Changes in proteoglycan composition during development of rat skin. The occurrence in fetal skin of a chondroitin sulfate proteoglycan with high turnover rate

Extraction of the skin of newborn rat yielded two populations of galactosaminoglycan-containing proteoglycan: a Mr = 111,000-200,000 dermatan sulfate proteoglycan (DS-PG) with a Mr congruent to 55,000 core glycoprotein and a Mr congruent to 10(6) chondroitin sulfate proteoglycan (CS-PGs) composed of two subpopulations with different size core-glycoproteins (Mr congruent to 480,000 and 520,000). Tryptic peptide mapping of chondroitinase-treated DS-PG and CS-PGs indicated that the peptide patterns observed with the two core molecules from CS-PGs were identical with each other but distinct from the peptide pattern of the DS-PG core molecule. It is likely therefore that the two forms of CS-PGs are derived from the same gene product by post-translational modification or partial degradation, but DS-PG is derived from a distinct gene product. Comparison of the concentration (hexuronate/DNA) of the proteoglycans in newborn and fetal rat skin showed an age-related change in proteoglycan composition; at 4 days before birth, the uronic acid proportions, DS-PG:CS-PGs, were about 14:1 and during the next 4 days, DS-PG increased 2.2-fold whereas CS-PGs decreased 4-fold. On a per DNA basis, the rate of [3H]serine incorporation into CS-PGs was 2.5 times the rate for DS-PG at 4 days before birth but decreased by 95% during the next 4 days. The rate for DS-PG also decreased but to a much lesser extent, so that by 2 days before birth, it began to exceed the rate for CS-PGs. The striking change in the concentration and labeling rate of CS-PGs can be interpreted either as a decrease of CS-PGs synthesis, or as an increase of CS-PGs breakdown, or both, a process which might be involved in the transition of extracellular matrix from a fetal type to a newborn or adult type.     

Stimulation of glycosaminoglycan sulfotransferase from chick embryo cartilage by basic proteins and polyamines

A soluble glycosaminoglycan sulfotransferase (3'-phosphoadenylylsulfate:chondroitin 4'-sulfotransferase, EC 2.8.2.5) from chick embryo cartilage has been prepared free from endogenous acceptor. The reaction with this enzyme preparation was stimulated by basic proteins and polyamines, the degree of stimulation being dependent on the chemical nature of both basic compounds and acceptor glycosaminoglycans. A maximum stimulation was obtained when protamine (basic compound) and chondroitin (acceptor) were involved in the reaction mixture at a molar ratio of protamine to repeating disaccharide units of chondroitin, 1:100. The stimulation of sulfotransferase activity by basic substances was much higher than that by Mn2+. However, increasing the Mn2+ concentration immediately reduced the stimulation by basic substances. The Km value for 3'-phosphoadenosine 5'-phosphosulfate of the sulfotransferase, when chondroitin was used as acceptor, was 1 . 10(-6) M in the presence of 25 microgram/ml protamine, compared to 2 . 10(-5) M in the absence of protamine. These observations indicate that the basic proteins and polyamines may interact with acceptor polysaccharide, thereby causing an increase in the affinity of the enzyme toward 3'-phosphoadenosine 5'-phosphosulfate.     

Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.     

The relationship between consumption of tyrosine and phenylalanine as precursors of catecholamine at breakfast and the circadian typology and mental health in Japanese infants aged 2 to 5 years

Background

This study aims to examine the relationship between tyrosine and phenylalanine intake at breakfast as precursors of dopamine, and scores on the Torsvall-Åkerstedt Diurnal Type Scale and of mental health in Japanese infants aged 2 to 5 years.

Results

An integrated questionnaire was administered to parents of 1,367 infants attending one of ten nursery schools governed by Kochi City or a kindergarten affiliated with the Faculty of Education at Kochi University (775 answers for analysis: 56.7%) in May and June 2008. Questionnaires included the Torsvall-Åkerstedt Diurnal Type Scale and questions on sleep habits (onset, offset, quality, quantity, and so on), meal habits (content and regularity of timing), and mental health (depressive states). Amount of tyrosine and phenylalanine intake was calculated based on a breakfast content questionnaire and data on the components of amino acids in foods. Infants who ingested more than 800 mg of tyrosine or phenylalanine at breakfast per meal were more morning-type than those who ingested less than 800 mg (ANOVA: P= 0.005). However, this relationship disappeared in the ANCOVA analysis (with the covariance of tryptophan intake, P= 0.894). Infants who ingested more than 800 mg of the two amino acids at breakfast showed significantly higher mental health scores (lower frequency of depressive states) than those who ingested less than 800 mg (ANOVA: P = 0.004). This relationship remained significant when ANCOVA analysis was performed with the covariance of tryptophan (ANCOVA: P= 0.017).

Conclusions

These results suggest that tyrosine and phenylalanine ingested at breakfast are not related with circadian phase, but are relate with mental health in infants.     

Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase

The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain. We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units [Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463-468]. In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST. Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho[(35)S]sulfate. Incubations with O-desulfated heparin, predominantly composed of [(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-](n)(), resulted in 2-O-sulfation of iduronic acid. When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure [(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-](n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid. Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid. In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM).     

Solution structure of human acidic fibroblast growth factor and interaction with heparin-derived hexasaccharide

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparansulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110–131 and 17–21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin- oligosaccharides in a similar manner.     

Effects of combination of Caiapo with other plant-derived substance on anti-diabetic efficacy in KK-Ay mice

B. Ludvik et al., have recently shown the effect of Caiapo (Ipomoea batatas L.) on reducing fasting blood glucose and insulin resistance in type-2 diabetic patients. It, however, was required 2-4 weeks after the single administration of Caiapo. The present study aimed to determine if the combination therapy of Caiapo with a mulberry leaf powder, which inhibits alpha-glucosidase, or with a loquat leaf extract, which shows an insulin-like effect, could make it possible to enhance the antidiabetic activities of Caiapo, and to shorten the time necessary for the inhibition of increasing blood glucose levels. A mixture of the pulverized tuber of Caiapo (357 mg/kg) and the mulberry leaf powder (143 mg/kg), or a mixture of the pulverized skin of Caiapo (194 mg/kg) and the powdered loquat leaf extract (6 mg/kg) was orally administered to 6 weeks-old male KK-Ay mice for 28 days and the glucose loading test was conducted every 7 days. In the glucose loading test after one week feeding, a reduction in blood glucose concentration after 60 minutes of the administration of glucose was observed in both mixture groups against the control group (p < 0.05) in the case of Caiapo only, similar delayed effects were seen in 2-3 weeks after feeding.     

Carbohydrate structure and differential binding of prostate specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy

Serum prostate-specific antigen (PSA) assay is widely used for detection of prostate cancer. Because PSA is also synthesized from normal prostate, false positive diagnosis cannot be avoided by the conventional serum PSA test. To apply the cancer-associated carbohydrate alteration to the improvement of PSA assay, we first elucidated the structures of PSA purified from human seminal fluid. The predominant core structure of N-glycans of seminal fluid PSA was a complex type biantennary oligosaccharide and was consistent with the structure reported previously. However, we found the sialic acid alpha2-3 galactose linkage as an additional terminal carbohydrate structure on seminal fluid PSA. We then analyzed the carbohydrate moiety of serum PSA from the patients with prostate cancer and benign prostate hypertrophy using lectin affinity chromatography. Lectin binding was assessed by lectin affinity column chromatography followed by determining the amount of total and free PSA. Concanavalin A, Lens culinaris, Aleuria aurantia, Sambucus nigra, and Maackia amurensis lectins were tested for their binding to the carbohydrates on PSA. Among the lectins examined, the M. amurensis agglutinin-bound fraction of free serum PSA is increased in prostate cancer patients compared to benign prostate hypertrophy patients. The binding of PSA to M. amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, was also confirmed by surface plasmon resonance analysis. These results suggest that the differential binding of free serum PSA to M. amurensis agglutinin lectin between prostate cancer and benign prostate hypertrophy could be a potential measure for diagnosis of prostate cancer.     

Rsp1p, a J domain protein required for disassembly and assembly of microtubule organizing centers during the fission yeast cell cycle

Regulation of microtubule organizing centers (MTOCs) orchestrates the reorganization of the microtubule (MT) cytoskeleton. In the fission yeast Schizosaccharomyces pombe, an equatorial MTOC (eMTOC) at the cell division site disassembles after cytokinesis, and multiple interphase MTOCs (iMTOCs) appear on the nucleus. Here, we show that, upon eMTOC disassembly, small satellites carrying MTOC components such as the gamma-tubulin complex travel in both directions along interphase MTs. We identify rsp1p, an MTOC protein required for eMTOC disassembly. In rsp1 loss-of-function mutants, the eMTOC persists and organizes an abnormal microtubule aster, while iMTOCs and satellites are greatly reduced. Conversely, rsp1p overexpression inhibits eMTOC formation. Rsp1p is a J domain protein that interacts with an hsp70. Thus, our findings suggest a model in which rsp1p is part of a chaperone-based mechanism that disassembles the eMTOC into satellites, contributing to the dynamic redistribution of MTOC components for organization of interphase microtubules.     

Heparan sulfate 6-o-sulfotransferase is essential for muscle development in zebrafish

Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein shows specific 6-O-sulfotransferase activity and preferentially acts on the iduronosyl N-sulfoglycosamine. In the developing embryo, hs6st is expressed in the brain, the somites, and the fins; the same structures that were affected upon morpholino-mediated functional knockdown. Morpholino injections significantly inhibited 6-O- but not 2-O-sulfation as assessed by HPLC. Morphants display disturbed somite specification independent of the somite oscillator mechanism and have impaired muscle differentiation. In conclusion, our results show that transfer of sulfate to specific positions on glycosaminoglycans is essential for muscle development.