Three-dimensional ultrasound evaluation of fetal tooth germs

Thirty children have had percutaneous endoscopic gastrostomy (PEG) inserted for feeding purposes. During a follow-up period of 5-45 months (mean 24 months), complications were recorded in 13 patients. Some patients had more than one complication. Complications included colocutaneous fistula, gastro-oesophageal reflux, wound infection, granulation tissue formation, tube leakage, tube blockage and bleeding. The authors present a retrospective review of the results and discuss the management of these complications.     

Distribution and expression of mRNAs for the proto-oncogenes c-fos and c-jun in bone cells in vivo

Aminopeptidase A is a homodimeric membrane-bound zinc metallopeptidase anchored at the plasma membrane by a 22-amino-acid hydrophobic segment. The anchor segment separates a small N-terminal cytoplasmic domain from a large ectodomain that contains the active site. Site-directed mutagenesis was performed to investigate the role of the cytoplasmic domain of aminopeptidase A in membrane anchoring and routing of the enzyme. Expression in COS-7 cells of a mutant lacking the N-terminal cytoplasmic domain resulted in the efficient secretion of a catalytically active enzyme in the medium. The soluble mutated aminopeptidase A, purified from the medium of a stable cell line, exhibited similar biochemical features to those of the wild-type enzyme. Pulse/chase metabolic labeling experiments revealed that the soluble form is generated intracellularly at an early stage of biosynthesis, suggesting that the signal peptide/membrane anchor domain of aminopeptidase A is removed in the endoplasmic reticulum through the action of the signal peptidase.     

In vitro utilization of exogenous procollagen by embryonic tooth germs

Embryonic mouse tooth germs treated with L-azetidine-2-carboxylic acid cease their development, undergo a regression of the enamel organ, and do not maintain the histological characteristics of the explanted dental organ. On the other hand if procollagen is added exogenously to explants continously treated with L-azetidine, the effects of the inhibitor are not seen and the tissue is maintained. Thus, exogenously supplied procollagen supports morphogenesis in tooth rudiments that are unable to synthesize procollagen.     

Effect of ascorbic acid deficiency on mouse second molar tooth germs cultivated in vitro

Mandibular second molar tooth germs from two-day old mice were cultured in vitro, on millipore membranes, for periods of up to 20 days in liquid medium with or without added ascorbic acid. Tooth germs grown in ascorbate medium were characterized by relatively normal growth, differentiation, morphology and histology. Cuspation patterns were maintained. The epithelial root sheath continued to grow along the millipore membrane. Tooth germs cultured in ascorbate-deficient medium manifested a consistent and striking failure in maintenance of differentiated odontoblastic and ameloblastic tissue with arrest of predentin synthesis, severe structural collapse and reduction in size. Cuspation patterns were lost in scorbutic molars, with sinking of surface layers into pulpal tissue and flattening of the entire organ. This resulted in a lack of recognizable morphology and in severe disorganization of tissues. Only growing areas of the root sheath with associated proliferation of preameloblasts and pre-odontoblasts and adjacent pulpal tissue remained normal and refractory to ascorbate deficiency. Odontoblastic as well as ameloblastic layers were disrupted and cells were dedifferentiated. Newly differentiated odontoblasts became highly vacuolated when they became polarized and started to secrete extracellular matrix.     

Ameloblast-lineage cells of rat tooth germs proliferate and scatter in response to hepatocyte growth factor in culture

Hepatocyte growth factor (HGF) is considered to be one of the mediators of epithelial-mesenchymal interactions during early organogenesis and to be involved in the development of murine molars. In this study, the immunohistochemical localization of HGF and of its receptor, c-Met, revealed that HGF was distributed in the proliferating mesenchymal cells in the dental papillae and that c-Met was continuously expressed in the epithelial cells during the development of rat incisors. These observations confirmed the involvement of HGF in the development of rat incisors, as demonstrated previously in molars. We then used a primary culture of ameloblast-lineage cells, prepared from mandibular incisors of young rats, to examine the direct effects of HGF on the growth and differentiation of ameloblasts. We found that HGF at 2-20 ng/ml induced a marked increase in the number of ameloblast-lineage cells and in the scattering of such cells. Our results suggest that HGF promotes the proliferation and scattering of ameloblast-lineage cells simultaneously.     

Biochemical and functional changes during the bovine fetal thyroid development

Despite improvements in critical care medicine and the development and aggressive use of potent broad-spectrum anti-microbial agents, mortality due to severe sepsis has not changed during the recent years and still comes to 35% to 45%. For quite a long time our understanding of the pathophysiology of sepsis was mainly focused on endotoxin and proinflammatory cytokines like tumor necrosis factor or interleukin-1. Now it is generally accepted that many signs and symptoms of sepsis are not directly mediated by cytokines but are transmitted through other mediator systems. The coagulation system comes into play especially when the septic process progresses to malperfusion and organ failure. Antithrombin III is an important inhibitor of the intrinsic, extrinsic and common pathway of coagulation. Recently, evidence has been accumulating that there is an additional anti-inflammatory potential of the drug. Currently there are several clinical trials ongoing to investigate whether this effect is of clinical relevance in the treatment of patients with severe sepsis.     

Structural changes in the intramuscular connective tissue during development of bovine semitendinosus muscle

Structural changes in the intramuscular connective tissue during development of bovine semitendinosus muscle were investigated using the cell-maceration method for scanning electron microscopy, by which cellular elements were eliminated and collagen fibrils and fibres were exposed. The endomysium was discontinuous and showed various shapes and sizes in the muscle of 7-month fetuses. The perimysium consisted of collagen fibres in loose contact with each other. In the muscle of neonatal calves, the endomysium consisted of cylindrical sheaths and displayed a honeycomb structure, and the perimysium was composed of several layers of collagen fibres. Collagen fibrils in the endomysium bound ever more closely with each other, and collagen fibres in the perimysium increased in thickness, and the wavy pattern of collagen fibres became more regular with growth of cattle. We have examined the mechanical strength of the intramuscular connective tissue by our new method, 'intramuscular connective tissue (IMCT) model'. The IMCT model is composed of collagen fibrils and fibres which maintains the organization in the endomysium and perimysium in situ. The shear-force value of the model increased rapidly from the 7th fetal month to the neonatal stage, and increased linearly with postnatal ageing thereafter. Changes in the arrangement of collagen fibrils and fibres seem to closely related to an increase in the mechanical strength of the intramuscular connective tissue during development of bovine skeletal muscle.     

Differential expression of the estrogen-regulated proto-oncogenes c-fos, c-jun, and bcl-2 and of the tumor-suppressor p53 gene in the male mouse chronically infected with Taenia crassiceps cysticerci

Chronic infection with Taenia crassiceps cysticerci produces a 200-fold increase in serum estradiol levels in male mice. The aim of this study was to investigate the expression pattern of c-fos and c-jun, two estradiol-regulated genes, as well as that of p53 and bcl2 in the testes, spleen, and thymus of male mice infected with T. crassiceps cysticerci. In parasitized animals the c-fos mRNA content was significantly increased in all tissues studied, whereas the c-jun mRNA content was increased only in the thymus. The p53 mRNA content was markedly reduced in all tissues of the parasitized animals analyzed, whereas bcl-2 gene expression was abolished in the thymus. On the other hand, thymic cell analysis performed by flow cytometry showed a diminution in the content of CD3+, CD4+, and CD8+ subpopulations in the parasitized mice. Our results suggest that the increase in estradiol levels of the host should change the expression pattern of several genes that participate in apoptosis regulation in the thymus of male mice during chronic infection with T. crassiceps cysticerci.     

Localization of estrogen-receptor-related antigen in human odontoblasts

Estrogen receptors have been demonstrated in many osteogenic cell lines. Recently, we showed that estrogen deficiency induced by ovariectomy caused enhanced dentin formation in adult rats, suggesting that estrogen receptors may be present in dental tissues. Nothing is known about estrogen receptors in human teeth. We used immunohistochemical staining and immuno-blotting to demonstrate the presence of estrogen receptors in human pulp and/or the pulpo-dentinal border. Unerupted human wisdom teeth were surgically removed, frozen in liquid nitrogen, and prepared for immunological studies. Western blot analysis with monoclonal antibodies specific for human estrogen-receptor-related antigens demonstrated an approximately 29-kDa clear double band in the material scraped from the predentin-odontoblast border and in the fluid that emerged into the pulpal chamber, evidently from the odontoblasts. A weaker double band was also present in pulpal tissue samples. By immunohistochemical staining, estrogen-receptor-related antigens were visualized in the predentinal-odontoblast region and in the pulpal blood vessels. Our results suggest the presence of estrogen receptors in human teeth, and thus the previously reported enhancement of the dentin formation in rats after ovariectomy may be mediated via these receptors.     

Development and cell fate in interspecific (Mus musculus/Mus caroli) orthotopic transplants of mouse molar tooth germs detected by in situ hybridization

Interpretation of results from previous tooth germ transplantation studies is limited by the inability to distinguish between donor and host cells unequivocally. Furthermore, ectopic transplantation sites have generally been used and the relevance of this to tooth development in situ is uncertain. The aim here was to determine cell fate in orthotopic tooth germ transplants using an interspecific mouse marker system. Mandibular first molar tooth germs were dissected from Mus musculus (CD1) and Mus caroli mice (age range 15-19 day embryo) and transplanted interspecifically into the alveolar crypt of extirpated first mandibular molars in neonatal M. musculus (CD1) and M. caroli hosts. Grafts were recovered at intervals up to 4 weeks postoperatively. Paraffin wax-embedded sections were examined using routine histological techniques and in situ hybridization with a biotinylated DNA probe (pmSat5) specific for M. musculus, to distinguish between donor and host cells. Development of M. musculus tooth germs in M. caroli mandibles and vice versa was similar and transplants progressed to incipient root formation. Vascularization of transplants was chimaeric, being donor-derived in the pulp and host-derived more peripherally. The investing soft tissues comprised a mixture of donor and host cells, predominantly donor. Donor cells were also found in the soft tissue of intertrabecular spaces in the surrounding bone, but alveolar osteocytes were almost entirely host-derived. Long-term survival of grafts was limited and few donor cells were present after 2 weeks. This study provides an unequivocal demonstration of the origin of all cells present in transplanted tooth germs.     

Quantitative detection of amplification of proto-oncogenes in breast cancer

In the present study, dot-blot hybridization, serial dilution analysis and densitometric scanning were used to detect amplification of proto-oncogenes including c-erbB2, c-myc, int-2 and c-Ha-ras in 101 paraffin-embedded breast cancers. Expression of c-erbB2 was also examined by immunohistochemistry. Amplification of c-erbB2, c-myc and int-2 genes was found in 34.7%, 17.8% and 11.9% of breast cancers respectively. However amplification of c-Ha-ras was not detected in all cases. In 11.9% of cases co-amplification of two or more oncogenes was observed. Positive immunostaining of c-erbB2 was seen in 23.8% of the cases and it was significantly associated, but not always corresponding to the amplification of the gene. There was no difference between primary and metastatic breast cancer in the alterations of proto-oncogenes examined in this study, which suggested that the amplification and overexpression of these proto-oncogenes occurred prior to and maintained in the process of metastasis of breast cancer. Statistical analysis showed that high-scale of immunopositive staining of c-erbB2 and high-fold co-amplification of proto-oncogenes were significantly correlated with large size of the tumour and the number of involved lymph nodes. Our results indicate that the alterations of multiple oncogenes are involved in the development of breast cancer and some of them may have prognostic importance for breast cancer patients.     

Doxorubicin-induced alterations of c-myc and c-jun gene expression in rat glioblastoma cells: role of c-jun in drug resistance and cell death

We studied the effect of doxorubicin on the expression of c-myc and c-jun in the rat glioblastoma cell line C6 and its doxorubicin-resistant variant C6 0.5, at equitoxic exposures. For quantitation, the mRNA levels of these oncogenes were related to those of two domestic genes, beta-actin and glyceraldehyde phosphate dehydrogenase. After a transient overexpression of the genes during the first hour of incubation, there was a selective, dose-dependent down-regulation of both genes by doxorubicin in the sensitive cells. In the resistant cell line, c-myc expression was also decreased in response to doxorubicin incubation, but the expression of c-jun remained unchanged over the whole range of concentrations. In contrast, vincristine had no effect on the amounts of c-myc and c-jun mRNAs in either line. The effect of doxorubicin on the mRNA levels of c-jun was also observed on the JUN proteins by immunoblotting, but the MYC protein levels remained unchanged upon doxorubicin treatment. There was a significant correlation between the levels of c-myc and c-jun gene expression and the degree of growth inhibition induced by doxorubicin. In addition, doxorubicin induced a fragmentation of DNA in sensitive cells, but not in resistant cells, thus revealing a resistance to apoptosis in this line. Doxorubicin-induced cell death did not appear to be mediated by p53 in either cell line.     

Expression of E- and P-cadherin during tooth morphogenesis and cytodifferentiation of ameloblasts

The mechanism of hyposensitization in bronchial asthma has not been fully elucidated. We established a hyposensitization model of bronchial asthma in rats and examined airway responses and immunological parameters. Brown Norway rats were sensitized by a subcutaneous injection of ovalbumin (OA) at day 1 and by the inhalation of 2% OA aerosol at day 15. Animals were hyposensitized by intraperitoneal injections of OA from day 17 to day 22. They were challenged with OA or acethylcholine (Ach) aerosol at day 23 and changes in intratracheal pressure were recorded. Lungs were lavaged and OA-induced proliferative responses by blood lymphocytes were examined for animals without aerosol challenge at day 23. OA-specific serum IgE levels were measured by enzyme-linked immunosorbent assay. Hyposensitization significantly reduced the OA-induced immediate airway response, accumulation of CD4+ lymphocytes and eosinophils recovered by bronchoalveolar lavage, and the OA-induced proliferative response by blood lymphocytes. The airway responses to Ach and serum OA-specific IgE levels in hyposensitized group were not significantly different from those in the sensitized group. These results indicate that amelioration of airway inflammation and hyporesponsiveness of lymphocytes against OA are involved in the attenuated immediate antigen-induced airway response following hyposensitization.     

Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium

The effects of NG-nitro-L-arginine (NOLAG), an inhibitor of nitric oxide synthase (NOS), and of indomethacin, an inhibitor of cyclooxygenase, on the rise in cerebral blood flow (CBF) accompanying increasing levels of hypercapnia (paCO2 = 40-135 mmHg) were studied in anesthetized rats. CBF was measured by intracarotid injection of 133Xe. Progressive increases in paCO2 of 10 mmHg, at intervals of about 8-10 minutes, were associated with gradual increases in CBF until a paCO2 level of 115 mmHg was reached. No further CBF changes (from the maximum value of 446 +/- 70 ml 100 g-1 min-1) were seen with additional step increase in paCO2. Intracarotid infusion of 7.5 mg/kg NOLAG significantly attenuated the CO2-elicited CBF increase by about 45-65% at paCO2 values below 115 mmHg. Beyond this level, there was a lesser inhibition of about 27-35%. 30 mg/kg NOLAG had essentially the same effect as 7.5 mg/kg NOLAG. 50 mg/kg NOLAG, given intraperitoneally (i.p.) twice daily for 4 days, also caused an attenuated CBF response to CO2, but the inhibitory effect was significantly less than with acute NOLAG administration in the paCO2 range of 61-90 mmHg. Infusion of L-arginine, 1 g/kg/h, prevented the effect of 7.5 mg/kg NOLAG. Indomethacin, 10 mg/kg, i.v. produced a more dramatic attenuation of the response, to the extent that the steady rising curve of CBF as a function of paCO2 was almost completely abolished. With indomethacin, a moderate increase (50%) in CBF was seen at the lowest level of hypercapnia, but raising paCO2 above this level did not result in further increases in CBF. This effect could not be prevented by L-arginine. When combining 7.5 mg/kg NOLAG with 10 mg/kg indomethacin, the response to hypercapnia was totally blocked. The results suggest that NOLAG and indomethacin act through different mechanisms on the hypercapnic CBF response, and that indomethacin is the more powerful inhibitor.     

Two proto-oncogenes that play dual roles in embryonal cell growth and differentiation

BACKGROUND AND PURPOSE: Clinical and experimental data indicate that hyperglycemia can aggravate the consequences of stroke and cerebral ischemia. The purpose of this study was to examine the effects of moderate hyperglycemia on the response of the blood-brain barrier to normothermic (37 degrees C) and hypothermic (30 degrees C) global forebrain ischemia. METHODS: Sixteen rats underwent 20 minutes of four-vessel occlusion followed by 30 minutes of postischemic recirculation. We used the protein tracer horseradish peroxidase as an indicator of increased vascular permeability, and rats were perfusion-fixed for microscopic analysis. To produce moderate hyperglycemia, we gave an intraperitoneal injection of 50% dextrose 15 minutes before the ischemic insult. RESULTS: After normothermic brain ischemia, normoglycemic rats (plasma glucose level, 115 +/- 3 mg/dl) demonstrated extravasated horseradish peroxidase mainly restricted to the cerebral cortex. In contrast, more severe and widespread protein extravasation was documented throughout the neuraxis of hyperglycemic (plasma glucose level, 342 +/- 27) rats. Sites of protein leakage included the cerebral cortex, striatum, hippocampus, thalamus, and cerebellum. Foci of protein extravasation were associated with pial and large penetrating vessels. Intraischemic hypothermia significantly attenuated the blood-brain barrier consequences of hyperglycemic brain ischemia. CONCLUSIONS: Under normothermic ischemic conditions, hyperglycemia significantly worsens the degree of acute blood-brain barrier breakdown compared with normoglycemia. Postischemic blood-brain barrier disruption may play an important role in the pathogenesis of increased brain damage associated with systemic hyperglycemia.