model in which the membrane potential is increased and, with it, the driving force on the coupled Na+-AIB movement. The effect is possibly mediated by a change in electrogenicity of the Na:K pump. TPA raises the steady-state accumulation levels of AIB. By altering external K+ in the presence of valinomycin, the membrane potential may be adjusted and, with it, the accumulation of AIB. Over a wide range of membrane potentials, TPA-treated cells accumulate more AIB than do controls, suggesting a possible change in the internal Na+ activity.

Nonsolvent water in human erythrocytes.

From the ability of a concentrated suspension of human erythrocytes to regulate the pH of unbuffered, anisotonic, external media it is possible to calculate the fractional cell volume in which chloride is dissolved. The difference between this volume and the total cell water gives the nonsolvent water (for chloride) of the cell. Nonsolvent water is less than 3% of the isotonic cell volume. The quantity of nonsolvent water per cell may increase as the cells shrink in hypertonic solutions.

Mechanism of in vitro photohemiolysis in erythropoietic protoporphyria (EPP).

Four photosensitive patients with EPP showed markedly elevated RBC protoporphyrin levels. In vitro exposure of. a monolayer of their RBC in buffered saline to 3 x 10(6) ergs/mm2 of 4000 Å irradiation (= 45 minutes under a bank of fluorescent black lights) produced complete hemolysis within 24 hours; normal RBC showed negligible hemolysis after similar exposure. All of the cellular potassium was released from the patients' cells before the onset of significant hemolysis. Hemolysis was largely prevented by suspending the patients' cells in a 1:1 mixture of isotonic buffered NaCl and isotonic sucrose. These findings suggest that the initial photochemical lesion is of a "small hole" nature, creating channels through which small ions (but not sucrose) can diffuse. Since the effect of the intracellular colloid is offset by addition of sucrose to the external medium, the lesions appear to be less than 10 Å in diameter.

Effects of maturational stage on insulin sensitivity during puberty.

During puberty, plasma insulin levels increase, and insulin sensitivity decreases along with multiple other physical and hormonal changes. To determine 1) the time course of the decrease in insulin sensitivity in relationship to Tanner stage of genital development, and 2) how this change relates to changes in GH secretion, insulin-like growth factor-I (IGF-I), IGF-binding protein-3, and gonadal steroid secretion, we studied 58 healthy children and adolescents (34 males and 24 females; age 7-15 yr) using overnight GH sampling and frequently sampled iv glucose tolerance tests. The insulin sensitivity index (ISI) was calculated using the program MINMOD. ISI differed significantly by Tanner stage (P < 0.05, by analysis of variance) with a decrease from Tanner stage 1 to 2 (P < 0.05). IGF-I and IGF-binding protein-3 followed opposite patterns to ISI, with lower levels in Tanner stage 1 than in stages 2-5 (P < 0.05). Mean GH levels did not increase until Tanner stage 4 (P < 0.05) and then fell during Tanner stage 5. Multiple linear regression analysis revealed negative relationships among ISI, IGF-I, and body mass index. No relationship was found with GH. We conclude that the pubertal change in ISI is not necessarily associated with increased GH secretion, but is associated with increased GH peripheral effect, as indicated by the relationship between ISI and IGF-I.

Assessment of depot leuprolide acetate dose-adequacy for central precocious puberty.

The development of GnRH analogs (GnRHa) has made it possible to treat children with central precocious puberty (CPP). This treatment may prevent adult short stature due to premature epiphyseal fusion. Achievement of this goal, however, is dependent upon adequate suppression of gonadal steroid production as a result of GnRHa-induced pituitary desensitization and decreased gonadotropin release. A depot formulation of a GnRHa [leuprolide acetate (dLA)] is being used by many clinicians for the treatment of CPP, but studies to establish the optimal dose of dLA have not been performed. In this study we evaluated the effectiveness of dLA (7.5 mg, im, every 4 weeks). Six children (7-10 yr old) with CPP treated with dLA were assessed clinically and divided into two groups: A (incompletely suppressed) and B (well suppressed). Each group had overnight blood sampling and a GnRH stimulation test the following morning. LH pulses were analyzed and compared to 11 normal prepubertal children. Mean LH concentration, LH curve area, LH pulse frequency, and mean LH pulse amplitude were significantly greater (P less than 0.03) in group A than in group B or the normal prepubertal children. There was no significant difference among the three groups in GnRH-stimulated peak LH release. These results indicate that dLA (7.5 mg, im, every 4 weeks) does not produce complete desensitization in all children with CPP and suggest that overnight monitoring of LH release is more sensitive than GnRH stimulation testing for the assessment of dLA dose adequacy.

Characterization of the RDC1 gene which encodes the canine homolog of a proposed human VIP receptor. Expression does not correlate with an increase in VIP binding sites.

We have isolated a portion of the canine gene encoding the orphan receptor RDC1 [1]. The complete coding sequence is contained in a single exon, and an intron divides the 5' untranslated region of RDC1 mRNA. The RDC1 protein is 94% homologous to the gene product of GPRN1, which has been proposed to serve as a VIP receptor when expressed in CHO-K1 and COS-7 cells (Sreedharan, S.P. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4986-4990). Northern analysis indicates that CHO-K1 cells endogenously express a 2.1 kb RDC1 mRNA. However, while CHO-K1 cells possess detectable low affinity [125I]VIP binding sites, VIP binding is not altered in membranes of CHO-K1 cells expressing varying amounts of the RDC1 gene construct. Further, endogenous VIP binding is not increased by transient expression of RDC1 in COS-7 cells. Taken together, the data suggest that RDC1 is not a canine homolog of the proposed VIP receptor.

Remote, semiautomated production of F-18-labeled 2-deoxy-2-fluoro-D-glucose.

A routine production system for 2-deoxy-2-[18F]fluoro-D-glucose (2-18FDG) for medical use is presented. The method involves the preparation of F-18-labeled F2 by the 20Ne(d, alpha) 18F reaction, subsequent fluorination of 3,4,6-tri-o-acetyl-D-glucal, purification, and hydrolysis of the appropriate diastereoisomer to obtain 2-18FDG. These operations are performed using a completely remote, semiautomatic system. There have been no failures in more than 220 consecutive production runs. Currently, two preparations per day of 15-25 mCi of 2-18FDG are routinely performed, with a radiochemical purity, measured by TLC, in excess of 95% and with less than 1 mR radiation exposure to the chemist. Reaction conditions, operational variables, and systems are described in detail.

Sebaceous gland activity and serum dehydroepiandrosterone sulfate levels in boys and girls.

BACKGROUND AND DESIGN: Increases in sebaceous gland activity are often the earliest sign of the approach of puberty in children. These increases have been attributed to increases in the secretion of adrenal androgens, but the supporting data are sparse and are based on measurements of urinary, rather than serum, androgen concentrations. In this study, we examined sebum composition, serum levels of dehydroepiandrosterone sulfate, and pubertal stage in 111 boys and girls, aged 2 to 15 years. Sebum composition was evaluated by measuring the ratio of wax esters/(cholesterol + cholesterol esters), a ratio known to increase with increasing sebaceous gland activity. RESULTS: Both wax esters/(cholesterol + cholesterol esters) ratios and serum dehydroepiandrosterone sulfate levels began to increase in children 7 to 10 years old. These changes occurred in many children before the appearance of any physical signs of puberty. Wax esters/(cholesterol + cholesterol esters) ratios were correlated with dehydroepiandrosterone sulfate levels in both boys and girls. In prepubertal children, the regression lines passed through the origin. In subjects who were in early or late puberty, the y intercepts of the regression lines had positive values. CONCLUSION: Adrenal androgens appear to be the major determinants of sebaceous gland activity during the prepubertal period and to be additive to another hormone or hormones during puberty.

Validity of near infrared body composition analysis in children and adolescents.

The purpose of this study was to evaluate the validity and reliability of the Futrex 5000A near infrared (NIR) spectrophotometer for the assessment of body composition in children and adolescents. Forty-eight subjects (24 boys and 24 girls) with a mean age of 12.7 +/- 2.7 yr underwent three methods of body composition testing: NIR, densitometry by hydrostatic weighing (HYDRO), and skinfold anthropometry (SKF). Percent body fat (%BF) and fat free mass (FFM) derived from the Lohman's age-adjusted Siri equation served as the criterion. Within session test-retest reliability was determined for the NIR device for all subjects and between-week reliability was evaluated for all test methods in 14 subjects. Based on the excellent within- and between-session reliability (ICC ranged from 0.907-0.999), the system offers the potential of obtaining longitudinal data in growth and development studies. However, the significant mean differences, moderate correlations with the criterion (r = 0.62-0.71) and inflated standard errors of estimate (SEE = 4.9-5.5% BF, 2.2-2.9 kg FFM) and total prediction errors (TE = 5.5-8.0% BF, 2.7-3.7 kg FFM) indicate that refinement of prediction equations is needed to establish the measurement validity. Continued research with expanded populations is needed to further demonstrate and evaluate the utility of this device.

Cross-validation of the Slaughter skinfold equations for children and adolescents.

Prior to sexual maturation, children and adolescents have more water and less bone mineral content than adults, resulting in less dense fat-free body mass (FFM). This suggests that previously established adult skinfold/density equations are inappropriate for use with children and adolescents for the prediction of body fatness (%BF) and FFM. To overcome this problem, Slaughter and colleagues have introduced new skinfold (SKF) equations that take into account the changing density of FFM in children and adolescents as they mature. The purpose of our study was to cross-validate a select set of the Slaughter SKF equations by comparing them with a criterion measurement (Lohman's Siri age-adjusted body density equation) in 122 subjects ranging in sexual maturation from pre- to post-pubescent and ranging in age from 8-17. Our cross-validation found very high intraclass (reliability) correlations (ICCs = 0.98-0.99) and high validity correlations (rs = 0.79-0.99). The standard errors of the estimate for %BF ranged from 3.5-4.6% and total errors for %BF ranged from 3.6-4.6%. The Slaughter equation using tricep and calf SKF for females was significantly different (P < 0.05) from the criterion measure in its prediction of %BF. In males, there was an interaction between the SKF equation and subject maturation level. The data indicate that the Slaughter SKF equations hold promise for estimating body composition in children and adolescents but are still in need of refinement.

Pubertal arrest associated with valproic acid therapy.

Valproic acid has previously been demonstrated to decrease the secretion of multiple pituitary hormones; however, clinical symptoms associated with decreased hormone secretion have not been described. A girl is reported with complex partial seizures who was treated with valproic acid from age 10 years, 7 months to 12 years, 1 month and during this time had an arrest of both growth and secondary sexual development. Two months after discontinuing valproic acid she had resumption of both pubertal growth and maturation. A prospective controlled study is needed to determine more precisely the effects of valproic acid on growth and development.

Herpes zoster radiculopathy.

Herpes zoster-related radiculopathy usually can be easily diagnosed in the presence of cutaneous lesions. Before development of the skin rash, the diagnosis may be in doubt, particularly if motor symptoms and signs are a major clinical feature. We report a patient with herpes zoster-related radiculopathy whose clinical features mimicked other spinal disorders.

Cross-validation of bioelectrical impedance analysis of body composition in children and adolescents.

BACKGROUND AND PURPOSE: The reliability and validity of measurements obtained with two bioelectrical impedance analyzers (BIAs), an RJL Systems model BIA-103 and a Berkeley Medical Research BMR-2000, were investigated using the manufacturers' prediction equations for the assessment of fat-free mass (FFM) (in kilograms) in children and adolescents. SUBJECTS: Forty-seven healthy children and adolescents (23 male, 24 female), ranging in age from 8 to 20 years (mean = 12.1, SD = 2.3), participated. METHODS: In the context of a repeated-measures design, the data were analyzed according to gender and maturation (Tanner staging). Hydrostatic weighing (HYDRO) and Lohman's Siri age-adjusted body density prediction equation served as the criteria for validating the BIA-obtained measurements. RESULTS: High intraclass correlation coefficients (ICC > or = .987) demonstrated good test-retest (between-week) measurement reliability for HYDRO and both BIA methods. Between-method (HYDRO versus BIA) correlation coefficients were high for both boys and girls (r > or = .97). The standard errors of estimate (SEEs) for FFM were slightly larger for boys than for girls and were consistently smaller for the RJL system than for the BMR system (RJL SEE = 1.8 kg for boys, 1.3 kg for girls; BMR SEE = 2.4 kg for boys, 1.9 kg for girls). The coefficients of determination were high for both BIA methods (r2 > or = .929). Total prediction errors (TEs) for FFM showed similar between-method trends (RJL TE = 2.1 kg for boys, 1.5 kg for girls; BMR TE = 4.4 kg for boys, 1.9 kg for girls). DISCUSSION AND CONCLUSION: This study demonstrated that the RJL BIA with the manufacturer's prediction equations can be used to reliably and accurately assess FFM in 8- to 20-year-old children and adolescents. The prediction of FFM by the BMR system was acceptable for girls, but significant overprediction of FFM for boys was noted.

Assessment of 18F gaseous releases during the production of 18F-fluorodeoxyglucose.

Fluorodeoxyglucose labeled with 18F (18F-FDG) is the most commonly used radiopharmaceutical in positron emission tomography (PET). Fluorine-18-labeled FDG is used as a diagnostic tool in PET studies to monitor the physiology of the brain, diagnose heart function and disease, and to image cancerous tumors. At the University of California, Los Angeles (UCLA), three cyclotrons produce [18F]-fluoride ion using 18O-enriched water targets. Fluorine-18, which has a half-life of 109.8 min, is produced using an 18O(p.n.)18F reaction and is chemically processed to yield 18F-FDG. This study presents data which demonstrate that during the radiochemical processes involved in the production of 18F-FDG, gaseous effluent containing 18F is released. Forty cyclotron production runs with average end of cyclotron bombardment activities of 15.9 +/- 1.88 GBq (430 +/- 50.8 mCi) and end of radiochemical synthesis activities of 5.40 +/- 1.27 GBq (146 +/- 34.3 mCi) yield 18F gaseous effluent releases ranging from 0 to 2560 MBq (0 to 69.2 mCi) with a mean of 437 MBq (11.8 mCi). Temporal correlation of the 18F gaseous releases during 18F-FDG radiochemical production has tied the 18F release to the addition of the glucose precursor (mannotriflate) and ethyl ether in the radiochemical processing. The results are presented in terms of activities released and dilution factors required from the release stack point to maintain controlled (occupational) and uncontrolled (public) area limits in accordance with the recommendations of the International Commission on Radiological Protection and the regulatory requirements of the federal government.