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.

Homozygous CDA*3 is a major cause of life-threatening toxicities in gemcitabine-treated Japanese cancer patients.

Among 242 Japanese pancreatic cancer patients, three patients (1.2%) encountered life-threatening toxicities, including myelosuppression, after gemcitabine-based chemotherapies. Two of them carried homozygous CDA*3 (CDA208G>A [Ala70Thr]), and showed extremely low plasma cytidine deaminase activity and gemcitabine clearance. Our results suggest that homozygous *3 is a major factor causing gemcitabine-mediated severe adverse reactions among the Japanese population.

A new statistical screening approach for finding pharmacokinetics-related genes in genome-wide studies.

Biomedical researchers usually test the null hypothesis that there is no difference of the population mean of pharmacokinetics (PK) parameters between genotypes by the Kruskal-Wallis test. Although a monotone increasing pattern with a number of alleles is expected for PK-related genes, the Kruskal-Wallis test does not consider a monotonic response pattern. For detecting such patterns in clinical and toxicological trials, a maximum contrast method has been proposed. We show how that method can be used with pharmacogenomics data to a develop test of association. Further, using simulation studies, we compare the power of the modified maximum contrast method to those of the maximum contrast method and the Kruskal-Wallis test. On the basis of the results of those studies, we suggest rules of thumb for which statistics to use in a given situation. An application of all three methods to an actual genome-wide pharmacogenomics study illustrates the practical relevance of our discussion.

Functional characterization of CYP3A4.16: catalytic activities toward midazolam and carbamazepine.

1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, K(m) and V(max) for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (V(max)/K(m)) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in K(m) without a difference in V(max). 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, K(m2) and V(max2) of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.

Local extensive granulomatous inflammation of the neck region and lymphangitis caused by Lichtheimia corymbifera infection in a Japanese Black calf.

A 7-month-old female Japanese Black calf developed elongated, nodular mass measuring 30 × 16 cm extended from the retropharyngeal region to mid lateral neck region. Histological examination revealed granulomatous lymphangitis with non-septate fungal hyphae recognized throughout the lesions. Fungal culture, DNA sequencing and molecular phylogenetic tree analysis confirmed the sequence of Lichtheimia corymbifera. The lymphogenous route was speculated to be the main route of fungal spread leading to the characteristic nodular appearance of this case.

cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs.

E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids of the N termini of E4TF1-47 and -53 were identical except for one amino acid insertion in E4TF1-53, and they differ from each other at the C terminus. These three recombinant cDNA clones were expressed in Escherichia coli, and the proteins behaved in the same manner as purified proteins in a gel retardation assay. Nucleotide and predicted amino acid sequences were highly homologous to GABP-alpha and -beta, which is further supported by the observation that GABP-specific antibody can recognize human E4TF1.

Retinoblastoma binding factor 1 site in the core promoter region of the human RB gene is activated by hGABP/E4TF1.

We previously reported two oncogenic point mutations present in the RB (retinoblastoma) gene promoter region, found at consensus Sp1 and ATF sites, respectively, and in two separate hereditary RB families. However, Sp1 protein was shown not to bind to the Sp1 site; this indicated that the Sp1 consensus site mutation was blocking the action of an alternative transcription factor, which we called RBF-1 (retinoblastoma binding factor 1). Subsequent purification of RBF-1 revealed it to be hGABP/E4TF1, a transactivator from the adenovirus early-region 4 promoter. In this study, we directly examined the effects of hGABP/E4TF1 on transactivation of the RB gene promoter through the RBF-1 site. As expected, hGABP/E4TF1 enhanced the core RB promoter activity, whereas it did not stimulate a mutant RBF-1 site. We therefore conclude that the most essential transcription factor in the human RB gene is likely to be hGABP/E4TF1.

Activation of the retinoblastoma gene expression by Bcl-3: implication for muscle cell differentiation.

The retinoblastoma (Rb) protein is a master regulator of cell cycle. Accumulating evidence suggests that elevation of Rb expression is a key event in differentiation of various cell types. However the mechanism of regulation of Rb expression is poorly understood. Here we report that the candidate oncoprotein Bcl-3, previously characterized as a member of the IkappaB family, activates transcription of the Rb gene, whose promoter has no typical kappaB sites. A target element for Bcl-3 that matches the consensus for the E4TF1/GABP transcription factor was identified. Bcl-3 was shown to promote tetramer formation of E4TF1. During muscle cell differentiation, increased bcl-3 expression was observed before the induction of the Rb mRNA. Transient expression of Bcl-3 in myoblasts was shown to induce expression of the endogenous Rb. Furthermore, expression of the antisense bcl-3 RNA in myoblasts suppressed induction of Rb and myogenic differentiation. These results suggest that Bcl-3 is an upstream regulator of Rb expression during differentiation of muscle cells.

The retinoblastoma binding factor 1 (RBF-1) site in RB gene promoter binds preferentially E4TF1, a member of the Ets transcription factors family.

The tumor suppressor retinoblastoma gene product, pRB, is a well known regulator of G1/S cell cycle progression. Moreover, mutational inactivations within the retinoblastoma gene (RB) are found in many human malignant tumors, and thus, believed to be an essential step in tumor formation. The human RB gene is considered as a housekeeping gene with no characteristic TATA or CAAT elements in its promoter region, but the sequence between 206 and 185 bases upstream of the initiation codon, essential for RB promoter activity, contains putative Sp1 and ATF recognition sites. We have previously reported that point mutations in this region, causing low penetrance retinoblastomas, completely reduced RB promoter activity, and that a nuclear factor, named RBF-1 (retinoblastoma binding factor 1), could specifically bind to this sequence, overlapping Sp1 recognition sequence. We show here, that RBF-1 can recognize a specific DNA sequence, 5'-GGCGGAAGT-3', overlapping the Sp1 and ATF sites and corresponding to the consensus DNA binding site for members of Ets transcription factors family. When RBF-1 site was used for sequence specific DNA affinity purification from erythroleukemia cells, reconstitution assays, immunoblotting analysis and peptide mapping show that the two major co-purified proteins are identical to human E4TF1-60 and -53 proteins. This reveals that E4TF1 can bind to the RBF-1 site of RB gene promoter, which, thus, constitutes a new target for this member of Ets transcription factors family.

Effects of herbimycin A and ST638 on Fc epsilon receptor-mediated histamine release and Ca2+ signals in rat basophilic leukemia (RBL-2H3) cells.

We examined the effect of the two protein tyrosine kinase inhibitors, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimycin A, on the activation processes of rat basophilic leukemia (RBL-2H3) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal IgE antibody were stimulated with multivalent antigen (DNP conjugate of bovine serum albumin). Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the tyrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 150 kDa) including phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inositol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individual cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobilization of calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic calcium ion concentration or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation following antigen stimulation regulates phosphatidylinositol hydrolysis and the influx of extracellular calcium.

Molecular and biochemical analyses of combining sites of monoclonal anti-morphine antibodies.

V region nucleotide sequences were determined by mRNA sequencing for 11 monoclonal anti-morphine antibodies with slightly different specificities for morphine-related opiates. The VH region nucleotide sequences of the antibodies MOR8, MOR33, MOR35, MOR44, MOR83, and MOR76 were classified into the VH-5 (7183) family, while the antibodies MOR39, MOR115, MOR131, MOR158 and MOR180 used VH-1 (J558) family genes. MOR39, MOR115 and MOR131 used the V lambda-1 gene for their L chain V region. MOR158 and MOR180 used the Vk-10 gene. MOR8, MOR33, OR35, MOR44, MOR76 and MOR83 used VK-21D. The antibody sets MOR158 and MOR180; MOR39 and MOR131; and MOR8, MOR33, MOR35, MOR44, MOR76 and MOR83 appeared to be somatic mutants derived from the same clones since they showed the same VH/VL usage and V(D)J recombination pattern. The pH-reactivity profiles for these antibodies revealed that the binding of morphine to the antibodies is highly dependent on the pH value of the assay solution, suggesting the importance of the electrostatic interaction between the positive charge of morphine and the negative charges at or near the combining sites. Direct UV-photoaffinity labeling with 3H-morphine was carried out in order to estimate the orientation of morphine in the combining sites. The H chains were preferentially labeled in MOR8, MOR33, MOR35, MOR76, and MOR83, whereas most of the crosslinked hapten was found in the L chains in MOR39, MOR115, MOR131, MOR158 and MOR180. Thus, these 11 antibodies were classified into two types in terms of reactivity in the photoaffinity labeling.

Production and characterization of high-affinity monoclonal antibodies against morphine.

Twelve hybridoma cell lines producing MAbs against morphine were established by using morphine hemisuccinate-conjugated bovine serum albumin as an immunogen. The MAbs belonged to the IgG1 subclass with kappa- or lambda-chains. The association constants of the antibodies ranged from 4.6 x 10(8) to 4.7 x 10(10) (M-1). These antibodies revealed slightly different cross-reactivities with various agonistic opiates and antagonists. In general, the antibodies were strongly cross-reactive with the opiate agonists, codeine, ethylmorphine, dihydromorphine and dihydrocodeine, while their cross-reactivities with norcodeine and the opiate antagonists, naloxone and naltrexone, were weak. The cross-reactivities with dihydromorphinone, dihydrocodeinone, naloxone, naltrexone, dextromethorphan and homatropine varied from clone to clone. Interestingly, certain MAbs displayed weak but significant cross-reactivities with the synthetic opiate, meperidine. However, none of the antibodies was cross-reactive with the opioid peptides, beta-endorphin, Met-enkephalin, and D-Ala2-D-Leu5-enkephalinamide. Radioimmunoassay for morphine using one of the antibodies (MOR 131.5.13) was shown to be sufficiently sensitive (IC50 = 0.1 nM) for the purposes of forensic analysis of morphine. This set of monoclonal anti-opiate antibodies is assumed to be suitable for analyzing the structure-function relationship in the hapten-antibody interaction, since the antibodies revealed similar but not identical cross-reactivities with various morphine related compounds.

Molecular characterization of monoclonal anti-steroid antibodies: primary structures of the variable regions of seven antibodies specific for 17 alpha-hydroxyprogesterone or 11-deoxycortisol and their pH-reactivity profiles.

The variable region nucleotide sequences of seven monoclonal anti-steroid antibodies that are specific for the closely related progesterone derivative, 11-deoxycortisol or 17 alpha-hydroxyprogesterone (17-OHP), have been determined by genomic cloning and DNA-sequencing or by direct mRNA-sequencing. As for their heavy chain variable regions, the nucleotide sequences of the SCET.M8.1 (SCET) and OHP 4B2.2.1 (4B2) antibodies were classified into the VH-9 family, while OHP 7D7.2.3 (7D7), 1E9.3.1 (1E9), 57.G6.1 (57) and 138.H8.1 (138) used VH-3 family genes. OHP 101.B11.1 (101) used a gene of the VH-1 family. For their light chain variable regions, SCET and 57 used VK-28 group genes, while 4B2, 7D7, 1E9, 101 and 138 antibodies used genes of the VK-21 subgroups (21A, 21B or 21C). All of the antibodies used different combinations of genes in the VH families and VK groups or subgroups. This indicates that the antibody response against the steroid hapten, 17-OHP, is fairly polyclonal, and several VH/VL combinations show high affinity for progesterone-related steroids. Although the primary structures of hypervariable loop regions of the mAbs were relatively diverse, generally, hydrophobic and aromatic amino acids were rich in these regions. Moreover, the length of heavy chain CDR3 was constant in all the antibodies investigated in this paper as well as the previously reported anti-progesterone monoclonal antibodies (mAbs). This suggests that the length of VH CDR3 in these mAbs has a considerable influence on the formation of antigen-combining pockets. The pH-reactivity profiles for the anti-17-OHP mAbs indicated that the the steroid-mAb binding was independent of pH between pH 4 and 11 in most of the mAbs. The results suggest that the steroid-mAb interactions are not largely affected by the electrostatic environments near the combining sites of these mAbs. Taken together, these data imply that the shape of hydrophobic depressions in the combining sites is important for the binding of relatively large, hydrophobic and rigid haptens like steroids.

Comparison of the antigenicities of native human growth hormone (hGH) and three forms of recombinant hGHs using monoclonal antibodies.

A panel of hybridomas producing antibodies specific for human growth hormone (hGH) were prepared by using a recombinant hGH [methionylsomatotropin (r-hGH)] as an immunogen. Thirteen representative monoclonal antibodies which showed different reactivity patterns were used to analyze the antigenicities of four different forms of hGHs by RIA inhibition studies. Native hGH and r-hGH showed almost the same antigenicities with these monoclonal antibodies. A Cys-substituted recombinant hGH (r-hGH-165) retained the epitopes recognized by 11 monoclonals but not those recognized by two monoclonals. All except one of the monoclonals showed little or no reactivity with a recombinant hGH fragment (r-hGH-AB). On the basis of these results, the differences in the structures and antigenicities of the recombinant hGH proteins were discussed.

A murine monoclonal anti-metallothionein autoantibody recognizes a chemically synthesized amino-terminal heptapeptide common to various animal metallothioneins.

A murine hybridoma clone (MT 189-14-7) producing an IgM-class monoclonal antibody specific for metallothionein (MT) was produced with rat Zn-MT 2-keyhole limpet hemocyanin conjugate as an immunogen. The reactivity of the monoclonal antibody with MTs obtained from various animal species was determined by the competitive radioimmunoassay. The MT 189-14-7 antibody bound equally to various MTs 1 and 2 including mouse MTs 1 and 2. This indicates that the antibody is a murine autoantibody reactive with various animal MTs and recognizes a common structure of the various MTs. Subsequently, the location of the antigenic determinant recognized by the MT 189-14-7 antibody was determined by using various synthetic human MT 2 peptides. Among the synthetic peptides examined, both the amino-terminal beta-domain, Ac-(hMT 2 1-29)-OH, and an amino-terminal heptapeptide, Ac-(hMT 2 1-7)-OH, but not the carboxy-terminal alpha-domain peptides, H-(hMT 2 29-35)-OH and H-(hMT 2 30-61)-OH, showed inhibitory activities slightly higher than the native MTs in the competitive radioimmunoassay. These results demonstrate that the MT 189-14-7 autoantibody recognizes the epitope located within the amino-terminal heptapeptide common to various MTs.

Assignment of the E4TF1-60 gene to human chromosome 21q21.2-q21.3.

The gene encoding human transcription factor E4TF1-60 was previously mapped to chromosome 21q21. We analyzed the localization of the E4TF1-60 gene in more detail by genomic Southern hybridization and determined the sequence of the exons and the regions surrounding the intron boundaries. We report here that E4TF1-60 locates in the long arm of chromosome 21 at q21.2-q21.3 and contains a total of ten exons.

Induction of metallothionein in a human astrocytoma cell line by interleukin-1 and heavy metals.

The effects of cytokines and heavy metals on the expression and localization of metallothioneins (MTs) within U373MG astrocytoma cells were analyzed by using indirect immunofluorescence using a monoclonal anti-MT antibody (MT45). IL-1, CdCl2 (50 microM) or ZnCl2 (500 microM) remarkably augmented intracellular MT levels, whereas IL-6 or 10 microM of ZnCl2 showed no inducing activity. From 24 to 48 h after the addition of CdCl2 or IL-1, immunoreactive MTs were found in the cytoplasm and the nucleus. After 72 h, immunoreactive MTs accumulated in a granular form near the cell surfaces in the presence of CdCl2 (50 microM) or IL-1 plus ZnCl2 (10 microM). However, this accumulation was not observed when only IL-1 was added. Thus, Zn2+ facilitated the appearance of the granular form of immunoreactive MTs at a concentration where they do not induce MTs by themselves.

Comparison of methods for freezing interleukin-dependent murine cell lines.

Various methods for freezing several interleukin-dependent murine cell lines have been compared and were shown to differ in their efficacy from line to line. An interleukin-2-dependent cell line, T572.C5, which was more sensitive to dimethylsulfoxide (DMSO) than the other cell lines, was the most difficult to freeze in a viable state. In order to define better conditions for freezing, the effects of the changes in freezing rates and concentrations of DMSO and of the addition of Ficoll were determined. The optimal freezing rates and DMSO concentrations varied from line to line. The addition of Ficoll increased the viability in some, but not all, cell lines, depending on the freezing conditions used. Thus, the optimal conditions for freezing interleukin-dependent cell lines varied from line to line. In general, the direct freezing procedure was better than the styrofoam-box freezing method. Although programmed freezing under direct temperature control was the most suitable among the freezing procedures examined, unprogrammed procedures also were of practical use.

Purification and characterization of guinea pig lymphotoxin produced by lymph node cells stimulated by phytohemagglutinin.

Guinea pig lymphotoxin (LT) produced by stimulation of lymph node cells with Phaseolus vulgaris phytohemagglutinin was purified approximately 1,000-fold in specific activity by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, and gel filtration on Sephadex G-100. The properties of LT tested at the various stages of purification revealed that the LT was a heat-sensitive, protease-sensitive proteinaceous substance, and its approximate molecular weight was estimated to be 50,000 by means of gel filtration. The most purified fraction (Fr. D) was found to be devoid of the activities of migration inhibitory and mitogenic factors, clearly indicating that these activities are mediated by different substances.

Gene expression patterns during enhanced periods of visual cortex plasticity.

During a critical period in its postnatal development the mammalian visual cortex displays susceptibility to experience-dependent alterations of neuronal response properties. Plasticity represents an integrated set of developmental processes controlled by a transcriptional hierarchy that coordinates the action of many genes. To illuminate the expression of these critical genes, we examined gene expression patterns of 18371 non-redundant cDNAs in the visual cortex of cats at birth, at eye opening, at the peak of the critical period of eye dominance plasticity and in the adult cat using filter-based cDNA arrays and software-based hierarchical cluster analysis. We identified a small set of genes that were selectively expressed during the peak of the critical period for plasticity. We further examined the patterns of expression of these genes by analyzing the gene expression pattern of dark-reared chronologically older animals that are known to retain this ocular dominance plasticity beyond the chronologically defined critical period. This additional cluster assessment allowed us to separate age-related changes in the patterns of gene expression from plasticity-related changes, thus identifying a subset of genes that we define as plasticity candidate genes. Those plasticity candidate genes that have previously characterized functions include participants in second messenger systems, in cell adhesion, in transmitter recycling and cytokines, among others. Comparison of cDNA array quantitation with reverse transcription-polymerase chain reaction showed almost identical expression profiles for three genes that we examined. The expression pattern of one identified gene, opioid binding cell adhesion molecule, from the cDNA array analysis, is also in agreement with immunocytochemical results. We conclude that the approach of high-density cDNA array hybridization can be used as a useful tool for examining a complex phenomenon of developmental plasticity since it is amenable to multiple developmental stage gene expression comparisons.