Solution studies of recombinant human stromal-cell-derived factor-1.

Stromal-cell-derived factor-1 (SDF-1alpha) is an 8-kDa chemokine that is constitutively expressed in bone-marrow-derived stromal cells and has been identified as a ligand for the CXCR4 receptor. We produced the chemokine recombinantly as methionine-SDF-1alpha in Escherichia coli without the leader peptide sequence. The protein was denatured, refolded, and further purified by reversed-phase HPLC. SDF-1alpha was shown to be >95% pure as judged by SDS-PAGE. The final yield of purified and refolded SDF-1alpha was 1-2 mg per gram of wet cell paste. The refolded protein is a ligand for the CXCR4 receptor and has been used to block HIV-mediated cell fusion and downmodulates the CXCR4 receptor. Our ability to purify hundreds of milligrams of refolded protein allowed us to conduct detailed studies of the biophysical properties of the protein. We have used a combination of biophysical techniques to study the solution properties of SDF-1alpha. The average mass of SDF-1alpha, as determined by static light scattering, gave us the first indications that the chemokine may self-associate. Further investigation with sedimentation velocity ultracentrifugation confirmed the existence of two species. The measured s(20, W) values defined two masses corresponding to monomer and dimer. Finally, sedimentation equilibrium ultracentrifugation and dynamic light scattering yielded a composite value of 150 +/- 30 microM for the dimerization constant. We conclude that SDF-1alpha exists in a monomer-dimer equilibrium.

Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells.

Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.

Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells.

Mouse melanocortin receptors, MC1-R, MC3-R, MC4-R, and MC5-R, when expressed in HEK293 cells and stimulated with either alpha-melanocyte-stimulating hormone (alpha-MSH) or desacetyl-alpha-MSH, mediate increases in intracellular free calcium concentration ([Ca(2+)](i)) with EC(50) values between 0.3 and 4.3 nM. The increase in [Ca(2+)](i) is cholera toxin sensitive and pertussis toxin insensitive. The mechanism involves calcium mobilization from intracellular stores without a transient rise in inositol trisphosphate. Mouse agouti protein (55 nM) is a competitive antagonist of alpha-MSH (6-fold) and desacetyl-alpha-MSH (8-fold), coupling the mMC1-R to increased [Ca(2+)](i). Agouti protein (55 nM) significantly increased the EC(50) for alpha-MSH (3-fold), and 550 nM agouti protein significantly increased the EC(50) for desacetyl-alpha-MSH (4-fold), coupling the mMC4-R to a rise in [Ca(2+)](i). However, agouti protein antagonism of the MC4-R may not be competitive since there was a trend for the maximum response to also increase. There was no significant antagonism of the MC3-R and MC5-R by agouti protein (55 nM). Understanding the physiological relevance of the transduction of a calcium signal by melanocortin peptides may be important for future development of therapeutic targeting of the melanocortin receptors.

Hydration changes implicated in the remarkable temperature-dependent membrane permeation of cyclosporin A.

Cyclosporin A is a cyclic peptide believed to exist as multiple conformers in aqueous solution. Two major conformations, distinguished by a single cis-trans isomerization and the presence of four either intramolecular or intermolecular hydrogen bonds, have been confirmed depending on whether CsA is characterized in organic solvents or bound in aqueous complex with cyclophilin. The relationship between CsA conformation and its ability to penetrate biological membranes is currently unknown. Using Caco-2 cell monolayers, we documented a remarkable increase (more than 2 orders of magnitude) in the membrane permeation of the peptide as temperature was increased from 5 to 37 degrees C. The solubility of CsA was 72 microM at 5 degrees C, but decreased by more than an order of magnitude at 37 degrees C. Moreover, CsA partitioned into non-hydrogen bond donating solvents linearly as a function of increasing temperature, suggestive of a significant conformational change. However, while NMR spectra of CsA confirmed the previously predicted presence of multiple conformers in aqueous solution, the equilibrium between the two major species was not affected by changes in temperature. These NMR data indicated that the observed temperature-dependent changes in the membrane permeability of CsA do not originate from changes in the peptide backbone conformation. Sedimentation equilibrium analysis revealed that CsA behaves in a highly nonideal manner over the temperature range tested. We interpret this behavior as a change in the hydration state with a smaller (or weaker) hydration shell surrounding the peptide at higher temperatures. Such a change would result in lower peptide desolvation energy, thereby promoting partitioning into cellular membranes. We contend that changes in membrane penetration result from alterations in the hydration state of CsA and are not related to the interconversion of the defined conformations.

Replication-associated activities of purified human papillomavirus type 11 E1 helicase.

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.

Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.

Agouti antagonism of melanocortin-4 receptor: greater effect with desacetyl-alpha-melanocyte-stimulating hormone (MSH) than with alpha-MSH.

Desacetyl-alpha-MSH is more abundant than alpha-MSH in the brain, the fetus, human blood, and amniotic fluid, but there is little information on its ability to interact with melanocortin receptors. The aim of this study is to compare and contrast the ability of desacetyl-alpha-MSH and alpha-MSH to couple melanocortin receptors stably expressed in HEK293 cells, to the protein kinase A (PKA) signaling pathway. Desacetyl-alpha-MSH activated mouse MC1, MC3, MC4 and MC5 receptors with EC50s = 0.13, 0.96, 0.53, and 0.84 nM, and alpha-MSH activated these receptors with EC50s = 0.17, 0.88, 1.05, and 1.34 nM, respectively. Mouse agouti protein competitively antagonized alpha-MSH and desacetyl-alpha-MSH coupling to the MC1-R similarly. In contrast, mouse agouti protein antagonized desacetyl-alpha-MSH much more effectively and potently than alpha-MSH coupling the MC4-R to the PKA signaling pathway. Furthermore, mouse agouti protein (10 nM) significantly reduced (1.4-fold) the maximum response of mMC4-R to desacetyl-alpha-MSH and 100 nM mouse agouti significantly increased (4.8-fold) the EC50. Minimal antagonism of alpha-MSH coupling mMC4-R to the PKA signaling pathway was observed with 10 nM mouse agouti, whereas both 50 and 100 nM mouse agouti appeared to reduce the maximum reponse (1.1- and 1.3-fold, respectively) and increase the EC50 (2.5- and 3.4-fold respectively). Mouse agouti protein did not significantly antagonize either alpha-MSH or desacetyl-alpha-MSH coupling mouse MC3 and MC5 receptors. Understanding the similarities and differences in activation of melanocortin receptors by desacetyl-alpha-MSH and alpha-MSH will contribute to delineating the functional roles for these endogenous melanocortin peptides.

Product release is the major contributor to kcat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA.

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3' single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3'-fluorescein-tagged 21-mer (F21). kcat for this reaction was 0.12 s-1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs ( approximately 2) were disrupted concertedly. The maximal value of the rate constant (keff) describing the late phase of the reaction (strand separation) was 0. 5 s-1, which was 4-fold greater than kcat. Release of HF31 from E. HF31 in the presence of ATP (0.21 s-1) was the major contributor to kcat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.

Human recombinant phosphodiesterase 4B2B binds (R)-rolipram at a single site with two affinities.

The interactions between (R)-rolipram and purified human recombinant low-Km, cAMP-specific phosphodiesterase (HSPDE4B2B) constructs were investigated using biochemical, kinetic, and biophysical approaches. The full-length protein (amino acids 1-564) and an N-terminal truncated protein (amino acids 81-564) exhibited high-affinity (R)-rolipram binding, whereas an N-terminal and C-terminal truncated protein (amino acids 152-528) lacked high-affinity (R)-rolipram binding. The 152-528 and 81-564 proteins had similar Km's and kcat/Km's and differed less than 4-fold compared with the 1-564 protein. (R)-Rolipram inhibition plots were biphasic for the 1-564 and 81-564 proteins and fit to two states, a high-affinity (Ki = 5-10 nM) state and a low-affinity (Ki = 200-400 nM) state, whereas the 152-528 protein fit to a single state (Ki = 350-400 nM). The stoichiometry for high-affinity binding using a filter binding assay was found to be <1 mol of (R)-rolipram per mole of 1-564 or 81-564 protein. Titration microcalorimetric studies revealed both a high-affinity state with a stoichiometry of 0.3 mol of (R)-rolipram per mole of protein and a low-affinity state with a stoichiometry of 0.6 mol of (R)-rolipram per mole of protein for the 81-564 protein. A single low-affinity state with a stoichiometry of 0.9 mol of (R)-rolipram per mole of protein was seen using the 152-528 protein. The data indicate that purified HSPDE4B2B 1-564 and 81-564 proteins contain a single binding site for (R)-rolipram and suggest that the proteins exist in two different states distinguishable by their affinity for (R)-rolipram. Furthermore, the high-affinity binding state of the protein requires amino acid residues at the N-terminus (81-151) of the protein and catalytic domain (152-528), whereas the low-affinity binding state only requires residues in the catalytic domain (152-528). Phosphorylation at residues 487 and 489 of the 81-564 protein does not appear to alter the substrate kinetics or the stoichiometry and binding affinity of (R)-rolipram.

Detailed characterization of a purified type 4 phosphodiesterase, HSPDE4B2B: differentiation of high- and low-affinity (R)-rolipram binding.

We have overexpressed in a baculovirus expression system, and purified to > 95% homogeneity, milligram quantities of a human recombinant rolipram-sensitive cAMP phosphodiesterase, HSPDE4B2B (amino acid residues 81-564). The protein expression levels were approximately 8 mg of HSPDE4B2B (81-564) per liter of Sf9 cells. The Km of the purified enzyme for cAMP was 4 microM and the Ki for the Type 4 phosphodiesterase-specific inhibitor (R)-rolipram was 0.6 microM. The specific activity of the purified protein was 40 mumol/min/mg protein. A nonequilibrium filter binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with a Kd of 1.5 nM and a stoichiometry of 0.05-0.3 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Equilibrium dialysis experiments revealed a single binding constant of 140 nM with a stoichiometry of 0.75 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Size exclusion chromatography and analytical ultracentrifugation experiments suggest that the protein exists in multiple association states larger than a monomer. Proteolysis experiments revealed a 43-kDa fragment that contained catalytic and rolipram-inhibitable activities, but the fragment showed no high-affinity (R)-rolipram binding. Based on the proteolytic cleavage studies a 43-kDa protein was constructed, expressed, and purified. This protein, HSPDE4B2B (152-528), had Km and Vmax similar to those of the HSPDE4B2B (81-564) protein, but did not exhibit high-affinity (R)-rolipram binding. The protein did show low-affinity (R)-rolipram binding using the equilibrium binding assay. These results show that a low-affinity binding site for (R)-rolipram is solely contained within the catalytic domain of HSPDE4B2B, whereas high-affinity (R)-rolipram binding requires residues within the catalytic domain and residues flanking N- and/or C-terminal to the catalytic region.

Mutations in the carboxyl terminus of the agouti protein decrease agouti inhibition of ligand binding to the melanocortin receptors.

Several mutations that cause ectopic expression of the agouti gene result in obesity, hyperinsulinemia, and yellow coat color. A candidate pathway for agouti induced obesity and hyperinsulinemia is through altered signaling by melanocortin receptors, as agouti normally regulates coat coloration through antagonism of melanocortin receptor 1. Furthermore, melanocortin peptides mediate functions including steroidogenesis, lipolysis, and thermoregulation. We report apparent inhibition dissociation constants for mouse and human agouti protein inhibition of ligand binding to the melanocortin receptors, to determine which of these receptors might be involved in agouti induced diabetes. The similarity in the apparent K(I) values for agouti inhibition of ligand binding to the brain melanocortin receptors 3 and 4 (mouse: K(I) app = 190 +/- 74 and 54 +/- 18 nM; human: K(I) app = 140 +/- 56 and 70 +/- 18 nM, respectively) suggests that the MC3-R is a potential candidate for a receptor mediating the effects of agouti protein overexpression. Agouti residues important for melanocortin receptor inhibition were identified through the analysis of deletion constructs and site-specific variants. Val83 is important for inhibition of binding to MC1-R (K(I) app for Val83Ala agouti increased 13-fold relative to wild-type protein). Arg85, Pro86, and Pro89 are important for selective inhibition of binding between MC1-R and MC3-R and MC4-R as their apparent K(I) values are essentially unchanged at MC1-R, while they have increased 6-10-fold relative to wild-type protein at MC3-R and MC4-R.

Interactions of alpha-melanotropin and agouti on B16 melanoma cells: evidence for inverse agonism of agouti.

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) and agouti control the switch between eumelanin and pheomelanin synthesis in mammalian melanocytes. Here we investigated interactions between alpha-MSH, agouti protein, cAMP elevating agents and phorbol ester on mouse B16 melanoma cells. Agouti (Kd 3.7 nmol/l) and alpha-MSH (Kd 2.3 nmol/l) had similar affinities to the MC1 melanocortin receptor. Both alpha-MSH and agouti induced MC1 receptor down-regulation. Agouti antagonized melanogenesis induced by alpha-MSH, forskolin, cholera toxin (CT), and pertussis toxin (PT). It also reduced the constitutive melanin formation of long-term cultures. Cell proliferation was inhibited by agouti (43% at 100 nM). This effect was reversed by alpha-MSH, forskolin, or CT. B16-G4F cells, a cell variant that lacks the MC1 receptor, did not respond to agouti. From these results we conclude that agouti shows the characteristics of an inverse agonist acting through the MC1 receptor.

Agouti protein inhibits growth of B16 melanoma cells in vitro by acting through melanocortin receptors.

Agouti protein is known to antagonize cAMP formation, tyrosinase activation and melanogenesis in mouse B16-F1 melanoma cells induced by alpha-melanocyte-stimulating hormone (alpha-MSH). We now demonstrate that although agouti binds to the melanocortin receptor MC1-R with an almost identical affinity to that of alpha-MSH, it does not antagonize the inhibitory action of alpha-MSH on the growth of B16-F1 cells. Instead it has a similar antiproliferative action with a half-maximal effective concentration of 13 nM. In G4F cells lacking MC1-R, agouti is without effect. Agouti was also found to induce MC1-R down-regulation with identical kinetics and magnitude as alpha-MSH. Thus, the different effects of agouti on B16-F1 cells proceed via interaction with MC1-R but are not exclusively antagonistic.

Catalytic activity of the SH2 domain of human pp60c-src; evidence from NMR, mass spectrometry, site-directed mutagenesis and kinetic studies for an inherent phosphatase activity.

During solution structural studies it was apparent that the human recombinant pp60c-src SH2 domain (srcSH2, residues 144-249) possessed an inherent phosphatase (Pase) activity. Complexes of U[13C,15N]srcSH2 with unlabeled Ac-pYEEIE (I) were examined using 31P and 1H-detected isotope filtered NMR methods. The presence of a high-affinity complex in equimolar solutions of I and U[13C, 15N]-srcSH2 was demonstrated by chemical shift perturbations, line broadening, and the observation of intermolecular nuclear Overhauser effects from the pY and Ile side-chain protons of I to protons on amino acid residues present in the binding pocket of srcSH2. Solutions containing excess I relative to srcSH2 revealed a slow hydrolysis of I to produce Ac-YEEIE and inorganic phosphate. The hydrolysis rate determined from NMR and HPLC-electrospray ionization mass spectrometry data at 30 degrees C for solutions containing excess I was 0.002-0.003 h-1. srcSH2 also catalyzed the hydrolysis of p-nitrophenyl phosphate (pNPP). Isoelectric focusing gels of a number of mutant srcSH2s demonstrated that this activity comigrated with srcSH2. Km, kcat, and kcat/Km were 3.7 +/- 0.4 mM, 3.1 +/- 0.2 x 10(-2) min-1, and 8.4 +/- 0.4 M-1 min-1, respectively, toward pNPP. The C188A mutant of the srcSH2 domain displayed 15% of the activity displayed by wild-type srcSH2, demonstrating that this residue is not absolutely required for activity. Two additional mutations in the known pY binding site, R178K and R158K, also resulted in decreased pNPPase activity, suggesting that the activity resides in or near this site. The inhibitor profile and pH dependence suggest that this is a novel protein Pase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Agouti structure and function: characterization of a potent alpha-melanocyte stimulating hormone receptor antagonist.

The murine agouti gene encodes for a novel 131 amino acid protein. The sequence includes a 22 residue putative secretion signal, an internal basic region, and a C-terminal domain containing 10 cysteines. Agouti has been found to antagonize the binding of certain pro-opiomelanocortin peptides, such as alpha-melanocyte stimulating hormone (alpha-MSH), to the murine melanocortin-1 receptor (MC1-R). We report the purification of a secreted murine agouti to homogeneity by a two-step procedure from baculovirus-infected Trichoplusia ni (T. ni). The protein is glycosylated and exhibits competitive, high-affinity antagonism (Ki = 0.8 nM) versus alpha-MSH in cell-based assays employing B16F10 cells. Association state analysis by analytical ultracentrifugation reveals that agouti exists in a monomer--dimer plus aggregate equilibrium at low micromolar concentrations. Data from secondary structure studies indicate that the protein is highly stable to thermal denaturation. Enzymatic digestion to probe disulfide bond arrangement yielded a discrete C-terminal (Val 83-Cys 131) domain. The isolated highly cysteine-rich C-terminal domain retains alpha-MSH antagonism equipotent with mature agouti. This bioactive domain contains all 10 cysteines which exhibit sequence homology when aligned with several conotoxins.

Solution structure of the human pp60c-src SH2 domain complexed with a phosphorylated tyrosine pentapeptide.

Human pp60c-src is a cellular nonreceptor tyrosine kinase that participates in cytosolic signal transduction and has been implicated in the development of malignant tumors in the human breast and colon. Signal transduction is mediated by highly specific interactions between the SH2 domain and receptor phosphorylated tyrosine binding motifs. To elucidate the molecular conformation and interactions in solution, a family of highly resolved nuclear magnetic resonance (NMR) structures was determined for the src SH2 domain complexed with a high-affinity phosphorylated pentapeptide, acetyl-p YEEIE-OH. The 23 structures, generated with a distance geometry (DG) and a dynamical simulated annealing (SA) procedure, satisfied 2072 experimental restraints derived from a variety of multifrequency/multidimensional and isotope-filtered NMR data. Superimposition of residues 143-245 upon the mean coordinate set yielded an atomic rmsd of 0.58 +/- 0.09 A for the N, C alpha, C' atoms and 1.04 +/- 0.08 for all the non-hydrogen atoms. Residues in the ordered secondary structure regions superimpose to 0.29 +/- 0.04 A for the N, C alpha, C' and 0.73 +/- 0.08 A for all the non-hydrogen atoms. The angular order parameter calculated for the phi, psi angles was > 0.9 for 81 of the 106 protein residues. The main protein conformational features are three antiparallel beta-strands that traverse a compact core with an alpha-helix on each side of the core near the N- and C-termini. The observed intermolecular nuclear Overhauser effects (NOE) from the pY, +1E, and +3I residues positioned the ligand in an extended conformation across the SH2 domain surface with the pY and +3I side chains inserted into the protein binding pockets. In general, the protein conformation is consistent with previously reported structures of different SH2 domain complexes determined by X-ray crystallography. However, inter- or intramolecular interactions involving the guanidinium side chains of the solvated R alpha A2 or the buried R beta B5 were not observed at pH = 5.5 or 7.0. If such interactions exist in solution, the absence of any confirming data probably arises from rapid exchange with solvent and/or undetermined dynamic components. Thus, the unrestrained R alpha A2 side chain did not show an amino-aromatic interaction or a hydrogen bond to the -1 carbonyl oxygen as observed in the crystal structures. This result is consistent with the solution structure of a different SH2 domain complex. A more detailed comparison between the crystal structure and the NMR-derived solution structures of the same src SH2 domain complex is presented.(ABSTRACT TRUNCATED AT 400 WORDS)

Disposition and effects of 85Kr in pregnant rats.

Pregnant rats were housed in 85Kr atmospheres at 10, 15, or 20 days of gestation (dg) and killed after 4 hr of exposure to 37-40 nCi/ml. The 85Kr was present in the components of the fetoplacental unit (FPU) at concentrations (nCi/g) equivalent to approximately 2% of the concentration (nCi/ml) in the exposure atmosphere. Tissue distribution of 85Kr and the distribution of radiation dose did not suggest any unusual hazard to the fetus associated with exposure of pregnant animals. This conclusion was tested using 5-day exposures to a 1000-fold increased concentration: 40 muCi/ml. The main effects observed in pregnant rats exposed to 85Kr from 7-12 or 12-17 dg (estimated radiation dose of 5 X 10(3) rad to maternal lung and 5 X 10(5) rad to maternal skin surface) were deaths, impaired weight gain and skin lesions. Secondarily, the maternal toxicity led to indications of embryotoxicity, although the incidence of malformations was not increased by the estimated 50-rad dose to the FPU.

Toxicology of 85Kr: chronic exposure studies.

Male and female Wistar rats were exposed chronically to graded doses of 85Kr to determine long-term biological effects of simulated environmental exposure. Rats were exposed in 4 groups, including room air-exposed controls and 3 groups exposed to 85Kr concentrations equivalent to 10(2), 10(3) and 10(4) times the maximum permissible concentration (MPC) for the general population (3 X 10(-7) microCi/ml). Exposures continued 24 hr/day, 7 days/week for 808 days, until only approximately 10% of the original 400 rats were alive. No effect of 85Kr exposure was observed on weight gain or tumor incidence, including leukemias. The results support the adequacy of established 85Kr exposure limits for the general population in light of the absence of evidence of build-up in tissues after chronic exposure and the lack of exposure-related biological effects in rats after near-lifespan exposure to concentrations equivalent to 10,000 times the MPC.

Toxicology of 85Kr: tissue distribution and retention in the rat.

Wistar rats were exposed to 85Kr gas atmospheres to determine saturation/desaturation kinetics in major tissues. Tissue partition coefficients [concentration 85Kr in tissue (muCi/g)/concentration of 85Kr in the atmosphere (muCi/cc)] were determined following equilibration with 85Kr gas. Tissue partition coefficients were highest for adrenals and fat; lowest for bone, heart and brain. Tissue partition coefficients for immature rats, 1-12 days of age, and for adults, were not significantly different. The highest concentrations of 85Kr were found in fatty tissues and in gas pockets in the intestinal lumen.

Communicating with the family of the child with a developmental disability.

As more and more families of handicapped children seek dental care, increased demands will be placed on the dentist and staff to communicate effectively and humanely, not only with the child but with the parents. Increased training is currently being provided through doctoral programs and continuing education to deal with special human needs of the patient and family in a dental setting. The dentist should address the thoughts and feelings of the parents as well as those of the child. The child and family present special problems but also special opportunities to fulfill professional responsibilities and experience personal and professional gratification.