Role of nerve growth factor in cutaneous wound healing: accelerating effects in normal and healing-impaired diabetic mice.

Four full-thickness skin wounds made in normal mice led to the significant increase in levels of nerve growth factor (NGF) in sera and in wounded skin tissues. Since sialoadenectomy before the wounds inhibited the rise in serum levels of NGF, the NGF may be released from the salivary gland into the blood stream after the wounds. In contrast, the fact that messenger RNA and protein of NGF were detected in newly formed epithelial cells at the edge of the wound and fibroblasts consistent with the granulation tissue produced in the wound space, suggests that NGF was also produced at the wounded skin site. Topical application of NGF into the wounds accelerated the rate of wound healing in normal mice and in healing-impaired diabetic KK/Ta mice. This clinical effect of NGF was evaluated by histological examination; the increases in the degree of reepithelialization, the thickness of the granulation tissue, and the density of extracellular matrix were observed. NGF also increased the breaking strength of healing linear wounds in normal and diabetic mice. These findings suggested that NGF immediately and constitutively released in response to cutaneous injury may contribute to wound healing through broader biological activities, and NGF improved the diabetic impaired response of wound healing.

Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytes.

The influence of transglutaminase 2 (TG2) activity on the proliferative effect of epidermal growth factor (EGF) and on EGF receptor affinity in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) has been investigated using a primary culture system. PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF-induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high-affinity EGF receptor expression was markedly up-regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF-induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high-affinity EGF receptor was down-regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down-regulation of high-affinity EGF receptors.

Effect of Propionibacterium acnes on the cellular immune responses to tumor-specific antigens on malignant rat liver cells.

The effect of Propionibacterium acnes on the cellular immune responses to tumor-specific membrane antigens was investigated by microcytotoxicity assays (MA) and 51Cr release assays (CRA) with use of mesenteric lymph node cells (LNC) of syngeneic BD IV and BD VI rats. BD rat liver cell lines transformed in vitro by chemical carcinogens were used as target cells with tumor-specific antigens. By MA, the LNC from rats that were inoculated with malignant liver cells under the adjuvant effect of heat-killed P. acnes showed significant cytotoxic response to the target cells but not to nonmalignant liver cells. By CRA, these LNC did not show specific cytolysis to the malignant liver cells. Assays with various target cells derived from BD rat liver and inhibition tests with syngeneic and xenogeneic antisera against tumor-specific antigens on the malignant liver cells proved that LNC reacted with tumor-specific individual or tumor-specific cross-reacting antigens on the malignant liver cells. Cytotoxic responses against the malignant liver cells were not demonstrated even by MA with use of the LNC from rats inoculated with either the malignant liver cells or P. acnes alone. LNC from the rats inoculated with both nonmalignant liver cells and P. acnes were not cytotoxic to malignant or nonmalignant liver cell lines.

Propionibacterium acnes-mediated humoral immune responses to tumor-specific antigens on rat liver cells transformed in vitro by chemical carcinogens.

Effects of Propionibacterium acnes on production of antibodies against tumor-specific membrane antigens were investigated in syngeneic inbred BD IV and BD VI rats. BD rat liver cell lines transformed in vitro by chemical carcinogens were used as target cells for tumor-specific antigens. By membrane immunofluorescence, antibodies against these rat liver cell lines were detected in syngeneic BD rat sera. Antibodies were produced in syngeneic rats under the adjuvant effect of heat-killed P. acnes only. In assays with various target cells and absorption experiments, the antibodies reacted with a tumor-specific individual antigen or tumor-specific cross-reacting antigen on the surfaces of transformed BD rat liver cells. No antibodies against these antigens were found in the sera obtained from syngeneic rats immunized with either the transformed cell lines or P. acnes but not with both. Freund's complete adjuvant did not induce antibodies against these tumor-specific antigens.

Emerging ST121/agr4 community-associated methicillin-resistant Staphylococcus aureus (MRSA) with strong adhesin and cytolytic activities: trigger for MRSA pneumonia and fatal aspiration pneumonia in an influenza-infected elderly.

The pathogenesis of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pneumonia in influenza-infected elderly individuals has not yet been elucidated in detail. In the present study, a 92-year-old man infected with influenza developed CA-MRSA pneumonia. His CA-MRSA was an emerging type, originated in ST121/agr4 S. aureus, with diversities of Panton-Valentine leucocidin (PVL)(-)/spat5110/SCCmecV(+) versus PVL(+)/spat159((etc.))/SCCmec (-), but with common virulence potentials of strong adhesin and cytolytic activities. Resistance to erythromycin/clindamycin (inducible-type) and gentamicin was detected. Pneumonia improved with the administration of levofloxacin, but with the subsequent development of fatal aspiration pneumonia. Hence, characteristic CA-MRSA with strong adhesin and cytolytic activities triggered influenza-related sequential complications.

Different responses to some stimulators of prolyl hydroxylase activities in various rat organs.

Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) of soluble fraction (105 000 X g supernatant) of rat granulation tissues was markedly enhanced by addition of nucleoside triphosphates to the assay medium. But the stimulatory activities of nucleoside triphosphates were very different in fractions derived from tissues of rat. In skin, lung or whole fetal tissues other than granuloma, GTP enhanced the enzymatic activity by 3-4 fold. On the other hand, in kidney, liver and spleen tissues it brought about no enhancement. The same results were obtained even if ATP regenerating system was added in the assay medium. The stimulatory effect of nucleoside triphosphates was not seen with the soluble fraction of liver, but it appeared with the enzyme fraction purified by affinity column chromatography. The same phenomenon was observed by addition of bovine serum albumin instead of nucleoside triphosphates as stimulator. We discuss the possible reasons as to why the responses of the enzyme to stimulators were quite different among various tissues.

Cloning and structural determination of human peptide YY cDNA and gene.

We have isolated two kinds of cDNAs and the gene encoding human peptide YY and determined their nucleotide sequences. The human peptide YY gene is composed of four exons and three introns spanning approx. 1.2 kbp. Two mRNA species are generated from the gene by alternative splicing of the third intron.

Biological activities of 24-fluoro-1 alpha,25-dihydroxyvitamin D-2 and its 24-epimer.

Biological activities of two epimeric 24-fluorinated vitamin D-2 analogs, 24-fluoro-1 alpha,25-dihydroxyvitamin D-2 [24-F-1,25-(OH)2D2] and its 24-epimer [24-epi-24-F-1,25-(OH)2D2], were studied and compared with 1 alpha,25-dihydroxyvitamin D-3 [1,25-(OH)2D3] and 1 alpha,25-dihydroxyvitamin D-2 [1,25-(OH)2D2]. 24-F-1,25-(OH)2D2 was nearly as active as 1,25-(OH)2D3 and 1,25-(OH)2D2 both in regulating calcium metabolism in vivo including bone mineral mobilization and intestinal calcium transport and in inducing differentiation of HL-60 cells. While 24-epi-24-F-1,25-(OH)2D2 showed distinct properties in these two types of the actions. Though the 24-epimer was nearly as potent as 1,25-(OH)2D3 in inducing differentiation of HL-60 cells, it showed little activity in regulating calcium metabolism in vivo. The fluorine atom introduced at the 24-position of either 1,25-(OH)2D2 or its 24-epimer had no potentiating effect. This is in sharp contrast with the cases of 24- and 26,27-multifluorinated analogs of active vitamin D-3.

Preliminary cryocrystallography analysis of an eumenine mastoparan toxin isolated from the venom of the wasp Anterhynchium flavomarginatum micado.

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 A resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 A. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation.

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis.

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

Synthesis and biological evaluation of all A-ring stereoisomers of 5,6-trans-2-methyl-1,25-dihydroxyvitamin D(3) and their 20-epimers: possible binding modes of potent A-ring analogues to vitamin D receptor.

BACKGROUND: The secosteroid 1 alpha,25-dihydroxyvitamin D(3) (1) has a wide variety of biological activities, which makes it a promising therapeutic agent for the treatment of cancer, psoriasis and osteoporosis. Insight into the structure-activity relationships of the A-ring of 1 is still needed to assist the development of more potent and selective analogues as candidate chemotherapeutic agents, as well as to define the molecular mode of action. RESULTS: All possible A-ring stereoisomers of 5,6-trans-2-methyl-1,25-dihydroxyvitamin D(3) (6a-h) and their 20-epimers (7a-h) were designed and efficiently synthesized. The dependence of the affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP), as well as the HL-60 cell differentiation-inducing activity, upon the stereochemistry of the A-ring and at C20 in the side chain was evaluated. CONCLUSIONS: The binding affinities and potency of the 5,6-trans and 5,6-cis analogues were enhanced by a 2-methyl substituent in a certain orientation. Molecular docking studies based upon the X-ray crystal structure of VDR suggested that the axial 2-methyl group would be accommodated in a pocket surrounded by hydrophobic amino acid residues in the ligand binding domain, resulting in enhanced interaction.

Stimulation of granulocytic cell iodination by pine cone antitumor substances.

Antitumor substances (Fractions VI and VII) prepared from the NaOH extract of pine cone significantly stimulated the iodination (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood adherent mononuclear cells, polymorphonuclear cells (PMN), and human promyelocytic leukemic HL-60 cells. In contrast, these fractions did not significantly increase the iodination of nonadherent mononuclear cells, red blood cells, other human leukemic cell lines (U-937, THP-1, K-562), human diploid fibroblast (UT20Lu), or mouse cell lines (L-929, J774.1). Iodination of HL-60 cells, which were induced to differentiate by treatment with either retinoic acid or tumor necrosis factor, were stimulated less than untreated cells. The stimulation of iodination of both PMN and HL-60 cells required the continuous presence of these fractions and was almost completely abolished by the presence of myeloperoxidase inhibitors. The stimulation activity of these fractions was generally higher than that of various other immunopotentiators. Possible mechanisms of extract stimulation of myeloperoxidase-containing cell iodination are discussed.

Stimulation of human peripheral blood polymorphonuclear cell iodination by lignin-related substances.

Lignin is a heterogenous natural product composed of phenylpropane units and is usually associated with hemicellulose in its native state. Until now little attention has been paid to the potential therapeutic utility of lignified products. Natural lignified products are demonstrated in the present study to stimulate iodination significantly (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood polymorphonuclear cells (PMN). This stimulation was significantly inhibited in the presence of myeloperoxidase inhibitors. These materials were almost completely deprived of their stimulation capacity by treatment with NaCIO2, but this capacity was not affected by severe treatment with H2SO4 or trifluoroacetic acid. Similar stimulating activity by chemically defined tannin-related polyphenolic compounds was observed. Degradation products or component units of lignin, and natural antitumor polysaccharides and their chemically modified derivatives (introduced with negatively or positively charged groups) and polysialoglycoproteins had little or no activity. The results indicate the importance of a polymerized phenolic structure for the stimulation of PMN iodination. Possible physiological relevance of the stimulation of iodination by lignified substances is discussed.

Coffee and caffeine and alcohol effects on psychomotor function.

Interactions of alcohol and caffeine were studied in two double-blind and crossover trials in which several psychophysiologic functions and subjective effects were measured in healthy men. The effects of alcohol (1 gm/kg) with and without caffeine (200 or 500 mg) were measured in 10 subjects. Two doses (0.7 or 1.5 gm/kg) of alcohol alone and in combination with caffeine (250 + 250 mg) were similarly studied in another 10 subjects. Alcohol impaired psychomotor functions to an extent dependent on dose whereas caffeinated and decaffeinated coffee did not. Both kinds of coffee also failed to modify alcohol effects. Subjectively, caffeine was indistinguishable from placebo and no particular interaction of alcohol and caffeine was detected. Alcohol did elevate serum caffeine concentrations. We conclude that coffee does not counteract alcohol inebriation.

Effects of alkyl substitutions of xanthine skeleton on bronchodilation.

Structure-activity relationships in a series of 1,3,7-trialkyl-xanthine were studied with guinea pigs. Relaxant actions in the tracheal muscle were increased with alkyl chain length at the 1- and 3-positions of the xanthine skeleton, but decreased by alkylation at the 7-position. Positive chronotropic actions in the right atrium were potentiated with 3-alkyl chain length but tended to decrease with 1-alkylation and diminish by 7-substitution. Consequently, while the 1- and 3-substitutions were equally important for the tracheal smooth muscle relaxation, the substitution at the 1-position was more important than the 3-substitution for bronchoselectivity. The 7-alkylation may be significant to cancel heart stimulation. There were good correlations between the smooth muscle relaxant action and the cyclic AMP-PDE inhibitory activity in 3-substituents and the affinity for adenosine (A1) receptors in 1-, 3-, and 7-substituents. This suggests that not only the cyclic AMP-PDE inhibitory activity but also the adenosine antagonistic activity is important in the bronchodilatory effects of alkylxanthines. Among these xanthine derivatives, 1-butyl-3-propylxanthine and its 7-methylated derivative showed high bronchoselectivity in the in vitro and in vivo experiments compared to theophylline and enprofylline and may be new candidates for bronchodilator.

Bronchodilator activity of xanthine derivatives substituted with functional groups at the 1- or 7-position.

Xanthine derivatives with several functional groups at the 1- or 7-position were synthesized, and their pharmacological activities in guinea pigs were studied. In general, the in vitro tracheal relaxant action and positive chronotropic action of 3-propylxanthines were increased by substitutions with nonpolar functional groups at the 1-position, but decreased by any substitution at the 7-position. On the other hand, because positive chronotropic actions of substituents with allyl, aminoalkyl, alkoxyalkyl, and normal alkyl groups were much less than tracheal muscle became very high with substitutions of 3'-butenyl, (dimethylamino)ethyl, 2'-ethoxyethyl, 3'-methoxypropyl, and n-propyl groups at the 1-position and of 2'-ethoxyethyl, 2'-oxopropyl, and n-propyl groups at the 7-position, compared with theophylline and the corresponding unsubstituted xanthines, 3-propylxanthine and 1-methyl-3-propylxanthine. When compounds were intraduodenally administered to the guinea pig, 1-(2'-ethoxyethyl)-, 1-(3'-methoxypropyl)-, 1-(3'-butenyl)-, and 1-[(dimethylamino)-ethyl]-3-propylxanthines, 1-methyl-7-(2'-oxopropyl)-3-propylxanthine, and denbufylline (1,3-di-n-butyl-7-(2'-oxopropyl)xanthine) effectively inhibited the acetylcholine-induced bronchospasm without heart stimulation or central nervous system-stimulation at the effective dosage range. Particularly, the bronchodilatory effect of 1-(2'-ethoxyethyl)-3-propylxanthine was much stronger and more continuous than those of theophylline and pentoxifylline. On the other hand, there were certain relationships among the in vitro tracheal relaxant activities of these compounds, their affinities for adenosine (A1) receptors in the brain membrane, and their inhibition of cyclic AMP-phosphodiesterase (PDE) in the tracheal muscle. The affinity for A2 receptors of these compounds was very low or negligible. This suggests that both the action on A1 receptors or interaction with adenosine and the cyclic AMP-PDE inhibitory activity contribute to the bronchodilator action of 1- and 7-substituted xanthines. This study indicates that the substitutions with none or low polar functional groups at the 1-position could improve the selectivity and duration of the bronchodilator effects of xanthines.

Synthesis, biological evaluation, and conformational analysis of A-ring diastereomers of 2-methyl-1,25-dihydroxyvitamin D(3) and their 20-epimers: unique activity profiles depending on the stereochemistry of the A-ring and at C-20.

All eight possible A-ring diastereomers of 2-methyl-1, 25-dihydroxyvitamin D(3) (2) and 2-methyl-20-epi-1, 25-dihydroxyvitamin D(3) (3) were convergently synthesized. The A-ring enyne synthons 19 were synthesized starting with methyl (S)-(+)- or (R)-(-)-3-hydroxy-2-methylpropionate (8). This was converted to the alcohol 14 as a 1:1 epimeric mixture in several steps. After having been separated by column chromatography, each isomer led to the requisite A-ring enyne synthons 19 again as 1:1 mixtures at C-1. Coupling of the resulting A-ring enynes 20a-h with the CD-ring portions 5a,b in the presence of a Pd catalyst afforded the 2-methyl analogues 2a-h and 3a-h in good yield. In this way, all possible A-ring diastereomers were synthesized. The synthesized analogues were biologically evaluated both in vitro and in vivo. The potency was highly dependent on the stereochemistry of each isomer. In particular, the alpha alpha beta-isomer 2g exhibited 4-fold higher potency than 1 alpha,25-dihydroxyvitamin D(3) (1) both in bovine thymus VDR binding and in elevation of rat serum calcium concentration and was twice as potent as the parent compound in HL-60 cell differentiation. Furthermore, its 20-epimer, that is, 20-epi-alpha alpha beta 3g, exhibited exceptionally high activities: 12-fold higher in VDR binding affinity, 7-fold higher in calcium mobilization, and 590-fold higher in HL-60 cell differentiation, as compared to 1 alpha,25-dihydroxyvitamin D(3) (1). Accordingly, the double modification of 2-methyl substitution and 20-epimerization resulted in unique activity profiles. Conformational analysis of the A-ring by (1)H NMR and an X-ray crystallographic analysis of the alpha alpha beta-isomer 2g are also described.

Mucociliary clearance mechanism in smoking and nonsmoking normal subjects.

Mucociliary clearance mechanisms were evaluated in 17 normal subjects visually and qualitatively by radioaerosol inhalation cinescintigraphy of the lung, and quantitatively by calculating the following indices: (a) overall or regional lung retention ratio; (b) airway deposition ratio; (c) airway retention ratio; (d) airway clearance efficiency; and (e) alveolar deposition ratio. The inhaled aerosol deposited homogeneously throughout the lungs, and mucus transport was always cephalad in direction and constant in velocity, although a temporary stasis of mucus was seen in smokers. Overall lung retention ratio was significantly smaller and airway deposition ratio was significantly larger in the smokers than in nonsmokers, but there was no difference between the groups in airway retention ratio or airway clearance efficiency. There was an inverse relationship between alveolar deposition ratio and cigarette consumption. Mucociliary clearance mechanisms were well maintained in the normal subjects, but in the smokers inhaled aerosol tended to deposit more proximally.

Effect of bronchodilation on the deposition and clearance of radioaerosol in bronchial asthma in remission.

Radioaerosol inhalation lung cine-scintigraphy and lung function tests were performed on ten patients with bronchial asthma in remission before and after inhalation of salbutamol following intravenously administered aminophylline. Radioaerosol inhalation lung cine-scintigraphy was very useful in revealing the changes not only in the deposition patterns of inhaled aerosol in the lungs but also in the dynamic transport of mucus on the airways. The bronchodilating effect of the combined treatment was significant; the inhaled aerosol deposited more homogeneously and less centrally in the lungs, the "penetration index" and the alveolar deposition ratio (ALDR) increased from 31 +/- 3 to 49 +/- 7%, and from 29 +/- 2% to 39 +/- 1%, respectively, while the airway deposition ratio (ADR) decreased from 72 +/- 2 to 61 +/- 1% immediately after the treatment. Lung function data including FVC, FEV 1.0, FEV 1.0%, MMF, V50 and Vp significantly improved after the treatment. There was, however, little visual or quantitative improvement in mucociliary clearance after the treatment.