Network organization of interstitial connective tissue cells in the human endolymphatic duct.

The human endolymphatic duct (ED) and sac of the inner ear have been suggested to control endolymph volume and pressure. However, the physiological mechanisms for these processes remain obscure. We investigated the organization of the periductal interstitial connective tissue cells and extracellular matrix (ECM) in four freshly fixed human EDs by transmission electron microscopy and by immunohistochemistry. The unique surgical material allowed a greatly improved structural and epitopic preservation of tissue. Periductal connective tissue cells formed frequent intercellular contacts and focally occurring electron-dense contacts to ECM structures, creating a complex tissue network. The connective tissue cells also formed contacts with the basal lamina of the ED epithelium and the bone matrix, connecting the ED with the surrounding bone of the vestibular aqueduct. The interstitial connective tissue cells were non-endothelial and non-smooth muscle fibroblastoid cells. We suggest that the ED tissue network forms a functional mechanical entity that takes part in the control of inner ear fluid pressure and endolymph resorption.     

Proteoglycans of human gingival epithelium and connective tissue.

Proteoglycans extracted from separated specimens of healthy human gingival epithelium and from connective tissue have been purified. The epithelial proteoglycans fractionated as a single included peak on Sepharose 4B-CL and contained heparan sulphate and dermatan sulphate glycosaminoglycans. The connective-tissue proteoglycans separated into three major populations on Sepharose 4B-CL, one of which was excluded from this gel under associative conditions (0.5 M-sodium acetate, pH 7.4). Subsequent fractionation of the excluded material under dissociative conditions (4 M-guanidinium chloride/0.05 M-sodium acetate, pH 7.4) revealed an absence of any aggregate formation of molecules within this population. The connective-tissue proteoglycans contained heparan sulphate, dermatan sulphate and chondroitin 4-sulphate, the proportions of which varied with the molecular size of the proteoglycans. Amino acid analysis of the protein cores of gingival-epithelial and connective-tissue proteoglycans revealed differences that were similar to the differences described between other types of proteoglycans such as those from skin.     

Comparative analysis of monoamine producers in the major duodenal papilla and lungs of rabbits]

By means of incubation of slices in 2% solution of glyoxylic, acid adrenergic nervous structures and endocrine cells (APUD cells) have been studied in the ampule wall of the duodenal large papilla and in the lungs in 6 mature male rabbits. Topographic proximity of APUD cells and adrenergic nervous fibers is noted. Contents of serotonin and catecholamines in the structures mentioned have been investigated microfluorometrically. Simultaneous revealing of producers of monoamines make possible to suppose that serotonin and catecholamines get paracrinically from the APUD cells into terminals of the adrenergic nervous fibers.     

The development of the connective tissue in the human orbit.

The development of the mesenchymal structures of the human orbit was studied using 10 mu or 60 mu serial sections of orbits of foetuses of 35 to 320 mm stages (C.R. length; 2-9 months). The ontogenesis of the orbital walls, the eye muscles, the blood vessels and the connective tissue was compared. This comparison revealed that the eye muscles and their fasciae together with the adventitial layers of the larger blood vessels and nerves develop first, closely linked in time to the progress of ossification and growth of most of the orbital bones. Only the (endochondral) ossification of the ethmoid starts much later. The orbital connective tissue septa development commences later, i.e. from the third month onwards, concurrently with the development of special mesenchymal condensations, a particular capillary system and adipose tissue. About five months later the adult configuration is attained.     

Stereo architecture of the interface of the epithelial cell layer and connective tissue core of the foliate papilla in the rabbit tongue.

The three-dimensional relationship between the epithelial cell layer and the underlying connective tissue core (CTC) of the foliate papilla of the rabbit tongue was studied by scanning electron microscopy after removal of the epithelial cell layer. The foliate papillae were fixed in Karnovsky's fixative, and the epithelial cell layers were exposed to long-term hydrochloric acid treatment (3.5 N HCl for 2-3 weeks at room temperature). The foliate papillae consisted of ridges and grooves located on the posterolateral margin of the tongue. They appeared as linear projections or ridges of lingual mucosa roughly perpendicular to the longitudinal axis of the tongue. These projections or ridges were parallel to one another and separated by grooves. After removal of the epithelium, two kinds of CTC folds appeared: one was the septal fold of CTC which runs in the central portion under each linear projection or ridge, and the other consisted of two sheets of groove side folds of CTC which run along both sides of the former and face the groove side epithelium. It was revealed that there are two sheets of septal epithelial processes, and each of them fits in between each septal fold and groove side fold of CTC. Numerous taste buds were located in the groove side epithelia, and their pores faced the surface of the groove. On the hollow surfaces that appeared on the surface of the groove side fold of CTC after removal of the epithelial cells with taste buds, nerve-terminal-like structures were encountered. Some openings of the ducts of small lingual glands were arranged linearly on the underside of the basal portion of each groove side epithelium.     

Involvement of cis-acting repressive element(s) in the 3'-untranslated region of human connective tissue growth factor gene.

To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it was fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR.     

Three-dimensional architecture of the connective tissue core and surface structures of the lingual papillae in the rabbit

Three-dimensional characteristics of the epithelial cell layer and connective tissue interface of the tongue were studied using scanning electron microscopy. In this study, the fragments of tongue were fixed in modified Karnovsky's fixative solution. Subsequently, the specimens were treated with 10% NaOH solution for 4-7 days at room temperature and postfixed in 1% OsO4 in 0.1 M phosphate buffer (pH 7.4) for 2 hours at 4 degrees C. They were dehydrated through a graded ethanol series, and critical-point dried with CO2. The specimens were coated with gold and observed in a scanning electron microscope, JEOL JSM-6100. The results showed numerous papillae on the dorsal surface of the tongue divided into four groups (filiform, fungiform, foliate and vallate papillae). Filiform papillae are conically shaped; fungiform papillae have an irregular round surface; foliate papillae are oval in shape and have some parallel projections; and vallate papillae are located in the posterior part of the tongue and have a depression around the center. After the treatment with 10% NaOH solution, the original arrangements of connective papillae could be seen. This characteristic three-dimensional distribution of the collagen fiber bundles is typical for each superficial papillae depending on whether it is filiform, fungiform, foliate or vallate.     

NMR and molecular modeling studies on two glycopeptides from the carbohydrate-protein linkage region of connective tissue proteoglycans.

Complete 1H and 13C NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, Ser(GlcpAbeta(1-->3) Galpbeta(1-->3)Galpbeta(1-->4)Xylpbeta)-Gly-Ser-Gly-Se r (GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)Xylp beta)-Gly (1), and the tetrasaccharide dipeptide, Ser(GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)X ylpbeta)-Gly (2). The vicinal coupling constant data show that the monosaccharide residues adopt4 C 1 chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The straight phi torsion angles of the glycosidic C1'-O1 bonds showed a strict preference for the -sc range whereas the psi torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for beta(1-->3) linkage, +ac for beta(1-->4) linkage). The predominant conformation about the glycopeptide bond is straight phi = -sc and psi = +ac. The presence of strong daN (i, i+1) NOE contacts, and the general absence of dNN (i, i+1) contacts (except for a weak Ser-5/Gly-6 dNN contact) and the dbN (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide.     

Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice

Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation.     

Development and structure of epithelial formations of the connective tissue stroma of human gingiva

Processes of development and structure of epithelial formations, localized in the connective tissue framework of the gingival mucous membrane have been studied. During the prenatal period of development they are presented as epithelial pearls of Serra, and after birth--as remnants of ++tooth-forming epithelium (RTE). They are remnants of sheaths of the destroyed pearls. The latter, like RTE in the gingival stroma, have a genetic connection with the epithelial islets of Malasser and can cause some diseases in the gingiva.     

Physical mapping of 14 new DNA markers isolated from the human distal Xp region.

We have isolated 14 new DNA markers from the human Xpter-Xp21 region distal to the Duchenne muscular dystrophy gene by targeted cloning, employing two somatic cell hybrids containing this region as their sole human material. High-resolution physical localization of these markers within this region was obtained by hybridization to two mapping panels consisting of DNA from patients carrying various translocations and deletions in distal Xp. Five markers were assigned to the pseudoautosomal region where their position on the long-range map of this region was further determined by pulsed-field gel electrophoresis. The other nine markers map to the X-specific region. Informative TaqI restriction fragment length polymorphisms were observed for four loci. One of these represents a region-specific low-copy repeated element. These 14 new markers represent useful tools for the understanding of distal Xp deletion and translocation mechanisms and for the positional cloning of disease genes in the region.     

EDTA separation and recombination of epithelium and connective tissue of human oral mucosa. Studies of tissue transplants in nude mice

A possible epithelial-mesenchymal interaction in determining epithelial histologic features of human oral mucosa was examined. The study comprised 74 biopsies of normal buccal mucosa and 54 biopsies of normal palatal mucosa. Epithelium was separated from connective tissue by the use of 1 mM ethylenediamine tetraacetate dihydrate. Self-recombined and cross-recombined epithelial and connective tissues and connective tissue sheets alone were transplanted to subcutaneous sites of nude mice. Histologic examination of cross-recombined palatal epithelium/buccal connective tissue transplants showed a change in keratinization pattern but no major change in number of epithelial cell layers as the result of connective tissue influence. Transplanted sheets of connective tissue after growth for 14 days showed that complete separation of biopsies from buccal mucosa had been obtained. However, palatal mucosa had been incompletely separated as evidenced by re-epithelialization of most of the connective tissue transplants. The consequences of the incomplete palatal epithelium-connective tissue separation are discussed.     

Intraspecific nucleotide sequence differences in the major noncoding region of human mitochondrial DNA.

Nucleotide sequences of the major noncoding region of human mitochondrial DNA (mtDNA) from 95 human placentas have been determined. These sequences include at least a 482-bp-long region encompassing most of the D-loop-forming region. Comparisons of these sequences with those previously determined have revealed remarkable features of nucleotide substitutions and insertion/deletion events. The nucleotide diversity among the sequences is estimated as 1.45%, which is three- to fourfold higher than the corresponding value estimated from restriction-enzyme analysis of whole mtDNA genome. A hypervariable region has also been defined. In this 14-bp region, 17 different sequences were detected. More than 97% of the base changes are transitions. A significantly nonrandom distribution of nucleotide substitutions and sequence length variations were also noted. The phylogenetic analysis indicates that diversity among the negroids is much larger than that among the caucasoids or the mongoloids. In fact, part of the negroids first diverged from other humans in the phylogenetic tree. A striking finding in the phylogenetic analysis is that the mongoloids can be separated into two distinct groups. Divergence of part of the mongoloids follows the earliest divergence of part of the negroids. The remainder of the mongoloids subsequently diverged together with the caucasoids. This observation confirmed our earlier study, which clearly demonstrated, by the restriction-enzyme analysis, existence of two distinct groups in the Japanese.     

Initial characterization of the microenvironment that regulates connective tissue degradation in amniochorion during normal human labor.

Extracellular matrix degradation in fetal membranes leading to its rupture is coupled to myometrial activity and cervical ripening during human normal labor. Mechanisms which modulate collagen degradation in amniochorion during labor have not been elucidated. Initial characterization of the effect of different blood compartments on connective tissue degradation in amniochorion during human labor was explored. Amniochorion explants were stimulated with plasma of maternal venous blood, umbilical cord blood or placental blood, obtained from women with pregnancies at term, with or without labor. MMP-2 and MMP-9 activities were quantified in conditioned media by gelatin-zymography as an index of connective tissue degradation. Collagen content was measured in tissue explants and collagen fibrils distribution was examined by electron microscopy. Placental plasma from term pregnancies, with or without labor, is enriched with soluble signals that enhance the in vitro MMP-9 production by amniochorion. Accompanying ultrastructural distortion of collagen fibers and demonstration of collagen degradation fragments confirmed induction of extracellular matrix degradation. Control experiments in which MMP-9 activity was blocked with TIMP-1 resulted in inhibition of all the above mentioned changes. These results suggest that placental intervillous space is a functional compartment in which mediators capable to induce collagen degradation in amniochorion are selectively expressed during human labor.     

Cytophotometric determination of the DNA content in epithelial and connective tissue cells of the human prostate

A study of the quantitative parameters of DNA in cells nuclei of epithelium and connective tissue allowed establishing certain periods which are characterized by an increase in the percentage of ploidy. The causes and importance of this phenomenon for the development of pathological processes in tissues of the prostate gland and the possibility of its use for the diagnosis are discussed.