Heavy chain variable (VH) region diversity generated by VH gene replacement in the progeny of a single precursor cell transformed with a temperature-sensitive mutant of Abelson murine leukemia virus.

Sequence analysis of a large number of DNA clones containing a functional heavy chain variable, diversity, and joining (VHDJH) complex generated by VH to VHDJH joining (VH gene replacement) in the progeny derived from a common precursor cell transformed with a temperature-sensitive (ts) Abelson murine leukemia virus (A-MuLV) indicates that endogenous VH gene replacement in vitro generates immunoglobulin gene joints distinct from those generated by the usual VH to DJH joining. Such joints keep the pentamer CAAGA at the 3' end of the donor VH segment and lack a recognizable D segment, as can be seen also in vivo. The results suggest that VH gene replacement participates in generating VH region diversity in vivo, as previously postulated. During the joining process, a unique VH gene was selected in all progeny cells, together with a single A nucleotide dominantly added to the junctional boundaries. The basis of these regulatory processes is discussed.

Mechanical regulation of bone homeostasis through p130Cas-mediated alleviation of NF-κB activity.

Mechanical loading plays an important role in bone homeostasis. However, molecular mechanisms behind the mechanical regulation of bone homeostasis are poorly understood. We previously reported p130Cas (Cas) as a key molecule in cellular mechanosensing at focal adhesions. Here, we demonstrate that Cas is distributed in the nucleus and supports mechanical loading-mediated bone homeostasis by alleviating NF-κB activity, which would otherwise prompt inflammatory processes. Mechanical unloading modulates Cas distribution and NF-κB activity in osteocytes, the mechanosensory cells in bones. Cas deficiency in osteocytes increases osteoclastic bone resorption associated with NF-κB-mediated RANKL expression, leading to osteopenia. Upon shear stress application on cultured osteocytes, Cas translocates into the nucleus and down-regulates NF-κB activity. Collectively, fluid shear stress-dependent Cas-mediated alleviation of NF-κB activity supports bone homeostasis. Given the ubiquitous expression of Cas and NF-κB together with systemic distribution of interstitial fluid, the Cas-NF-κB interplay may also underpin regulatory mechanisms in other tissues and organs.

Clinical and experimental features of MuSK antibody positive MG in Japan.

We investigated the presence of antibodies (Abs) against muscle-specific tyrosine kinase (MuSK) in Japanese myasthenia gravis (MG) patients. MuSK Abs were found in 23 (27%) of 85 generalized seronegative MG (SNMG) patients but not in any of the ocular MG patients. MuSK Ab-positive patients were characterized as having female dominance (M:F, 5:18), age range at onset 18 to 72 (median 45) years old, and prominent oculobulbar symptoms (100%) with neck (57%) or respiratory (35%) muscle weakness. Limb muscle weakness was comparatively less severe (52%), thymoma absent. Most patients had good responses to simple plasma exchange and steroid therapy. MuSK IgG from all 18 patients was exclusively the IgG 4 subclass and bound mainly with the MuSK Ig 1-2 domain. Serial studies of 12 individuals showed a close correlation between the variation in MuSK Ab titers and MG clinical severity (P = 0.01 by Kruskal-Wallis). MuSK Ab titers were sharply decreased in patients who had a good response to early steroid therapy or simple plasma exchange, but there was no change, or a rapid increase on exacerbation after thymectomy. Measurement of MuSK Ab titers aids in the diagnosis of MG and the monitoring of clinical courses after treatment.

Myasthenia gravis induced by autoantibodies against MuSK.

Myasthenia gravis (MG) is caused by the failure of neuromuscular transmission mediated by autoantibodies. That is, the binding of autoantibodies to postsynaptic membranes in neuromuscular junctions (NMJ) results in weakening of the ocular, bulbar and limb muscles and produces the characteristic syndrome of MG. This relatively rare disease serves as a model not only for study of the pathogenesis and treatment of all autoimmune disorders but also for understanding the basic mechanisms of neuromuscular transmission at the NMJ. About 80 to 85% of patients with MG have autoantibodies against acetylcholine receptors (AChR). Although a number of studies have shown the possible existence of other autoantibodies in the remaining approximately 20% of MG patients, the responsible autoantigens have remained elusive. However, antibodies against muscle-specific kinase (MuSK) have been found in 30% of MG patients without AChR antibodies. MuSK, a tyrosine kinase receptor, is required for the development of NMJ's postsynaptic membranes. Still, the pathogenicity of MuSK antibodies as a cause of muscle weakness in patients with MG remains a matter of dispute, because the experimental autoimmune MG caused by MuSK antibodies in animals was absent. Here we describe recent progress toward understanding the pathogenic role of MuSK antibodies in the decline of muscle strength that typifies MG.

Identification and characterisation of a developmentally regulated mammalian gene that utilises -1 programmed ribosomal frameshifting.

Translational recoding of mRNA through a -1 ribosomal slippage mechanism has been observed in RNA viruses and retrotransposons of both eukaryotes and prokaryotes. Whilst this provides a potentially powerful mechanism of gene regulation, the utilization of -1 translational frameshifting in regulating mammalian gene expression has remained obscure. Here we report a mammalian gene, Edr, which provides the first example of -1 translational recoding in a eukaryotic cellular gene. In addition to bearing functional frameshift elements that mediate expression of distinct polypeptides, Edr bears both CCHC zinc-finger and putative aspartyl protease catalytic site retroviral-like motifs, indicative of a relic retroviral-like origin for Edr. These features, coupled with conservation of Edr as a single copy gene in mouse and man and striking spatio-temporal regulation of expression during embryogenesis, suggest that Edr plays a functionally important role in mammalian development.

The Eck receptor tyrosine kinase is implicated in pattern formation during gastrulation, hindbrain segmentation and limb development.

Members of the protein superfamily of transmembrane receptor tyrosine kinases are key components of intercellular signal transduction pathways that elicit appropriate cellular responses to environmental cues during development of multicellular organisms. In a search for additional receptor tyrosine kinases expressed during mouse embryogenesis we cloned the murine homolog of Eck, a member of the Eph subfamily, that maps to the distal region of mouse chromosome 4. Specific antisera defined Eck in murine embryonic cells as a glycoprotein of 130 kDa with an intrinsic autophosphorylation activity. Immunohistochemical staining and laser scanning microscopy revealed a dynamic and tightly regulated distribution of Eck receptor protein in the developing mouse embryo. During gastrulation, a high transient distribution of Eck was seen in mesodermal cells aggregating in the midline as notochordal plate. A similar restriction of Eck receptor protein was apparent along the rostrocaudal axis of the developing neural tube. In hindbrain neuroepithelia, Eck protein localised specifically to cells of rhombomere 4 and was also seen transiently in cells populating second and third branchial arches and neurogenic facial crest VII-VIII and IX-X. Receptor distribution also implicated Eck in development of the proximodistal axis of the limb, expression being restricted to distal regions of limb bud mesenchyme. At later stages, additional sites of Eck protein expression were seen in the cartilaginous model of the skeleton, tooth primordia, infundibular component of the pituitary and various fetal tissue epithelia. Taken together, our data suggest pleiotropic functions for the Eck receptor, initially in distinctive aspects of pattern formation and subsequently in development of several fetal tissues, and reveal possible allelism with known mouse developmental mutant loci.

ATF-2-binding regulatory element is responsible for the Ly49A expression in murine T lymphoid line, EL-4.

To understand the mechanism of Ly49A-expression and its significance in T-cell differentiation, we analyzed the 5'-flanking region of the Ly49A gene in a search for the Ly49A-regulatory element. Since very few known regulatory elements have been found in this region, presumably a novel regulatory sequence(s) could exist. Accordingly, we defined the 13-bp regulatory element, 5'-ATGACGAGGAGGA-3', restricted to Ly49A-expression in EL-4 cells in comparison with two other representative cell lines tested. This element, designated as EL13, proved to be previously undiscovered by homology search and is highly homologous with several virus DNAs. Using EL13 as a probe we have cloned a cDNA encoding a binding protein to EL13. Its deduced nucleotide sequence revealed that EL13-binding protein is almost identical with rat ATF-2. Although ATF-2 is known to bind to cyclic AMP responsive element (CRE), EL13 shares five out of eight nucleotides with this consensus sequence. Our results suggested that ATF-2 may play an important role via binding to EL13 for the expression of Ly49A. These data will provide useful information for understanding T-cell and NK-cell differentiation in murine immune system.

The strain-dependent constitutive expression of murine serum amyloid-P component is regulated at the transcriptional level.

The strain-dependent expression of murine serum amyloid P-component (SAP) has been known to be linked to the Sap locus. We have quantified the SAP mRNA in several inbred strains including DBA/2 and C57BL/6 mice which represent high and low producers of SAP at resting state, respectively, and found that the mRNA levels correlated well with the amount of SAP protein. Interestingly, the SAP mRNA level of F1 mouse between DBA/2 and C57BL/6 was low and similar to that of C57BL/6. Primer extension and ribonuclease (RNAase) protection analyses demonstrated that a single type of transcript was generated from the SAP gene and that the cap sites were identical regardless mouse strains tested under unstimulated and stimulated (by lipopolysaccharide (LPS) or interleukin-6 (IL-6)) conditions. To investigate possible structural difference of the SAP gene including 5' flanking region, we have cloned, sequenced and compared the SAP genes from DBA/2 and C57BL/6 mice. Sequence analyses revealed that the 5' flanking regulatory regions, as well as the coding regions, were well-conserved between the two strains. These results demonstrate that the strain-dependent SAP expression occurs at the transcriptional level but seems to be affected by neither different type of the transcripts nor structural difference of the 5' flanking and coding regions of the SAP gene. It was suggested that a possible transcription factor with suppressive activity, which is encoded by a gene linked to Sap, may be involved.

Identification and characterization of 5' extension of mammalian agrin cDNA, the exons and the promoter sequences.

Agrin, which is secreted from motor neurons, is essential for the formation and maintenance of the vertebrate neuromuscular junctions. Here we show the complete N-terminal sequence of the mammalian cDNA required for the expression and secretion as well as the intron/exon structure and the 5'-flanking sequence required for basal promoter activity. The 5'-flanking region and the first exon are extremely GC rich and contain a CpG island. These features may account for hindrance in identification of the 5' end of the cDNA and the promoter region of the mammalian agrin gene.

Characterization of a new subgroup of human Ig V lambda cDNA clone and its expression.

From a human bone marrow cDNA library, we have cloned and sequenced a gene which cross-hybridized with murine pre-B cell-specific gene 8HS-20 cDNA under the low-stringent condition. Sequence analysis predicted that this gene (YM-1) encoded 240 amino acids which had the basic structure of immunoglobulin lambda light chain. The 3' half of the YM-1 sequence was identical to the J lambda 2 C lambda 2 region except for four nucleotides. The 5' part of the gene had 87.6% sequence homology with the reported V lambda gene called T1. Comparison of the deduced amino acid sequences with representative members of the seven other V lambda subgroups showed considerable structural homology, but the maximum homology with these chains was 44%. Therefore, we conclude that YM-1 belongs to a new V lambda subgroup. Interestingly YM-1 showed higher homology with VpreB1 (56%) than with any of the other V lambda subgroups. By Southern blot analysis four to six cross-hybridizing V lambda bands were detected at high stringency. Expression of the V lambda gene was observed in immature as well as mature B cell lines without accompanying V-JC gene joining, suggesting that V lambda of the YM-1 locus is activated at the early stage of maturation.

Expression and structure of serum gp70 as an acute phase protein in NZB mice.

A cDNA corresponding to a serum gp70 synthesized as an acute phase protein in mouse hepatocytes was cloned and analyzed. This cloned cDNA had the characteristics of an endogenous xenotropic murine leukemia virus. Synthesized oligo-DNA specific for this cDNA reacted strongly with liver RNA derived from NZB mice injected with LPS as a trigger of an acute phase inflammatory response. There was also low level of gp70 in the kidney in response to LPS injection. The LTR structure of the cDNA showed that this clone is the immediate precursor of an infectious xenotropic virus in the proposed evolutionary scheme of murine leukemia virus.

Guidelines for pre-clinical animal and cellular models of MuSK-myasthenia gravis.

Muscle-specific tyrosine kinase (MuSK) autoantibodies are the hallmark of a form of myasthenia gravis (MG) that can challenge the neurologist and the experimentalist. The clinical disease can be difficult to treat effectively. MuSK autoantibodies affect the neuromuscular junction in several ways. When added to muscle cells in culture, MuSK antibodies disperse acetylcholine receptor clusters. Experimental animals actively immunized with MuSK develop MuSK autoantibodies and muscle weakness. Weakness is associated with reduced postsynaptic acetylcholine receptor numbers, reduced amplitudes of miniature endplate potentials and endplate potentials, and failure of neuromuscular transmission. Similar impairments have been found in mice injected with IgG from MG patients positive for MuSK autoantibody (MuSK-MG). The active and passive models have begun to reveal the mechanisms by which MuSK antibodies disrupt synaptic function at the neuromuscular junction, and should be valuable in developing therapies for MuSK-MG. However, translation into new and improved treatments for patients requires procedures that are not too cumbersome but suitable for examining different aspects of MuSK function and the effects of potential therapies. Study design, conduct and analysis should be carefully considered and transparently reported. Here we review what has been learnt from animal and culture models of MuSK-MG, and offer guidelines for experimental design and conduct of studies, including sample size determination, randomization, outcome parameters and precautions for objective data analysis. These principles may also be relevant to the increasing number of other antibody-mediated diseases that are now recognized.

Genomic DNA with transformation-related activity and melanoma antigen expression.

We have analyzed the mouse melanoma antigen with monoclonal antibodies established by syngeneic immunization. To further understand the structure of this antigen at a molecular level, we have cloned the genomic DNA controlling the expression of melanoma antigen by cosmid library transfection and a monoclonal antibody. In the process of analyzing this DNA fragment we found that it contained a gene related with transformation, which was proved by tumor formation in nude mice inoculated with NIH/3T3 transfectants of this DNA fragment. We discuss the structure of the gene product based on the deduced amino acid sequence of cDNA, which maps the genome bearing transformation-related activity.

A case of CREST syndrome and myeloperoxidase-specific anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis.

We report the first case of myeloperoxidase-specific anti-neutrophil cytoplasmic autoantibody (MPO-ANCA)-associated glomerulonephritis in a patient with CREST syndrome. A 74-year-old Japanese man with CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia) developed rapidly progressive renal failure without elevation of blood pressure. Renal biopsy revealed glomerular sclerosis and fibrous crescents. The MPO-ANCA titer was elevated to 145 EU/ml. When patients with collagen diseases develop rapidly progressive glomerulonephritis, the possibility of MPO-ANCA-associated glomerulonephritis should be kept in mind.

Missense mutations in the phosphomannomutase 2 gene of two Japanese siblings with carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein syndrome type I (CDG1) is an autosomal recessive disorder characterized by severe nervous system involvement and a carbohydrate moiety deficiency in N-linked glycoproteins. Clinical symptoms are psychomotor retardation, stroke-like episodes or hemorrhagic episodes, hepatic dysfunction, polyneuropathy, and cerebellar ataxia. Marked atrophy of the cerebellar hemispheres and pons is recognizable on CT scan or MRI. CDGI has been mapped to human chromosome 16p by linkage studies. Recently, missense mutations in the gene for phosphomannomutase (PMM2) have been detected in Caucasian patients with CDG1. We studied DNA mutations in PMM2 in a Japanese family with CDG1. DNA sequencing of PMM2 in the siblings showed missense mutations of maternal origin in exon 5 and of paternal origin in exon 8. No such mutations were detected in 50 unrelated healthy Japanese. These findings suggest that the PMM2 is responsible for CDG1 in the Japanese as well as in Caucasians, and CDG1 may be the diagnosis in OPCA of neonatal onset, more often than currently thought.

Radial bone mineral density in hemodialysis patients with adynamic bone disease.

Adynamic bone disease (ABD) has attracted attention as the most frequent type of renal osteodystrophy, but there are few reports about the bone mineral density (BMD) in ABD patients. This study investigated the BMD in hemodialysis patients with ABD and with relatively normal bone turnover. We measured the BMD of the distal one-third of the radius by dual-energy X-ray adsorptiometry. In the ABD group (intact PTH<65 pg/ml, intact osteocalcin<30 ng/ml), there were 19 men and 17 women with a mean age of 56.4 +/- 12.0 years. In the relatively normal bone turnover group (intact PTH: 120-250 pg/ml), there were 24 men and 16 women with a mean age of 57.1 +/- 14.7 years. Although there were no significant differences between the two groups with respect to age, gender, and duration of hemodialysis, a significant increase of the BMD and the calcium x phosphate product was observed in the ABD group (radial BMD: 0.648 +/- 0.137 g/cm2 versus 0.572 +/- 0.132 g/cm2, calcium x phosphate product: 57.53 +/- 14.92 mg2/dl2 versus 49.76 +/- 12.13 mg2/dl2). These findings suggest that an increase in radial BMD may not be a useful marker of the improvement in bone lesions in ABD patients.

A case of antiphospholipid antibody syndrome diagnosed after thrombosis of an arteriovenous shunt.

A 32-year-old male dialysis patient with lupus nephritis was admitted because of shunt obstruction. The arteriovenous fistula was reconstructed, but obstruction recurred twice within several hours after surgery. A high blood level of anticardiolipin beta2-glycoprotein I antibody suggested that shunt obstruction was caused by a thrombotic tendency related to the antiphospholipid antibody syndrome. Accordingly, for the third shunt procedure, antiplatelet therapy (which had been commenced for systemic lupus erythematosus) was combined with dalteparin sodium from before surgery and warfarin was added postoperatively. This regimen prevented shunt obstruction. In conclusion, hemodialysis patients who suffer repeated shunt obstruction should be examined for antiphospholipid antibody syndrome.

A randomized multicenter trial to evaluate the effects of UV-Flash system on peritonitis rates in CAPD.

A number of systems and devices have been developed to reduce peritonitis in CAPD patients. One of the newer developed systems in the UV-Flash. A retrospective study was conducted in five Japanese hospitals to measure the efficiency of the UV-Flash system in reducing the peritonitis rate for CAPD patients. This study took place between January 1983 to January 1992. The UV-Flash system had a significantly lower peritonitis rate (1/46.6 patient months) compared to the Standard system (1/27.5), but showed nominal significance when compared to the Disconnect system (1/47.6). Due to the lack of differentiation in peritonitis rates between the UV-Flash and Disconnect system, patient types for both systems were analyzed. The study found more impaired patients on UV-Flash (37.2%) as opposed to the Disconnect (9.8%). These impaired patients treated on the UV-Flash system showed a significantly lower peritonitis rate (1/27.5) when compared to the Disconnect (1/9.7) system. The UV-Flash system proved able to achieve sufficient prevention of peritonitis in CAPD patients. Thus, this system can be successfully applied to high risk CAPD patients.

Renal osteodystrophy in hemodialysis patients.

Patterns of bone loss in the axial and appendicular skeleton were studied in 88 chronic hemodialysis patients (59 males and 29 females) and 60 normal volunteers (30 males and 30 females). The hemodialysis patients were properly medicated with phosphate binders and 1 alpha-OH D3 where necessary. The metacarpal index (MCI), sigma gray scale/diameter (sigma GS/D) and bone mineral content (BMC) were measured as bone mass indices, and the relationship investigated between clinical factors [age, duration of hemodialysis, serum phosphate (P), calcium (Ca), carboxy-terminal fragments of parathyroid hormone (C-PTH), osteocalcin (OC), alkaline phosphate (ALP) and Ca x P]. The bone loss in the hemodialysis patients was greater than that in the normal controls and was accelerated after menopause in women. However, the bone mass indices in a few of the hemodialysis patients of advanced age (over 60) showed higher values than those of the controls. The bone mass indices in male hemodialysis patients showed a negative correlation with the hemodialysis duration, C-PTH and OC, as did those in female patients with hemodialysis duration. On the other hand, BMC in female hemodialysis patients showed a negative correlation with P, C-PTH and Ca x P. In conclusion, age and the duration of hemodialysis are the most essential factors in skeletal and trabecular bone loss in male and female hemodialysis patients. Subsequent factors responsible for skeletal bone loss in male patients are C-PTH and OC, and those for trabecular bone loss in female patients are P, C-PTH and Ca x P. Control of the levels of C-PTH, OC, P and Ca x P is recommended for prevention of bone loss in hemodialysis patients.

Effect of recombinant human erythropoietin administration on immunological indices in patients undergoing chronic hemodialysis.

In patients undergoing chronic hemodialysis, marked anemia may cause decreased immunological function. To improve this anemia, we investigated the effect on immunological indices of recombinant human erythropoietin (rHuEPO) administration in 24 hemodialysis patients (13 males and 11 females) with renal anemia complications. Their mean age was 54.9 +/- 14.8 years and the mean duration of dialysis was 100.5 +/- 54.9 months. The subjects were treated with rHuEPO for 12 months, which helped to maintain a hematocrit value elevated by 5% from the baseline. Cell-mediated immunity and humoral immunity were assessed prior to as well as throughout the treatment period. Of the total number of patients studied, the anemia of 16 improved while 8 did not show signs of sufficient improvement. The improved group showed an increase in in vitro lymphocytic response to phytohemagglutinin (PHA), CD4/CD8, CD16 and serum IgM levels, while the CD8 level decreased significantly. Improvement in the general physical condition with rHuEPO treatment appeared to be associated with the changes in immunological indices, but the precise mechanism remains obscure.