An improved method for the structural profiling of keratan sulfates: analysis of keratan sulfates from brain and ovarian tumors

A previously developed method for the structural fingerprinting of keratan sulfates (Brown et al., Glycobiology, 5, 311-317, 1995) has been adapted for use with oligosaccharides fluorescently labeled with 2-aminobenzoic acid following keratanase II digestion. The oligosaccharides are separated by high-pH anion-exchange chromatography on a Dionex AS4A-SC column. This methodology permits quantitative analysis of labeled oligosaccharides which can be detected at the sub-nanogram ( approximately 100 fmol) level. Satisfactory calibration of this method can be achieved using commercial keratan sulfate standards. Keratan sulfates from porcine brain phosphocan and human ovarian tumors have been examined using this methodology, and their structural features are discussed.     

Ocular distribution of keratan sulfates during pre- and postnatal development in rabbits

Twenty-six monoclonal antibodies (MAbs) developed against rabbit corneal proteokeratan sulfate (PKS), were used to evaluate immunohistochemically the ocular distribution of PKS during prenatal and early postnatal development in rabbits. These MAbs were directed against epitopes located in the keratan sulfate (KS) chains of the proteoglycan (SundarRaj et al., 1985). Staining of cryostat sections of the eyes was carried out using an indirect peroxidase-conjugated technique. Only one of the MAbs reacted with the presumptive corneal region at day 13 or 16 of fetal development. By day 20, more MAbs reacted with the corneal stroma. There were distinct differences, however, in the distribution of the epitopes recognized by the various MAbs. A few of them stained only the posterior region of the cornea, whereas others showed a decreasing staining gradient from the posterior to the anterior region. By day 24, all of the MAbs reacted with the corneal stroma, but some reacted also with the limbal region and with the conjunctival stromal matrix. One MAb also reacted with the conjunctival epithelial layer, but only at this stage of development. Conjunctival staining was more intense at day 28 of fetal development and at day 2 postnatally. KS was not detectable in the conjunctiva of adult rabbits with any of the MABs. These results suggest that although KS synthesis starts at very early stages of fetal development, there are progressive changes in its antigenic structure in specific regions of the cornea and conjunctiva during corneal development.     

Detection of age-related changes in the distributions of keratan sulfates and chondroitin sulfates in developing chick limbs: an immunocytochemical study

A panel of four separate monoclonal antibodies, all known to specifically recognize epitopes on keratan sulfate glycosaminoglycans, were employed in an immunocytochemical study of developing chick hind limbs. In addition, two monoclonal antibodies specific for epitopes on chondroitin/dermatan sulfate glycosaminoglycans were employed on equivalent sections to determine the degree of colocalization of keratan and chondroitin/dermatan sulfates. The spatial distributions of keratan sulfate and chondroitin/dermatan sulfate differed to some extent. In younger embryos, high extracellular concentrations of keratan sulfate occurred in joints and articular cartilages, with diminishing amounts being present in epiphyseal and diaphyseal regions. The high concentration of keratan sulfate in joints and articular cartilage corresponded to equally high concentration of chondroitin-6 sulfate. With advancing age, the above mentioned distribution was modified, most notably by increased amounts of keratan sulfate within diaphyseal regions. Finally, the use of four different anti-keratan sulfate monoclonal antibodies made it possible to compare keratan sulfate epitope expression. Differences in keratan sulfate epitopes were noted in some regions of bones, mostly in diaphyseal regions of younger bones and epiphyseal regions of older bones. This pattern of keratan sulfate expression suggests that different types of keratan sulfate may be present and their expression may be developmentally regulated.     

Insect tissue culture systems: models for study of hormonal control of development

Summary The regulation of growth and development of insects is under endocrine control and involves both juvenile hormones and ecdysteroids. Neuropeptides are master regulators which control the secretion of these hormones. Most experiments in insect endocrinology have been conducted in vivo, but tissue culture methodology is playing an increasing role due to the great interest in simpler model systems for the study of complex processes that occur in vivo. The availability of appropriate media has allowed the culture of a variety of insect organs and cell lines of defined origin which have kept certain properties of the parent tissues. Tissue culture approaches have been useful for studying hormonal control of morphogenetic processes. Cell lines are particularly suited to the study of hormonally regulated mechanisms of macromolecular biosynthesis and gene expression. Thus, the value of in vitro analysis in studies of regulation of hormone production is now recognized. Results obtained from tissue culture allow more precise definition of the hormonal requirements of insect cells and tissues for growth and differentiation and might make possible the discovery of new growth regulators.     

Purification of Keratan Sulfate-endogalactosidase and its action on keratan sulfates of different origin.

A glycosidase which attacks corneal keratan sulfate was purified from extracts of Pseudomonas sp. IFO-13309. When corneal keratan sulfate was degraded by the purified enzyme, Sephadex G-50 chromatography indicated the presence of a number of oligosaccharides differing in size and sulfate content. The characterization of two major fractions of the oligosaccharides indicated that the point of enzyme attack is limited to the endo-beta-D-galactoside bonds in which nonsulfated D-galactose residues participate. The enzyme, unlike ordinary exo-beta-D-galactosidases, did not catalyze the hydrolysis of phenyl beta-D-galactoside. Moreover, beta-D-galactosyl-(1 leads to 3)-2-acetamido-2-deoxy-beta-D-glucosyl-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-D-glucose ("lacto-N-tetraose") was completely refractory to the action of this enzyme, suggesting that a structure of the type, X-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-Y, is not the only specificity-determining factor, i.e. neighboring sugars, X and Y, or even larger portions of substrate molecule must have an important effect. Compared with corneal keratan sulfate, keratan sulfates from human nucleus pulposus and shark cartilage were attacked at lower rates with a resultant production of oligosaccharides of relatively large size. The result is in agreement with the view that considerable variations exist in the structure of keratan sulfates of different origin, and further suggests that the enzyme may serve as a useful reagent in studying these variations.     

Endo-beta-galactosidase of Escherichia freundii. Purification and endoglycosidic action on keratan sulfates, oligosaccharides, and blood group active glycoprotein.

Endo-beta-galactosidase was purified 4400-fold from a culture filtrate of Escherichia freundii with 45% recovery. The enzyme preparation was practically free of exoglycosidases, sulfatase, and proteases. This enzyme hydrolyzed several keratan sulfates, endoglycosidically releasing oligosaccharides of various molecular sizes. Among the digestion products of the corneal keratan sulfate, the structure of a disaccharride and a tetrasaccharride were shown to be 2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-D-galactose and 2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-6-O-sulfo-beta-D-galactosyl-(1 leads to 4)-2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-D-galactose, respectively. These oligosaccharide structures indicate that this enzyme specifically hydrolyzes the galactosidic bonds in which nonsulfated galactose residues participate. The enzyme could also hydrolyze a small oligosaccharide such as lacto-N-neotetraitol as follows: Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 3)Gal(beta 1 leads to 4) sorbitol leads to Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 3)Gal + sorbitol AB active blood group substance could be hydrolyzed by this enzyme only after Smith degradation. After enzymatic digestion small oligosaccharides and resistant macromolecules were produced. These findings indicate that the enzyme should be useful in studying the precise structures of keratan sulfates, related glycoproteins, and oligosaccharides.     

Immunocytochemical distribution of E-cadherin in normal and injured lung tissue of the rat

Affinity purified rabbit anti-mouse E-cadherin antibodies, reacting with diverse rat epithelia, were used to characterize epithelial changes in a radiation-induced fibrosis model of rat lung by immunoblotting techniques, immunoperoxidase and immunofluorescence microscopy. Immunostaining of normal rat lung tissues revealed a predominant staining of type II pneumocytes. Immunoelectron microscopy confirmed the immunohistochemical data of normal lung tissue obtained at the light microscopic level. In severely injured rat lung, we found enhanced immunoreactivity for E-cadherin at the surface of type I alveolar epithelial cells. The results suggest that E-cadherin is an adhesion molecule that is modulated after pathological alteration of the alveolar epithelium and that the antiserum may be useful for the characterization of normal and diseased rat epithelia.     

Immunochemical characterization and ultrastructural localization of chondroitin sulfates and keratan sulfate in embryonic chick bone marrow

Summary Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.     

Immunocytochemical study of YB-1 nuclear distribution in different cell types

In the present work we studied the distribution of YB-1 in the nuclei of mouse hepatocytes, early embryos and human skin fibroblasts with the use of light and electron microscopy. To reveal YB-1, we applied rat polyclonal antibody against the C-terminal fragment of YB-1 molecule and rabbit polyclonal antibody against full-length YB-1 molecule. YB-1 distribution patterns varied significantly in different cell types. YB-1 was found to be colocalized with RNA polymerase I in mouse hepatocytes and embryos. Besides, YB-1 was revealed in a population of Cajal bodies in 2-cell mouse embryos but not in other cells studied.     

Immunocytochemical study of the spinal distribution of substance P, enkephalins, cholecystokinin and serotonin in amyotrophic lateral sclerosis

Using immunocytochemical methods, a severe loss of substance P, but not of enkephalin, cholecystokinin and serotonin containing fibers was observed in lamina IX of the spinal cords from 4 amyotrophic lateral sclerosis cases. Substance P-fibers were decreased before degeneration of motoneurons. They were normal in the remaining spinal gray matter.     

Sexual differentiation of the rodent hypothalamus: hormonal and environmental influences

Brain sexual differentiation is a complex developmental phenomenon influenced by the genetic background, sex hormone secretions and environmental inputs, including pollution. The main hormonal drive to masculinize and defeminize the rodent brain is testosterone secreted by the testis. The hormone does not influence sex brain differentiation only in its native configuration, but it mostly needs local conversion into active metabolites (estradiol and DHT) through the action of specific enzymatic systems: the aromatase and 5alpha-reductase (5alpha-R), respectively. This allows the hormone to control target cell gene expression either through the estrogen (ER) or the androgen (AR) receptors. The developmental profile of testosterone metabolizing enzymes, different in the two sexes, is therefore of the utmost importance in affecting the bioavailability of the steroids active in brain differentiation. Widely diffused pollutants, like polychlorinated biphenyls (PCBs) are able to affect the production and/or action of testosterone metabolites, exerting detrimental influences on reproduction and sex behavior. The main studies performed in our and other laboratories concerning the pattern of expression and the control of the enzymatic systems involved in brain androgen action and metabolism are shortly reviewed. Some recent data on the influence exerted by PCBs on these metabolic systems are also reported.     

Immunocytochemical study of the distribution of cytoskeletal proteins in neurons from mouse embryo spinal cord in culture

Monoclonal FITC and Rhodamine-labeled antibodies were used to investigate distribution of tubulin, actin, and neurofilament protein with a molecular weight of 160 kDa in neurons from mouse embryo spinal cord cultivated in a monolayer. It was found that migration of tubulin from the neuronal soma occurs during the course of nerve cell development. The extent to which neuronal processes became filled with tubulin varied according to the stage of cell differentiation. Neuronal actin concentration was negligible and was found at points along the processes close to focal contacts with lining cells. The main body of neurofilament protein is concentrated in the neuronal soma and proximal portions of neurites.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 618–623, 1988.     

南方鲇两种生长激素受体的结构分析及其组织分布和激素调节

采用生物信息学方法,从东方纯（Fugurubripes）基因组中分离出两种不同基因编码的GHR1（fGHR1）和GHR2（fGHR2）。随后设计引物,采用RT-PCR和Smart^TM Race相结合的方法从南方鲇（Silurus meridionalis）肝脏中克隆出两种不同基因编码的生长激素受体,即GHR1（scGHR1）和GHR2（scGHR2）cD-NA,其全长分别为1985bp（编码602个氨基酸）和2300bp（编码553个氨基酸）。这两种受体都含有生长激素受体典型的标志性基序FGDFS,以及细胞内属于细胞因子受体家族共有的Box1和Box2序列框。同时两者又存在差异,表现在胞外半胱氨酸残基和胞内酪氨酸残基数目的不同。采用半定量RT-PCR方法研究了scGHR1和scGHR2在南方鲇各组织中表达量的差异,结果表明：scGHRl和scGHR2有广泛的组织分布,在肝脏中两者的表达量最高。用17p雌二醇（E2）、17α-甲基睾酮（MT）和可的松（conisol）对南方鲇进行药物处理,结果表明E2能够下调肝脏scGHR1和scGHR2 mRNA水平,而MT上调scGHR1和scGHR2 mRNA水平。同时,经cortisol处理后scGHR1 mRNA表达水平上升,而scGHR2 mRNA表达水平不变。     

Immunocytochemical study of the distribution of a ganglioside in sea urchin eggs.

An antibody against M5 ganglioside (NeuGc alpha 2-6Glc beta 1-1Cer), the dominant ganglioside in the eggs of the sea urchin, Anthocidaris crassispina, was purified by affinity chromatography from rabbit antiserum against crude ganglioside of the eggs. The specificity of the antibody was verified by enzyme-linked immunosorbent assay and TLC immunostaining. M5 ganglioside was also the major one in the eggs of another sea urchin, Hemicentrotus pulcherrimus, as judged from TLC analyses including immunostaining. Cryostat-sections of H. pulcherrimus eggs were examined to determine the distribution of M5 ganglioside by indirect immunofluorescence microscopy with the antibody. Before fertilization, the egg cortex was highly stained, while the other part of cytoplasm was uniformly but much more weakly stained. After fertilization, the staining rapidly decreased in the cortex and was restricted to a very thin peripheral layer and to cytoplasmic patches. The immunoreactivity was also observed in the esophagus and the somatic cells of the testis, but the spermatozoa were never stained with the antibody.     

Sequential distribution of keratan sulphate and chondroitin sulphate epitopes during ameloblast differentiation

Proteoglycans are complex macromolecules containing one or more glycosaminoglycan chains and exhibiting a variety of biological functions in connective tissues. The aim of the present study was to immunolocalize the distribution of keratan sulphate and chondroitin sulphate epitopes during initial enamel formation in order to study temporo-spatial expression patterns of these macromolecules. Third molars of four-months-old pigs were used for immunolocalization of keratan sulphate and chondroitin sulphate epitopes in the developing enamel layer. Tooth organs were prepared for paraffin sections in order to perform indirect immunohistochemistry. The results demonstrated a mutually exclusive positioning between these two epitopes. Keratan sulphate epitopes were observed in pre-secretory pre-ameloblasts and adjacent stratum intermedium while chondroitin sulphate epitopes were demonstrated in secretory ameloblasts and adjacent stratum intermedium. Our findings suggest that proteoglycans containing glycosaminoglycan chains may play a regulatory role during enamel mineralization.     

Source,sink and hormonal control of translocation in wheat

Summary An analysis of the pattern of movement of ¹⁴C-labelled flag leaf assimilates in wheat (Triticum aestivum l. c.v. Gabo) during grain development, indicated that the greater the requirement for assimilates by the ear the more rapid was the speed of movement of these through the peduncle to the ear and also the lower their concentration. Experiments with [¹⁴C] indoleacetic acid ([¹⁴C]IAA) suggested that auxin production by the grains was not responsible for the control of assimilate translocation through the peduncle. Limiting the supply of available assimilates by shading the lower parts of the plant, did not significantly alter the speed of movement of ¹⁴C-photosynthate through the peduncle, while severing half of the vascular tissue in the peduncle altered the pattern of movement of ¹⁴C to the ear and enhanced the speed of movement of ¹⁴C through the remaining functional conducting tissue. These results are discussed in relation to the mechanism of translocation.