Localization of sex steroid receptor cells, with special reference to thymulin (FTS)-producing cells in female rat thymus

Using monoclonal antibodies against progestin receptors (PR) and estrogen receptors (ER), and polyclonal antibodies to thymulin (FTS) and keratin, localization of the sex steroid receptors was studied immunohistochemically in ovariectomized estrogen-treated rat thymus, with special reference to FTS-producing cells. Both ER- and PR-immunostained cells were mainly localized in the medullary region, especially at its periphery (i.e., the corticomedullary junction). A few cells were also situated in the subcapsular area. They were medium- to large-sized and had a dendritic cell process, some of which were immunohistochemically keratin- and FTS-positive, indicative of reticuloepithelial (RE) cells. Hassall's corpuscles were also receptor-positive and FTS-positive. T-cells were not immunostained with anti-ER, anti-PR or anti-FTS. Light microscopically, both ER and PR immunostainings were localized in the cytoplasm and/or nucleus of keratin-stained RE cells. Electron microscopically, both steroid receptors were shown more precisely to distribute as aggregates of osmiophilic black dots on polysomes and perinuclear space in the cytoplasm and on the euchromatin area in the nucleus. These results suggest that the sex steroids E and P exert their effects through receptors within RE cells which produce FTS to regulate T-cell differentiation.     

Myopathy phenotype in transgenic mice expressing mutated PABPN1 as a model of oculopharyngeal muscular dystrophy

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD)is a late-onset disorder characterized clinically by progressiveptosis, dysphagia and limb weakness, and by unique intranuclearinclusions in the skeletal muscle fibers. The disease is causedby the expansion of a 10-alanine stretch to 12–17 alanineresidues in the poly(A)-binding protein, nuclear 1 (PABPN1;PABP2). While PABPN1 is a major component of the inclusionsin OPMD, the exact cause of the disease is unknown. To elucidatethe molecular mechanism and to construct a useful model fortherapeutic trials, we have generated transgenic mice expressingthe hPABPN1. Transgenic mice lines expressing a normal hPABPN1with 10-alanine stretch did not reveal myopathic changes, whereaslines expressing high levels of expanded hPABPN1 with a 13-alaninestretch showed an apparent myopathy phenotype, especially inold age. Pathological studies in the latter mice disclosed intranuclearinclusions consisting of aggregated mutant hPABPN1 product.Furthermore, some TUNEL positive nuclei were shown around degeneratingfibers and a cluster of it in the lesion in necrotic musclefibers. Interestingly, the degree of myopathic changes was moreprominent in the eyelid and pharyngeal muscles. Further, muscleweakness in the limbs was apparent as shown by the fatigabilitytest. Nuclear inclusions seemed to develop gradually with aging,at least after 1 week of age, in model mouse muscles. We establishedthe first transgenic mouse model of OPMD by expressing mutatedPABPN1, and our model mice appear to have more dramatic alternationsin myofiber viability. ^* To whom correspondence should be addressed. Tel: +81 963736083; Fax: +81 963736599; Email: yamamura{at}gpo.kumamoto-u.ac.jp     

Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese

Molecular diagnosis makes a substantial contribution to precise diagnosis, subclassification, prognosis, and selection of therapy. Mutations in the PDS (SLC26A4) gene are known to be responsible for both Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct, and the molecular confirmation of the PDS gene has become important in the diagnosis of these conditions. In the present study, PDS mutation analysis confirmed that PDS mutations were present and significantly responsible in 90% of Pendred families, and in 78.1% of families with nonsyndromic hearing loss associated with enlarged vestibular aqueduct. Furthermore, variable phenotypic expression by the same combination of mutations indicated that these two conditions are part of a continuous category of disease. Interestingly, the PDS mutation spectrum in Japanese, including the seven novel mutations revealed by this study, is very different from that found in Caucasians. Of the novel mutations detected, 53% were the H723R mutation, suggesting a possible founder effect. Ethnic background is therefore presumably important and should be noted when genetic testing is being performed. The PDS gene mutation spectrum in Japanese may be representative of those in Eastern Asian populations and its elucidation is expected to facilitate the molecular diagnosis of a variety of diseases.     

Improved affinity of a human anti-Entamoeba histolytica Gal/GalNAc lectin Fab fragment by a single amino acid modification of the light chain

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser91 and Arg96 in the third complementarity-determining region of the light chain with other amino acids. The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser91 and nine clones for Arg96 reacted strongly with E. histolytica trophozoites. Sequence analyses revealed that the substituted amino acids at Ser91 were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones. The remaining eight clones exhibited no amino acid change at position 91 or 96. These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin. Pro or Gly substitution for Ser91 caused an increased affinity of the Fab, but substitution with Ala or Val did not. The replacement of Arg96 with Leu did not affect affinity. These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin.     

Clear cell adenocarcinoma with endobronchial polypoid growth

Clear cell adenocarcinoma of the lung is extremely rare. On radiography, a 45-year-old female with fever was found to have an abnormal shadow in the left lower lung field. Bronchoscopy revealed a polypoid tumor in the left bronchus. On biopsy, the tumor was determined to be adenocarcinoma. Preoperative examination found no tumors outside of the lung. The patient underwent left lower lobectomy with bronchial wedge resection. The tumor had completely obstructed and dilated the left lower bronchus, but had not invaded the tissue outside the bronchial wall. Microscopically, the cytoplasm of the tumor cells contained abundant glycogen, and the tumor had solid and glandular structures. The tumor was diagnosed as clear cell adenocarcinoma of the lung.     

Cisplatin-induced long-term dynorphin A-immunoreactivity in cell somata of rat area postrema neurons

We evaluated long-term dynorphin A-immunoreactivity in the rat area postrema (AP) after the administration of cisplatin. First, rats were given 1, 5 and 10 mg/kg body weight cisplatin (i.p.) and their behavior was monitored for 72 h. We observed a delayed increase in pica 24-72 h after injection, compared to the 24 h before injection. We attributed this to the cisplatin injection. Pica was defined as an increase in the intake of non-nutritional matter such as kaolin. Administration of 1, 5 and 10 mg/kg cisplatin led to an increase in kaolin intake on day 1. Administration of 5 and 10 mg/kg of cisplatin led to decreased intake of laboratory chow (MF) on days 1–3, but 10 mg/kg cisplatin causes an excessive aggravation of their condition. Following this behavioral experiment, we immunohistochemically examined the induction of dynorphin A in the AP at 24, 48 and 72 h post-administration of 1 and 5 mg/kg cisplatin. Administration of 5 mg/kg cisplatin caused dynorphin A to accumulate gradually in the neurosoma of the AP neurons, and the numbers of positive AP neurosomata at 48 and 72 h post-administration were higher than following an equal dosage of 0.9% NaCl. These findings suggest that dynorphin A increases in the central nervous system for a long time following administration, and causes certain behavioral and clinical changes, including those related to appetite and nausea.     

Arpp,a new homolog of carp,is preferentially expressed in type 1 skeletal muscle fibers and is markedly induced by denervation

In this study, we isolated and characterized a murine counterpart of the human Arpp (hArpp) gene. Sequence analysis revealed that the murine Arpp (mArpp) gene is almost identical to the Ankrd2 gene, which has recently been isolated as a mouse gene induced in stretched skeletal muscle. The mArpp gene encodes a protein of 332 amino acids that contains four well-conserved ankyrin-repeat domains in the central portion of the protein. The amino acid sequence of mArpp protein (mArpp) is highly homologous to that of mouse cardiac-restricted ankyrin-repeat protein (Carp), which is proposed to be a putative genetic marker for cardiac hypertrophy. Immunohistochemical analysis revealed that mArpp is preferentially expressed in type 1 skeletal muscle fibers, and that mArpp is localized in both the nucleus and the sarcomeric I-band of muscle fibers, suggesting that Arpp may function as a nuclear and sarcomeric protein. Furthermore, mArpp was also expressed in neurons of the cerebellum and cerebrum, the islets of Langerhans in the pancreas, and the esophageal epithelium, suggesting that mArpp may play a functional physiologic role in brain, pancreas, and esophagus as well as in type 1 muscle fibers. Interestingly, although mArpp was localized in both nucleus and cytoplasm in neurons, its localization was restricted to nucleus in pancreas and esophagus, suggesting that intracellular localization of mArpp is regulated in a tissue-specific manner. Furthermore, we found that mArpp- and Carp-expression in skeletal muscle were markedly up-regulated after denervation. Although the elevated expression level of Carp was kept only for two weeks after denervation, that of Arpp was kept at least for 4 weeks, suggesting that mArpp and Carp may play distinct functional roles in denervated skeletal muscle.