Pathological role of excessive DNA as a trigger of keratinocyte proliferation in psoriasis.

Psoriasis is characterized by excessive growth and aberrant differentiation of epidermal keratinocytes due to persistent inflammation. However, the underlying mechanism that triggers immune activation in psoriasis is not clear. In this study, we explored excessive DNA as a potential trigger of psoriasis using cultured human keratinocytes and psoriatic skin tissues. We demonstrated that human genomic DNA fragments induced tumour necrosis factor (TNF)-α expression, hyperproliferation and over-expression of heparin-binding epidermal-like growth factor (HB-EGF) and transforming growth factor (TGF)-α, accompanied by defective expression of keratins 1 and 10 in cultured normal human epidermal keratinocytes, which have a similar phenotype to that of keratinocytes in psoriatic skin lesions. In psoriatic lesions, we found high levels of double-stranded (ds)DNA fragments, accompanying keratinocytes expressing Ki-67, HB-EGF and TNF-α. In addition, we showed that 1,25-dihydroxyvitamin D3 inhibited genomic DNA fragment-induced TNFA and interleukin-1ß (IFNB) expression in human keratinocytes, and an intact function of cathelicidin anti-microbial peptide (CAMP) was required for this effect. These results suggest that excessive dsDNA fragments probably act as a risk factor for immune activation in psoriasis, and the active form of vitamin D can prevent genomic DNA-mediated skin inflammation via CAMP.

Genomic copy-number aberrations related to lymph-node metastasis of colon cancer.

Lymph-node metastasis is an important indicator in the diagnosis of colon cancer. In order to determine the genes involved in metastasis, genomic copy-number aberrations in the primary tumours and lymph-node metastases were analysed in 12 patients using comparative genomic hybridization. This method detects genomic copy-number changes at the chromosomal level and the identification of the regions of aberration on any chromosome. Copy-number gains at 6p12 and losses at 8p12 were observed in a greater number of the primary tumours than in the metastases. These aberrations appear to be involved in lymph-node metastasis of colon cancer, and may allow measurement of the risk of lymph-node metastasis from a given colon cancer.

Analysis of the relationship between sex and chromosomal aberrations in colorectal cancer by comparative genomic hybridization.

Colorectal cancer is thought to be more common in men than in women. The chromosomal locations of DNA gains and losses in surgical specimens of colorectal tumours were detected by comparative genomic hybridization and were compared by gender. Five chromosomal regions, 7p, 8p, 8q, Xp and Xq, contained multiple gains that were significantly more common in males than in females, and within these regions, the differences were significant for Xp21, Xp11.3, Xp11.4 and Xq26. Regions 1p, 3q, 11q, 12p, 12q and 15q contained multiple sites of gain that were significantly more common in females than in males. Tumours from male and female patients showed significantly more losses at 11p and 15q, and at 4q and Xq, respectively. The fact that gains in X-chromosomal regions were detected with a significantly higher frequency in tumours from male patients suggests that the difference between the genders might be explained by X-chromosomal inactivation.

Enzymatic synthesis of p-nitrophenyl N,N',N'',N'',N''''-pentaacetyl-beta-chitopentaoside in water-methanol system; significance as a substrate for lysozyme assay.

Transchitooligosylation from (GlcNAc)5 to the 4-position of PNP-GlcNAc was efficiently induced through lysozyme catalysis in an aqueous solution containing methanol with a high concentration. Use of the aqueous methanol system in this reaction not only guaranteed solubility of PNP-GlcNAc substrate, but also resulted in a remarkable increase in PNP-(GlcNAc)5 production. PNP-(GlcNAc)5 was substrate for lysozyme assay compared with PNP-(GlcNAc)4.

Transglycosylation reaction of a chitinase purified from Nocardia orientalis.

Chitinase from the culture filtrates of Nocardia orientalis IFO 12806 was purified to apparent homogeneity by precipitation with ammonium sulfate followed by successive chromatography on CM-Sephadex and Bio-Gel P-60, and finally by affinity chromatography on a phenyl-Sepharose CL-4B column. The enzyme, which is essentially a hydrolase, also catalyzed a transglycosylation reaction on tetra-N-acetyl-chitotetraose (GlcNAc)4 and penta-N-acetyl-chitopentaose (GlcNAc)5. The enzyme converted the tetrasaccharide into hexa-N-acetyl-chitohexaose (GlcNAc)6 (21%) and di-N-acetyl-chitobiose (GlcNAc)2 (63%) as the major products. The corresponding values for penta-N-acetyl-chitopentaose (GlcNAc)5 were hepta-N-acetyl-chitoheptaose (GlcNAc)7 23% and tri-N-acetyl-chitotriose (GlcNAc)3 59%. The rate of the transglycosylation depended on the temperature, the concentration of substrate and the pH.

Oligonucleotide synthesis using the 2-(levulinyloxymethyl)-5-nitrobenzoyl group for the 5'-position of nucleoside 3'-phosphoramidite derivatives.

A comparative study on the utility of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) and 2-(levulinyloxymethyl)benzoyl (LMBz) protecting groups for the 5'-positions of nucleoside 3'-phosphoramidite derivatives in the oligonucleotide synthesis is presented in terms of the syntheses of TpTpT, TpTpTpT, and UpCpApGpUpUpGpG. In addition we describe the synthesis, using the LMNBz protecting group, of the CpCpA terminus triplet of tRNAs bearing exocyclic amino groups with 15N-labeling, and the trimer Gp[A*]pG containing 2'-O-(beta-D-ribofuranosyl)adenosine ([A*]), the latter of which is found at position 64 in the yeast initiator tRNA(Met).

Histochemical and immunohistochemical study on the skin of patients with ossification of the posterior longitudinal ligament in the cervical spine.

We present an immunohistochemical study on extracellular matrix (ECM), decorin, in the epidermis and dermis of patients with ossification of the posterior longitudinal ligament (OPLL) to clarify the abnormality in the extracellular matrix of the skin of OPLL. Localization of decorin and TGF-beta in the skin was studied. All patients with OPLL showed a diffuse strong staining of decorin in the entire epidermal layer. The epidermis was also positive for TGF-beta antibody staining. Patients with cervical spondylotic myelopathy showed weak or no staining of decorin. Abnormality in the regulating system of TGF-beta may be involved in the precipitation of extracellular matrix.

Efficient methods for the synthesis of [2-15N]guanosine and 2'-deoxy[2-15N]guanosine derivatives.

The nucleophilic addition-elimination reaction of 2',3',5'-tri-O-acetyl-2-fluoro-O6-[2-(4-nitrophenyl)ethyl]inosine (8) with [15N]benzylamine in the presence of triethylamine afforded the N2-benzyl[2-15N]guanosine derivative (13) in a high yield, which was further converted into the N2-benzoyl[2-15N] guanosine derivative by treatment with ruthenium trichloride and tetrabutylammonium periodate. A similar sequence of reactions of 2',3',5'-tri-O-acetyl-2-fluoro-06-[2-(methylthio)ethyl]inosine (9) and the 6-chloro-2-fluoro-9-(beta-D-ribofuranosyl)-9H-purine derivative (11), which were respectively prepared from guanosine, with potassium [15N]phthalimide afforded the N2-phthaloyl [2-15N]guanosine derivative (15; 62%) and 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-6-chloro-2-[15N]phthalimido-9H-purine (17; 64%), respectively. Compounds 15 and 17 were then efficiently converted into 2',3',5'-tri-O-acetyl [2-15N]guanosine. The corresponding 2'-deoxy derivatives (16 and 18) were also synthesized through similar procedures.

Sensory nerve conduction velocities in the cutaneous afferents of the ulnar and peroneal nerves of the dog: tissue temperature-dependent reference ranges.

Sensory nerve conduction velocities in the cutaneous afferents of the ulnar and peroneal nerves of the neurologically normal adult dog were determined by stimulation at stimulus intensities of 15, 20 and 25 V through subcutaneously placed electrodes and by the averaged evoked response technique. Stimulus intensities of 15 V for the ulnar nerve and 20 V for the peroneal nerve were adequate to measure the sensory nerve conduction velocities of these nerves. A linear relationship was seen between sensory nerve conduction velocity (y in m/s) and tissue temperature (x in degree C) and the regression equations were expressed as follows: y = 1.6x + 12.3 at a stimulus intensity of 15 V for the ulnar nerve and y = 2.0x - 10.6 at 20 V for the peroneal nerve, respectively. The 95% confidence limits of the regressions of the sensory nerve conduction velocities against tissue temperature, obtained at a stimulus intensity of 15 V for the ulnar nerve or at a stimulus intensity of 20 V for the peroneal nerve, were proposed for a tissue temperature-dependent reference range to enable the clinician to evaluate graphically the sensory nerve conduction velocity in a diseased dog.

[Efficient synthesis of nucleosides labeled with stable isotopes and their application to structural biology].

1) A series of synthetic works on nucleosides appropriately labeled with stable isotopes of deuterium, carbon-13, and nitrogen-15 has been undertaken, confronting the strong demands for the nucleosides in the NMR spectroscopic study deeply related to structural biology, and the synthetic methods of (2'R)- and (2'S)-2'-deoxy[2'-2H]ribonucleosides, 2'-deoxy[5'-2H]ribonucleosides, [5'-13C]ribonucleosides, 2'-deoxy[5'-13C]ribonucleosides, 2'-deoxy[4-15N]cytidine, [4-15N]cytidine, 2'-deoxy[6-15N]adenosine, and [6-15N]adenosine were developed more efficiently than ever; some oligodeoxyribonucleotides were constructed by the use of these materials and found to be extraordinarily feasible for the NMR spectroscopic studies. 2) A novel approach to oligonucleotide synthesis on CPG has been established by the use of a base-labile protecting group, i.e., 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting group for the 5'-hydroxyl group of nucleoside 3'-phosphoramidites.

Synthesis of [5'-11C]ribonucleoside and -2'-deoyribonucleoside derivatives from D-ribose.

An approach to the synthesis of 2'-deoxy[5'-13C]ribonucleosides was achieved by the coupling reaction of a nucleic acid base derivative with D-[5-13C]ribose derivative (8). Compound 8 was derived from D-ribose (1) by way of methyl 2,3-di-O-benzyl(Bn)-D-ribofuranoside (2), 2,3-di-O-Bn-D-ribose diethyl dithioacetal (3), 2,3-di-O-Bn-D-ribose dibenzyl acetal (4), and 4-aldehydo-2,3-di-O-Bn-D-erythrose dibenzyl acetal (5), which was then successively subjected to Wittig reaction using Ph3P13CH3I-BuLi, highly stereoselective hydroxylation with OsO4 to give 2,3-di-O-Bn-D-ribose dibenzyl acetal (7), debenzylation with H2-Pd/C. The resulting 8 was subjected to coupling reaction with a nucleic acid base to give [5'-13C]ribonucleosides. The products were derived into the corresponding 2'-deoxy[5'-13C]ribonucleoside derivatives by the established manner.

Highly diastereoselective synthesis of 2'-deoxy[2'-2H]ribonucleoside derivatives by the use of tris(trimethylsilyl)[2H]silane.

The use of (Me3Si)3SiH or (Me3Si)3Si2H-Et3B system in place of Bu3SnH or Bu3Sn2H-Et3B system for a reductive protylation or deuteration reaction of 2'-Br-2'-deoxy-3',5'-O-TIPDS-[2'-2H]ribonucleosides or 2'-Br-2'-deoxy-3',5'-O-TIPDS-ribonucleosides was confirmed to improve the reactions to afford an excellent diastereoselectivity, i.e., the diastereoselectivity of (2'S)- or (2'R)-2'-deoxy-[2'-2H]ribonucleosides obtained by the reaction at 0 degree C is equivalent to the diastereoselectivity obtained by that using Bu3SnH or Bu3Sn2H-Et3B system at < -70 degrees C; the reductive deuteration of 2'-O-phenoxythiocarbonyl-3',5'-O-TIPDS-ribonucleosides through (Me3Si)3Si2H-Et3B system at 0 degree C also gave (2'R)-2'-deoxy[2'-2H]ribonucleosides with such a high diastereo-selectivity as above.

Difference of the 2',5'-oligoadenylate and the 3',5'-oligoadenylate in the formation of base pairings.

The 2',5'-oligoadenylates (tetramer, pentamer, and hexamer) were synthesized and their structural properties were compared with the corresponding 3',5'-oligoadenylates. Stacking effect observed with the 2',5'-oligoadenylates was weaker than that observed with the corresponding 3',5'-oligo-adenylates. With two polyuridylate (3',5') strands, a 2',5'-hexaadenylate formed a triple strand only in the presence of Mg++ion at room temperature, while a 3',5'-hexaadenylate did even without Mg++ion.

Regioselective phenylcarbamoylation of hydroxy-groups of ribonucleosides with bis(tributyltin) oxide--phenyl isocyanate system.

Partial phenylcarbamoylation of ribonucleosides by means of bis(tributyltin) oxide--phenyl isocyanate system is described herein; the reaction was found to occur regioselectively to give the corresponding 5'-, 3'-, and 2'-O-phenylcarbamoyl derivatives due to the conditions used.

Simple synthetic procedure for 2'- and 3'-tetrahydropyranyl ribonucleoside derivatives involving regioselective acylation with acyl chlorides, and synthesis of ribonucleotide oligomers.

Acylation of ribonucleosides through acyl chlorides in pyridine was induced with high regioselectivity under a controlled condition to give the corresponding 2',5'-diacylates in excellent yields and 2'-acylates, depending on the amount of the acyl chlorides used. On the other hand, a treatment of these resulting acylates on a silica gel (Wakogel C-300, Wako Pure Chemical Co. Ltd.) conducted their transformation into the corresponding 3',5'-diacylates or 3'-acylates effectively. These resulting 2',5'- and 3',5'-diacylates were further derived, by the known method, into the 3'- and 2'-O-tetrahydropyran-2-yl ribonucleoside derivatives, respectively, with which some ribonucleotide oligomer syntheses have been performed.

Oligonucleotide synthesis on a cellulose acetate functionalized with a spacer.

4-(2-Hydroxyethylsulfonyl)dihydrocinnamate derivative of cellulose acetate (D.S. 1.77) was used for polymer support synthesis of oligonucleotides bearing a phosphodiester function at the 3'-terminal position, taking into consideration that the cellulose derivative is readily soluble in pyridine, but slightly soluble in a solvent such as ethanol.

Preparation and heteronuclear 2D NMR spectroscopy of a DNA dodecamer containing a thymidine residue with a uniformly 13C-labeled deoxyribose ring.

[13C5]-2-Deoxy-D-ribose, synthesized from [13C6]-D-glucose (98% 13C), was coupled with thymine to give [1',2',3',4',5'-13C5]-thymidine (T) in an 18% overall yield. The thymidine was converted to the 3'-phosphoramidite derivative and was then incorporated into a dodecamer 5'-d(CGCGAATTCGCG)-3' by solid-phase DNA synthesis. Preparation of 0.24 mumole of the labeled dodecamer, which is sufficient for a single NMR sample, consumed only 25 mg of glucose. By virtue of the 13C labels, all of the 1H-1H vicinal coupling constants in the sugar moieties were accurately determined by HCCH-E.COSY.

Study on highly diastereoselective synthesis of (2'R)-2'-deoxy[2'-2H]guanosine.

To develop an efficient method for the synthesis of a highly diasteroselective (2'R)-2'-deoxy[2'-2H]guanosine (1), studies of organic chemical conversion from 2'-bromo-2'-deoxy-N2-Isobutyryl-3',5'-O-TIPDS-guanosine (2) to 1 and a biological transdeoxyribofuranosylation of (2'R > 98% de)-2'-deoxy[2'-2H]uridine (4) were carried out. As the results, a highly diastereoselective synthesis of 1 was achieved by a biological transdeoxyribofuranosylation between 2,6-diaminopurine and 4 by the use of Enterobacter aerogenes AJ-11125, followed by treatment with adenosine deaminase. The results will be described in detail.

Further improvement in protecting method for the oligonucleotide synthesis in terms of a cellulose acetate derivative as a polymer support.

For further improvement in the investigation to utilize a cellulose acetate derivative as a novel type of polymer-support for the synthesis of oligonucleotides, the investigations on utilizing another spacer; on protecting groups for O6-position of guanosine unit, ribothymidine, and pseudouridine; and on a novel protecting group for the introduction of phosphate function at 5'-terminal position, targeting the syntheses of 13-mer, ApApGpGpApApApApUpUpApUpG, 11-mer, pCpUpCpGpUpCpCpApCpCpA, and 12-mer, UpCpCpGpGprTp- psipCpGpApUpU, found in the partial structures of a yeast tRNA(Ala), will be described in detail.