Low-intensity pulsed ultrasound stimulates cell proliferation, proteoglycan synthesis and expression of growth factor-related genes in human nucleus pulposus cell line.

Low-intensity pulsed ultrasound (LIPUS) stimulation has been shown to effect differentiation and activation of human chondrocytes. A study involving stimulation of rabbit disc cells with LIPUS revealed upregulation of cell proliferation and proteoglycan (PG) synthesis. However, the effect of LIPUS on human nucleus pulposus cells has not been investigated. In the present study, therefore, we investigated whether LIPUS stimulation of a human nucleus pulposus cell line (HNPSV-1) exerted a positive effect on cellular activity. HNPSV-1 cells were encapsulated in 1.2% sodium alginate solution at 1x10(5) cells/ml and cultured at 10 beads/well in 6-well plates. The cells were stimulated for 20 min each day using a LIPUS generator, and the effects of LIPUS were evaluated by measuring DNA and PG synthesis. Furthermore, mRNA expression was analyzed by cDNA microarray using total RNA extracted from the cultured cells. Our study revealed no significant difference in cell proliferation between the control and the ultrasound treated groups. However, PG production was significantly upregulated in HNPSV cells stimulated at intensities of 15, 30, 60, and 120 mW/cm(2) compared with the control. The results of cDNA array showed that LIPUS significantly stimulated the gene expression of growth factors and their receptors (BMP2, FGF7, TGFbetaR1 EGFRF1, VEGF). These findings suggest that LIPUS stimulation upregulates PG production in human nucleus pulposus cells by the enhancement of several matrix-related genes including growth factor-related genes. Safe and non-invasive stimulation using LIPUS may be a useful treatment for delaying the progression of disc degeneration.

Healing of burn wounds in transgenic mice overexpressing transforming growth factor-beta 1 in the epidermis.

Transforming growth factor-beta (TGF-beta) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-beta in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-beta on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-beta in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-beta. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-beta 1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-beta 1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO(2) laser. Using this model, we demonstrated activation of latent TGF-beta after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-beta1 gene into keratinocytes markedly increases the release and activation of TGF-beta after burn injury. Elevated local TGF-beta significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls, P < 0.05) and homozygous (25% versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.

Flavonol glycosides from Asplenium foreziense and its five related taxa and A. incisum.

The flavonoids of Asplenium foreziense, A. fontanum subsp. fontanum and subsp. pseudofontanum, A. obovatum subsp. obovatum var. obovatum and var. protobillotii, A. obovatum subsp. lanceolatum, and A. incisum were isolated and identified for chemotaxonomic survey. A major constituent of all taxa was kaempferol 3-O-gentiobioside. As minor compounds, kaempferol 3,7-O-glycoside and/or kaempferol 3-O-glycoside were found in A. fontanum, A. obovatum and A. foreziense, and kaempferol 3-O-gentiobioside-4'-O-glucoside, kaempferol 3-O-glucoside and quercetin 3-O-diglucoside in A. incisum. It was suggested that A. foreziense, A. fontanum including subsp. pseudofontanum and A. obovatum including subsp. lanceolatum are not only morphologically but also chemotaxonomically related. The East Asian A. incisum was chemically and geographically different from these taxa.

Chalcone and flavonol glycosides from Asarum canadense (Aristolochiaceae).

Two chalcone glycosides were isolated, together with seven known flavonol glycosides, from the leaves of Asarum canadense. The structures of the chalcone glycosides were established as chalcononaringenin 2',4'-di-O-glucoside and chalcononaringenin 2'-O-glucoside-4'-O-gentiobioside by chemical, UV, FAB MS, 1H and 13C NMR evidence.

Transforming growth factor-beta in thermally injured patients with hypertrophic scars: effects of interferon alpha-2b.

Hypertrophic scarring is a common dermal fibroproliferative disorder that leads to poor quality wound healing, prolongs rehabilitation, and increases morbidity following major thermal and other injuries to the deep dermis. Local and systemic transforming growth factor (TGF)-beta has been implicated as a fibrogenic cytokine in the pathogenesis of many fibrotic disorders, whereas interferon (IFN) alpha-2b may improve the pathologic features of dermal fibrosis directly or by antagonizing the effects of TGF-beta and histamine. Nine patients with severe hypertrophic scarring were evaluated for 8 weeks before treatment with subcutaneous recombinant IFN alpha-2b, 2 x 10(6) IU three times per week for 24 weeks. Clinical assessment was performed using standardized photography, a burn scar assessment tool, and serial scar volume measurements. Monthly measurements of serum TGF-beta and plasma Ntau-methylhistamine were made prior to, during, and after IFN alpha-2b therapy and compared with 27 age-matched controls. Serial biopsies of the hypertrophic scars and normal skin were performed for evaluation of mast cell numbers. Significant improvement in scar assessment occurred in 7 of 9 patients, and 3 of 9 demonstrated significant reductions in scar volume with interferon therapy beyond that occurring during the 8-week control period. For the entire group, mean rates of improvement were significantly better during interferon therapy with no recurrence following treatment. Before interferon therapy, serum TGF-beta was significantly higher in the burn patients with hypertrophic scarring than in a control population (123.04 +/- 36.48 vs. 56.85 +/- 8.38 ng/ml, p < 0.05). Within 3 months of IFN alpha-2b therapy, serum TGF-beta levels fell significantly and remained within the normal range during therapy and after interferon therapy was stopped. Plasma Ntau-methylhistamine levels were also significantly elevated in the hypertrophic scar patients as compared with age and sex-matched controls (153.6 +/- 92.07 vs. 48.3 +/- 28.9 pg/ml, p < 0.05), and significant reductions were achieved with interferon therapy and maintained after interferon was discontinued. Paired biopsies of hypertrophic scarring and normal tissue demonstrated increased numbers of mast cells in hypertrophic scars compared with normal uninjured skin from the same patients (2.65 +/- 1.63 vs. 1.04 +/- 0.62 cells/high power field, p < 0.001); however, no significant change in mast cell content of the hypertrophic scars accompanied interferon therapy. Patients with severe hypertrophic scarring demonstrate increased levels of serum TGF-beta and plasma Ntau-methylhistamine following thermal injury. A significant clinical improvement in scar quality and volume occurred during IFN alpha-2b therapy, which was associated with normalization of serum TGF-beta and plasma Ntau-methylhistamine levels. A double-blind, placebo-controlled trial will be required to further assess the usefulness of subcutaneous treatment with IFN alpha-2b for the treatment of hypertrophic scarring.

IFN-alpha2b suppresses the fibrogenic effects of insulin-like growth factor-1 in dermal fibroblasts.

The interferon (IFN) proteins, including IFN-alpha2b have been used as antifibrogenic factors to modulate the expression of extracellular matrix (ECM) proteins associated with fibroproliferative disorders in skin. This study was conducted to determine if IFN-alpha2b can counteract the fibrogenic effects of insulin-like growth factor-1 (IGF-1), which is present in large quantity in fibrotic dermis. Human dermal fibroblasts were established in culture and treated with either vehicle (control), 2000 U/ml IFN-alpha2b alone, 100 ng/ml IGF-1 alone, or both IFN-alpha2b and IGF-1. The results showed that treatment with IFN-alpha2b inhibited the proliferation of dermal fibroblasts, reduced the steady-state levels of type I procollagen mRNA in the cells, and reduced the production of collagen as measured by hydroxyproline in conditioned medium. However, this treatment also increased levels of collagenase mRNA in the cells and collagenase activity in the medium. Cells treated with IGF-1 showed increased proliferation and collagen production and decreased collagenase. Cells treated with both IFN-alpha2b and IGF-1 exhibited a 44% reduction in hydroxyproline production (p < 0.05) and a 363% increase in collagenase activity over cells treated with IGF-1 alone (p < 0.01). These results indicate that when IGF-1 and IFN-alpha2b are used individually, they function as fibrogenic and antifibrogenic factors for dermal fibroblasts, respectively, and that fibrogenic effects of IGF-1 on cell proliferation, collagen, and collagenase expression can be counteracted by IFN-alpha2b. These findings support the potential use of IFN-alpha2b as a therapeutic agent for treatment of fibroproliferative disorders, such as postburn hypertrophic scarring.

Determination of plasma Ntau-methylhistamine in vivo by isotope dilution using benchtop gas chromatography-mass spectrometry.

A practical sensitive and specific method for determination of the stable metabolite of histamine, Ntau-methylhistamine, in human plasma using benchtop gas chromatography-stable isotope dilution mass spectrometry has been developed. Ntau-Methylhistamine, a principal metabolite of histamine in humans, was extracted and purified from human plasma using a two-step procedure with Sep-Pak silica cartridges. Quantitation of Ntau-methylhistamine was made possible by the synthesis of Ntau-[2H3]methylhistamine used as an internal standard. Derivatization with pentafluoropropionyl anhydride of extracts of human plasma yielded the bis-pentafluoropropionyl derivative of Ntau-methylhistamine for measurement using selected ion monitoring of the m/z 417/420 ion pair after electron impact on a benchtop gas chromatography-mass spectrometry (GC-MS). By improvements in the plasma extraction technique, inclusion of a synthetic internal standard and the development of a sensitive and stable derivative of the histamine metabolite, Ntau-methylhistamine was found to be significantly elevated in the plasma of patients with the dermal fibroproliferative disorder, hypertrophic scarring as compared to age-matched normal volunteers (98.5+/-29.5 pg/ml, n=9, versus 43.3+/-16.5 pg/ml, n=8, p<0.05). As such, this method affords a sensitive, specific and practical approach to measurement of histamine metabolites in plasma and other biological fluids.

Synthesis of tritium-labelled N tau-methylhistamine for the improvement of extraction efficiency of N tau-methylhistamine from biological fluids.

In order to trace the loss of N tau-methylhistamine, a principal metabolite of histamine, during extraction and purification from human plasma and urine samples, N tau-[3H]methylhistamine was prepared in two steps from N alpha t-butoxycarbonylhistamine (II). In the first step, compound II was deprotonated with NaH in an aprotic solvent and treated with [3H]methyl iodide. The products, N alpha t-butoxycarbonyl-N tau-[3H]methylhistamine (III) and N alpha t-butoxycarbonyl-N pi-[3H]methylhistamine (IV), were then hydrolysed with iodotrimethylsilane under mild and short reaction conditions. Facile purification with Sep-Pak silica cartridges gave the combined two isomers of N tau-[3H]methylhistamine and N pi-[3H]methylhistamine in 10.7% radiochemical yield with a radiochemical purity of > 94% and a ratio of approximately 2:1. Improvements in the extraction of methylhistamine using chromatography on Sep-Pak silica cartridges led to an overall recovery of 82.5 +/- 0.3% (n = 3) based upon total [3H]methylhistamine from normal human plasma.

The synthesis of N-acetyl-4-S-cysteaminyl [U-14C]phenol as a basis for the development of an antimelanoma and melanoma-radioimaging agent.

This study reports the synthesis of 14C-labelled N-acetyl-4-S-cysteaminylphenol ([14C]N-Ac-4-S-CAP), a potent melanocytotoxic and anti-melanoma agent. A reaction of [U-14C]phenol with NH2CH2CH2S+ cation in HBr solution was used for the synthesis of (4-S-cysteaminyl[14C]phenol[14C]4-S-CAP). The excess cystamine was removed, and [14C]4-S-CAP and 2-S-cysteaminyl[14C]phenol ([14C]2-S-CAP) were separated on preparative thin layer chromatography. [14C]4-S-CAP was acetylated with acetic anhydride in pyridine and subsequently O-deacetylated with NH3 in methanol, resulting [14C]N-Ac-4-S-CAP (3.8% radiochemical yield; a sp. act. of 450 kBq/mumol; radiochemical purity > 99%).

Selective in vivo accumulation of N-acetyl-4-S-cysteaminylphenol in B16F10 murine melanoma and enhancement of its in vitro and in vivo antimelanoma effect by combination of buthionine sulfoximine.

In order to develop a new chemotherapeutic agent based on exploitation of the specific metabolic pathway of malignant melanoma, a phenolic thioether, N-acetyl-4-S-cysteaminylphenol (NA-CAP), the substrate of melanin-forming enzyme, tyrosinase was developed. Our previous in vivo studies have clearly shown that this compound has a significant and selective melanocytotoxicity and antimelanoma effect. This study further examined the specificity of the antimelanoma effect of NA-CAP through the study of biodistribution and accumulation of NA-CAP in B16F10 melanoma-bearing mice. We also tested the antimelanoma effect of NA-CAP by combination treatment with buthionine sulfoximine on the growth of in vitro culture cells and in vivo B16F10 melanoma lung colonies. We found a selective accumulation of 14C-labeled NA-CAP into s.c. transplants and lung colonies of melanoma grown in C57BL mice. This accumulation was mediated by selective covalent binding of NA-CAP to the melanoma tissues. The combination of NA-CAP and buthionine sulfoximine significantly increased the chemosensitivity of B16F10 melanoma cells in vitro and reduced the number of in vivo melanoma lung colonies. We conclude that NA-CAP acts as an alkylating agent to melanoma tissue and that the combination of buthionine sulfoximine enhances the therapeutic index of this potent melanoma-specific drug through the depletion of tissue glutathione.

Exploitation of pigment biosynthesis pathway as a selective chemotherapeutic approach for malignant melanoma.

Human malignant melanoma represents a difficult therapeutic challenge to both medical scientists and practicing physicians. However, the biologic uniqueness of the tumor may provide opportunities for exploitation in therapeutics. This study proposed to undertake a systemic approach to the chemotherapy of malignant melanoma based upon the uniqueness of pigment-cell metabolic pathway pertaining to conversion of tyrosine and dopa with subsequent formation of melanin by tyrosinase and its related enzymes. The sulphur homologue of tyrosine, cysteinylphenol (CP), its amine derivative, cysteaminylphenol (CAP), and their N-acetyl and alpha-methyl derivatives have been synthesized and tested in in vivo and in vitro melanocytotoxicity and antimelanoma effects. These phenolic thioethers (PTEs) and phenolic thioether amine (amides) (PTEAs), which are substrates of tyrosinase, showed significant cytotoxicity that is selective to melanocytes and melanoma cells. Most previous attempts to impair the melanin pathway as a therapeutic strategy have been of limited success because they have been directed to catecholic compounds that are unstable and insufficient in lethality at physiologically tolerable doses. By contrast, our approach relies on phenolic compounds, PTEs and PTEAs, which are more stable than catechols and become toxic only after oxidation by tyrosinase. We found PTEA as the most promising agent for the future development of chemotherapeutic agents. The possible biologic, chemical, and pharmacologic reactions of these synthetic compounds within the melanoma cells are studied and discussed.

Drug-induced perturbations in the in vivo distribution of oncological radiotracers--II. 5-[125I]iodo-2'-deoxyuridine influenced by nitrobenzylthioinosine-5'-phosphate (NBMPR-P) and acyclothymidine (ACT).

Nitrobenzylthioinosine (NBMPR), a potent inhibitor of facilitated nucleoside transport in vitro and in vivo, and acyclothymidine (ACT), a potent inhibitor of pyrimidine nucleoside phosphorylase in vitro, have been used in an attempt to modulate the biodistribution of 125I-labelled iododeoxyuridine ([125I]IUdR). ACT or NBMPR-P (a water-soluble prodrug of NBMPR) were injected into BDF1 mice bearing implanted Lewis lung tumors, according to protocols which would provide high and low plasma levels of the inhibitor. Compared with controls, both inhibitors induced transient, marginal increases in hepatic, renal and blood levels of [125I]IUdR, and decreased levels in tumors at short time intervals after injection. It is concluded that there is a mild tumor-sparing effect when either NBMPR or ACT are administered together with single i.v. diagnostic doses of [125I]IUdR.

Flavone glycosides from Asplenium normale.

Eight flavone glycosides were isolated from fronds of Asplenium normale, its two varieties, var. boreale and var. shimurae, and related species, A. oligophlebium. Six of the glycosides were identified: apigenin 7-O-dirhamnoside and 7-O-glucosylrhamnoside, luteolin 7-O-dirhamnoside and 7-O-glucosylrhamnoside, genkwanin 4'-O-glucosylrhamnoside, and vicenin-2. The remaining two glycosides were tentatively characterized as genkwanin 4'-O-glycoside and 6,8-di-C-glycosylluteolin. The four taxa analysed in this survey all had distinctive flavonoid profiles.

Synthesis and antiviral activity of IVFRU, a potential probe for the non-invasive diagnosis of herpes simplex encephalitis.

Synthesis of (E)-5-(2-iodovinyl)-1-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)uracil (IVFRU; 4a), and its [125I] and [131I] derivatives (4b and 4c) are described. Compared with IVDU, IVFRU had comparable antiviral activity (MIC50 = 0.01-0.1 microgram/ml), and a greater ethanol/water partition coefficient; its affinity for the murine erythrocyte nucleoside transporter system was greater than that of 2'-deoxyuridine. The [125I] derivative (4b) was selectively trapped within rabbit kidney cells (27.9 and 41.2% at 4 and 24 hr, respectively) infected in vitro by thymidine kinase-positive (TK+) herpes simplex virus (HSV-1), but not within either HSV (TK-) or mock-infected cells where uptake was less than 1%. It was resistant to glycosidic bond cleavage by pyrimidine nucleoside phosphorylases, but underwent catabolism to an unidentified metabolite and iodide during in vivo plasma and urinary excretion studies in mice. We conclude that the [123I] derivative of IVFRU warrants further evaluation as a non-invasive radiopharmaceutical agent for the diagnosis of herpes simplex encephalitis (HSE), and that IVFRU warrants further evaluation as an antiviral agent.

Pharmacokinetics and metabolism of E-5-(2-[131I]iodovinyl)-2'-deoxyuridine in dogs.

E-5-(2-Iodovinyl)-2'-deoxyuridine (IVdU) is a potent inhibitor of herpes simplex virus type 1 replication in vitro. The selective antiviral activity of IVdU is due to preferential phosphorylation by the herpes simplex virus type 1-encoded thymidine kinase. This selective sequesteration provided the rationale for the development of radioiodinated IVdU as a potential radiopharmaceutical compound for use in noninvasive diagnosis of herpes simplex virus encephalitis. We studied the pharmacokinetics and the in vivo metabolism of [131I]IVdU in dogs. The radioactive components in plasma were characterized and quantitated by radio high-pressure liquid chromatography. During incubation with dog blood, [131I]IVdU was metabolized to the corresponding base (E)-5-(2-iodovinyl)uracil. 131I-labeled (E)-5-(2-iodovinyl)uracil accounted for 73% of the total radioactivity present in plasma after 2 h of incubation, suggesting that phosphorolysis of the nucleoside is the major degradation pathway of IVdU in blood. The in vivo studies showed that there was an initial rapid clearance of the tracer from blood, followed by a second very slow clearance phase. Evaluation of the renal excretion of the radiotracer showed that only 8% of the injected dose was excreted by kidneys over an 8-h period. IVdU was rapidly metabolized to three radioactive compounds. Two of these metabolites, the base (E)-5-(2-iodovinyl)uracil and iodide, were characterized. The radioactivity associated with these metabolites was responsible for the slow clearance phase. Our results suggest that the development of [131I]IVdU as a radiopharmaceutical compound will require measures to prevent its rapid degradation in vivo.

Tumor uptake of radiolabelled pyrimidine bases and pyrimidine nucleosides in animal models--VIII. Synthesis and tissue distribution of N-[2-(hydroxyethoxy) methyl]-5-[3H]methyluracil.

The tritium-labelled acyclonucleoside, N-[2-(hydroxyethoxy)methyl]-5-[3H]methyluracil (3H-3), was synthesized for evaluation as a tumor diagnostic agent. 5-[3H]-Methyluracil, 3H-1, was converted to the 2,4-bis-trimethylsilyl intermediate which was coupled with 2-acetoxyethoxymethyl bromide to afford 1-[(2-acetoxyethoxy)methyl-5-[3H]methyluracil (3H-2). Treatment of 3H-2 with sodium methoxide in methanol afforded 3H-3 (specific activity 188 MBq mmol-1. The tissue distribution of 3H-3 was examined in male BDF1 mice bearing Lewis Lung (LL) carcinomas. Long bone exhibited the highest tumor: tissue ratios. The kidney contained the highest radioactivity level relative to the tumor. This suggested a major urinary route of excretion. The major radioactive blood component (89.21%) was found to have a biological half-life of 0.19 min. The title compound is unsuitable for use as a diagnostic agent for LL carcinoma because of low tumor uptake and rapid urinary elimination of injected radioactivity from the body.