Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint.

OBJECTIVE: To determine whether the joint space is a suitable environment for embryonic stem (ES) cells to grow and form cartilage. METHOD: We transplanted ES cells into the knee joint and a subcutaneous space of mice with severe combined immunodeficiency. RESULTS: Teratomas formed in both areas. Those in the joints grew and destroyed the joints. The incidence of cartilage formation was the same in the knee joint and subcutaneous space, but the ratio of cartilage to teratoma was higher in the knee joint than in the subcutaneous space. The teratomas were proved to have been derived from the transplanted ES cells by detection of the neomycin-resistance gene that had been transfected into the ES cells. CONCLUSIONS: It is currently not possible to use ES cells to repair joint tissues. Further optimization of donor ES cells to differentiate as well as inhibit tumour growth may help to meet these challenges.

Substitution of Dmo1 with normal alleles results in decreased manifestation of diabetes in OLETF rats.

AIM: Dmo1 (Diabetes Mellitus OLETF type I) is a major quantitative trait locus for dyslipidaemia, obesity and diabetes phenotypes in the Otsuka Long Evans Tokushima Fatty (OLETF) rat strain. To evaluate possible metabolic and pathological improvements generated by correction of the Dmo1 genetic pathway, we produced congenic lines, in which both OLETF Dmo1 alleles are replaced by the F344-derived genome. METHODS: Congenic animals were produced by introgressing F344-derived Dmo1 alleles into the OLETF rat. Congenic animals of the fourth generation (BC4) were intercrossed to obtain F1 animals (BC4:F1). Animals of the next generation, BC4:F2, were used for this study. We used 23 BC4:F2 males harbouring homozygous replacement of the OLETF Dmo1 region with the F344-derived genome. Seven animals with OLETF-derived Dmo1 alleles were used as controls. RESULTS: Dmo1-F344/F344 congenic rats showed significant decreases in body weight, abdominal fat weight, serum triacylglycerols, total cholesterol, food consumption and blood glucose after glucose loading (13%, 39%, 45%, 27%, 18% and 27% respectively; p < 0.05) compared with Dmo1-OLETF/OLETF animals. Furthermore, histopathological analysis of the kidney showed that mesangial sclerosis, hyalin deposits and deposition of PAS-positive substance were significantly lower in Dmo1-F344/F344 animals (p < 0.05). CONCLUSION: Improvements in metabolic parameters and histopathological scores show that correction of the Dmo1 genetic pathway in the diabetic and mildly obese OLETF rat strain produces wide-ranging therapeutic effects. Thus, this pathway might represent a new drug target also applicable to humans.

Mouse chromosome 19 and distal rat chromosome 1: a chromosome segment conserved in evolution.

Through a combination of radiation hybrid mapping and studies by FISH and zoo-FISH we have made a comparative investigation of the distal portion of rat chromosome 1 (RNO1) and the entire mouse chromosome 19 (MMU19). It was found that homologous segments of RNO1 and MMU19 are similar in banding morphology and in length as determined by several different methods, and that the gene order of the 46 genes studied appears to be conserved across the homologous segments in the two species. High-resolution zoo-FISH techniques showed that MMU19 probes highlight only a continuous segment on RNO1 (1q43-qter), with no detectable signals on other rat chromosomes. We conclude that these data suggest the evolutionary conservation of a chromosomal segment from a common rodent ancestor. This segment now constitutes the entire MMU19 and a large segment distally on RNO1q in the mouse and rat, respectively.

Genetic analysis of pancreatic duct hyperplasia in Otsuka Long-Evans Tokushima Fatty rats: possible association with a region on rat chromosome 14 that includes the disrupted cholecystokinin-A receptor gene.

An Otsuka Long-Evans Tokushima Fatty (OLETF) strain of rat spontaneously developed hyperglycemia, hyperinsulinemia, insulin resistance and mild obesity, which had been studied as animal model for type II diabetes mellitus (T2DM). Recently, we observed that this strain coincidentally developed atypical hyperplasia of the choledocho-pancreatic ductal epithelium with a complete incidence. In an effort to locate genes responsible for this hyperplasia, we prepared 288 backcross progeny from a mating between OLETF rats and BN rats (which do not develop hyperplasia), and performed a genome-wide scan using 207 polymorphic genetic markers. We observed a prominent association of hyperplasia with a region involving a marker locus D14Mit4 (P = 0.00020, Fisher's exact test) and Cckar (the cholecystokinin-A receptor gene; P = 0.00025, Fisher's exact test) which is known to be disrupted in an OLETF strain. Our findings indicated that epithelial hyperplasia of the choledocho-pancreatic duct is associated with a region on rat chromosome 14 around the Cckar gene in an additive fashion with another two susceptible loci, each on chromosome 9 and 7. This implied the possibility that Cckar deficiency could result in a predisposition towards pancreatic duct hyperplasia.

Effects of Dmo1 on obesity, dyslipidaemia and hyperglycaemia in the Otsuka Long Evans Tokushima Fatty strain.

Whole-genome scans have identified Dmo1 as a major quantitative trait locus (QTL) for obesity and dyslipidaemia in the Otsuka Long Evans Tokushima Fatty (OLETF) rat. We have produced congenic rats for the Dmo1 locus, using marker-assisted speed congenic protocols, enforced by selective removal of other QTL regions (QTL-marker-assisted counterselection), to efficiently transfer chromosomal segments from non-diabetic Fischer 344 (F344) rats into the OLETF background. In the third generation of congenic animals, we observed a substantial therapeutic effect of the Dmo1 locus on lipid metabolism, obesity control and plasma glucose homeostasis. We conclude that single-allele correction of an impaired genetic pathway can generate a substantial therapeutic effect, despite the complex polygenic nature of type II diabetic syndromes.

Single-allele correction of the Dmo1 locus in congenic animals substantially attenuates obesity, dyslipidaemia and diabetes phenotypes of the OLETF rat.

1. Whole-genome scans have identified Dmo1 as a major quantitative trait locus for dyslipidaemia and obesity in the Otsuka Long Evans Tokushima Fatty (OLETF) rat. 2. We have produced congenic rats for the Dmo1 locus through successive back-cross breeding with diabetic OLETF rats. Marker-assisted speed congenic protocols were applied to efficiently transfer chromosomal segments from non-diabetic Brown Norway (BN) rats into the OLETF background. 3. In the fourth generation of congenic animals, we observed a substantial therapeutic effect of the Dmo1 locus on lipid metabolism, obesity control and plasma glucose homeostasis. 4. We have concluded that Dmo1 primarily affects lipid homeostasis, obesity control and/or glucose homeostasis at fasting and is secondarily involved in glucose homeostasis after loading. 5. The results of the present study show that single-allele correction of a genetic defect of the Dmo1 locus can generate a substantial therapeutic effect, despite the complex polygenic nature of type II diabetic syndromes.

Analysis of chromosomal aberrations involving chromosome 1q31-->q53 in a DMBA-induced rat fibrosarcoma cell line: amplification and overexpression of Jak2.

In a study of DMBA-induced rat fibrosarcomas we repeatedly found deletions and/or amplifications in the long arm of rat chromosome 1 (RNO1). Comparative genome hybridization showed that there was amplification involving RNO1q31-->q53 in one of the DMBA-induced rat fibrosarcoma tumors (LB31) and a cell culture derived from it. To identify the amplified genes we physically mapped rat genes implicated in cancer and analyzed them for signs of amplification. The genes were selected based on their locations in comparative maps between rat and man. The rat proto-oncogenes Ccnd1, Fgf4, and Fgf3 (HSA11q13.3), were mapped to RNO1q43 by fluorescence in situ hybridization (FISH). The Ems1 gene was mapped by radiation hybrid (RH) mapping to the same rat chromosome region and shown to be situated centromeric to Ccnd1 and Fgf4. In addition, the proto-oncogenes Hras (HSA11p15.5) and Igf1r (HSA15q25-->q26) were mapped to RNO1q43 and RNO1q32 by FISH and Omp (HSA11q13.5) was assigned to RNO1q34. PCR probes for the above genes together with PCR probes for the previously mapped rat genes Bax (RNO1q31) and Jak2 (RNO1q51-->q53) were analyzed for signs of amplification by Southern blot hybridization. Low copy number increases of the Omp and Jak2 genes were detected in the LB31 cell culture. Dual color FISH analysis of tumor cells confirmed that chromosome regions containing Omp and Jak2 were amplified and were situated in long marker chromosomes showing an aberrant banding pattern. The configuration of the signals in the marker chromosomes suggested that they had arisen by a break-fusion-bridge (BFB) mechanism.

Quantitative trait loci for lipid metabolism in the study of OLETF x (OLETF x Fischer 344) backcross rats.

1. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is a model of type II diabetes with accompanying dyslipidaemia and obesity. 2. To define chromosomal intervals associated with obesity (abdominal fat weight and plasma leptin levels), dyslipidaemia (plasma triglyceride, cholesterol and free fatty acids) and hyperglycaemia (plasma glucose levels), we have performed genome-wide quantitative traits loci (QTL) analyses of 115 male OLETF x (OLETF x Fischer 344) backcross animals at 16 weeks of age. 3. The Diabetes Mellitus OLETF type I (Dmo1) locus on rat chromosome 1 showed statistically significant involvement in elevations of plasma levels of triglycerides (P = 4.87 x 10(-6) at D1Rat90) and total cholesterol (P = 1.16 x 10(-5) at D1Rat306). 4. No other loci produced significant linkage to these observed phenotypes. 5. These analyses have confirmed the importance of Dmo1 in lipid homeostasis at younger ages as well as during overt diabetes, which appears later. Thus, alterations at the Dmo1 locus are a major risk factor for pathogenesis in the strain, a finding that agrees with physiological studies that indicate a role for dyslipidaemia in the type II diabetic syndrome of OLETF rats.

Characterization of newly developed SSLP markers for the rat.

We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27-36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F2 progeny of (OLETFxBN)F2 crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species.

Functional outcome in knee osteoarthritis after treatment with hylan G-F 20: a prospective study.

OBJECTIVE: To assess functional change in patients with knee osteoarthritis (OA) after treatment with intra-articular hyaluronic acid (Hylan G-F 20; Synvisc). DESIGN: Prospective case series with 6-month follow-up. SETTING: Outpatient community orthopedic practice. PARTICIPANTS: Eighty-four consecutive patients referred to private orthopedic group with symptoms and radiographic evidence of unilateral or bilateral knee OA who had either failed or could not tolerate the side effects of conservative treatment. Sixty-one patients completed the study. Nineteen patients were lost to follow-up. Four patients withdrew from study due to subsequent knee arthroplasty. INTERVENTION: Three weekly injections of Hylan G-F 20 into one or both (if bilaterally symptomatic) knees (110 knees total). OUTCOME MEASURE: SF-36 Health Survey was completed before treatment and 6 months after treatment. RESULTS: Statistically significant improvement (p < .001) in Physical Functioning, Role-Physical, Bodily Pain, Social Functioning, and Role-Emotional categories on 6-month follow-up survey. Age and percent above ideal body weight were not significant predictors of functional change. CONCLUSION: Efficacy of intra-articular injection of Hylan G-F 20 for knee OA 6 months after injection is demonstrated in several categories of the SF-36, indicating a measurable improvement in overall functioning in these patients.

Molecular cloning, expression, and mapping of a novel human cDNA, GRP17, highly homologous to human gadd45 and murine MyD118.

From a human placenta cDNA library, we isolated a novel gene whose predicted product is highly homologous in amino acid sequence to human gadd45 and murine MyD118 proteins (about 55% and 52% identity, respectively). The cDNA clone, designated GRP17 (gadd-related protein, 17 kDa), contained a 477-bp open reading frame encoding 159 amino acids. Northern blot analysis revealed strong expression of a 1.35-kb transcript in heart, placenta, liver, skeletal muscle, prostate, testis, and ovary. A 1.7-kb additional transcript was detected in liver. We assigned the GRP17 gene to chromosome 9q22.1-q22.2 by fluorescence in situ hybridization (FISH).

Genetic dissection of "OLETF," a rat model for non-insulin-dependent diabetes mellitus: quantitative trait locus analysis of (OLETF x BN) x OLETF.

To identify genetic determinants relevant to non-insulin-dependent diabetes mellitus (NIDDM), we performed a genome-wide analysis for quantitative trait loci (QTLs) using 359 backcross progeny of the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The OLETF strain is a well-studied animal model of obese NIDDM, with features of hyperinsulinemia, hyperglycemia, insulin resistance, and abundant abdominal fat. Our extensive genomic scanning with 218 markers revealed nine significant QTLs, including a strong determinant of obesity on chromosome 1 (Dmo1: LOD = 13.99, for body weight). Two highly significant QTLs for glucose homeostasis were found, one on chromosome 1 (Dmo4 LOD = 7.16, for postprandial glucose level) and the other on chromosome X (Dmo11/Odb1: LOD = 7.81, for postprandial glucose level). These data are comparable to results of our previous studies of the OLETF rat.

A radiation hybrid map of the rat genome containing 5,255 markers.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

Structural and genetic studies of the proliferation disrupter genes of Drosophila simulans and D. melanogaster.

To examine whether structural and functional differences exist in the proliferation disrupter (prod) genes between Drosophila simulans and D. melanogaster, we analyzed and compared both genes. The exon-intron structure of the prod genes was found to be the same--three exons were interrupted by two introns, although a previous report suggested that only one intron existed in D. melanogaster. The prod genes of D. simulans and D. melanogaster both turn out to encode 346 amino acids, not 301 as previously reported for D. melanogaster. The numbers of nucleotide substitutions in the prod genes was 0.0747 +/- 0.0180 per synonymous site and 0.0116 +/- 0.0039 per replacement site, both comparable to those previously known for homologous genes between D. simulans and D. melanogaster. Genetic analysis demonstrated that D. simulans PROD can compensate for a deficiency of D. melanogaster PROD in hybrids. The PRODs of D. simulans and D. melanogaster presumably share the same function and a conserved working mechanism. The prod gene showed no significant interaction with the lethality of the male hybrid between these species.

Genetic determinants of plasma triglyceride levels in (OLETF x BN) x OLETF backcross rats.

Altered lipid metabolism is closely associated with diabetes in humans, although predisposing genetic factors that affect hyperlipidemia have not yet been clarified. Our previously established OLETF strain is an obese rat model of type II diabetes, exhibiting hypertriglycemia as well as hyperinsulinemia, hyperglycemia, insulin resistance, and abundant abdominal fat. To identify genetic factors responsible for dyslipidemic phenotypes in OLETF rats, we performed a whole-genome scan using 293 male (OLETF x BN) x OLETF backcross rats. Our analysis identified two significant quantitative trait loci (QTLs), on rat chromosomes 1 and 8, that are related to fasting triglyceride levels. The chromosome 1 QTL colocalized with Dmo1 (diabetes mellitus, OLETF type 1), a locus previously shown to associate strongly with both fat levels and body weight. The other significant QTL localizes to the chromosome 8 marker D8Mit2, in a region where several apo-lipoprotein genes are clustered.

Further genetic studies on the Katsunuma population of Drosophila melanogaster.

Changes in the genetic structure of the Katsunuma natural population of Drosophila melanogaster have been examined during the past 35 years. The frequency of recessive lethal genes on the second chromosome once increased from 15% to 30% in the early 1970s, then decreased to about 24% in the late 1970s, and thereafter showed no significant changes. Sterility genes, the frequency of which is always less than the lethals, showed a similar tendency. The SD (segregation distorter) mutant gene disappeared but some others such as rbl (reduced bristle) and bw (brown) persisted in the population. The frequency of inversion-carrying chromosomes gradually decreased in the period, such that the standard chromosome frequency in the second and third chromosomes increased from about 40% to more than 80%. Coincident with these frequency changes is the invasion of a transposable element P into the Katsunuma population. The P element should have invaded into Katsunuma in the late 1960s. It spread over the population apparently inducing deleterious mutations, causing the decrease in the allelism rate, and hence increasing the effective population size. Soon, however, most flies became resistant to the P element-mediated transposition as they began to harbor defective P elements. During the course of spreading, the P element must also have induced deleterious mutations on the polymorphic inversions, breaking up the heterotic gene complexes along the chromosomes, which probably caused the reduction in the frequency of inversion chromosomes. Temporal invasion of D. simulans, a sibling species of D. melanogaster, into Katsunuma occurred several times after 1978, and the species seems to have been settled since 1990. This, however, did not have any effect on the genetic structure of D. melanogaster population.

cDNA cloning and characterization of a human proteasomal modulator subunit, p27 (PSMD9).

We have employed cDNA cloning to deduce the complete primary structure of a new subunit, designated p27, of the modulator trimer complex that stimulates the association of the PA700 regulator with the catalytic 20S proteasome to form the ATP-dependent active 26S proteasome. We found two distinct cDNAs encoding two highly homologous proteins except in the C-terminal region, which are termed tentatively p27-1 and p27-2. The short p27-2 cDNA has a deletion of 65 bp near the 3'-end region of the long p27-1 cDNA, which encodes a large protein with an extended C-terminal region, designated p27-L, whereas the long p27-1 cDNA encodes a small protein named p27-S. The polypeptides of p27-L and p27-S consist of 223 and 209 amino acid residues with calculated molecular masses of 24,852 and 22,764 and isoelectric points of 6.50 and 5.28, respectively. Immunoblot analysis with anti-p27 antibody revealed that p27, together with two other ATPase components, TBP1 and p42, was associated with not only the modulator complex but also significantly with the 26S proteasome complex, suggesting that the three are common/sharing subunits in these two complexes. By the fluorescence in situ hybridization method, the p27 (PSMD9) gene was mapped to the q24.2-q24.3 band of human chromosome 12. Computer-assisted homology analysis revealed the high sequence similarities of p27-L with a possible counterpart in Caenorhabditis elegans and Saccharomyces cerevisiae whose function is yet unknown, the yeast gene that is here termed NAS2 (non-ATPase subunit 2). Disruption of NAS2 had no effect on cell viability, indicating that the subunit is not essential for proliferation of yeast cells.

Diagnosis and rehabilitation strategies for patients with hysterical hemiparesis: a report of four cases.

Conversion disorder is a psychological disturbance that produces subconscious alterations in sensorimotor function. Hysterical hemiparesis is a relatively rare, and difficult to diagnose, form of conversion disorder presenting as unilateral motor weakness with or without sensory deficits. We report four patients who required inpatient rehabilitation for hysterical hemiparesis, a diagnosis for which there is little information regarding rehabilitation management. In all cases, an extensive acute care evaluation including multiple imaging studies failed to identify a new neurologic lesion. All patients had rapid functional improvement using functional and behavioral therapies and extensive psychosocial support (mean length of stay of 11 days; mean Functional Independence Measure [FIM] gain of 22; mean discharge FIM of 112), consistent with other published reports of rehabilitation of conversion disorder. Evaluation of these cases reveals consistencies regarding presentation, psychosocial history, and rehabilitation course that can aid clinicians in making the diagnosis. Rehabilitation strategies for hysterical hemiparesis are reviewed.