A Prospective Longitudinal Study of Early Childhood Caries Onset in Initially Caries-Free Children.

INTRODUCTION: Early childhood caries (ECC) is a complex oral disease that is prevalent in US children. OBJECTIVES: The purpose of this 2-y prospective cohort study was to examine baseline and time-dependent risk factors for ECC onset in initially caries-free preschool children. METHODS: A cohort of 189 initially caries-free children aged 1 to 3 y was recruited. At each 6-mo study visit, children were examined using the ICDAS index; salivary samples were collected to assess mutans streptococci (MS), lactobacilli, Candida species, salivary cortisol (prior and after a stressor), and salivary IgA. Diet and oral health behavior were assessed from parent report. Child and family stress exposure was assessed from measures of psychological symptoms, stressful life event exposure, family organization and violence exposure, and social support. Sociodemographic factors were also considered. A Kaplan-Meier estimator of survival function of time to ECC and a Cox proportional hazards model were used to identify predictors of ECC onset. RESULTS: Onset of ECC was associated with high salivary MS levels at baseline (log-rank test, P < 0.0001). Cox proportional hazards regression showed that the risk of dental caries significantly increased with salivary MS in log scale over the 6-mo period (hazard ratio, 1.08; P = 0.01). Other risk factors in the model did not reach statistical significance. CONCLUSION: Our results provide prospective evidence that an increase in salivary MS predicts ECC onset in young, initially caries-free children, confirming that a high salivary MS count likely plays a causal role in ECC onset, independent of covariates. KNOWLEDGE TRANSFER STATEMENT: These results suggest that we must focus on reducing salivary MS counts in young children and preventing or delaying MS colonization in infants and young children determined to be at risk for ECC.

Social, Psychological, and Behavioral Predictors of Salivary Bacteria, Yeast in Caries-Free Children.

INTRODUCTION: Early childhood caries (ECC) is a complex, multifactorial oral disease that is a major public health concern because it is prevalent, profoundly alters a child's quality of life, is difficult to treat effectively, and has a distressing tendency to recur following treatment. OBJECTIVES: The purpose of the study was to examine social, psychological, and behavioral predictors of salivary bacteria and yeast in young children at risk for ECC. METHODS: A sample of 189 initially caries-free preschool children was assessed for child stress physiology from salivary cortisol, child and family stress exposure, diet, oral health behaviors, and sociodemographic risks. Multiple logistic regression analysis was implemented to examine the associations between these risk factors and cariogenic microorganisms: mutans streptococci (MS), lactobacilli (LB), and Candida species. RESULTS: Higher baseline salivary cortisol (odds ratio [OR] = 6.26; 95% confidence level [CL], 1.69-23.16) and a blunted response to an acute laboratory stressor (OR = .56; 95% CL, .37-.83) were associated with an increased likelihood of elevated salivary MS (≥105 colony-forming units/mL) in caries-free children. Sociodemographic risk for cariogenic microorganisms was also found. Specifically, lower education attainment of the parent/primary caregiver was associated with children being more likely to carry salivary Candida species and elevated salivary MS; in addition, children from households with an unemployed parent/primary caregiver were more likely (OR = 3.13; 95% CL, 1.2-8.05) to carry salivary Candida species and more likely (OR = 3.03; 95% CL, 1.25-7.33) to carry elevated levels of MS and/or salivary Candida and/or LB. CONCLUSIONS: The impact of sociodemographic risk and stress physiology on cariogenic disease processes are evident prior to ECC onset. The findings provide novel data on the early onset of cariogenic processes in children and the importance of considering sociodemographic, psychosocial, and behavioral factors when judging ECC risk. KNOWLEDGE TRANSFER STATEMENT: The findings provide valuable and novel findings that, pre-ECC onset, the caries disease process is explicable from a detailed assessment of behavioral, sociodemographic, and psychosocial stress variables.

Associations between maternal inflammation during pregnancy and infant birth outcomes in the Seychelles Child Development Study.

PROBLEM: Markers of maternal inflammation may determine infant birth outcomes. METHOD OF STUDY: Maternal serum samples were collected at 28 weeks gestation (n = 1418) in the Seychelles Child Development Study Nutrition Cohort 2 and analyzed for immune markers by MSD multiplex assay, including cytokines from the Th1 (IFN-γ, IL-1ß, IL-2 and TNF-α) and Th2 (IL-4, IL-5, IL-10) subsets, with IL-6, MCP-1, TARC, sFlt-1 and VEGF-D. Associations of log-transformed immune markers with birthweight, length, head circumference and gestational age were assessed by multiple linear regression models, which were adjusted for maternal age, BMI, parity, child sex, gestational age and socioeconomic status. RESULTS: Neither total Th1, Th2 nor Th1:Th2 were significantly associated with any birth outcome. However, the angiogenesis marker VEGF-D was predictive of a lower birthweight, (ß = -0.058, P = 0.017) and birth length (ß = -0.088, P = 0.001) after adjusting for covariates. Higher concentrations of CRP were predictive of a lower birthweight (ß = -0.057, P = 0.023) and IL-2 (ß = 0.073, P = 0.009) and the chemokine MCP-1 (ß = 0.067, P = 0.016) were predictive of a longer gestational age. CONCLUSIONS: In our cohort of healthy pregnant women, we found no evidence for associations between the Th1 or Th2 inflammatory markers with birth outcomes. However, VEGF-D and CRP appear to predict lower birthweight and IL-2 and MCP-1 a longer gestation. Greater understanding is required of the variation in these immune markers at different gestational stages, as well as the factors which may regulate their balance in healthy pregnancy. n = 233.

Prenatal methyl mercury exposure in relation to neurodevelopment and behavior at 19 years of age in the Seychelles Child Development Study.

BACKGROUND: Fish are important sources of protein and contain a variety of nutrients, such as n-3 long-chain polyunsaturated fatty acids (PUFA), essential for normal brain development. Nevertheless, all fish also contain methyl mercury (MeHg), a known neurotoxicant in adequate dosage. Our studies of the Seychelles Child Development Study (SCDS) Main Cohort enrolled in 1989-1990 (n=779) have found no consistent pattern of adverse MeHg effects at exposures achieved by daily fish consumption. Rather, we have observed evidence of improved performance on some cognitive endpoints as prenatal MeHg exposure increases in the range studied. These observations cannot be related to MeHg and may reflect the role of unmeasured covariates such as essential nutrients present in fish. To determine if these associations persist into young adulthood, we examined the relationship between prenatal MeHg exposure, recent PUFA exposure and subjects' neurodevelopment and behavior at 19 years of age. METHODS: We examined 533 participants using the following test battery: the Profile of Mood States-Bipolar (POMS-Bi); Finger Tapping; Kaufman Brief Intelligence Test (K-BIT); measures of Fine Motor Control and Complex Perceptual Motor Control; and Visual Spatial Contrast Sensitivity. We collected the following covariates: maternal IQ, family life course stressors, socioeconomic status, and subjects' recent postnatal MeHg, sex, and computer use. Primary analyses (based on N=392-475) examined covariate-adjusted associations in multiple linear regression models with prenatal MeHg as the primary exposure measure. Secondary analyses additionally adjusted for total n-6 and fish-related n-3 PUFA measured in the subjects' serum at the 19-year examination. RESULTS: Study participants had a mean prenatal MeHg exposure of 6.9 ppm, and a mean recent postnatal exposure of 10.3 ppm. There were no adverse associations between prenatal MeHg and any of the measured endpoints. For recent postnatal MeHg exposure, however, adverse associations were observed for Finger Tapping (non-dominant hand) among women and for the K-BIT Matrices for both sexes, with or without adjustment for PUFA. CONCLUSION: Our findings continue to provide no evidence for an adverse effect of prenatal MeHg exposure on development in a cohort that consumes fish daily. Observations for postnatal MeHg exposure will need to be confirmed using more comprehensive exposure measures.

Influence of cranberry proanthocyanidins on formation of biofilms by Streptococcus mutans on saliva-coated apatitic surface and on dental caries development in vivo.

Cranberry crude extracts, in various vehicles, have shown inhibitory effects on the formation of oral biofilms in vitro. The presence of proanthocyanidins (PAC) in cranberry extracts has been linked to biological activities against specific virulence attributes of Streptococcus mutans, e.g. the inhibition of glucosyltransferase (Gtf) activity. The aim of the present study was to determine the influence of a highly purified and chemically defined cranberry PAC fraction on S. mutans biofilm formation on saliva-coated hydroxyapatite surface, and on dental caries development in Sprague-Dawley rats. In addition, we examined the ability of specific PAC (ranging from low-molecular-weight monomers and dimers to high-molecular-weight oligomers/polymers) to inhibit GtfB activity and glycolytic pH drop by S. mutans cells, in an attempt to identify specific bioactive compounds. Topical applications (60-second exposure, twice daily) with PAC (1.5 mg/ml) during biofilm formation resulted in less biomass and fewer insoluble polysaccharides than the biofilms treated with vehicle control had (10% ethanol, v/v; p < 0.05). The incidence of smooth-surface caries in rats was significantly reduced by PAC treatment (twice daily), and resulted in less severe carious lesions compared to the vehicle control group (p < 0.05); the animals treated with PAC also showed significantly less caries severity on sulcal surfaces (p < 0.05). Furthermore, specific A-type PAC oligomers (dimers to dodecamers; 0.1 mg/ml) effectively diminished the synthesis of insoluble glucans by GtfB adsorbed on a saliva-coated hydroxyapatite surface, and also affected bacterial glycolysis. Our data show that cranberry PAC reduced the formation of biofilms by S. mutans in vitro and dental caries development in vivo, which may be attributed to the presence of specific bioactive A-type dimers and oligomers.

Carbon nanotubes randomly decorated with gold clusters: from nano2hybrid atomic structures to gas sensing prototypes.

Carbon nanotube surfaces, activated and randomly decorated with metal nanoclusters, have been studied in uniquely combined theoretical and experimental approaches as prototypes for molecular recognition. The key concept is to shape metallic clusters that donate or accept a fractional charge upon adsorption of a target molecule, and modify the electron transport in the nanotube. The present work focuses on a simple system, carbon nanotubes with gold clusters. The nature of the gold-nanotube interaction is studied using first-principles techniques. The numerical simulations predict the binding and diffusion energies of gold atoms at the tube surface, including realistic atomic models for defects potentially present at the nanotube surface. The atomic structure of the gold nanoclusters and their effect on the intrinsic electronic quantum transport properties of the nanotube are also predicted. Experimentally, multi-wall CNTs are decorated with gold clusters using (1) vacuum evaporation, after activation with an RF oxygen plasma and (2) colloid solution injected into an RF atmospheric plasma; the hybrid systems are accurately characterized using XPS and TEM techniques. The response of gas sensors based on these nano(2)hybrids is quantified for the detection of toxic species like NO(2), CO, C(2)H(5)OH and C(2)H(4).

In vivo expansion of the endogenous B-cell compartment stimulated by radiation and serial bone marrow transplantation induces B-cell leukaemia in mice.

Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5(+) B cells. This B-cell lineage is established during ontogeny and replenished by the process of self-renewal. Spontaneous and induced leukaemias that frequently affect this lineage are thought to arise as a result of the frequent cell division required to maintain the population throughout adulthood and in response to repeated exposure to environmental antigens. In a series of bone marrow transplant (BMT) experiments performed in B6D2F1 mice, B-cell leukaemia occurred in recipients of serially transplanted syngeneic bone marrow. This study was therefore designed to determine the frequency and phenotype of the observed leukaemia. Male donor cells were initially transplanted into lethally irradiated female hosts and secondary (2 degrees ) BMT was performed at 3 months. At 1, 2, 3 and 16 months following primary (1 degrees ) BMT, and when 2 degrees BMT recipients developed leukaemia, animals were sacrificed and their tissues extensively examined. These analyses confirmed a host-derived CD5(+) transplantable B-cell leukaemia that was initiated in 50% of 1 degrees BMT recipients. With serial passage, the leukaemia became more aggressive and lost CD5 expression, suggesting transformation to a high-grade leukaemia/lymphoma. This previously unreported observation suggests that the combination of radiation and subsequent serial transplantation induces a proliferative stress to the host B-cell compartment that is causative in leukaemic transformation.

Defective fluid secretion and NaCl absorption in the parotid glands of Na+/H+ exchanger-deficient mice.

Multiple Na(+)/H(+) exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to study the in vivo functions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na(+)/H(+) exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1(-/-)). Salivation in response to pilocarpine stimulation was reduced significantly in both Nhe1(-/-) and Nhe2(-/-) mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na(+)/K(+)/2Cl(-) cotransporter mRNA increased dramatically in Nhe1(-/-) parotid glands but not in those of Nhe2(-/-) or Nhe3(-/-) mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2(-/-) or Nhe3(-/-) mice were comparable with those of wild-type mice. In contrast, Nhe1(-/-) mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na(+) absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na(+)/K(+)/2Cl(-) cotransporter activity.

In vivo chromosomal instability and transmissible aberrations in the progeny of haemopoietic stem cells induced by high- and low-LET radiations.

PURPOSE: To study stable and unstable chromosomal aberrations in the haemopoietic cells of CBA/H mice after exposure to both high- and low-LET radiations. MATERIALS AND METHODS: Chromosomal aberrations were scored in the clonal progeny of X-, alpha- or non-irradiated short-term repopulating stem cells using the spleen colony-forming unit (CFU-S) assay, 12 days post-transplantation and in the bone marrow reconstituted by X-, neutron- or non-irradiated exogenous (transplanted) or endogenous (X- or neutron whole-body-irradiated) long-term repopulating stem cells for up to 24 months. RESULTS: Chromosomal instability was demonstrated in 3-6% of cells in all cases. After transplantation of X- or neutron-irradiated bone marrow approximately 8% of cells with stable aberrations were recorded at all times. After 3Gy X- or 0.5 Gy neutron- whole-body irradiation stable aberrations were detected in approximately 17 and 5% of cells respectively. CONCLUSIONS: Chromosomal instability induced in vitro can be transmitted in vivo by transplantation of haemopoietic stem cells exposed to high- or low-LET radiations. Comparable instability can be induced and shown to persist for the remaining lifetime after whole-body irradiation. There was no direct relationship between the expression of stable and unstable aberrations and significant interanimal variation in the expression of both stable and unstable aberrations.

Chromosomal instability in unirradiated cells induced in vivo by a bystander effect of ionizing radiation.

Using a bone marrow transplantation protocol in which we transplanted a mixture of irradiated and nonirradiated bone marrow cells that were distinguishable by a cytogenetic marker, we have demonstrated chromosomal instability in the progeny of nonirradiated hemopoietic stem cells. This first demonstration of a link between a bystander effect of ionizing radiation and the induction of genomic instability in vivo clearly poses a major challenge to current views of the mechanisms of radiation-induced DNA damage with mechanistic implications for the health consequences of radiation exposure particularly in the context of the induction of malignancy.

Severe impairment of salivation in Na+/K+/2Cl- cotransporter (NKCC1)-deficient mice.

The salivary fluid secretory mechanism is thought to require Na(+)/K(+)/2Cl(-) cotransporter-mediated Cl(-) uptake. To directly test this possibility we studied the in vivo and in vitro functioning of acinar cells from the parotid glands of mice with targeted disruption of Na(+)/K(+)/2Cl(-) cotransporter isoform 1 (Nkcc1), the gene encoding the salivary Na(+)/K(+)/2Cl(-) cotransporter. In wild-type mice NKCC1 was localized to the basolateral membranes of parotid acinar cells, whereas expression was not detected in duct cells. The lack of functional NKCC1 resulted in a dramatic reduction (>60%) in the volume of saliva secreted in response to a muscarinic agonist, the primary in situ salivation signal. Consistent with defective Cl(-) uptake, a loss of bumetanide-sensitive Cl(-) influx was observed in parotid acinar cells from mice lacking NKCC1. Cl(-)/ HCO(3)(-) exchanger activity was increased in parotid acinar cells isolated from knockout mice suggesting that the residual saliva secreted by mice lacking NKCC1 is associated with anion exchanger-dependent Cl(-) uptake. Indeed, expression of the Cl(-)/ HCO(3)(-) exchanger AE2 was enhanced suggesting that this transporter compensates for the loss of functional Na(+)/K(+)/2Cl(-) cotransporter. Furthermore, the ability of the parotid gland to conserve NaCl was abolished in NKCC1-deficient mice. This deficit was not associated with changes in the morphology of the ducts, but transcript levels for the alpha-, beta-, and gamma-subunits of the epithelial Na(+) channel were reduced. These data directly demonstrate that NKCC1 is the major Cl(-) uptake mechanism across the basolateral membrane of acinar cells and is critical for driving saliva secretion in vivo.

The effect of chronic clonidine administration on salivary glands and caries in the rat.

Clonidine is a widely prescribed, centrally acting antihypertensive with proposed pharmacologic mechanisms of stimulation of central postsynaptic alpha(2)-adrenergic receptors and agonist activity at presynaptic alpha(2)-adrenergic receptors, interfering with peripheral regulation of norepinephrine and acetylcholine release. Both of these mechanisms are capable of adversely influencing salivary output and composition, potentially leaving an individual with increased caries risk. The purpose of the present study was to determine the effect of chronic administration of clonidine on saliva, salivary glands, and caries in rats. Sprague-Dawley rat pups were infected with Streptococcus sobrinus, given Diet 2000 and 10% sucrose water ad libitum, and either desalivated, or treated with clonidine HCl (125 or 250 microg/kg administered daily for 28 days by means of osmotic minipumps), or assigned as controls. There were no statistical differences in stimulated parotid or submandibular gland salivary output or sublingual gland weights among the groups. The weight of the submandibular glands as a percent of total body weight was significantly decreased in animals that received clonidine when compared with controls. Sulcal caries scores in both clonidine groups and smooth surface caries scores in the high clonidine group were increased when compared with control animals. Positive control animals (desalivated) had significantly higher caries scores than all other groups. These data show that chronic administration of clonidine significantly decreases submandibular gland weight and increases susceptibility to dental caries.

Litter effects on caries in rats and implications for experimental design.

The purpose of this study was to obtain quantitative estimates of litter effects on caries development in rats and to examine the implications for design of rat caries experiments. Twelve female Sprague-Dawley rats, aged 60 days, were bred with 4 male rats. Nine of the 12 dams had litters in close proximity. The litters were culled to 10 pups. One pup from each litter was placed with each of the other dams for nursing, leaving 2 pups from each litter with the birth dam. This design allowed the litter effect to be separated into a prenatal component, reflecting the shared genetic makeup and in utero environment of littermates, and a postnatal component reflecting a shared environment from shortly after birth to weaning. Pups were infected with Streptococcus sobrinus and fed Diet 2000 and 10% (w/v) sucrose water for 5 weeks. There was no significant evidence of a postnatal litter effect for smooth surface caries (p = 0.37) or sulcal caries (p = 0.43). The prenatal litter effect was significant for both smooth surface caries and sulcal caries (p<0. 01). When litter effects are present, the statistical power of caries studies is improved if animals from the same litter are divided evenly among experimental groups. In addition, if litter effects are present but not allowed for in data analysis, incorrect statistical inferences may be drawn. Based on our results and other reports of litter effects, we recommend planning for litter effects in the design and analysis of rat caries studies.

The efficacy of an intraoral fluoride-releasing system in irradiated head and neck cancer patients: a preliminary study.

This study compared the anticaries effectiveness of an intraoral fluoride-releasing system, or IFRS, with a standard regimen of daily application of a 1.1 percent neutral sodium fluoride gel in custom trays. Caries protection in subjects in the IFRS group was comparable to that in subjects in the 1.1 percent neutral sodium fluoride group. The subjects all had head or neck cancer and had received radiation therapy, but no more recently than three months before taking part in the study. Overall, IFRS devices were well-tolerated and patient satisfaction was high. The IFRS appears to offer several advantages over the daily application of fluoride gels in custom trays.

Influence of maternal lead ingestion on caries in rat pups.

Lead is one of the most toxic and pervasive pollutants in society, and although there has been some lowering of blood lead levels in recent years, the levels continue to be of concern for African Americans, central city residents, residents in the Northeast region of the United States, persons with low income, and those with low educational attainment. Notably, these are the persons and the region where the highest prevalence of dental caries is observed. Information relating lead toxicity to oral health is sparse, but the preponderance of epidemiological data shows a relation between lead in the environment and the prevalence of dental caries. Using our well-defined rat caries model we found that pre- and perinatal exposure to lead results in an almost 40% increase in the prevalence of caries and a decrease in stimulated parotid function of nearly 30%. Levels of lead in milk from lead-treated dams were approximately 10 times as high as the corresponding blood lead levels, suggesting that lead is being concentrated by mammary glands. These findings may help in part to explain the comparatively high levels of dental caries observed in the inner cities of the United States where exposure to lead is common. Environmental sources of lead include drinking water, lead-based paint and, to a lesser extent, automobile and industrial emissions. In humans lead is accumulated and stored in bones (half-life of approximately 62 years), and even maternal exposure to lead decades before pregnancy can subsequently result in exposure of the developing fetus to elevated levels of lead. Moreover, lead concentration in maternal blood has been shown to increase during pregnancy and lactation because of mobilization of stored lead from bone, and typically, lead is found in milk at a higher concentration than the level found in maternal plasma at the same time point.

Genetic factors influencing alpha-particle-induced chromosomal instability.

Alpha-particle-induced chromosomal instability in haemopoietic cells obtained from the CBA/H, DBA/2 and C57BL/6 inbred strains of mouse has been demonstrated at frequencies dependent on genotype. The CBA/H and DBA/2 strains may be regarded as 'sensitive' and the C57BL/6 strain as 'resistant'; resistance was dominant in cells from F1 hybrids. Previously, in cultures where we demonstrated radiation-induced chromosomal instability we also demonstrated an enhanced and persisting oxyradical activity. Quantitative differences in superoxide generation have now been correlated with genetically determined differences in the expression of chromosomal instability. Our findings demonstrate an important influence of genetic factors in alpha-particle-induced chromosomal instability.

Biochemical and immunological studies and assay of rat sublingual mucins.

Original studies of rat sublingual mucins raised questions as to the existence of a second mucin species as distinguished by binding to hydroxyapatite. The existence of multiple mucin species is of concern in pharmacological studies of mucous-cell secretion as each species could represent distinct mucous-cell populations that respond differently to secretagogues. Thus a separate hydroxyapatite-bound mucin pool expressed in rat sublingual glands was isolated and characterized. Biochemical comparison of hydroxyapatite-bound mucins to total and hydroxyapatite-unbound sublingual mucins demonstrated no substantial differences in either amino acid and carbohydrate contents or in size distributions. In addition, a radioimmunoassay was developed using antisera prepared previously against unbound mucins. The three mucin pools exhibited equal specificities in displacement of radiolabelled unbound mucin tracer in the radioimmunoassay. Thus, bound and unbound mucins are indistinguishable, both immunologically and in biochemical composition. The radioimmunoassay was then evaluated for use in pharmacological studies of acinar mucous-cell secretion. Measurement by radioimmunoassay of secretion from isolated acini in response to carbachol was concentration-dependent (EC50 approx. 0.3 microM and maximal stimulation at 1 microM carbachol). In immunolocalization studies the antiserum was highly selective for mucous cells, recognized all mucous cells within histological sections, and was localized subcellularly to mucous-cell secretion granules and trans-Golgi, further validating the radioimmunoassay as a method to detect exocrine secretion from the entire pool of acinar mucous cells. Moreover, the radioimmunoassay was compared and found equivalent to an acid-precipitation method to assess relative secretion, suggesting the acid-precipitation method is also valid for pharmacological studies of isolated acini.

Both M1 and M3 receptors regulate exocrine secretion by mucous acini.

We investigated the role of M1 and M3 receptors in regulating exocrine secretion from acini isolated from rat sublingual glands. In secretion experiments, we derived affinity values (KB) from Schild regression analysis for the antagonists pirenzepine (61.0 nM) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 1.06 nM). The KB for 4-DAMP is similar to its affinity value [equilibrium dissociation constant from competition studies (Ki); 1.81 nM] determined from radioligand competition experiments. In contrast, the KB for pirenzepine is between its high-affinity (17.6 nM) and low-affinity (404 nM) Ki values. In separate secretion experiments, we found that the M1 receptor antagonist, M1-toxin, induces a rightward shift in the concentration-response curve to muscarinic agonist and inhibits maximal secretion by 40%. The inhibitory effect of M1-toxin appears specific for M1 receptor blockade, since the toxin abolishes acinar high-affinity pirenzepine-binding sites and does not inhibit secretion induced by nonmuscarinic agents. Additional pharmacological studies indicate muscarinic receptors do not function through putative neural elements within isolated acini. Our combined results are consistent with both M1 and M3 receptors directly regulating mucous acinar exocrine secretion and indicate M3 receptors alone are insufficient to induce a maximal muscarinic response.

Long-term in vivo transmission of alpha-particle-induced chromosomal instability in murine haemopoietic cells.

We have previously reported non-clonal cytogenetic aberrations in the clonal descendants of murine haemopoietic stem cells irradiated in vitro with alpha-particles. The data are consistent with a transmissible chromosomal instability induced in a stem cell resulting in a diversity of aberrations in its clonal progeny many cell divisions later. To determine whether this instability is transmissible in vivo, bone marrow cells obtained from the male mouse have been irradiated with alpha-particles in vitro and transplanted into female recipients. In the repopulated haemopoietic system we have observed persisting chromosomal instability up to 1 year post-transplantation. The findings demonstrate a long-lived effect of the alpha-particle-induced lesion in the donor repopulating stem cells.

Muscarinic cholinergic receptor subtypes in rat sublingual glands.

Mucin glycoprotein secretion by rat sublingual glands is regulated primarily by muscarinic cholinergic receptors. Studies were conducted to identify muscarinic receptor subtypes in whole glands as well as in isolated acinar structures. In radioligand binding studies, we used subtype-selective antagonists in competition studies to initially determine receptor subtype heterogeneity. In membranes from whole glands, both pirenzepine and methoctramine displayed two affinity sites (M1 and M3) of nearly equal proportions. In contrast, acinar membranes contained a 1:2 and 2:1 ratio of M1 to M3 sites for pirenzepine and methoctramine, respectively. In all cases, p-fluoro-hexahydro-siladifenidol and 4-diphenylacetoxy-N-methylpiperidine each bound to a single class of binding sites. Northern analysis using oligonucleotide probes specific for the 5' ends of the translated regions of m1 through m5 receptors detected only m1 and m3 subtypes in poly(A)+ RNA from whole glands. We also used antisera specific for each receptor subtype to immunoprecipitate solubilized receptors from membrane preparations. Only m1 (51.7 and 64.9%) and m3 (48.3 and 34.7%) subtypes were found consistently in membranes from whole sublingual glands and isolated acini, respectively. Studies with other exocrine glands generally described the predominance of m3 receptors, and m1 receptors, if present, were presumably associated with contaminating neural structures. Our results therefore demonstrate that mucous acini from rat sublingual glands contain abundant amounts of both m1 and m3 receptors.