Regulation of the epithelial-mesenchymal transformation through gap junction channels in heart development.

Analyses of mice lacking the gap junction protein, connexin45 (Cx45), have provided new insights into the essential roles of gap junction channels in early embryogenesis. Of great surprise is the function of Cx45 in the endothelium, where it is essential for synchronized activation of the transcription factor Nfatc1. This laterally synchronized regulation model extends the generally accepted vertical model, in which interactions between the endocardium and the myocardium induce endocardial cushion formation through the epithelial-mesenchymal transformation.

Diversity and molecular anatomy of gap junctions.

In animal tissues, most cells are connected via intercellular cytoplasmic channels called gap junctions. Various electron microscopy techniques have made a crucial contribution to our understanding of the function and structure of gap junction channels. Tracer studies and freeze-fracture replica observations indicate that the connexon, the unit gap junction channel, is a pair of hemichannels apposed in the narrow intercellular gap between neighboring cell membranes. Recent advances in cellular biology have shown that connexon hemichannels are composed of hexamers of connexin proteins. Purification of the gap junction membrane and cDNA cloning analysis indicate the diversity of the connexin protein family, which contains more than 18 members, and their tissue- and cell type-specific distributions. Defects in some connexin genes may cause various hereditary diseases, such as X-linked Charcot-Marie-Tooth disease (Cx32), nonsyndromic autosomal deafness (Cx26), and cataract (Cx50). Analysis of gene knockout mice indicates that certain types of connexin play important roles in differentiation and development at crucial times in specific tissues and cell types.

Loss of connexin45 causes a cushion defect in early cardiogenesis.

At around embryonic day 9, the primitive heart of a mouse embryo undergoes spectacular alterations within 24 hours. We created mice harboring an nls-lacZ gene in place of connexin45, which encodes the only known gap junction protein in the primitive heart before embryonic day 9, using the Cre-loxP system. Connexin45-deficient mice died of heart failure at around embryonic day 10. They initiated heart contractions, but conduction block appeared within 24 hours after the first contractions. Their cardiac walls displayed an endocardial cushion defect, while the cardiac jelly was present. These abnormalities were caused by impairment of the epithelial-mesenchymal transformation of the cardiac endothelium. Activation of the cardiac endothelium depended on the presence of the connexin45 gap junctions since signaling through Ca(2+)/calcineurin and NF-ATc1 (originally named NF-ATc) was disrupted in the mutant hearts. These results indicate a requirement for gap junction channels during early cardiogenesis and hence implicate connexin45 in congenital heart diseases. http://www. biologists.com/Development/movies/dev4369.html

Relation between self-paced and synchronized movement in persons with mental retardation.

The relation between self-paced and synchronized tapping in 64 persons with mental retardation whose mental ages ranged from 2 to 11 years and chronological ages from 13 to 23 years was investigated. In a self-paced tapping task no stimulus was presented, and subjects' easy and spontaneous tapping was measured. In a synchronized tapping task their synchronous tapping with an auditory stimulus present at a quick or slow tempo was measured. Under both tempo conditions, the lower the subjects' mental age, the larger the errors in the intertap interval they made. The subjects of low mental age showed significantly larger errors in the intertap interval in the Slow than in the Quick Tempo condition and tended to tap at a rate near the self-paced tapping. These results may suggest that ability to adjust one's self pace is one of the key factors in the development of motor synchronization in persons with mental retardation.

Abnormalities of developmental cell death in Dad1-deficient mice.

BACKGROUND: Dad1, the defender against apoptotic cell death, comprises the oligosaccharyltransferase complex and is well conserved among eukaryotes. In hamster BHK21-derived tsBN7 cells, loss of Dad1 causes apoptosis which cannot be prevented by Bcl-2. RESULTS: To determine the role of Dad1 function in vivo, we prepared by gene targeting, mice harbouring a disrupted Dad1 gene. Homozygous mutants died shortly after they were implanted with the characteristic features of apoptosis. In an in vitro blastocyst culture system, Dad1-null cells displayed abnormalities which were comparable to those obtained in vivo. However, oligosaccharyltransferase activity was apparently retained even after the Dad1-null cells were destined to die. Some live-born heterozygous mutants displayed soft-tissue syndactyly. Mild thymic hypoplasia was also indicated in heterozygotes. CONCLUSION: These results suggest the involvement of the Dad1 gene in the acquisition of a common syndactyly phenotype, as well as in the control of programmed cell death during development.

Relation between synchronized and self-paced response in preschoolers' rhythmic movement.

Regulation of the rhythmic movement of 29 preschoolers ages 3 to 6 years was studied in connection with self-paced response. An Auditory Pulse condition presented the pulse audibly, a Visual Pulse condition presented the pulse visibly, and a Moving Visual Target condition presented the repetitive movement of a visual target. We used a Quick Tempo condition in which the interstimulus interval was slightly different from the average self-paced tapping rate at which each subject felt comfortable, and a Slow Tempo in which the interval was considerably different. The error in the interresponse interval of tapping, i.e., the time gap between the mean interresponse and interstimulus intervals, was calculated as an indicator of regulation. The error in the former decreased across age groups only in the Slow Tempo condition. In the Slow-Tempo Visual-Pulse condition in which the error in the interresponse interval was particularly large, the younger subjects tended to respond at a rate near the self-paced response. In both tempos, the error in the interresponse interval in the Moving Visual Target condition was much the same as in the Auditory Pulse condition and was statistically smaller than in the Visual Pulse condition. These results may suggest that one of the important factors in the development of preschoolers' synchronization with physical rhythm is an ability to modify or restrain the self-paced response and that additional information from movement of the visual target could assist them externally in regulating movement.

Detection of allergen- and mitogen-induced human cytokine transcripts using a competitive polymerase chain reaction.

Human cytokines, IL-4, IL-5, and IFN-gamma play an important role in the regulation of IgE synthesis and atopic diseases. In this communication, we describe the development of a quantitative assay of steady-state cytokine mRNAs (IL-4, IL-5, and IFN-gamma) from a variety of cell sources, including peripheral blood mononuclear cells (PBMCs) stimulated with either a mitogen (PHA) or ragweed pollen allergen extract, and cells from allergen-challenged inflammatory sites. Quantitative analysis of IL-5, IL-4 and IFN-gamma transcripts was achieved by a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique using internal standard (IS) cRNAs in the presence of specific oligonucleotide primers. Each IS was generated from a plasmid vector containing the respective cytokine cDNA modified by insertion with an SV40-DNA fragment. Both test RNA and IS were reverse-transcribed and subjected to the 'competitive' PCR in the same tube. We first demonstrate the linearity and reproducibility of this technique; second, we apply this competitive PCR assay to analyze quantitatively the expression of IL-4, IL-5, and IFN-gamma transcripts in PBMCs before and after stimulation with PHA or crude ragweed allergen. Finally, we analyzed cells isolated from the lung lavage fluids of an atopic subject following allergen challenge, and showed a significant increase of IL-4 and IL-5 transcripts, but not IFN-gamma, in the allergen-challenged site when compared to the control. This technique of PCR quantitation provides an easy and efficient tool to study the expression of cytokine genes in allergic inflammatory diseases.

Immunogenetic aspects of IgE-mediated responses.

The reported significant HLA association is consistent with the codominant expression of MHC-linked Ir genes. Similar studies are needed in the case of the other HLA-immune response associations. It seems unlikely that any unique HLA-D genetic sequence will be found only among subjects responding to a particular Ag. It is likely, however, that we will find that a particular sequence is a necessary, but not sufficient, requirement for responsiveness to a particular antigenic epitope. Previous family studies, in which failed to observed parent-to-child transmission of specific immune responsiveness to a particular allergen, suggest that further genetic and/or environmental factors are required for the expression of specific immune responsiveness. These factors include variations in the degree of antigenic exposure. Of particular importance is the need for Ag-specific TcR genes to be expressed in the mature T-cell repertoire. In study of the specific immune response, there have been several studies of the protein and DNA sequences of allergenic proteins; therefore, in regard to understanding the genetics of specific immune responsiveness to allergens and its relationship to atopic diseases, rapid advances can be anticipated over the next several years.

Analysis of cytokine transcripts in the bronchoalveolar lavage cells of patients with asthma.

A panel of steady-state cytokine mRNAs was analyzed in the bronchoalveolar lavage (BAL) cells from asthmatic subjects or patients challenged with ragweed allergen. This was achieved by combining both qualitative and quantitative assays using the reverse transcription-polymerase chain reaction (RT-PCR). Analysis of BAL cells from six mild allergic asthmatic and five nonasthmatic, nonallergic subjects showed no qualitative differences in the profile of cytokine mRNAs (including interleukin [IL]-1 beta, IL-2, IL-5, IL-6, IL-8, and granulocyte/macrophage colony-stimulating factor), except for tumor necrosis factor-alpha, which was detected in three out of six asthmatic BAL samples but in none of the controls. A key cytokine, IL-5, has been implicated in the pathogenesis of allergic inflammation through the recruitment of eosinophils. We found a significant enhancement of steady-state IL-5 transcripts in the BAL cells from allergen-challenged as compared with the saline-challenged control sites of four asthmatic patients; furthermore, the cellular source for IL-5 mRNA was identified in the mononuclear cell fraction, but not in the purified eosinophils, of the allergen-challenged BALs. These results suggest that the significant increase of IL-5 transcripts is primarily from the infiltrating mononuclear cells. Our study also demonstrates the power of qualitative and quantitative PCR analysis in determining the molecular basis of allergic inflammatory diseases.

Detection of specific IgE-secreting cells with an enzyme-linked immunospot assay.

BACKGROUND: An in vitro assay system for monitoring the induction of human allergen-specific IgE synthesis with an enzyme-linked immunospot assay is described. METHODS: IgE-secreting cells, responding specifically to short ragweed pollen allergens. Amb a V and Amb a VI, were directly enumerated after the coculture of high-density, resting B cells with either autologous T cells or allergen-specific T-cell clones from the peripheral blood of two ragweed-allergic individuals. Both T-cell clones were type 2-like T helper cells (TH2) as determined by reverse transcription polymerase chain reaction. The IgE-producing cells were detected on a nitrocellulose-based paper disc coated with antigen after treatment with biotinylated, anti-human IgE and avidin-peroxidase conjugate. RESULTS: We demonstrated the induction of Amb a V- or Amb a VI-specific IgE synthesis from either peripheral blood B cells or purified resting B cells. Specific IgE-secreting B cells could be detected only when T cells were present in the culture, providing that they were not separated by a membrane. The addition of interleukin-4 had an enhancing effect on the overall numbers of IgE-secreting cells and allergen-specific T cells and a synergistic effect in the coculture of B cells and an autoreactive T-cell line. CONCLUSIONS: These results suggest that cognate interaction T and B cells is required for the de novo induction of IgE responses. This in vitro system could thus provide a useful model for analysis of the molecular and cellular mechanisms that regulate the allergen-specific IgE responses in human beings.

The effect of interferon-gamma, interleukin-4 and immunoglobulin receptor cross-linking of monocytes on allergen-specific T-cell response.

Modulation of the proliferative responses of an allergen-specific human Th2 cell line by cytokine-treated monocytes was examined. The response of this cell line to the specific allergen, Amb a V (from short ragweed pollen), increased following the addition of interleukin-1 beta (IL-1 beta). However, in the presence of exogenous interferon-gamma (IFN-gamma), there was greater than 40% reduction in the responsiveness of these T cells. The addition of IL-1 beta did not reverse the inhibitory effect of IFN-gamma. To determine the primary target cell type for IFN-gamma, autologous monocytes were pretreated with IL-4, IFN-gamma, or medium alone, and used as antigen-presenting cells (APC). We showed that the responses of T cells to Amb a V were significantly down-regulated in the presence of autologous monocytes pretreated with IFN-gamma, but not for monocytes pretreated with IL-4. Similar inhibitory effect of IFN-gamma was confirmed using a human T-cell line specific for a ragweed allergen, Amb a I, and a human T-cell clone raised against ragweed extract. Cross-linking of CD23 (Fc epsilon RII) on monocytes pretreated with IFN-gamma increased this inhibitory effect in an additive fashion, but, in the absence of IFN-gamma treatment, such cross-linking had no effect. These inhibitory effects were not due to alterations in the surface expression of HLA-DR on the monocytes, and the addition of exogenous IL-1 beta was unable to reverse these effects. In similar experiments, cross-linking of CD64 (Fc gamma R) on monocytes showed no significant effects. In conclusion, IFN-gamma is important in regulating the function of monocytes involved in Th2 cell responses to allergens. IL-4 treatment, as well as cross-linking of FcR of monocytes, have no direct effect on such response.

[Involuntary smoking in a workplace situation--discomfort of nonsmokers and interpersonal awareness of smokers].

A questionnaire survey was conducted to examine the socio-behavioral interaction between smokers and nonsmokers in a workplace situation. A socio-behavioral interaction is hypothesized to be centered around the subjective discomfort such as 'bothered feelings' towards involuntary smoking among nonsmokers. Study subjects were 1,134 nonsmokers and 1,457 smokers working in 21 small-scale production companies in Japan. Among nonsmokers, a combined 93.3% reported 'perceived exposure to smoking', 'always', 'often', or 'sometimes' compared to 6.6% reporting 'never'. Similarly, 74.6% experienced 'bothered feelings' 'sometimes', 'always', or 'often' compared to 25.3% reporting 'never'. A combined total of 39.4% expressed their discomfort to smokers 'sometimes', 'often', and 'always' compared to 60.6% who 'never' did. In contrast, 59.1% of smokers responded that they smoked with consideration of nonsmokers' presence (interpersonal awareness) 'sometimes', 'often', and 'always' compared to 41.0% for 'never'. 'Bothered feelings' among nonsmokers was significantly lower among those with past smoking history and correlated positively with 'perceived exposure to smoking' (rs = 0.22, p less than 0.001) and with 'expression of discomfort among both male (rs = 0.32, p less than 0.001) and female nonsmokers (rs = 0.34, p less than 0.001). Among male smokers 'interpersonal awareness' was significantly higher for those with less cigarette consumption and with past history of smoking cessation. A regression analysis was performed using mean scores calculated for smokers and nonsmokers of each workplace. Lower 'bothered feelings' of nonsmokers significantly correlated with higher 'interpersonal awareness' of smokers (r = -0.59, p less than 0.005). These finding imply the presence of socio-behavioral interactions between smokers and nonsmokers and thus suggest the significance of workplace education on involuntary smoking.

[Immunological study of the HLA class II antigen associated with birch pollen allergy].

Pollinosis is known as a hereditary disease. Recently, association of HLA antigens and pollinosis caused by several pollen allergens has been reported. Furthermore, HLA antigens are considered to have a very important role in the development of pollinosis. We performed an HLA population study and tested the lymphocyte proliferative response (LPR) to birch pollen allergens with birch pollen allergic patients and healthy control subjects. The results of the HLA population study indicated that HLA-DR9 and HLA-DQw3 correlated with the development of birth pollinosis (relative risk [R. R] = 6.37 for DR9 and R. R = 7.92 for DQw3). DNA typing revealed that serotype DQw3 in patients were DQw9 specific. Two sources of birch pollen allergens, betula pendula and betula platyla var. japonica, were used in LPR. Lymphocytes from the patients, but not from the healthy control subjects, proliferated in response to these two allergens. Anti-HLA-DR antibody, but not anti-HLA-DQ antibodies, inhibited the patients' LPR. These results suggest that the HLA-DR molecules are responsible to present the pollen specific antigen to T lymphocytes inducing LPR in the patients. Furthermore, in some of the healthy control subjects, anti HLA-DQ antibodies enhanced this LPR almost to the same level as that of the patients'. This result indicates that HLA-DQ molecules might be associated with the suppression of T cell proliferation.

[Clinico-pathological findings of recurrent pleomorphic adenomas of the parotid gland].

We analyzed the clinical courses and pathological findings of six recurrent benign pleomorphic adenomas of the parotid gland, compared with fifty-nine primary pleomorphic tumors. Primary cases were treated with extirpation of a tumor nodule with a surrounding area of parotid tissue with preservation of the facial nerve. Of the 59 patients, 42 who had follow-up information have had no recurrent tumors during a average 6.6 years follow up period. On the other hand, all six recurrent cases had received surgical enucleation or excisions elsewhere. To avoid the recurrence of the primary pleomorphic adenoma it was considered to be very important to remove a tumor completely without damaging the tumor capsule. CT scan is very useful to know the relationship between the recurrent tumor and the surrounding important organs. In order to determine the surgical approach and procedures for those cases, it is important to obtain the information of the previous pathological diagnosis or surgical procedures. For the recurrent tumor, complete extirpation of the tumor and surrounding operative scar was necessary for the management of the tumor. When the tumor was tightly adhesive with the facial nerve, sacrifice of the nerve and immediate nerve transplantation might be required.

Impaired glucagon secretion to insulin-induced hypoglycemia in anorexia nervosa.

In order to clarify the role played by pancreatic alpha-cell dysfunction in the impaired glucose recovery from hypoglycemia in patients with anorexia nervosa, the response of pancreatic alpha-cells to insulin-induced hypoglycemia was investigated in 16 patients with anorexia nervosa before and after treatment. The results were compared with those obtained after loading with arginine. Before treatment, despite comparable falls in plasma glucose levels, glucagon secretion was significantly reduced in the anorectic patients compared with control subjects. In addition, glucose recovery from hypoglycemia in the patients was attenuated. However, after treatment, both glucagon secretory activity and plasma glucose recovery following insulin-induced hypoglycemia were restored to normal. Plasma glucagon responses to arginine infusion were not significantly different in the untreated anorectic patients and control subjects. However, the plasma insulin response in the patients was significantly lower than in the control group. These results suggest that the impaired recovery of plasma glucose levels from insulin-induced hypoglycemia in patients with anorexia nervosa is primarily attributable to impaired pancreatic alpha-secretory capability. In addition, this abnormality in pancreatic alpha-cell function is reversible with treatment leading to improved nutrition and weight gain.

Glucagon secretion in anorexia nervosa.

Patients with anorexia nervosa frequently manifest impaired glucose tolerance. However, alterations in pancreatic glucagon secretion have also been associated with alterations in diabetes mellitus. For this reason, pancreatic alpha- and beta-cell responses to glucose load were measured in 25 anorexic patients both before and after treatment. The baseline glucose challenge failed to suppress plasma glucagon levels in the patients. However, in the control subjects and patients after treatment, glucagon levels were suppressed after glucose ingestion. Plasma glucose levels during the baseline challenge were significantly higher than those of the control subjects; however, after treatment glucose responses were nearly normal. Finally, insulin responses at baseline and after treatment were lower in the patients than in control subjects. These results suggest that the impaired glucose tolerance manifested by anorexic patients may be attributable to significant alterations in both pancreatic alpha- and beta-cell secretions and in pancreatic alpha-cell and glucose interrelationships.

Enhanced degradation of low density lipoprotein in human monocyte-derived macrophages associated with an increase in its free fatty acid content.

Low density lipoprotein (LDL) rich in oleic acid (designated FFA-rich LDL) was produced by the reconstitution technique. FFA-rich LDL, like acetyl LDL, moved faster than native LDL in agarose gel electrophoresis. While FFA-rich LDL was observed to degrade far less than natural LDL in lymphocytes, its degradation in monocyte-derived macrophages was three times higher than that of natural LDL or LDL reconstituted without the addition of oleic acid. A competitive study showed that the catabolism of FFA-rich LDL in macrophages may be influenced by systems other than the acetyl LDL receptor.

Pharmacokinetics of methimazole in children and adolescents with Graves' disease. Studies on plasma and intrathyroidal concentrations.

Methimazole concentrations in plasma and in the thyroid glands were measured by means of high-performance liquid chromatography. Pharmacokinetics of methimazole were studied after a single oral dose (175 mumol/m2) in nine children and adolescent who were in the thyrotoxic state. Plasma levels of methimazole showed peak concentrations of 4.4 to 12.6 (median 9.2) mumol/l at 0.5 to 4 h after drug administration. Plasma half-life, area under the curve, and distribution volume ranged from 2.73 to 6.04 h, 32.8 to 77.9 mumol X l-1 X h-1, and 0.516 to 0.913 l/kg, respectively. These pharmacokinetic parameters showed a wide variation among the patients, but were quite reproducible in the same subject. Intrathyroidal concentrations of methimazole were measured in another nine subjects including four adolescents and five adults who underwent thyroidectomy. The drug concentrations in the thyroid glands ranged between 3.5 and 23.8 mumol/kg tissue and were far higher than those in the plasma obtained at the time of surgery. In this series of experiments, the dose of the drug varied from 76 to 319 mumol/m2, time after the last dose to surgery from 5 to 24 h, and the mode of drug administration from a single to three divided doses. Among these variable factors, only the daily dose of methimazole corrected by body surface area showed significant correlation with the intrathyroidal concentration, whereas the time after the last dose of the drug and the mode of drug administration did not.(ABSTRACT TRUNCATED AT 250 WORDS)