[Current findings about bone organic connective tissue and its regulation].
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Bone Development↗
Biomedical subjects
M Sandberg
Publications and source records attributed to M Sandberg.
Expression of epidermal growth factor and epidermal growth factor receptor genes in healing colonic anastomoses in rats.
This study uses complementary DNA probes to find out if epidermal growth factor (EGF) and EGF receptor genes are activated in healing colonic anastomoses in rats. 35 rats had a colocolonic anastomosis which was removed for study 1, 2, 3, 5, 7, or 14 days after operation. Six animals without surgery served as controls. The specimens were examined by northern blotting, in situ hybridisation, and conventional microscopy. Microscopic healing progressed normally. The expression of the EGF gene was minimal and surgery did not activate it. However, surgical trauma did increase the expression of the EGF receptor gene by 2.2 times when compared to day one. In situ hybridisations localised a strong EGF receptor expression in mucosal epithelial cells in all specimens, and a moderate reaction in fibroblasts in the repair tissue in the anastomotic line. The enhanced EGF receptor gene expression suggests that anastomotic healing is associated with the presence of EGF or an EGF-like substance and its activity increases during the first postoperative week.
Anastomosis, Surgical↗
Localization of type I and III collagen and fibronectin production in injured gastrocnemius muscle.
The healing of muscle rupture consists of two simultaneous processes, regeneration of disrupted muscle fibers and production of connective tissue scar. These two processes are at the same time supportive to and competitive with each other. Their balanced progression is necessary for optimal healing. Synthesis of three connective tissue proteins, collagen types I and III and fibronectin, was analyzed during the regeneration process from 2 days to 3 weeks by Northern blot and in situ hybridization and immunohistochemistry. For this purpose a partial standard rupture of the gastrocnemius muscle was induced in 56 rats by a strike with a blunt spring-loaded hammer. Northern blot analysis of the specific mRNAs during the healing process revealed distinctly different expression patterns for fibronection and type I and III collagens. During the early stages (days 2 and 3) fibronectin, derived mainly from plasma, was abundant in the traumatized area, but local production of fibronectin mRNA by fibroblasts had also already started by day 2, closely followed by that of type III collagen. This early active synthesis of type III collagen and fibronectin was followed by a decrease after 1 week. The production of type I collagen mRNAs was activated somewhat later and remained elevated for at least 3 weeks. The muscle cells did not contain procollagen mRNAs. The observed sequence of connective tissue proteins reflects the particular function that each carries out during muscle wound healing (e.g., fibronectin in fibroblast trapping, type III collagen in plasticity/flexibility, and type I collagen in tensile strength).
Animals↗
Cytoskeletal gene expression in normal and neoplastic human odontogenic epithelia.
In situ and Northern hybridization was carried out to study cytokeratin (Ck) 1, 4, 8, 18, and 19 and vimentin (Vim) gene expression in 13- to 24-week-old human fetal tooth germs, including overlying oral epithelium and odontogenic tumors (N = 6) of epithelial (ameloblastoma) and epithelial-ectomesenchymal (ameloblastic fibroma) origin. The results were compared with immunocytochemistry using monoclonal antibodies. A relatively strong expression of simple epithelial Ck 19 mRNA, together with low, but significant expression of Ck 8 and 18 mRNAs, was demonstrated in all normal and neoplastic odontogenic epithelia studied. Transcripts for squamous differentiation marker, Ck 4, and for terminal differentiation marker, Ck 1, were detected suprabasally in the fetal oral epithelium, focally in the dental lamina but not in the enamel organ. Ck 4 mRNA was expressed variably in most odontogenic tumors studied, whereas Ck 1 mRNA was detected in one ameloblastoma only. Vim mRNA was not found in the fetal oral epithelia, dental lamina or the enamel organ, but a distinct immunoreactivity with monoclonal antibodies to Vim was seen in the stellate reticulum cells of the enamel organ. The epithelium of most ameloblastomas showed a focal Vim mRNA and polypeptide expression. In addition to Vim, the neoplastic ectomesenchymal cells of ameloblastic fibroma coexpressed low amounts of simple epithelial Cks 8, 18, and 19. The results indicate that the differentiation and cytoskeletal gene expression programs of odontogenic epithelia upon neoplastic transformation are not fully retained. Most ameloblastomas and ameloblastic fibromas show differentiation parameters reminiscent of dental lamina. Ameloblastomas seem to form a heterogenous group of tumors, which may originate from odontogenic epithelial cells at various differentiation levels. The origin of ameloblastic fibroma is more closely related to the tooth germ proper.
Ameloblastoma↗
Collagen biosynthesis in lichen sclerosus et atrophicus studied by biochemical and in situ hybridization techniques.
Collagen was studied by biochemical and immunohistochemical techniques and by in situ hybridization in four patients with lichen sclerosus et atrophicus (LSA). The solubility of collagen in acetic acid and pepsin was increased in the lesional skin of the LSA patients when compared with their non-affected skin or samples from control subjects, indicating that a marked proportion of the collagen was newly synthesized. Collagen synthesis was slightly increased in fibroblasts derived from the lesional skin of the LSA patients. In situ hybridization with human sequence-specific cDNAs to type I procollagen demonstrated active fibroblasts in lesional skin of LSA patients, further confirming a high level of collagen synthesis in LSA. Remarkably, active fibroblasts were not associated with inflammatory cell infiltrates noted in LSA skin nor did they express transforming growth factor beta(TGF beta 1). Our results indicate that despite the degeneration of connective tissue, there is an active regeneration process in LSA with significant collagen synthesis.
Aged↗
Synthesis of laminin and type IV collagen by trophoblastic cells and fibroblastic stromal cells in the early human placenta.
In situ hybridization was used on both routinely processed paraffin embedded tissue sections and cryosections to study the synthesis of the basement membrane (BM) proteins laminin and type IV collagen in early human placentas from 8 to 11 weeks of gestation. Complementary DNA and RNA probes coding for the pro alpha 1 (IV) chain of human type IV collagen and the B1 chain of human laminin were used to detect respective mRNA. The clusters of cytotrophoblastic cells (i.e., cytotrophoblastic cell columns) contained cells that expressed both laminin and type IV collagen mRNA. Nearly all the stromal cells of developing villi also expressed mRNA for these proteins, type IV collagen mRNA levels being more pronounced than those of laminin. Conversely, the cytotrophoblastic cells of the villous trophoblastic epithelium synthesized mRNA for laminin more actively than that for type IV collagen. Decidual cells, endometrial stromal cells, and cells in the wall of spiral arteries all expressed mRNA for both laminin and type IV collagen. The results of in situ hybridization were found to correlate well with immunohistochemical stainings with specific antibodies to laminin and type IV collagen. The trophoblastic cells of the columns and the stromal fibroblasts are shown to contribute significantly to BM protein synthesis in the developing human placenta. The abundance of BM proteins may indicate their significance in guiding tissue organization in the placenta.
Antisense Elements (Genetics)↗
The neurotoxicity of zinc in the rat hippocampus.
Intrahippocampal injections of zinc chloride (5-10 nmol) caused a discrete lesion in the rat hippocampus, involving all neuronal perikarya. In addition to the necrosis, the lesion was also characterized by a decrease in staining of the neuropil, the presence of pyknotic neurons, and occasionally infarction. Pathological changes occurred within 8 h of an injection, and neuronal loss, as judged by the loss of Nissl staining, was complete within 24 h. On the other hand, the loss of acidophilic staining of the neurons was more gradual, as acid fuchsin staining was still present in neurons in the periphery of the damaged area 4 days later. In comparison with an excitotoxic lesion, glial infiltration into the damaged area was minimal, even up to 3 weeks later, suggesting that some glial cell toxicity also occurred.
Animals↗
The neurotoxicity of ouabain, a sodium-potassium ATPase inhibitor, in the rat hippocampus.
Intrahippocampal injection of 1 nmol ouabain, a sodium/potassium (Na+,K(+)-)ATPase inhibitor, produced a necrotic lesion within 4 days, characterised by a massive invasion by foaming macrophages. A lower dose of ouabain (0.1 nmol) produced a more discrete lesion of all groups of neuronal perikarya in the hippocampus, with only a minimal degree of glial infiltration. The neuronal perikaryal death produced in the subicular, CA1 and CA2 regions was only partially decreased by intraperitoneal injections of the anticonvulsants diazepam and MK-801; these drugs were without effect in the CA3 or hilar interneuronal regions. At neither dose of ouabain was there any indication of neuronal loss in brain regions outside the hippocampus, typically produced by prolonged seizure activity. It is suggested that ouabain has a two-fold action, a release of toxic acidic amino acids and a prolonged depolarization of neurons leading to osmolysis or calcium necrosis.
Animals↗
Bursectomy of chicken embryos at 60 hours of incubation leads to an oligoclonal B cell compartment and restricted Ig diversity.
Chickens that have been surgically bursectomized at 60 h of embryonic development usually generate Ig producing B cells; however, the bursectomized chickens are incapable of specific antibody responses, even after repeated immunization. In the present work, we analyzed the molecular basis of this immunodeficiency. In the bursectomized chickens, DNA sequencing revealed a repertoire of Ig L and H chains with a low number of different V-J and V-D-J joints, indicating an oligoclonal B cell compartment. In addition, the L and H chains belonging to each B cell clone had similar gene conversion events in the V region. In situ hybridization to Harderian gland tissue sections showed, that B cells of the bursectomized chickens were, however, capable of terminal plasma cell maturation. Thus, in chickens that were lacking the bursal microenvironment, 1) only a few B cell precursors differentiated into mature Ig-producing B cells, 2) low rate of gene conversion resulted in restricted Ig diversity. Regarding the chicken B cell differentiation, the present data support a model that the induction of B cell differentiation is a bursa-independent event, whereas the bursa of Fabricius has a crucial role in the amplification and diversification of the embryonic B cell repertoire.
Amino Acid Sequence↗
[An international case-control study. Aplastic anemia is an unusual disease previously overdiagnosed].
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Adolescent↗
The two mRNA forms for the type I alpha regulatory subunit of cAMP-dependent protein kinase from human testis are due to the use of different polyadenylation site signals.
Using a cDNA probe for the type I alpha regulatory subunit, two mRNA species (1.5 and 3.0 kb in length) were detected in human testis. From a human testis cDNA-library a 3.0 kb clone, containing the entire reading frame of the protein, was isolated. Comparison of the nucleotide sequence of this clone to the sequence of a 1.5 kb cDNA clone earlier reported, showed that the longer clone was identical to the shorter but extended another 1.5 kb in the 3' end. Sequencing data together with Northern blot analysis indicated that the two mRNA species for human type I alpha regulatory subunit were generated from the same gene by the use of different polyadenylation site signals.
Amino Acid Sequence↗
Rearrangement of immunoglobulin light chain genes in the chicken occurs prior to colonization of the embryonic bursa of Fabricius.
We have applied polymerase-chain-reaction-directed immunoglobulin gene analysis to study the embryonic differentiation of chicken B cells. Immunoglobulin light chain DNA segments in the rearranged configuration were amplified from cells of the intraembryonic mesenchyme as early as day 7 of incubation. We showed by sequencing that the rearranged variable region genes in these early B-cell progenitors were not different from the germ-line V lambda 1 gene (the single functional light chain variable region gene in chickens). In the bursal B lymphocytes, on the other hand, clear gene conversion events were first observed at day 15 of embryonic development. The present data indicate that rearrangement of light chain genes in the chicken occurs independently of the bursa of Fabricius and that diversification of the variable region begins only later, when the surface immunoglobulin-positive B cells are proliferating in the bursal follicles.
Animals↗
In vivo substitution of choline for sodium evokes a selective osmoinsensitive increase of extracellular taurine in the rat hippocampus.
Recent investigations have demonstrated that taurine and phosphoethanolamine (PEA) are the amino acids most sensitive to microdialysis-perfusion with reduced concentrations of NaCl. The aim of the present work was to assess the importance of Na+ deficiency in evoking this response. Further, the previously described selectivity of replacement of Cl- with acetate with respect to amino acid release was reinvestigated. The hippocampus of urethane-anesthetized rats was dialyzed with Krebs-Ringer bicarbonate buffer, and amino acid concentrations of the perfusate were determined. Choline chloride was then stepwise substituted for NaCl, and, in some cases, mannitol (122 mM) was included in low sodium-containing media. In other experiments, NaCl was replaced with sodium acetate. The dialysate levels of taurine increased selectively in response to Na+ substitution. The elevation of taurine was linearly related to the increase in choline chloride, and maximal levels amounted to 335% of basal levels. The increase in extracellular taurine was not inhibited by perfusion with medium made hyperosmotic with mannitol. Replacement of Cl- with acetate stimulated the release of taurine to 652% of resting levels. In addition, PEA levels increased to 250% of control concentration. Other amino acids were unaffected by Cl- substitution. The results show that taurine transport is considerably more sensitive to Na+ depletion than glutamate transport, which also is known to be Na+ dependent. The taurine increase evoked by low Na+ is not caused by cellular swelling as it was unaffected by hyperosmolar medium. Finally, substitution of acetate for Cl- causes a specific elevation of extracellular taurine and PEA, possibly as a result of cytotoxic edema.
Animals↗
On the use of diazepam and pro-diazepam (2-benzoyl-4-chloro-N-methyl-N-lysylglycin anilide), as adjunct antidotes in the treatment of organophosphorus intoxication in the guinea-pig.
Diazepam and pro-diazepam (2-benzoyl-4-chloro-N-methyl-N-lysylglycin anilide) have been used as adjunct antidotes to pyridostigmine and atropine against the organophosphate, soman, in the guinea-pig. Both added significant protection to the pyridostigmine/atropine treatment. Animals pretreated with diazepam, 60 min before soman, were "better" protected than animals given an equimolar dose of pro-diazepam therapeutically 1 min after soman. A pretreatment with diazepam for three days further increased the protection. A therapeutic dose of pro-diazepam, 1 min after soman, gave no further protection, to the three day diazepam pretreatment. The serum concentrations of diazepam (given i.p.) and desmethyldiazepam (given i.m.) were determined by GLC after diazepam (i.p.) and pro-diazepam (i.m.) were given. The protection, relative to the control, provided by the diazepam pretreatment (60 min before and for three days before soman) correlated linearly, r = 0.9898, with the serum values of diazepam achieved at these times. Our data suggest that diazepam as adjunct to pyridostigmine and atropine administered as pretreatment gives a "safer" protection, than an equimolar dose of pro-diazepam given therapeutically.
Animals↗
Molecular cloning of a tissue-specific protein kinase (C gamma) from human testis--representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase.
Two different mammalian genes for the catalytic subunit (C) of cAMP-dependent protein kinase have previously been characterized (C alpha, C beta). In the present study, we report the molecular cloning of a third isoform of C, from a human testis cDNA library, as well as the isolation of human cDNAs for C alpha and C beta. This third form of C, which we will designate C gamma, is clearly derived from a distinct gene and shows a tissue-specific expression. A close evolutionary relation between C gamma and C alpha was suggested by nucleotide homologies (86% inside the open reading frame, 81% in the 3'-untranslated region). Thus, the C gamma cDNA cross-hybridized with the 2.8 kilobase (kb) C alpha mRNA, present at high levels in most human tissues, as well as with a 1.8 kb C gamma-specific mRNA, which was only found at detectable levels in human testis. However, at the amino acid level, C alpha and C beta showed a close relationship (93% homology), whereas C gamma diverged significantly from both C alpha (83%) and C beta (79%). Taken together with the tissue-specific expression of C gamma, this suggests a pressure on C gamma during evolution, acting to modulate it in a functionally specific way. Certain amino acid substitutions make C gamma a distinct member of the cAMP-dependent subfamily of protein kinases, and suggest that C gamma may be distinct in its protein substrate specificity or its interaction with the different regulatory subunits.
Amino Acid Sequence↗
Expression of the myc proto-oncogenes in developing human fetal brain.
We have analysed c-myc, N-myc and L-myc gene expression in developing human fetal brain by Northern hybridization, RNAase protection and in situ hybridization. The unique zonal organization of the developing fetal brain allows a particularly good assessment of the coupling of myc gene expression to cell proliferation and differentiation in vivo. By Northern and in situ hybridization, L-myc as well as c-myc and N-myc transcripts in the brain were found in the post-mitotic cortical and intermediate layers, as well as in the mitotically active layers containing the neuroepithelial precursor cells. Consistent results were also obtained for L-myc using RNAase protection analysis. Both the 3.6 and 3.8kb forms of the L-myc mRNA, resulting from alternative splicing of intron I, were detected in layers of neuroectodermal origin, but not in the meninges or choroid plexus. We also extended L-myc expression and splicing analyses to other developing human fetal tissues. L-myc mRNA was expressed in several other fetal tissues, particularly in fetal skin. Predominantly intron I containing L-myc mRNA was observed in fetal striated and cardiac muscle. Thus, L-myc is expressed in a wider spectrum of developing tissues than previously known. Our findings also, show that L-myc as well as N-myc and c-myc expression is uncoupled from cell division in developing brain.
Blotting, Northern↗
Differential localization of type I and type III procollagen messenger ribonucleic acids in inflamed periodontal and periapical connective tissues by in situ hybridization.
Inflammatory lesions of periodontal and periapical connective tissue were studied by in situ hybridization to detect cells responsible for type I and type III collagen production. Formalin-fixed and paraffin-embedded tissue specimens from patients with oral lesions of various stages of inflammation were hybridized with cDNA probes specific for human pro alpha 1(I) and pro alpha 1(III) collagen mRNAs, and with bacteriophage lambda DNA as a control probe. This technique permitted us to localize fibroblasts active in type I collagen synthesis in the vicinity of inflammatory infiltrates in all the samples studied. Cells containing high levels of type III collagen mRNA were seen in early abscess formation and they were particularly abundant in pyogenic granuloma and irritation fibroma. Type I collagen mRNA was prominent in gingival fibrosis. In the infrabony lesions with active inflammatory infiltrations the production of collagen was confined mostly to the periphery of the lesions. These findings give indirect evidence that cytokines liberated during the early stages of the inflammatory process stimulate expression of the type III collagen gene by fibroblasts. In chronic lesions a gradual switch from type III to type I collagen gene expression occurs. The change in collagen types appears to underlie the observed isolation of the inflammation by a collagenous capsule. In all the samples studied fibroblasts exhibited marked variation in their levels of procollagen mRNAs, supporting previous views about their heterogeneity in connective tissues. The approach presented here offers new possibilities to study cellular interactions and metabolic activities in inflammatory lesions.
Adolescent↗
[Drug-induced agranulocytosis. A case control study].
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Adolescent↗