Effect of low-level laser irradiation on osteoglycin gene expression in osteoblasts.

Many studies have attempted to elucidate the mechanism of the biostimulatory effects of low-level laser irradiation (LLLI), but the molecular basis of these effects remains obscure. We investigated the stimulatory effect of LLLI on bone formation during the early proliferation stage of cultured osteoblastic cells. A mouse calvaria-derived osteoblastic cell line, MC3T3-E1, was utilised to perform a cDNA microarray hybridisation to identify genes that induced expression by LLLI at the early stage. Among those genes that showed at least a twofold increased expression, the osteoglycin/mimecan gene was upregulated 2.3-fold at 2 h after LLLI. Osteoglycin is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix which was previously called the osteoinductive factor. SLRP are abundantly contained in the bone matrix, cartilage cells and connective tissues, and are thought to regulate cell proliferation, differentiation and adhesion in close association with collagen and many other growth factors. We investigated the time-related expression of this gene by LLLI using a reverse transcription polymerase chain reaction (RT-PCR) method, and more precisely with a real-time PCR method, and found increases of 1.5-2-fold at 2-4 h after LLLI compared with the non-irradiated controls. These results suggest that the increased expression of the osteoglycin gene by LLLI in the early proliferation stage of cultured osteoblastic cells may play an important role in the stimulation of bone formation in concert with matrix proteins and growth factors.

Hsp27 suppresses the formation of inclusion bodies induced by expression of R120G alpha B-crystallin, a cause of desmin-related myopathy.

The R120G mutation in the small heat shock protein (sHSP) alpha B-crystallin has been identified in a family suffering from desmin-related myopathy. In this study, we characterized the features of transiently expressed R120G alpha B-crystallin in mammalian cells. In addition, we examined interactions of this mutant alpha B-crystallin with Hsp27, another representative sHSP. In HeLa cells, transiently expressed R120G alpha B-crystallin was mainly fractionated in the insoluble fraction, although wild-type alpha B-crystallin was predominantly found in the soluble fraction. In immunofluorescence studies, we found 15-25% of R120G alpha B-crystallin-expressing cells to contain multiple cytosolic inclusion bodies, in which Hsp27 was also localized. When R120G alpha B-crystallin and Hsp27 were transiently co-expressed in HeLa cells, the amount of R120G alpha B-crystallin in the soluble fraction was greater than with expression of R120G alpha B-crystallin alone. Moreover, co-expression resulted in reduced formation of inclusion bodies, suggesting that Hsp27 acts as a molecular chaperone for R120G alpha B-crystallin.

Late infantile Hirschsprung disease-mental retardation syndrome with a 3-bp deletion in ZFHX1B.

A 48-year-old woman with late infantile onset mental retardation developed megacolon. Although the patient had no typical clinical features of Hirschsprung disease-mental retardation syndrome, a new 3-base pair deletion, eliminating an Asn, was identified in the responsible gene ZFHX1B. This suggests that screening for ZFHX1B mutations is warranted even in the absence of typical clinical features of the syndrome.

Selective localization of G protein gamma5 subunit in the subventricular zone of the lateral ventricle and rostral migratory stream of the adult rat brain.

G proteins play important roles in transmembrane signal transduction, and various isoforms of each subunit, alpha, beta and gamma, are highly expressed in the brain. The Ggamma5 subunit is a minor isoform in the adult brain, but we have previously shown it to be highly expressed in the proliferative region of the ventricular zone in the rat embryonic brain. We show here that Ggamma5 is also selectively localized in a proliferative region in the adult rat brain, including the subventricular zone of the lateral ventricle and rostral migratory stream. The Galphai2 subunit colocalized with Ggamma5 in these regions, the two subunits being present in neuronal precursors and ependymal cells but not in proliferating astrocytes. In addition, intense staining of Ggamma5 was seen in axons of the olfactory neurons, which are known to regenerate. These results suggest specific roles for Ggamma5 in precursor cells during neurogenesis so that this isoform might be a useful biological marker.

A solitary bronchial papilloma with malignant changes.

We describe a case of solitary papilloma of the bronchus and provide a review of 38 similar cases reported in Japan. A 70-year-old man complained of cough and sputum. Chest X-rays and CT scans revealed atelectasis of the right middle lobe. On bronchoscopy, a polypoid tumor was found at the orifice of the bronchus of the right middle lobe. The tumor was histologically diagnosed as a squamous papilloma with moderate atypia. Because of elevated tumor markers and the reported high incidence of malignant changes in papillomas, the tumor was endoscopically resected by electrosurgical snare. While this procedure resulted in improvement of atelectasis, the chest CT taken subsequently revealed a mass adjacent to the resected polypoid tumor in the middle lobe bronchus. Percutaneous needle biopsy followed by histopathological examination confirmed the tumor to be a squamous cell carcinoma. Only three cases of malignant changes in papillomas have been previously reported in Japan. Electrosurgical snare, which allows the identification of tissue at the tumor base, should be the treatment of choice rather than YAG laser surgery.

Ser-59 is the major phosphorylation site in alphaB-crystallin accumulated in the brains of patients with Alexander's disease.

The phosphorylation state of alphaB-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against alphaB-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25-28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of alphaB-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. alphaB-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. AlphaB-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, alphaB-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in alphaB-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59.

Evaluation of the electroosmotic medium pump system for preparative disk gel electrophoresis.

This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921-1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the "electroosmotic medium pump system." Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step.

Identification of 40-kDa outer membrane protein as an aggregation factor of Porphyromonas gingivalis to Streptococcus gordonii.

Porphyromonas gingivalis, an important pathogen in periodontitis, aggregates with other oral microorganisms such as Streptococcus gordonii. We previously succeeded in gene cloning the 40-kDa outer membrane protein (OMP) from P. gingivalis. Although recombinant (r) 40-kDa OMP itself did not show aggregation activity, the affinity-purified antibody against 40-kDa rOMP inhibited the aggregation activity of P. gingivalis cells toward S. gordonii which is one of the first oral bacteria to colonize on tooth surfaces and can be expected to support subsequent colonization of other bacteria. In this study, in order to clarify the pathological role of 40-kDa OMP, we used a cross-linking reagent to construct a polymeric form of r40-kDa OMP and examined its aggregation activity. The polymeric r40-kDa OMP significantly expressed aggregation activity with S. gordonii cells. Moreover, the antibody against r40-kDa OMP inhibited the aggregation activity of the polymeric r40-kDa OMP. These findings clearly demonstrate that 40-kDa OMP, as a multivalent form, is one of the important aggregation factors on the cell surface of P. gingivalis.

Manipulation of the somatosensory cortex modulates stimulus-induced repetitive ear movements in a seizure-sensitive strain of gerbil.

Some Mongolian gerbils (Meriones unguiculatus) respond to stimulation by seizures, the pattern of which changes progressively during development. We previously established a seizure-sensitive strain, MGS/Idr, in which all animals exhibit such stimulus-induced seizures. We have now noted that all adults of this strain also show repetitive backward movements of the ears at the ears at the beginning of stimulus-induced seizures, although the incidence varies with the individual. We examined whether the cerebral cortex was involved in these movements and found that electrical stimulation of an area of the somatosensory cortex elicited strong backward movement of the ear on the contralateral side, and that unilateral application of bicuculline, a GABAA receptor antagonist, induced spontaneous repetitive backward movements of the same ear. In this area, sharp waves appeared in the electrocortigram during the repetitive ear movements induced by seizure-inducing stimuli. Unilateral ablation of this area abolished stimulus-induced repetitive movements of the contralateral ear, but had no effects on those of the ipsilateral ear. These results suggest that, in certain types of seizure-susceptible subjects, it may be possible to modify stimulus-induced repetitive movements by manipulating a certain area of the somatosensory cortex which is related to these movements and that this gerbil strain may be useful in research on this subject.

Primary intestinal T-cell lymphoma resembling lymphomatous polyposis: report of a case.

We report an interesting case of primary intestinal T-cell lymphoma (ITL) resembling lymphomatous polyposis (LP) in a 24-year-old man. The neoplasm macroscopically showed numerous small polyps throughout the colon and microscopically showed diffuse proliferation of small-sized tumor cells with occasionally cleaved or irregularly shaped nuclei. The tumor cells were immunohistochemically positive for CD3, CD8, TIA-1, and CD56, and a polymerase chain reaction study showed a single band, indicating monoclonal rearrangement of the T-cell receptor beta gene. The phenotypic features in the current case are consistent with those of ITL derived from cytotoxic CD56+ CD8+ intraepithelial lymphocytes. This is the second documented case of primary ITL with a morphologic pattern of LP.

[Diagnosis of neurodegenerative disease by mass spectrometry].

We analyzed wild-type and variant transthyretins (TTRs) by mass spectrometry and reported that all TTR preparations demonstrated free TTR, TTR conjugated with thiol compounds and several minor components. We previously described a component with a molecular mass 80 Da larger than free TTR, which was proven to be TTR conjugated with bisulfite. The amyloid fibril formation of purified TTR was monitored by the turbidity at 330 nm, and by a Congo red binding assay as a function of pH. The S-sulfonated TTR showed clear elevation of the turbidity and Congo red binding under acidic conditions. In contrast, TTR reduced by dithiothreitol, which was free of the S-sulfonated component, did not show evidence of amyloid fibril formation. We analyzed rabbit serum TTR obtained from a rabbit fed a diet containing sulfite and from a rabbit on a sulfite-free diet. Compared to that in the rabbit fed a sulfite-containing diet, sulfonated TTR was decreased on the 7th day of a sulfite-free diet. These results suggested that the S-sulfonated wild-type TTR is highly amyloidogenic, and that prolonged ingestion of antimicrobial and antioxidant agents containing sulfite/bisulfite, may cause senile systemic amyloidosis.

Detection by mass spectrometry of highly increased amount of S-sulfonated transthyretin in serum from a patient with molybdenum cofactor deficiency.

Serum transthyretin has several isoforms, most of which are caused by disulfide linkage with cysteine residue at position 10. We found an ion peak 80 D larger than unmodified transthyretin by electrospray ionization mass spectrometry and assigned it to S-sulfonated transthyretin. The peak height was <2% of total transthyretin in control sera from more than 200 individuals including infants. Transthyretin from a patient with molybdenum cofactor deficiency was analyzed, and the peak was prominent, higher than 85% of total transthyretin. In patients with this disease, the presence of elevated levels of sulfite leads to the formation of S-sulfonated cysteine. The peak can be used as a diagnostic marker for molybdenum cofactor deficiency, although more sera from patients with this disease should be tested.

Muscle develops a specific form of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during myogenic differentiation.

Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, alphaB-crystallin, p20, MKBP/HSPB2, and HSPB3, omitting lens-specific alphaA-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, alphaB-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and alphaB-crystallin, but not for MKBP/HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are alphaB-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and that these systems work independently in muscle maintenance and differentiation.

A new amyloidogenic transthyretin variant, [D38A], detected by electrospray ionization/mass spectrometry.

A new variant of transthyretin (TTR) was detected by mass spectrometry (MS) in a 63-year-old Japanese female patient suffering from amyloidosis. TTR was analyzed by 2-dimensional liquid chromatography coupled with electrospray ionization MS. Variant TTR showed extra peaks in addition to normal TTR peaks. The extra peaks were about 44 Da smaller than normal TTR peaks, and the abundance of variant peaks showed about 80% of the corresponding normal free and adduct peaks. Direct genomic DNA sequencing of TTR exon 2 showed both adenine and cytosine in the position corresponding to the second base of codon 38. This codes for a variant alanine (GCT) as well as the normal aspartic acid (GAT), indicating that the case is heterozygous for the substitution, [D38A].

Enhanced amyloidogenicity of sulfonated transthyretin in vitro, a hypothetical etiology of senile amyloidosis.

Genetic variants of transthyretin (TTR) cause systemic amyloidosis and wild-type TTR may also in some situations produce amyloid fibrils. We have analyzed wild-type and variant TTRs by mass spectrometry and found that TTR preparations from all individuals demonstrated free TTR, TTR conjugated with thiol compounds and several minor components. We previously described a component which had a molecular mass 80 Da larger than free TTR and was proved to be TTR conjugated with sulfite. Here, the amyloid fibril formation of the TTR isoforms was monitored by the turbidity at 330 nm, and by a Congo red-binding assay as a function of pH, according to the method of Lai et al. The S-sulfonated TTR showed the highest level of amyloid fibril formation. In contrast, TTR reduced by dithiothreitol, which was free of the S-sulfonated component, showed neither elevation of the turbidity nor the Congo red binding. Commercially purchased TTR without further treatment containing free, S-sulfonated and other species of TTR molecules showed an intermediate elevation. These results suggested that the S-sulfonated wild-type TTR is highly amyloidogenic. Although further experiments are needed to apply the observation to in vivo phenomenon, exogenous sulfite may be a cause of senile systemic amyloidosis.

Bromocriptine protects dopaminergic neurons from levodopa-induced toxicity by stimulating D(2)receptors.

Neuroprotective properties of bromocriptine, a D(2) receptor agonist, were investigated using the in vitro neurotoxicity of levodopa for dopaminergic neurons from rat embryonic ventral mesencephalon. Levodopa, when added to the culture medium, showed toxicity which was specific for dopaminergic neurons. Bromocriptine was found to protect dopaminergic neurons from levodopa toxicity. Another D(2) agonist, 2-(N-phenethyl-N-propyl-amino-5-hydroxytetralin, showed similar protective effects. The neuroprotective effect of bromocriptine was inhibited by supplementation of the culture medium with sulpiride, a D(2) antagonist, or by D(2) receptor knockdown with an antisense oligonucleotide. Dopaminergic neurons treated with levodopa showed an increase in free radicals. These data suggest that neuroprotective properties of bromocriptine seen in this cellular model of neurotoxicity are dependent on dopamine D(2) autoreceptor binding and that levodopa toxicity may be related to increased free radical generation in dopaminergic neurons.

Expression of the Ets-1 proto-oncogene correlates with malignant potential in human astrocytic tumors.

The protein encoded by the Ets-1 proto-oncogene is a transcription factor that regulates expression of matrix proteases. It has been associated with tumor invasion and angiogenesis. Glioma progression is characterized by increased invasiveness and neovascularization, so we hypothesized that expression of Ets-1 proto-oncogene might play a role in the progression of these tumors. Therefore, we examined the expression of Ets-1 protein by immunohistochemical means and in situ hybridization in tissues obtained from 81 primary and 20 recurrent astrocytic tumors. Twenty-eight (65%) of 43 glioblastomas (Grade IV astrocytomas) stained for Ets-1. The percentage of positive cells in glioblastomas varied from 10 to 90%. Of the 16 anaplastic astrocytomas (Grade III), 4 (25%) were moderately positive (<50% of cells) for Ets-1. None of 22 cases of low-grade astrocytomas (Grade II) expressed endogenous Ets-1. The staining score was significantly associated with tumor grade (P < .0001). Normal brain tissues did not express Ets-1 protein, whereas recurrent astrocytoma cases expressed significantly more positivity for Ets-1 than did primary tumors (P = .03). The Ets-1 protein was observed mainly in the nucleus and corresponded to the cytoplasmic Ets-1 mRNA localization by in situ hybridization. Western and Northern blot analyses confirmed overexpression of Ets-1 protein and mRNA in high-grade tumors. We conclude that Ets-1 protein expression correlates with the malignant potential of tumors of astroglial origin.