Purification and identification of lactoperoxidase in milk basic proteins as an inhibitor of osteoclastogenesis.

A milk protein fraction with alkaline isoelectric points (milk basic protein, MBP) inhibits both bone resorption and osteoclastogenesis for in vitro models. We previously identified bovine angiogenin as a component of MBP that inhibits bone resorption. However, purified angiogenin had no effect on osteoclastogenesis, suggesting that MBP contains unidentified component(s) that inhibit osteoclast formation. In this study, we purified lactoperoxidase (LPO) as the predominant inhibitor of osteoclastogenesis in MBP. The LPO treatment downregulated levels of reactive oxygen species in osteoclasts. Signaling by receptor activator of NF-kappa-B ligand/receptor activator of NF-kappa-B (RANKL/RANK) was downregulated in LPO-treated cells, and, in particular, the ubiquitination of tumor necrosis factor receptor associate factor 6 (TRAF6) and activation of downstream signaling cascades (JNK, p38, ERK, and NFκB) were suppressed. Ultimately, LPO treatment led to decreased expression of c-Fos and NFAT2. These results suggest that MBP contains at least 2 components that independently suppress bone resorption through a unique mechanism: angiogenin inhibits bone resorption and LPO inhibits RANKL-induced osteoclast differentiation. These data explain many of the positive aspects of milk consumption on bone health.

Substitution of Ser-17 of pp60c-src: biological and biochemical characterization in chicken embryo fibroblasts.

pp60c-src is phosphorylated mainly on Ser-17 and Tyr-527 in vivo. In this study, we examined the effect of the phosphorylation of Ser-17 on the properties of pp60c-src by introducing Rous sarcoma virus variants carrying pp60c-src in which Ser-17 had been substituted, into chicken embryo fibroblasts. The Ala-17 substitution in wild-type pp60c-src and pp60c-src carrying Phe-527 caused a two- to threefold elevation in the kinase activity in vitro of these proteins; the former variant resulted in no morphological changes of infected cells, whereas the latter variant transformed chicken embryo fibroblasts. Since the substitution of Tyr-527 per se has been reported to activate pp60c-src, these results suggest that the abolishment of the phosphorylation of Ser-17 does not affect noticeably the properties of pp60c-src in chicken embryo fibroblasts.

Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.

Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes.

Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein.

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.

Isolation of chicken-bek and a related gene; identification of structural variation in the ligand-binding domains of the FGF-receptor family.

cDNA clones carrying the chicken-bek gene and a related gene were isolated. Deducing the amino acid sequence of chicken-bek allowed us to predict that it encodes for a receptor tyrosine kinase related to the fibroblast growth factor (FGF) receptor, and that the chicken-bek gene and Cek3 are closely related. However, a significant structural difference was identified between chicken-bek and Cek3 within the putative extracellular region, in such a manner that the structure of the immunoglobulin-like domain was conserved. A probe specific to the altered structure detected mRNA in the tissues as did a probe common to bek and Cek3, indicating heterogeneity in the FGF-receptor family in a novel manner. Furthermore, another bek-like gene was isolated and the expressions of its mRNA and protein product were analysed in tissues and cultured cells.

Nuclease S1-sensitive sites on superhelical DNA molecules carrying the LTR region of Moloney murine leukemia virus.

The long terminal repeat (LTR) from proviral DNA of Moloney murine leukemia virus (Mo-MLV) was cloned on a derivative of pBR322, and after introducing superhelical torsions into the resulting recombinant, the sites of conformational transition were investigated by the nuclease S1-digestion method. With an increase in the negative linking differences, fourteen dominant cutting sites were identified, of which two were mapped inside the LTR and one at the 3' end of the LTR. By searching the sequence data, all these sites were localized in the regions having either palindromic sequences or AT-rich sequences. Free energy calculation for the local secondary structure on one strand indicated that nuclease S1 attacked the palindromic sequence regions which could form relatively stable hairpin structures. Under the conditions used, no correlation was found between the S1-sensitive sites and the potential Z-DNA-forming regions, including those within the enhancer sequence.

Reconstitution of nucleosomes in vitro with a plasmid carrying the long terminal repeat of Moloney murine leukemia virus.

The potential of nucleosome assembly along the sequence of a plasmid carrying the long terminal repeat (LTR) and its flanking region of Moloney murine leukemia virus was analyzed by in vitro reconstitution experiments with histones from chicken erythrocytes. The results of electrophoretic mobility-shift and micrococcal nuclease-digestion assays indicated that the plasmid DNA contained four preferred sites for nucleosome formation. However, all of these sites were mapped on the vector moiety but not on the LTR moiety. Computer analysis of the sequences in the four preferred sites, each spanning about 150 bp, indicated that short runs of (dA,dT) containing two kinds of triplets, AAA/TTT and AAT/ATT, occurred frequently. Furthermore, many of these triplets tended to occur in the same side of the DNA helix, suggesting that DNA curvature was involved in the preferred sites for nucleosome assembly. Consistent was the observation that DNA fragments carrying these preferred sites showed anomalous electrophoretic mobilities at a low temperature.

Differential expression of four connexin genes, Cx-26, Cx-30.3, Cx-32, and Cx-43, in the porcine ovarian follicle.

Connexin genes expressed in porcine ovaries were isolated by reverse transcription-PCR, and the expression of four connexin genes (Cx-43, -32, -30.3, and -26) was detected by in situ hybridization in internal and surrounding compartments of large antral follicles. Cx-43 and Cx-30.3 were expressed in the theca interna as well as in the granulosa cell compartment. However, two zones were observed in the theca interna by probing with Cx-43; about a quarter of the region close to the lamina basalis appeared devoid of Cx-43 messenger RNA (mRNA) and protein as well. Cx-30.3, in contrast, seemed to be expressed ubiquitously in these two compartments. Cx-26 and Cx-32 were expressed only in the thecal cells. Both Cx-43 and -30.3 also gave positive signals in the cumulus cells to the same extent as obtained in the granulosa cells with the respective probes, whereas the expression of Cx-32 and -26 was undetectable. Cx-43 mRNA drastically decreased in the functioning stage of the corpus luteum, whereas Cx-30.3 mRNA increased significantly in the early stage of luteinization in the estrus cycles. These results suggest that Cx-43 is not the only gap junction protein to be expressed in the ovarian follicle; other proteins, such as Cx-32, -30.3, and -26 seem to be expressed, and their expressions are regulated differently.

Ovarian glycosaminoglycans potentiate angiogenic activity of epidermal growth factor in mice.

Epidermal growth factor (EGF) has been shown to induce capillary proliferation. In the course of our investigation on the interaction of ovarian components with EGF, we observed that partially purified glycosaminoglycans (GAGs) isolated from mouse ovaries enhanced the angiogenic activity of EGF when applied simultaneously to the lateral wall of the sheath of musculus rectus abdominis. Mouse EGF from submandibular glands embedded in Elvax 40 implanting on the musculus rectus abdominis induced neovascularization in a dose-dependent manner. When 0.5 micrograms ovarian GAGs was embedded in the implant with a low amount of EGF that induced only slight neovascularization (0.5 or 1 microgram/implant), the angiogenic activity of the growth factor was markedly enhanced. The active GAG component was isolated by chromatography on Dowex 1-x2. The fraction eluted with 0.5 M NaCl possessed the greatest activity to potentiate the angiogenic activity of EGF. When the reaction mixture of GAGs and EGF was treated with 1% cetylpyridinium chloride, the angiogenic activity was identified with the supernatant. On the other hand, after incubating EGF with 0.5 M NaCl fraction, the angiogenic activity of EGF was identified with the precipitate (GAG fraction) of the cetylpyridinium chloride-treated reaction mixtures. These findings show that ovarian GAGs potentiate the angiogenic activity of EGF by interacting or complexing with EGF.

Isolation and characterization of a novel connexin gene, Cx-60, in porcine ovarian follicles.

A new member of the connexin family was isolated from the porcine ovary. The amino acid sequence deduced from the nucleotide sequence of genomic as well as complementary DNA clones predicted the reading frame encoding the 60-kDa protein product, indicating the largest molecular mass among the connexin family genes analyzed to date; we named this gene Cx-60 based on its predicted molecular mass. The features of its primary structure were compared with those of other connexin genes, and the characteristics of its expression profile were examined in mammalian ovarian follicles. Cx-60 shares significant similarities with other connexins in the transmembrane and extracellular domains, but showed a highly unique primary structure in the cytoplasmic and carboxyl-terminal domains. The Cx-60 gene was unique in that its messenger RNA was detected in both the theca interna compartment and cumulus cells in the ovary; of other tissues, Cx-60 expression was relatively evident in the colon, thymus, and spleen. Cx-60, in contrast to Cx-43, was expressed constitutively upon gonadotropin stimulation when examined in hypophysectomized rats. Taken together, these results indicate that at least the connexin genes Cx-60, Cx-43, Cx-32, Cx-30.3, and Cx-26 are expressed in porcine ovarian follicles with differing expression profiles, including cell specificity.

Application of fluorescently labeled poly(dU) for gene expression profiling on cDNA microarrays.

The membrane filter hybridization technique has been widely used for gene expression profiling. The preparation of sensitive and reliable probes is critical for quantitative analysis in this technique. We report a method in which fluorescently labeled poly(dU) is used to detect poly(A)-containing mRNA that hybridizes to specific gene targets. The probe can be used commonly for every sample, alleviating problems encountered in preparing cDNA probes by reverse transcription, particularly when many samples are to be analyzed. Moreover, the sensitivity is at least comparable to cDNA probes prepared by conventional protocols, and intensities of signals after hybridization are independent of mRNA sizes and solely dependent on copy numbers. This method was also shown to be applicable to DNA chip technology.

Differential screening of ovarian cDNA libraries detected the expression of the porcine collagenase inhibitor gene in functional corpora lutea.

cDNA libraries were constructed from porcine granulosa cells of antral follicles as well as functional corpus luteum, and clones encoding stage-specific genes have been isolated by differential screening. A clone specific to the functional stage of corpus luteum was found to encode the porcine collagenase inhibitor gene and the stage-specific expression in luteinizing tissue was confirmed by Northern blot analysis. The complete open reading frame of the porcine collagenase inhibitor was deduced from the nucleotide sequence, and the localization of the product was examined by immunohistochemical staining as well in pig ovary; the inhibitor was detected in the intercellular space of luteal cells and in the connective tissue around blood vessels in the functional corpus luteum.

Three soluble form messages of murine CD46 are produced through alternative mRNA splicing.

Murine CD46 (mCD46) is a type 1 membrane protein expressed predominantly in testicular germ cells, the distribution profile of which is in contrast to that of human CD46 showing a ubiquitous tissue distribution. We have identified an additional message of mCD46 that encodes a putative secretory form [Nomura et al. (1999) Immunogenetics 50, 245-254]. Here, we cloned three cDNAs encoding putative soluble CD46 from murine testis. These soluble form messages were yielded on insertion of unidentified nucleotide sequences, 77, 179, and 73 ntds, into the junctions between the SCR3 and SCR4 (variant 2), ST(c) and UK (variant 3), and SCR4 and ST(c) (variant 1) domains, respectively, the last one corresponding to the reported soluble form. The exons corresponding to these three inserts were identified in the murine CD46 genome, indicating that the alternative splicing of mRNA participates in the generation of these various CD46 messages. In normal mouse sera and cell lines, however, virtually no soluble CD46 was detected on immunoblotting. On Northern blotting analysis with specific probes, on the other hand, variant 1 was found to be predominantly expressed in the liver and heart. In addition, all variant messages were detected on PCR in all organs examined. When a rabbit cell line, RK13 cells, was transfected with cDNA of variant 1, protein synthesis was detected on immunoblotting. Although the mCD46 protein production was inefficient, this variant 1 exhibited factor I-cofactor activity as to inhibition of the complement cascade. Since the mCD46 protein was reported to be markedly up-regulated on infection of murine cells with mCMV, the soluble mCD46 proteins may act as a complement regulator that controls the systemic complement system under the conditions of a viral infection.

A quantitative analysis of rat central nervous system myelination using the immunohistochemical method for MBP.

The temporal order of the myelination of the nervous pathways in 0-42-day-old Wistar rats was quantitatively analyzed using immunohistochemistry with anti-myelin basic protein (MBP) antibody. Immunohistochemistry was performed on paraffin-embedded tissue according to the standard ABC technique. For the objective evaluation of myelination, we converted the level stained with the immunohistochemical method into continuous numbers of 0-256 giving the intensity of myelination, using an image analyzing system. We analyzed nine nervous pathways: corpus callosum, optic tract, internal capsule, spinal tract of trigeminal nerve, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract, medial longitudinal fasciculus, and cuneate fasciculus. The onset of the myelination of the spinal tract of the trigeminal nerve, inferior cerebellar peduncle, medial longitudinal fasciculus and cuneate fasciculus was day 7 (postnatal). That of the corpus callosum, optic tract, internal capsule and cerebellar white matter was day 14, and that of the pyramidal tract was day 21. The time required to reach the level of myelination of day 42 was day 21 for the spinal tract of the trigeminal nerve and the inferior cerebellar peduncle, day 28 for the internal capsule, day 35 for the corpus callosum, optic tract, cerebellar white matter and pyramidal tract, and day 42 for the medial longitudinal fasciculus. Our method using immunohistochemistry with anti-MBP antibody provided a highly sensitive and objective criterion for judging precisely the time and the progress of myelination in each nervous pathway and compare one nervous pathway with another.

A quantitative study of the progress of myelination in the rat central nervous system, using the immunohistochemical method for proteolipid protein.

The temporal changes in intensity of myelination of the nervous pathways in 0 to 42-day-old Wistar rats were quantitatively analyzed by immunohistochemistry with anti-proteolipid protein and compared with that obtained by immunohistochemistry with anti-myelin basic protein. Immunohistochemistry was performed on paraffin-embedded tissue according to the standard ABC technique. Intensity of myelination was examined by an image analyzing system. We analyzed nine nervous pathways: corpus callosum, optic tract, internal capsule, spinal tract of the trigeminal nerve, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract, medial longitudinal fasciculus, and cuneate fasciculus. The presence of immunoreactive fibers for proteolipid protein (PLP) in the spinal tract of the trigeminal nerve, medial longitudinal fasciculus and cuneate fasciculus was noted on postnatal day 0. Those of the corpus callosum, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract and internal capsule were noted on day 7, and that of optic tract on day 14. The time required to reach the intensity of myelination of day 42 was day 14 for the cuneate fasciculus, day 21 for the spinal tract of the trigeminal nerve, inferior cerebellar peduncle and medial longitudinal fasciculus, day 28 for the optic and pyramidal tracts, day 35 for the corpus callosum and day 42 for the internal capsule and cerebellar white matter. The appearance of immunoreactive fibers for PLP was usually earlier than that for myelin basic protein (MBP) and the pattern of difference between PLP and MBP can be classified into three groups: (1) their time of appearance and progress are almost the same, as in the optic tract; (2) the appearance and progress of PLP occurs earlier than those of MBP, as in the pyramidal tract; (3) the appearance of PLP occurs earlier than that of MBP, but their progress is the same. Our findings revealed that the time of appearance and progress of myelination as measured by PLP are different among the nervous pathways, and that there is also a difference between PLP and MBP. This difference between PLP and MBP may indicate a functional difference between them.

Clinical significance of periodic lateralized epileptiform discharges in children with relation to level of consciousness.

Six children with acute cerebral insult, ranging in age from 3 days to 8 years, revealed periodic lateralized epileptiform discharges in their electroencephalographic recordings. Their etiologic factors were cerebral infarction, intracranial bleeding, purulent meningitis, acute infantile hemiplegia, and encephalitis. Each patient exhibited a different type of convulsive seizure. Computer tomography or magnetic resonance imaging revealed diffuse lesions covering the cerebral cortex and subcortical white matter in 2 patients, a lesion of the subcortical white matter in 1 patient, a linear lesion in the cortex and along the borderline between the cortex and the subcortical white matter in 1 patient, and localized lesions in the cortex and basal ganglia in 1 patient. There were findings indicating the disconnection of the cerebral cortex with deeper structures in 3 patients. The appearance rate of periodic lateralized epileptiform discharges increased at levels of consciousness from 5 to 7 on a pediatric modification of the Glasgow Coma Scale. At levels of consciousness from 8 to 14 and below 4, the rate was very low.

Analysis of dystrophin in muscular diseases by two-dimensional gel electrophoresis using agarose gels in the first dimension.

We analyzed dystrophin in case of normal control, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and infectious muscular disease using two-dimensional gel electrophoresis and immunoblotting with 3 monoclonal dystrophin antibodies: Dys 1, a mid-rod-domain antibody; Dys 2, a C-terminal-domain antibody; and Dys 3, an N-terminal-domain antibody. In cases of normal control, a clearly separated doublet of bands was observed for Dys 1 and 3 at molecular weights 400 and 420 kDa. The isoelectric point was between pH approximately 5.7-approximately 5.9, similar to that for the myosin heavy chain. In one DMD case, a single faint band was observed for Dys 2. BMD presented a single-band pattern for each antibody. Infectious diseases cases showed 3- to 5-band patterns for Dys 1 and single or no bands for Dys 2 and 3. The pI of the Dys 1 band was almost identical. These results suggest coexistence of normal dystrophin and its proteolytic products, both containing triple helical segment, and show that two-dimensional gel electrophoresis may be applicable in the analysis of dystrophin in muscular disease.

[Weitz' aminium salt initiated electron transfer reactions and application to the synthesis of natural products].

A stable cation radical Weitz' aminium salt, tris(4-bromophenyl)aminium hexachloroantimonate (BAHA) initiated electron transfer has been found to efficiently promote a great variety of reactions on electron-rich substrates: e.g. the cation radical pericyclic reactions, the chain-induced cation radical oxygenation of strained olefines and dienes, and several other intriguing reactions on a great variety of electron-rich substrate. The discovery of the Weitz' aminium salt catalysed Diels-Alder reaction has stimulated interest in cation radical chemistry and facilitated the development of a wide range of cation radical pericyclic chemistry. Applications of the method to the synthesis of natural products utilizing BAHA are also presented. Reaction of a lignan precursor, cinnamyl alcohols with BAHA in tetrahydrofuran (THF) gave a furofuran lignan, (+/-)-sesamin in one step. Podophyllum lignans, (+/-)-isopodophyllotoxin and (+/-)-isopicropodophyllin were synthesized by a biomimetic procedure from the doubly unsaturated esters by means of the BAHA induced cation-radical cycloaddition reaction. A new general synthesis for (+/-)-dibenzocyclooctadiene lignans was established utilizing novel synthetic method for quinone derivatives by oxidation with BAHA. Reactions of phenol derivatives using BAHA gave the corresponding quinone derivatives, which may be useful synthon for obtaining quassinoids such as quassin.

[Clinicoencephalographic study of periodic lateralized epileptiform discharges in infants].

The mechanism of periodic lateralized epileptiform discharges (PLEDs) still remains unclear, although it has been the subject of a number of theories. We investigated the relationship between the level consciousness and PLEDs in order to clarify both the clinical significance and the mechanism of PLEDs. We studied two neonates and two infants with acute organic lesions of the brain (cerebral infarction, meningoencephalitis, cerebral hemorrhage, acute infantile hemiplegia), of whom all showing PLEDs in EEG. In each case, we analyzed sequential EEG records and the level of consciousness, and measured the periodicity, voltage and duration of PLEDs by a computer controlled digitizer. In each case, the periodicity and voltage of the discharges were related to the level of consciousness. The appearance rate of PLEDs was relatively high at the level of consciousness from III-100 to III-200. The inverse correlation between the frequency (1/periodicity) and the voltage of PLEDs was significant in 53% of the total records of PLEDs; it was also frequently observed at the level of consciousness between III-100 and III-200. From above findings we concluded that an appropriate degree of damage to produce PLEDs is required in both the cerebral cortex and subcortical white matter corresponding to the level of consciousness from III-100 to III-200.

[Facioscapulohumeral muscular dystrophy (FSH) and hearing loss].

It has been reported that cochlea is the lesion of hearing loss in FSH. However, the details of this lesion are not yet sufficiently known. We performed detailed audiologic studies to examine hearing loss in FSH. We experienced 2 cases of FSH associated with hearing loss. Case 1 was a girl aged 5 years, and case 2 a boy aged 15 years. Clinical findings, EMG and muscle biopsy gave a diagnosis of FSH in both cases. Hearing loss was evaluated by pure tone audiography, speech audiography, tympanometry, stapedial reflex, auditory brain stem response and electrocochleography. In case 1, pure tone audiograms revealed high tone hearing loss without an A-B gap. On speech audiography, the maximum articulation score was 100% and proved normal. The tympanogram was type A. Stapedial reflex was normal bilaterally. The threshold of the 5th wave increased markedly on auditory brain stem response. On electrocochleography, the H-curve of the input-output function curves of action potential was recorded, but the L-curve was absent. There were no complaints of hearing loss in case 2, but pure tone audiograms revealed high-tone hearing loss without an A-B gap. The tympanogram was type A. Stapedial reflex was normal bilaterally. On auditory brain stem response, threshold was increased and latency was prolonged when intensity was lowered. The electrocochleograms were almost normal. It has been reported that, in electrocochleography, the L-curve represents the function of the outer hair cells and the H-curve that of the inner hair cells. The electrocochleograms in case 1 showed damage to the outer hair cells.(ABSTRACT TRUNCATED AT 250 WORDS)