Developmentally regulated expression of the calcitonin gene related peptide (CGRP) in rat lung endocrine cells

Calcitonin (CT) and the calcitonin gene-related peptide (CGRP) are generated by alternative RNA processing from a single CT/CGRP gene. Recently, we reported the existence of CGRP-immunoreactivity and CGRP mRNA in endocrine cells or Kulchitsky (K) cells of human and rat lung [Wada et al. 1987b]. In this report, an examination was made of developmental changes in the expression of the CGRP gene in rat lungs by immunohistochemistry, radioimmunoassay (RIA) and Northern hybridization. CGRP-positive K-cells in lung tissue appeared on the 18th day of gestation. Their number was greatest on the 20th day of gestation and then decreased postnatally. The level of CGRP in rat lung was found to be highest in a 1-day-old neonate by RIA. In the Northern hybridization of rat lung using the CGRP 3' non-coding region (exon 6) of the first human CT/CGRP gene as the probe, 1.0 kilobase (kb) CGRP mRNA was found to be abundant on the 20th day of gestation and in a 1 day-old neonate. It thus appears that CGRP in rat lung is essential for pulmonary adaptation at birth and/or from the last intrauterine stage to the early neonatal period.     

Apparent 'glucokinase' activity in non-hepatic tissues due to N-acetyl-D-glucosamine kinase

1. Electrophoretic examination of tissue extracts from rat intestinal mucosa, kidney, lung, spleen, mammary gland, adipose tissue, heart muscle and placenta in agarose gels did not reveal the presence of any glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.2) activity corresponding to that present in rat liver. 2. All these tissues do contain an enzyme that possesses very high-Km glucose-phosphorylating activity but which has a slightly lower electrophoretic mobility than glucokinase and can be separated from it by various means. 3. This phosphotransferase activity is due to N-acetyl-D-glucosamine kinase (ATP:2-acetamido-2-deoxy-D-glucose 6-phosphotransferase, EC 2.7.1.59), which has been partialyy purified from intestinal mucosa tissue and shown to have similar kinetic properties to the same enzyme previously purified more extensively from liver and kidney. 4. It is suggested that many of the effects reported in the literature of 'glucokinase' activity in non-hepatic tissues are probably due to N-acetyl-D-glucosamine kinase.     

Expressions of sulfated glycoprotein 2 and pSvr-1 genes and involution of steroid hormone-dependent rat tissues.

To further survey the molecular mechanisms underlying the involution of steroid hormone-dependent rat tissues, we undertook experiments to test whether or not any significant correlation between the tissue involution and expressions of rat sulfated glycoprotein 2 (SGP-2) and pSvr-1 genes, which had been initially cloned from the Sertoli cells and the seminal vesicles, respectively, and then identified as androgen repressed messages both in the ventral prostate and in the seminal vesicles, could be observed in steroid hormone-dependent rat tissues. Expressions of these genes were stimulated within 48 h after castration of animals both in the ventral prostate and in the seminal vesicles as reported previously, but not significantly altered by ovariectomy in the uterus. Expressions of these genes in the thymus were significantly repressed by the administration of dexamethasone and/or cycloheximide. Although the roles of expressions of SGP-2 and pSvr-1 genes in steroid hormone-dependent tissues remain unclear, their presence might become useful molecular markers of tissue involution not only in androgen-dependent rat tissues but also in glucocorticoid-dependent ones, and also provide excellent model systems for the study of negative regulation mechanism of gene expression by steroid hormones.     

Thin bio-artificial tissues in plane stress: the relationship between cell and tissue strain, and an improved constitutive model

Constitutive models are needed to relate the active and passive mechanical properties of cells to the overall mechanical response of bio-artificial tissues. The Zahalak model attempts to explicitly describe this link for a class of bio-artificial tissues. A fundamental assumption made by Zahalak is that cells stretch in perfect registry with a tissue. We show this assumption to be valid only for special cases, and we correct the Zahalak model accordingly. We focus on short-term and very long-term behavior, and therefore consider tissue constituents that are linear in their loading response (although not necessarily linear in unloading). In such cases, the average strain in a cell is related to the macroscopic tissue strain by a scalar we call the "strain factor". We incorporate a model predicting the strain factor into the Zahalak model, and then reinterpret experiments reported by Zahalak and co-workers to determine the in situ stiffness of cells in a tissue construct. We find that, without the modification in this article, the Zahalak model can underpredict cell stiffness by an order of magnitude.     

An autoradiographic study of the role of satellite cells and myonuclei during myogenesis in vitro

Satellite cells and myonuclei of neonatal rat muscles were differentially labeled with 3H-thymidine according to the procedure of Moss and Leblond (1971). Minced muscles fragments containing either labeled satellite cells or labeled myonuclei were cultured until multinucleated myotubes grew out from the explants. Reutilzation of isotope released from degenerating nuclei was competitively inhibited by using a culture medium containing excess (0.32-0.41 mM) cold thymidine. after an 8-10 day growth period, the explants were fixed and prepared for autoradiographic (ARG) examination to determine whether labeled satellite cells or myonuclei had contributed to the myonuclear population of the developing myotubes. Counts were made of the number of labeled myotubes in the explants and compared with the number of labeled satellite cells and myonuclei in samples of the original muscle tissues fixed at the time of explantation. The original muscles showed a mean satellite cell labeling index of 51.7% and gave rise to myotubes with a mean labeling incidence of 40%. In contrast, myonuclear labeling in the original muscle tissues showed no correlation with subsequent myotube labeling. Only 3.4% myotube labeling was found in explants in which over 30% of the original tissue myonuclei had been labeled. Under conditions controlled for isotope reutilization, these observations confirm results of in vivo ARG studies indicating that satellite cells are the only significant source of regenerating myoblasts in injured muscle tissue.     

盆腔结缔组织炎大鼠模型的层黏连蛋白表达

目的建立混合菌液致SD大鼠盆腔结缔组织炎模型,研究大鼠盆腔黏连组织的病理学改变与层黏连蛋白表达。方法用注射器将细菌混悬液0．5mL,注人大鼠盆腔,并结合子宫穿孔手术,建立SD大鼠盆腔结缔组织炎模型。采用Philips分级评分法对大鼠盆腔黏连程度分级评分,切取黏连组织,用多聚甲醛固定,进行病理检测,并采用免疫组化法测定层黏连蛋白表达情况。结果模型组大鼠盆腔黏连Philips分级评分以Ⅲ级为主,与正常对照组比较差异极显著。HE染色镜检模型组可见大量炎细胞浸润,脓肿形成,伴有多少不等的纤维组织增生。免疫组化染色镜检模型组可见层黏连蛋白高表达,纤维结缔组织增生程度越高,层黏连蛋白表达越多。结论盆腔注射混合菌液并结合子宫穿孔手术,可作为建立大鼠盆腔结缔组织炎模型的方法。层黏连蛋白在盆腔结缔组织炎发病中起重要作用,参与整个炎症过程并维持盆腔黏连组织纤维化,可作为判断盆腔结缔组织炎的炎症和纤维化程度的参考指标。     

Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy

Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM.     

Electromechanical integration of cardiomyocytes derived from human embryonic stem cells

Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.     

Evidence from immunoblotting studies on uncoupling protein that brown adipose tissue is not present in the domestic pig

Adipose tissues and other tissues of the pig have been examined for the presence of the mitochondrial "uncoupling protein," characteristic of brown adipose tissue, in order to assess whether brown fat is present in this species. Mitochondria were prepared from various tissues and the proteins separated on the basis of molecular weight by sodium dodecyl sulphate--polyacrylamide gel electrophoresis. Immunoblotting procedures were then used to probe for uncoupling protein, employing a rabbit anti-(rat uncoupling protein) serum. Pigs were examined at 4 days, 4 weeks, and 8 weeks of age. No evidence for the presence of uncoupling protein was found at any of these ages. The protein was, however, readily detected in brown adipose tissue from rats, mice, golden hamsters, guinea pigs, Richardson's ground squirrel, and lambs. An additional group of pigs was acclimated to the cold (10 degrees C) for a period of 10 days prior to the examination of tissues, but again uncoupling protein was not detected in any tissue. These results indicate that uncoupling protein is either absent from adipose tissues of the pig or is present at such a low concentration that it is unlikely to support thermogenesis. It is concluded that the pig does not contain adipose tissue that is functionally "brown;" adipose tissues in this species appear to be exclusively "white."     

Transmission and scanning electron microscopy of cell monolayers grown on polymethylpentene coverslips.

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized, separated from the coverslip by liquid nitrogen immersion, and sectioned either "en face" or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.     

Transmission and Scanning Electron Microscopy of Cell Monolayers Grown on Polymethylpentene Coverslips

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized. separated from the coverslip by liquid nitrogen immersion, and sectioned either “en face” or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.     

Cloning and sequencing of a rat CuZn superoxide dismutase cDNA. Correlation between CuZn superoxide dismutase mRNA level and enzyme activity in rat and mouse tissues

The molecular cloning and nucleotide sequence of a cDNA clone (pR SOD) for rat CuZn superoxide dismutase (CuZnSOD) is reported. Nucleotide sequence homology with human superoxide dismutase is 86% for the coding region and 71% for the 3' untranslated region. The deduced amino acid sequence is given and the homologies with the sequences reported for other species are presented. Northern blot analysis of total RNA from various rat and mouse tissues and from two mouse cell lines show that pR SOD hybridizes with one mRNA species of about 0.7 kb. The amount of CuZnSOD mRNA in each tissue, measured by densitometry of the Northern blot autoradiograms, correlates with the enzymatic activity based on protein content. These results indicate that the control of CuZnSOD activity in mammalian tissues is largely dependent on the regulation of CuZnSOD mRNA levels. In human liver, fibroblasts and FG2 hepatoma cells, two CuZnSOD mRNAs (0.7 kb and 0.9 kb) are observed. The level of CuZnSOD mRNA in FG2 is 25% that of the liver and four times more abundant than in fibroblasts.     

Calcium-mediated breakdown of glial filaments and neurofilaments in rat optic nerve and spinal cord

Disruptive effects of calcium upon neurofilaments and glial filaments were studied in white matter of rat optic nerve and spinal cord and in rat peripheral nerve. Filament ultrastructure and tissue protein composition were compared following a calcium influx into excised tissues. A calcium influx was induced by freeze-thawing tissues in media containing calcium (5 mM) while control tissues were freeze-thawed in the presence of EGTA (5 mM). Experimental and control tissues were either fixed by immersion in glutaraldehyde and processed for electron microscopic examination or homogenized in a solubilizing buffer and analyzed for protein content by SDS-polyacrylamide gel electrophoresis. Morphological studies showed that calcium influxes led to the loss of neurofilaments and glial filaments and to their replacement by an amorphous granular material. These morphological changes were accompanied by the loss of neurofilament triplet proteins and glial fibrillary acidic (GFA) protein from whole-tissue homogenates. In addition, a calcium-sensitive 58,000-mol-wt protein was identified in rat optic and peripheral nerve. The findings indicate the widespread occurrence of neurofilament proteolysis following calcium influxes into CNS and PNS tissues. The parallel breakdown of glial filaments and loss of GFA protein subunits suggest the presence of additional calcium-activated proteases(s) in astroglial cells.     

Reconstitution of the malignant phenotype of genitourinary cancer in gradient culture

Summary The diagnosis of cancer is made on histologic examination by recognizing the characteristics of the malignant phenotype, i.e. abnormal cells in abnormal groupings, often in abnormal locations. Histophysiologic gradient culture reconstitutes conditions that meet the spatial imperatives of tissues in nature. A variety of carcinomas arise in the genitourinary system involving both glandular and stratified epithelium. To be considered here are the contrasting polarizations of proliferation of normal and neoplastic rat urothelium, the continuity of sheets of epithelium in nature, the poorly understood stable and unstable interepithelial boundaries, and the formation of organoid endocrinelike tissue in histophysiologic gradient culture of normal human amnion epithelium. This work was supported by research grant CA-14137 from the National Cancer Institute, Bethesda, MD, grant 1793 from the Council for Tobacco Research, and grants from the American Fund for Alternatives to Animal Research.     

Dissociation of single cells from lung or kidney tissue with elastase

Summary Experiments were conducted to determine the capacity of various enzyme preparations to dissociate single cells from guinea pig lung tissue. The number of cells separated from tissue progressively increased as the concentration of crude trypsin was increased from 25 to 250 mg per 100 ml. This action could be inhibited by soy bean trypsin inhibitor. Elastase, but not ethylenediaminetetraacetate (disodium salt), crystalline trypsin, nor chymotrypsin, dissociated cells from lung tissues. Crude trypsin (Trypsin 1∶300) was found to contain 3.0 Sachar units of elastase per mg. Elastase was also inhibited by soy bean trypsin inhibitor. Only some collagenase preparations dissociated cells from lung tissue. Impure bacterial proteases dissociated lung cells. Our data suggest that the term “trypsinization” to denote dissociation of cells from tissue with crude preparations of trypsin is misleading and should be discontinued. Partially supported bv Armour-Baldwin Laboratories and the National Institute of Health, Grant, AM 12919.     

Rapid preparation of uncoated biological specimens for scanning electron microscopy.

We have developed a relatively rapid glutaraldehyde-tannic acid (GTA) and osmium tetroxide (OsO4) fixation procedure which permits many types of uncoated biological specimens to be examined in the scanning electron microscope (SEM) at 20 kV without the occurrence of charging. Most specimens taken one day can be examined in the SEM the following afternoon. Types of specimens successfully treated were perfused adult and embryonic rat tissues, confluent human skin fibroblast tissue cultures, plant roots, flowers, seeds, some garden insects, and microcolonies of salivary streptococci. Cells in suspension and extracted human teeth did become electron conductive when treated with the GTA procedure. Most suspended cells must be centrifuged between each solution and the GTA procedure increases the preparation time for these cells. Extracted teeth are usually simply dried and coated. Therefore, the usual SEM preparation techniques are shorter and perhaps more useful for these types of specimens.     

Rapid Preparation of Uncoated Biological Specimens for Scanning Electron Microscopy

We have developed a relatively rapid glutarddehyde-tannic acid (GTA) and osmium tetroxide (OsO₄) fixation procedure which permits many types of uncoated biological specimens to he examined in the scanning electron microscope (SEM) at 40 kV without the occurrence of charging. Most specimens taken one day can be examined in the SEM the following afternoon. Types of specimens successfully treated were perfused adult and embryonic rat tissues, confluent human skin fibroblast tissue cultures, plant roots, flowers, seeds, some garden insects, and microcolonies of salivary streptococci. Cells in suspension and extracted human teeth did become electron conductive when treated with the GTA procedure. Most suspended cells must he centrifuged between each solution and the GTA procedure increases the preparation time for these cells. Extracted teeth are usually simply dried and coated. Therefore, the usual SEM preparation techniques are shorter and perhaps more useful for these types of specimens.     

Lipoprotein lipase in lungs, spleen, and liver: synthesis and distribution.

Lipoprotein lipase (LPL, E C 3.1.1.34) is the enzyme responsible for hydrolysis of triacylglycerols in plasma lipoproteins, making the fatty acids available for use by subjacent tissues. LPL is functional at the surface of endothelial cells, but it is not clear which cells synthesize the enzyme and what its distribution within tissues and vessels is. In previous studies we reported that in the major LPL-producing tissues (muscles, adipose tissue, and mammary gland) the enzyme is made by the major cell types. In the present work we have studied in adult guinea pigs some tissues that present LPL activity but in lower amounts (lung, spleen, and liver). On cryosections of these tissues we have searched for specific cell expression of the LPL gene (by in situ hybridization using a RNA probe) and for the corresponding protein distribution (by immunocytochemistry). Based on morphological criteria we can suggest that, contrary to the main LPL-producing tissues, in these tissues the enzyme is made by scattered cells, such as macrophages in the lung and spleen and Kupffer cells in the liver; endothelial cells present but do not synthesize the enzyme, indicating that the endothelial LPL originates in other cells. In the liver strong immunoreaction was detected in the sinusoid in contrast to the low level of mRNA expression, suggesting that liver takes up circulating LPL from blood.     

Protein Synthesis in Hybrid Cells Derived from Fetal Rat × Mouse Chimeric Organs

Abstract. Malignant hybrid cells (As3) derived from fusion of rat hepatoma cells (Fu5AH) with mouse teratocarcinoma cells (OTT6050) were injected into genetically marked mouse blastocysts which were subsequently transferred into pseudopregnant surrogate mothers. From a total of 61 fetuses developed, four normally differentiated fetuses at day 18 of gestation showed hybrid cell contributions in their livers and a few other organs of endo-mesodermal origin. The chimeric tissues were briefly cultured in vitro and then further investigated for their protein synthesis using two-dimensional gel electrophoresis. After comparison of the protein patterns obtained from the corresponding normal rat and mouse organs, several rat-specific polypeptides were detected in the cultured chimeric tissues illustrating functional xenogeneic gene expression during in situ differentiation. In addition, some other rat proteins characteristic of the parental hybrid cell line disappeared. The tumorigenicity of the chimeric tissues was tested by subcutaneous transplantation into immunodeficient nude mice. Tumors originating from two of the four chimeric organs differed histologically from those formed by cells of the hybrid As3 line since they also contained muscle-like structures resembling rhabdomyosarcomas. The tumors were analyzed for their protein synthesis and compared with the three malignant cell lines of parental origin. The morphologic differences between the tumors derived from the chimeric organs and those developed from the As3 cell line were also reflected in characteristic differences of their protein synthesis patterns. Our results demonstrate that interspecific rat × mouse hybrid cells, when implanted into early mouse embryos, participate in fetal tissue differentiation and selectively repress certain rat gene products typical of the malignant parental cells as well as functionally reactivate other rat genes presumably required for normal development.     

The presence of hepatocyte growth factor in the developing rat.

Hepatocyte growth factor (HGF), a heparin-binding polypeptide mitogen, stimulates DNA synthesis in adult rat and human hepatocytes and in several other cells of epithelial origin. Recently, it was determined that scatter factor (SF), a protein that has been shown to cause the dispersion and migration of epithelial cells in culture, is identical to HGF. Moreover, the receptor for HGF was identified as the product of the proto-oncogene, c-MET, a tyrosine kinase-containing transmembrane protein. c-MET expression has been reported in a variety of adult and embryonic mouse tissues. Similarly, we and others have demonstrated that HGF is expressed in various adult rat and human tissues. In the present study, the tissue distribution of HGF during rat development was determined by immunohistochemistry using an HGF-specific polyclonal antiserum. Between day 12 and day 19, immunoreactivity for HGF was present in various locations such as hematopoietic cells, somites, squamous epithelium of the esophagus and skin, periventricular germinal matrix of the brain, bronchial epithelium, renal collecting tubules and chondrocytes. After day 19, HGF immunoreactivity was also present in the pancreas, submaxillary glands and neural tissues. In addition to immunolocalizing HGF in tissue sections, bioreactive and immunoreactive HGF was extracted and purified from rat fetuses. Other studies demonstrated the presence of HGF and c-MET mRNA in total fetal rat, and in fetal and neonatal rat liver. Addition of purified HGF to fetal and neonatal rat liver cultures enriched for hepatocytes stimulated DNA synthesis up to six-fold over controls. These findings strongly suggest a pivotal role for this potent regulator of growth and development.