A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA.

The xeroderma pigmentosum group A protein (XPA) plays a central role in nucleotide excision repair (NER). To identify proteins that bind to XPA, we screened a HeLa cDNA library using the yeast two-hybrid system. Here we report a novel cytoplasmic GTP-binding protein, designated XPA binding protein 1 (XAB1). The deduced amino acid sequence of XAB1 consisted of 374 residues with a molecular weight of 41 kDa and an isoelectric point of 4.65. Sequence analysis revealed that XAB1 has four sequence motifs G1-G4 of the GTP-binding protein family in the N-terminal half. XAB1 also contains an acidic region in the C-terminal portion. Northern blot analysis showed that XAB1 mRNA is expressed ubiquitously, and immunofluorescence analysis revealed that XAB1 is localized mainly in the cytoplasm. Consistent with the GTP-binding motif, purified recombinant XAB1 protein has intrinsic GTPase activity. Using the yeast two-hybrid system, we elucidated that XAB1 binds to the N-terminal region of XPA. The deletion of five amino acids, residues 30-34 of XPA, required for nuclear localization of XPA abolished the interaction with XAB1. These results suggest that XAB1 is a novel cytoplasmic GTPase involved in nuclear localization of XPA.

Complex formation between lamin A and the retinoblastoma gene product: identification of the domain on lamin A required for its interaction.

The retinoblastoma susceptibility gene product (pRB) has been known to function as a negative regulator of cell growth. Recent observations suggest that its biological activity might be modulated by an interaction with nuclear structures. By using in vitro binding assays, we have found that pRB can associate with lamin A, which has been known to be one of the major nuclear matrix proteins. A series of GST-lamin A deletion mutants was constructed to define the amino acid sequence required for binding to pRB. A GST-lamin A (247-355) contained an activity to associate with pRB, while the other constructs, such as GST-lamin A (37-244) or GST-lamin A (356-571), could not bind to pRB. Within the pRB-binding domain of lamin A, there exists the short amino acid sequence which is also present in the pRB-binding region of the transcription factor E2F-1. The similar experiments using a set of GST-RB deletion mutants revealed that a region containing the E1A-binding pocket B and the carboxy-terminal portion of pRB was responsible for binding to lamin A.

The interactions of E2F with pRB and with p107 are regulated via the phosphorylation of pRB and p107 by a cyclin-dependent kinase.

It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evident for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk. Therefore, we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays. We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as well as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.

Characterization of recombinant monoclonal antithyrotropin receptor antibodies (TSHRAbs) derived from lymphocytes of patients with Graves' disease: epitope and binding study of two stimulatory TSHRAbs.

Anti-TSH receptor autoantibodies (TSHRAbs) are known to be involved in Graves' disease. To elucidate the molecular mechanism of the pathogenesis of Graves' disease, we previously isolated and reconstituted the Ig genes of two B cell clones (101-2 and B6B7) producing a monoclonal thyroid-stimulating antibody (TSAb), a stimulating type of TSHRAb, obtained from patients with Graves' disease. In the present study, we produced a large amount of recombinant monoclonal TSAbs in eukariotic cells using these genes and characterized them. First, we tried to identify their epitopes in the TSHR, by using a panel of mutants of the extracellular domain of the TSH receptor (TSHR). Substantial cell surface expression level of each mutant was confirmed by fluorescence-activated cell sorter analysis using a TSHRAb. Mutations in the N-terminal (but not C-terminal) region of the extracellular domain of TSHR abrogated or reduced TSAb activities of both antibodies, whereas they had opposite effects on TSH activity; cAMP generation by 101-2 significantly decreased in the receptors mutated in amino acids 52-56 and 58-61, and that by B6B7 decreased in amino acids 34-37 and 58-61. Secondly, purified antibodies were radiolabeled and tested for binding to cells expressing high levels of TSHR. Although their affinities were lower than that of TSH, their binding was not displaced by TSH. The antibody binding was not mutually competitive. These findings suggest that these antibodies interact with the N-terminal region of the receptor and transduce a signal through binding sites different from TSH. We believe that this is the first report of the characterization of human monoclonal TSHRAbs on their epitopes and bindings, confirming previous reports using patient sera or murine monoclonal antibodies.

Hashimoto's thyroiditis with heterogeneous antithyrotropin receptor antibodies: unique epitopes may contribute to the regulation of thyroid function by the antibodies.

Blocking-type TSH-binding inhibitor Igs (TBIIs) are known to cause hypothyroidism and an atrophic thyroid gland in patients with primary myxedema. They can block the activity of thyroid-stimulating antibodies (TSAbs) in Graves' patients as well as the activity of TSH. The majority of the epitopes for these blocking-type TBIIs have been, and are shown herein, to be present on the C-terminal region of the extracellular domain of the human TSH receptor (TSHR), whereas those for Graves' TSAbs are on the N-terminus. We report on a patient with Hashimoto's thyroiditis who suffered from mild hypothyroidism and a moderately sized goiter. Her serum had a potent blocking-type TBII and a weak TSAb in human and porcine TSHR systems. Using human TSHR/lutropin-CG receptor chimeras, we determined that the functional epitope of her blocking-type TBII was uniquely present on the N-terminal, rather than the C-terminal, region of the extracellular domain of the TSHR, unlike the case for blocking-type TBIIs in primary myxedema patients. The epitope of her TSAb was also unusual. Although the functional epitopes of most TSAbs are known to involve the N-terminal region of the receptor, her TSAb epitope did not seem to be present solely on the N- or C-terminus of the extracellular domain of the receptor. Blocking-type TBIIs from patients with primary myxedema blocked her TSAb activity as well as stimulation by TSH; her blocking-type TBII was able to only partially block her TSAb. In contrast, her blocking-type TBII almost completely blocked TSAbs from Graves' patients. Thus, we suggest that the unique epitopes of this patient's heterogeneous population of TSH receptor antibodies, at least in part, contribute to regulation of her thyroid function.

A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans.

The synergistic relationship between GH-releasing secretagogue (GHS) and GH-releasing hormone (GHRH) with respect to GH secretion is well known. In the present study, we report a similar relationship between GHRH and ghrelin, a recently identified endogenous ligand for the GHS receptor. In normal male adults, various doses of ghrelin were intravenously administered alone or together with 1.0 microg/kg GHRH. At small doses of 0.08 and 0.2 microg/kg ghrelin, combined administration of the two peptides significantly stimulated GH release in a synergistic manner; the mean GH response values of the two peptide combinations were more than the summed mean GH response values of each peptide alone (P < 0.05). In addition, at 1.0 microg/kg ghrelin, the tendency of the synergistic effect was observed, although the comparison was not statistically significant probably due to a submaximal dose ceiling effect. No synergistic effects with respect to ACTH or prolactin secretion were observed. In conclusion, the synergistic interaction between ghrelin and GHRH was clearly shown and might be useful for a provocation test to diagnose GH deficiency.

Substantial production of ghrelin by a human medullary thyroid carcinoma cell line.

Ghrelin, an endogenous ligand for the GH secretagogue receptor, is a novel acylated peptide produced in the gastrointestinal endocrine cells as well as neuroendocrine cells in the hypothalamus. The Ser(3) residue of ghrelin is modified by n-octanoic acid, a modification necessary for hormonal activity. Human medullary thyroid carcinoma is known to produce a variety of gastrointestinal and neuroendocrine peptides. In the present study we investigated ghrelin production in the thyroid gland, especially in human medullary thyroid carcinoma. PCR amplification demonstrated prepro-ghrelin gene transcripts in normal human thyroid tissue and two medullary thyroid carcinoma cell lines (human TT cells and rat 6-23 cells), but not in a rat thyroid follicular cell line. TT cells showed the expression of prepro-ghrelin mRNA of about 0.6 kb by Northern blot analysis. Furthermore, production of ghrelin in TT cells was demonstrated by RIA and immunocytochemistry. Accumulation of des-n-octanoyl ghrelin in the cultured medium of the cells was confirmed. Finally, human medullary thyroid carcinoma surgical specimens showed significantly higher des-n-octanoyl ghrelin contents than normal thyroid tissues. In conclusion, we revealed that ghrelin was produced by the human thyroid parafollicular carcinoma cell line, TT cells. These findings suggest that ghrelin is produced in the thyroid C cells as well as in medullary thyroid carcinoma and may provide opportunities to investigate its physiological role in the thyroid gland.

Inactivation of oncoprotein binding by a single Cys706-to-Tyr substitution in the retinoblastoma protein.

We previously found a new single amino acid substitution at codon 706 (Cys-to-Tyr) of the retinoblastoma (RB) gene in a sporadic retinoblastoma patient. The glutathione S-transferase-RB fused protein containing this mutation was here tested for binding to SV40 large T antigen and adenovirus E1A protein, and was shown to have lost its binding affinity. Thus, Tyr, as well as Phe, residues substituted for Cys706 were found to abolish the RB protein activity.

Characterization of the secretable ectodomain of thyrotropin receptor produced by the recombinant baculovirus system.

Thyrotropin receptor (TSHR) is a member of the glycoprotein hormone receptor family and an autoantigen of Graves' disease. Various attempts have been made to obtain a large amount of soluble ectodomain of TSHR in insect or mammalian cells, but most of them failed to secrete the overexpressed ectodomain. In the present study, we observed that about one-third of the ectodomain protein (sTSHR-gp), in which the signal peptide of TSHR was replaced by the baculovirus-encoded glycoprotein 67-signal peptide, was secreted into the culture medium and the remainder stayed within cells in the recombinant baculovirus system. Microsequencing the N-terminal of the purified protein confirmed that the baculovirus signal peptide was cleaved at the expected site. Carbohydrate studies using several glycosidases and lectins revealed that the secreted form of the ectodomain had biantennary carbohydrate, whereas the non-secreted form had high-mannose. Moreover, the secreted form of sTSHR-gp exhibited high-affinity ligand binding, whereas the non-secreted form did not show any significant ligand binding. Regarding the interactions of TSHR ectodomains with anti-TSHR antibodies, both the secreted and non-secreted forms of sTSHR-gp, almost completely neutralized the stimulatory and inhibitory anti-TSHR antibody activities. In conclusion, we succeeded in secreting the ectodomain of TSHR into culture medium, which was capable of binding to TSH and neutralizing anti-TSHR antibody activities.

Emergence and spread of a new clone of M type 1 group A Streptococcus coincident with the increase in invasive diseases in Japan.

BACKGROUND: In Japan invasive group A streptococcal infections such as sepsis and toxic shock syndrome (TSS) have increased since 1992. As is the case in the United States and Europe, M1 serotype is predominant among the isolates from Japanese patients. METHODS: By restriction enzyme digestion and pulsed field gel electrophoresis, we investigated the whole genomic DNA profiles of 95 M type 1 group A streptococcal strains isolated from patients with serious diseases including sepsis, toxic shock syndrome, necrotizing fasciitis and nonsuppurative complications and with uncomplicated pharyngitis during 1979 through 1996 in Japan. RESULTS: The genome profiles among 8 of 10 isolates from patients with serious diseases in 1979 through 1991 were all the same and were shared by the profiles of the 35 of 48 isolates from patients with uncomplicated pharyngitis in 1982 through 1991. All 18 strains isolated from patients with invasive diseases in 1992 to 1996 had a unique profile, which was shared by the profiles of 18 of 19 isolates from uncomplicated pharyngitis during the same period. This genomic profile was distinct from the predominant or any other profiles before 1992, and it was found to be a new clone. CONCLUSIONS: The emergence and spread of this new clone of M type 1 Streptococcus after 1991 may be associated with the increase in invasive streptococcal infections that occurred during the same period in Japan. Genomic profiles as well as serotypes of streptococcal isolates are important for the epidemiology of clinical relevance in streptococcal diseases.

Possible direct method to determine the radius of a star from the spectrum of gravitational wave signals

We computed the spectrum of gravitational waves from a dust disk star of radius R inspiraling into a Kerr black hole of mass M and specific angular momentum a. We found that when R is much larger than the wave length of the quasinormal mode, the spectrum has several peaks and the separation of peaks Deltaomega is proportional to R-1 irrespective of M and a. This suggests that the radius of the star in coalescing binary black hole-star systems may be determined directly from the observed spectrum of gravitational waves. This also suggests that the spectrum of the radiation may give us important information in gravitational wave astronomy as in optical astronomy.

Resonance assignments, solution structure, and backbone dynamics of the DNA- and RPA-binding domain of human repair factor XPA.

XPA is involved in the damage recognition step of nucleotide excision repair (NER). XPA binds to other repair factors, and acts as a key element in NER complex formation. The central domain of human repair factor XPA (residues Met98 to Phe219) is responsible for the preferential binding to damaged DNA and to replication protein A (RPA). The domain consists of a zinc-containing subdomain with a compact globular structure and a C-terminal subdomain with a positively charged cleft in a novel alpha/beta structure. The resonance assignments and backbone dynamics of the central domain of human XPA were studied by multidimensional heteronuclear NMR methods. 15N relaxation data were obtained at two static magnetic fields, and analyzed by means of the model-free formalism under the assumption of isotropic or anisotropic rotational diffusion. In addition, exchange contributions were estimated by analysis of the spectral density function at zero frequency. The results show that the domain exhibits a rotational diffusion anisotropy (Dparallel/Dperpendicular) of 1.38, and that most of the flexible regions exist on the DNA binding surface in the cleft in the C-terminal subdomain. This flexibility may be involved in the interactions of XPA with various kinds of damaged DNA.

Association of autoimmune thyroid disease with microsatellite markers for the thyrotropin receptor gene and CTLA-4 in Japanese patients.

In a previous study we identified a microsatellite marker near the thyrotropin receptor (TSHR) gene. Studies with this marker, TSHR-CA, revealed a significant association between autoimmune thyroid disease (AITD) in Japanese patients and one specific allele (allele 1; 180 base pair [bp]) of the microsatellite sequence. In addition, weak evidence for association of AITD with two alleles of the CTLA-4 gene was observed. In the present study, TSHR-CA has been mapped to approximately 600 kb of the TSHR gene using radiation hybrid mapping. TSHR-CA and another TSHR microsatellite marker, TSHR-AT, which is located in intron 2 of TSHR gene, were genotyped in a set of 349 unrelated Japanese AITD patients and 218 Japanese controls. The TSHR-AT marker showed association in this Japanese AITD population with a significant increase in allele 5 (294 bp; p < 0.05) and a significant decrease in allele 7 (298 bp; p < 0.05). The association of allele 5 of TSHR-AT was also significant in hypothyroid patients (thyrotropin-binding inhibitory immunoglobulin-positive [TBII+], P < 0.01; thyrotropin-binding inhibitory immunoglobulin-negative [TBII-], p < 0.05). The association of allele 7 of TSHR-AT were also significant for the hypothyroid TBII+ patients (p < 0.05). The CTLA-4 gene was also genotyped in this expanded set of Japanese AITD patients and controls. Association between AITD susceptibility and allele 2 (102 bp; p < 0.01) and allele 4 (106 bp; p < 0.01) were observed. These associations were also observed with GD patients (allele 2, p < 0.01; allele 4, p < 0.01). Associations with TSHR-CA were observed for Hashimoto's thyroiditis (HT) patients with respect to alleles 3 (179 bp; p < 0.05) and 5 (175 bp; p < 0.05) and with hypothyroid TBII- patients for allele 4 (177 bp; p < 0.05). The presence of specific alleles of TSHR-CA, TSHR-AT, and CTLA-4 contribute significant increase in risk of development of AITD. These results confirm and expand on our previous study suggesting that alleles of the TSHR and CTLA-4 genes, or genes near them contribute to AITD susceptibility and set the stage for future studies of interactions between these genes and AITD.

Emotional disturbances in trauma patients during the rehabilitation phase: studies of posttraumatic stress disorder and alexithymia.

Recent studies have shown a partial similarity between posttraumatic stress disorder (PTSD) and alexithymia. In this study, the authors examined the relationship between PTSD and alexithymia in two samples of 26 patients with burn injury and 27 patients with digit amputation during rehabilitation. The prevalence rates of DSM-III-R PTSD and alexithymia assessed by the Toronto Alexithymia Scale (TAS) were significantly higher for injury patients than for healthy volunteers. The rate of PTSD symptoms of avoidance and emotional numbing was significantly and positively correlated with the TAS scores in injury patients. The PTSD symptoms of avoidance and emotional numbing had a significant relationship with function after digit replantation. Alexithymia also had a similar relationship with physical conditions. These results suggest that 1) in some cases, alexithymia may be evident when PTSD emotional symptoms appear in injury patients, and 2) emotional disturbances (i.e., PTSD symptoms of avoidance and emotional numbing and alexithymia) may be influenced by the level of functional recovery after digit replantation.

Cranio-nasal median splitting for radical resection of craniopharyngioma.

A new surgical approach for radical resection of craniopharyngioma is presented. This approach (cranio-nasal median splitting) involves craniotomy in the centre of the frontal bone, removal of the median portion of the supraorbital bar that incorporates the nasal bone, and detachment of the medial canthal ligaments. The frontal lobes, the cribriform plates, the planum sphenoidale, and the upper nasal cavities are split in the midline. The extraventricular surface of the hypothalamus, the pituitary stalk, and the posterior portion of the Willis' arterial ring are well visualized through the midline infrachiasmatic route. The intraventricular surface of the hypothalamus is also visible in the same operative field through the lamina terminalis and/or the anterior portion of the corpus callosum. This excellent visualization is quite helpful for minimizing operative injury to the hypothalamus and the pituitary stalk whichever surface of the third ventricular floor the tumour is situated upon. Three cases of craniopharyngioma operated upon by this approach are presented. Discussions are focused not only on the indication, but on the advantages and disadvantages of this approach. The surgical techniques for reconstruction of the cranial base are also described, together with some precautions that should be taken to prevent possible postoperative complications.

Identification of a damaged-DNA binding domain of the XPA protein.

The XPA (xeroderma pigmentosum group A) protein is a zinc metalloprotein consisting of 273 amino acids which binds preferentially to UV- or chemical carcinogen-damaged DNA, suggesting that it is involved in the recognition of several types of DNA damage during nucleotide excision repair processes. Here we identify a DNA binding domain of the XPA protein. The region of the XPA protein responsible for preferential binding to DNA damaged by UV or cis-diammine-dichloroplatinum(II) (cisplatin) is contained within a truncated derivative of the XPA protein, MF122, consisting of 122 amino acids and containing a C4 type zinc finger motif. CD (circular dichroism) measurements of the MF122 protein showed that it has a helix-rich secondary structure, suggesting that it is a discretely folded, functional mini-domain. The MF122 protein should be useful for structural investigation of the XPA protein and of its interaction with damaged DNA.

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency.

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.

Successful closure of recurrent traumatic csf rhinorrhea using the free rectus abdominis muscle flap.

BACKGROUND: We present two patients in whom a free rectus abdominis muscle flap was used to close recurrent traumatic CSF rhinorrhea. CASE DESCRIPTION: CT scan of both patients showed frontal lobe atrophy and porencephaly after contusional hematoma. In the first patient, because the site of CSF leakage was not identified and the patient underwent three unsuccessful attempts to close the fistula using the fascia lata, we treated the patient by unifying all paranasal sinuses and by filling them with a free rectus abdominis muscle flap. In the second patient, CSF rhinorrhea recurred 6 years after closure of the fistula using the fascia lata. The patient underwent separation of a porencephalic cyst from the paranasal sinus and a free muscle flap was placed extradurally, because the CSF pulse pressure in the enlarged left anterior horn eroded the previously repaired fascia lata, resulting in the recurrence of CSF leakage. CONCLUSION: Although duraplasty is the primary procedure for repairing dural fistulas, the vascularized free muscle flap is an alternative method when the location of the fistula is not identified or the patient with recurrent CSF rhinorrhea has severe frontal lobe atrophy and porencephaly.

Lymphangioma. A long-term follow-up study.

At the Hospital for Sick Children 177 patients with lymphangioma, exclusive of those who had intra-abdominal lesions, were seen between 1927 and 1964. Forty-nine of these (aged 8 to 41 years) were available for follow-up examination. The results of treatment by surgical excision, aspiration, incision and drainage, and radiation are reported. No cases confirmed histologically as lymphangioma underwent spontaneous regression. Two histologically unconfirmed cases underwent partial regression. Four of the 11 who were left with lymphangioma tissue at operation never had significant recurrences. Therefore, this clinical study does not clarify the possible role of spontaneous regression in lymphangioma. Extensive surgery is the treatment of choice whenever feasible, but in unilocular or bilocular cystic lymphagnioma subsequent regression can be expected after palliative treatment (aspiration, or incision and drainage). Clinical and pathological criteria should be established for differentiation between lymphangioma and primary lymphedema. Lesions involving subepithelial, subdermal, and subcutaneous or internodal networks of lymphatics will produce lymphagioma; lesions of the collecting lymphatic trunks will result in lymphedema.

Lymphedema. A clinical review and follow-up study.

In a retrospective study of primary lymphedema, we found that the age of onset ranged from 0 to 16 years; sex did not appear to be a factor; and treatment was preventive rather than surgical. Thirteen patients were available for follow-up studies, the time interval being at least 6 years. The degree of swelling at the time of the follow-up examination was defined as mild, moderate, or severe. In patients with mild and moderate swelling the edema was not progressive and surgical treatment was not indicated. Severe swelling appeared to be associated with congenital lymphedema and with repeated infection. Pressure-support treatment can be effective in the reduction of swelling or in the prevention of further swelling, if applied constantly over a long period of time. The authors discuss the histological similarity between lymphedema and lymphagioma, and present an anatomical classification for the two entities.