Muscle and Myotendinous Tissue Properties at the Distal Tibia as Assessed by High-Resolution Peripheral Quantitative Computed Tomography.

High-resolution peripheral quantitative computed tomography (HR-pQCT) quantifies bone microstructure and density at the distal tibia where there is also a sizable amount of myotendinous (muscle and tendon) tissue (MT); however, there is no method for the quantification of MT. This study aimed (1) to assess the feasibility of using HR-pQCT distal tibia scans to estimate MT properties using a custom algorithm, and (2) to determine the relationship between MT properties at the distal tibia and mid-leg muscle density (MD) obtained from pQCT. Postmenopausal women from the Hamilton cohort of the Canadian Multicenter Osteoporosis Study had a single-slice (2.3 ± 0.5 mm) 66% site pQCT scan measuring muscle cross-sectional area (MCSA) and MD. A standard HR-pQCT scan was acquired at the distal tibia. HR-pQCT-derived MT cross-sectional area (MTCSA) and MT density (MTD) were calculated using a custom algorithm in which thresholds (34.22-194.32 mg HA/cm3) identified muscle seed volumes and were iteratively expanded. Pearson and Bland-Altman plots were used to assess correlations and systematic differences between pQCT- and HR-pQCT-derived muscle properties. Among 45 women (mean age: 74.6 ± 8.5 years, body mass index: 25.9 ± 4.3 kg/m2), MTD was moderately correlated with mid-leg MD across the 2 modalities (r = 0.69-0.70, p < 0.01). Bland-Altman analyses revealed no evidence of directional bias for MTD-MD. HR-pQCT and pQCT measures of MTCSA and MCSA were moderately correlated (r = 0.44, p < 0.01). Bland-Altman plots for MTCSA revealed that larger MCSAs related to larger discrepancy between the distal and the mid-leg locations. This is the first study to assess the ability of HR-pQCT to measure MT size, density, and morphometry. HR-pQCT-derived MTD was moderately correlated with mid-leg MD from pQCT. This relationship suggests that distal MT may share common properties with muscle throughout the length of the leg. Future studies will assess the value of HR-pQCT-derived MT properties in the context of falls, mobility, and balance.

A comparison of peripheral imaging technologies for bone and muscle quantification: a technical review of image acquisition.

The choice of an appropriate imaging technique to quantify bone, muscle, or muscle adiposity needs to be guided by a thorough understanding of its competitive advantages over other modalities balanced by its limitations. This review details the technical machinery and methods behind peripheral quantitative computed tomography (pQCT), high-resolution (HR)-pQCT, and magnetic resonance imaging (MRI) that drive successful depiction of bone and muscle morphometry, densitometry, and structure. It discusses a number of image acquisition settings, the challenges associated with using one versus another, and compares the risk-benefits across the different modalities. Issues related to all modalities including partial volume artifact, beam hardening, calibration, and motion assessment are also detailed. The review further provides data and images to illustrate differences between methods to better guide the reader in selecting an imaging method strategically. Overall, investigators should be cautious of the impact of imaging parameters on image signal or contrast-to-noise-ratios, and the need to report these settings in future publications. The effect of motion should be assessed on images and a decision made to exclude prior to segmentation. A more standardized approach to imaging bone and muscle on pQCT and MRI could enhance comparability across studies and could improve the quality of meta-analyses.

Peripheral quantitative computed tomography-derived muscle density and peripheral magnetic resonance imaging-derived muscle adiposity: precision and associations with fragility fractures in women.

PURPOSE: To determine the degree to which muscle density and fractures are explained by inter and intramuscular fat (IMF). METHODS: Women ⋝50 years of age (Hamilton, ON, Canada) had peripheral magnetic resonance imaging and peripheral quantitative computed tomography scans at 66% of the tibial length. Muscle on computed tomography images was segmented from subcutaneous fat and bone using fixed thresholds, computing muscle density. IMF was segmented from muscle within magnetic resonance images using a region-growing algorithm, computing IMF volume. Fracture history over the last 14 years was obtained. Odds ratios for fractures were determined for muscle density, adjusting for IMF volume, total hip BMD, age and body mass index. RESULTS: Women with a history of fractures were older (N=32, age:75.6±8.3 years) than those without (N=39, age: 67.0±5.2 years) (<0.01). IMF volume explained 49.3% of variance in muscle density (p<0.001). Odds for fractures were associated with lower muscle density even after adjusting for IMF volume but were attenuated after adjusting for age. CONCLUSIONS: Muscle adiposity represents only 50% of the muscle density measurement. Properties of muscle beyond its adiposity may be related to fractures, but larger and prospective studies are needed to confirm these associations.

Bone-muscle indices as risk factors for fractures in men: the Osteoporotic Fractures in Men (MrOS) Study.

OBJECTIVE: To assess bone-muscle (B-M) indices as risk factors for incident fractures in men. METHODS: Participants of the Osteoporotic Fractures in Men (MrOS) Study completed a peripheral quantitative computed tomography scan at 66% of their tibial length. Bone macrostructure, estimates of bone strength, and muscle area were computed. Areal bone mineral density (aBMD) and body composition were assessed with dual-energy X-ray absorptiometry. Four year incident non-spine and clinical vertebral fractures were ascertained. B-M indices were expressed as bone-to-muscle ratios for: strength, mass and area. Discriminative power and hazards ratios (HR) for fractures were reported. RESULTS: In 1163 men (age: 77.2±5.2 years, body mass index (BMI): 28.0±4.0 kg/m(2), 4.1±0.9 follow-up years, 7.7% of men ⋝1 fracture), B-M indices were smaller in fractured men except for bending and areal indices. Smaller B-M indices were associated with increased fracture risk (HR: 1.30 to 1.74) independent of age and BMI. Strength and mass indices remained significant after accounting for lumbar spine but not total hip aBMD. However, aBMD correlated significantly with B-M indices. CONCLUSION: Mass and bending B-M indices are risk factors for fractures in men, but may not improve fracture risk prediction beyond that provided by total hip aBMD.

Do athletic ECG changes predict athletic performance in Gurkha recruits?

INTRODUCTION: ECG changes are associated with regular long-term intensive exercise due to electrical manifestations of increased vagal tone, increased ventricular wall thickness and enlarged chamber size. The aim of this study was to further understand the relationship of athletic ECG changes and athletic performance in an athletic population. METHODS: A retrospective cohort study was performed in 195 Nepali civilian males undergoing selection to the Gurkhas. V̇O2max (maximal oxygen consumption) was estimated from a 1.5-mile run time using Cooper's formula and correlated with athletic ECG adaptations. Variables were explored with univariable and multivariable linear regression. RESULTS: The median number of athletic changes on ECG was 2 (IQR 1-2). There was no significant correlation (p=0.46) between the number of ECG adaptations and the degree of cardiovascular fitness by estimated V̇O2max (estV̇O2max). We found a negligible but significant correlation between the presence of inferior T wave inversion (TWI) and estV̇O2max (R2=0.03, p=0.02). The multivariable-fitted regression model was: estV̇O2max~Intercept+presence of RVH (right ventricular hypertrophy) voltage criteria+absence of sinus arrhythmia+T wave axis+inferior TWI. The overall regression was statistically significant: R2=0.10, F(df=4, df=189)=[5.4], p=0.0004). All variables in the multivariable model significantly predicted estV̇O2max (p<0.04). CONCLUSION: ECG changes of athleticism negligibly predict and differentiate athletic performance in our athletic population. The most predictive ECG markers being voltage criteria for left ventricular hypertrophy and RVH. Markers of increased vagal tone were not predictive. TWI, being a marker for disease, was also a marker for athletic performance in this cohort. The number of athletic ECG adaptations did not predict increased athletic performance.

A de novo mutation in KIT causes white spotting in a subpopulation of German Shepherd dogs.

Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss-of-function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1-bp insertion in the second exon that co-segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c-Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species.

Shyness in late childhood: relations with attributional styles and self-esteem.

BACKGROUND: Shyness in late childhood is related to social and psychological problems. The present study examined the relations among shyness, attributional styles and self-esteem. It was hypothesized that self-esteem mediated the effects of attributional styles on shyness. METHODS: Self-reported data on degree of shyness, attributional styles and self-esteem were obtained from 326 Chinese children with mean age of 10.85 years. RESULTS: It was found that positive attributional styles predicted shyness in the negative direction and the effects were fully mediated by self-esteem, and negative attributional styles predicted shyness in the positive direction both directly and through self-esteem. CONCLUSION: The results imply that how children attribute positive and negative outcomes affect both self-esteem and shyness. It is suggested that practitioners should conduct attribution-retraining workshops for shy children and help teachers and parents learn how to mitigate negative attributional style and foster positive attributional styles in children.

Low incidence of BRCA2 mutations in breast carcinoma and other cancers.

Inherited mutant alleles of familial tumour suppressor genes predispose individuals to particular types of cancer. In addition to an involvement in inherited susceptibility to cancer, these tumour suppressor genes are targets for somatic mutations in sporadic cancers of the same type found in the familial forms. An exception is BRCA1, which contributes to a significant fraction of familial breast and ovarian cancer, but undergoes mutation at very low rates in sporadic breast and ovarian cancers. This finding suggests that other genes may be the principal targets for somatic mutation in breast carcinoma. A second, recently identified familial breast cancer gene, BRCA2 (refs 5-8), accounts for a proportion of breast cancer roughly equal to BRCA1. Like BRCA1, BRCA2 behaves as a dominantly inherited tumour suppressor gene. Individuals who inherit one mutant allele are at increased risk for breast cancer, and the tumours they develop lose the wild-type allele by heterozygous deletion. The BRCA2 coding sequence is huge, composed of 26 exons that span 10,443 bp. Here we investigate the rate of BRCA2 mutation in sporadic breast cancers and in a set of cell lines that represent twelve other tumour types. Surprisingly, mutations in BRCA2 are infrequent in cancers including breast carcinoma. However, a probable germline mutation in a pancreatic tumour cell line suggests a role for BRCA2 in susceptibility to pancreatic cancer.

The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds.

Breast carcinoma is the most common malignancy among women in developed countries. Because family history remains the strongest single predictor of breast cancer risk, attention has focused on the role of highly penetrant, dominantly inherited genes in cancer-prone kindreds (1). BRCA1 was localized to chromosome 17 through analysis of a set of high-risk kindreds (2), and then identified four years later by a positional cloning strategy (3). BRCA2 was mapped to chromosomal 13q at about the same time (4). Just fifteen months later, Wooster et al. (5) reported a partial BRCA2 sequence and six mutations predicted to cause truncation of the BRCA2 protein. While these findings provide strong evidence that the identified gene corresponds to BRCA2, only two thirds of the coding sequence and 8 out of 27 exons were isolated and screened; consequently, several questions remained unanswered regarding the nature of BRCA2 and the frequency of mutations in 13q-linked families. We have now determined the complete coding sequence and exonic structure of BRCA2 (GenBank accession #U43746), and examined its pattern of expression. Here, we provide sequences for a set of PCR primers sufficient to screen the entire coding sequence of BRCA2 using genomic DNA. We also report a mutational analysis of BRCA2 in families selected on the basis of linkage analysis and/or the presence of one or more cases of male breast cancer. Together with the specific mutations described previously, our data provide preliminary insight into the BRCA2 mutation profile.

Necrotizing meningoencephalitis of Pug dogs associates with dog leukocyte antigen class II and resembles acute variant forms of multiple sclerosis.

Necrotizing meningoencephalitis (NME) is a disorder of Pug Dogs that appears to have an immune etiology and high heritability based on population studies. The present study was undertaken to identify a genetic basis for the disease. A genome-wide association scan with single tandem repeat (STR) markers showed a single strong association near the dog leukocyte antigen (DLA) complex on CFA12. Fine resolution mapping with 27 STR markers on CFA12 further narrowed association to the region containing DLA-DRB1, -DQA1 and, -DQB1 genes. Sequencing confirmed that affected dogs were more likely to be homozygous for specific alleles at each locus and that these alleles were linked, forming a single high risk haplotype. The strong DLA class II association of NME in Pug Dogs resembles that of human multiple sclerosis (MS). Like MS, NME appears to have an autoimmune basis, involves genetic and nongenetic factors, has a relatively low incidence, is more frequent in females than males, and is associated with a vascularly orientated nonsuppurative inflammation. However, NME of Pug Dogs is more aggressive in disease course than classical human MS, appears to be relatively earlier in onset, and involves necrosis rather than demyelination as the central pathobiologic feature. Thus, Pug Dog encephalitis (PDE) shares clinical features with the less common acute variant forms of MS. Accordingly, NME of Pug Dogs may represent a naturally occurring canine model of certain idiopathic inflammatory disorders of the human central nervous system.

Differences in fracture prevalence and in bone mineral density between Chinese and White Canadians: the Canadian Multicentre Osteoporosis Study (CaMos).

Fracture determinants differ between Canadians of Chinese and White descent, the former constituting the second largest visible minority group in Canada. The results of this study support the importance of characterizing bone health predictors in Canadians of different ethnicity to improve population-specific fracture prevention and treatment strategies. PURPOSE: We aimed to compare clinical risk factors, bone mineral density, prevalence of osteoporosis, and fractures between Chinese and White Canadians to identify ethnicity-specific risks. METHODS: We studied 236 Chinese and 8945 White Canadians aged 25+ years from the Canadian Multicentre Osteoporosis Study (CaMos). The prevalence of osteoporosis using ethnicity-specific peak bone mass (PBM), and of prior and incident low trauma fractures were assessed and compared between groups. Linear regressions, adjusting for age and anthropometric measures, were used to examine the association between baseline and 5-year changes in BMD and ethnicity. RESULTS: Chinese participants had shorter stature, lower BMI, and lower rate of falls than White participants. Adjusted models showed no significant differences in baseline BMD between ethnic groups except in younger men where total hip BMD was 0.059 g/cm2 (0.009; 0.108) lower in Chinese. Adjusted 5-year BMD change at lumbar spine was higher in older Chinese women and men compared with Whites. When using Chinese-specific PBM, the prevalence of osteoporosis in Chinese women was 2-fold lower than when using that of White women The prevalence of fractures was higher in White women compared with Chinese with differences up to 14.5% (95% CI 9.2; 19.7) and 10.5% (95% CI 4.5-16.4) in older White men. Incident fractures were rare in young Chinese compared with White participants and not different in the older groups. CONCLUSION: Our results support the importance of characterizing bone strength predictors in Chinese Canadians and the development of ethnicity-specific fracture prediction and prevention strategies.

Reproducibility of computer-assisted joint alignment measurement in OA knee radiographs.

OBJECTIVES: (1) To investigate the reproducibility of computer-assisted measurements of knee alignment angle (KA) from digitized radiographs of osteoarthritis (OA) participants requiring total knee arthroplasty (TKA) and (2) to determine whether landmark choice affects the precision of KA measurements on radiographs. METHODS: Using a custom algorithm, femoral, central, and tibial measurement-guiding rules were interactively placed on digitized posteroanterior fixed-flexion knee radiographs by mouse control and positioned according to different anatomic landmarks. The angle subtended by lines connecting these guiding rules was measured by three readers to assess interobserver, intraobserver and experience-inexperience reproducibility. Test-retest reproducibility was evaluated with duplicate radiographs from a healthy cohort. Reproducibility was assessed using root-mean square coefficients of variation (RMSCV%). The Bland-Altman method was performed on data obtained from varying anatomic landmarks (confidence interval, CI= 95%). RESULTS: From 16 healthy and 30 TKA participants, reproducibility analyses revealed a high degree of intraobserver (n=38, RMSCV=0.56%), interobserver (n=38, RMSCV=0.72%), test-retest (n=16, RMSCV=0.87%) and experience-inexperience (n=38, RMSCV=0.73%) reproducibility with variances below 1%. Varying the orientation of tibial and femoral rules according to anatomic landmarks produced a difference that exceeded an a priori limit of agreement of -1.11 degrees to +1.67 degrees. CONCLUSION: Our custom-designed software provides a robust method for measuring KAs within digitized knee radiographs. Although test-retest analyses were only performed in a healthy cohort, we anticipate a similar degree of reproducibility in an OA sample. A standardized set of anatomic landmarks employed for KA measurement is recommended since arbitrary selection of landmarks resulted in imprecise KA measurement even with a computer-assisted technique.

Quantitative analysis of subchondral sclerosis of the tibia by bone texture parameters in knee radiographs: site-specific relationships with joint space width.

OBJECTIVES: To determine the ability of radiographic bone texture (BTX) parameters to quantify subchondral tibia sclerosis and to examine clinical relevance for assessing osteoarthritis (OA) progression. We examined the relationship between BTX parameters and each of (1) location-specific joint space width (JSW) [JSW(x)] and minimum JSW (mJSW) of the affected compartment, and (2) knee alignment (KA) angle in knee radiographs of participants undergoing total knee arthroplasty (TKA). DESIGN: Digitized fixed-flexion knee radiographs were analyzed for run-length and topological BTX parameters in a subchondral region using an algorithm. Medial JSW(x) was computed at x=0.200, 0.225, 0.250 and 0.275 according to a coordinate system defined by anatomic landmarks. mJSW was determined for medial and lateral compartment lesions. KA angles were determined from radiographs using an anatomic landmark-guided algorithm. JSW measures and the magnitude of knee malalignment were each correlated with BTX parameters. Reproducibility of BTX parameters was measured by root-mean square coefficients of variation (RMSCV%). RESULTS: Run-length BTX parameters were highly reproducible (RMSCV%<1%) while topological parameters showed poorer reproducibility (>5%). In TKA participants (17 women, 13 men; age: 66+/-9 years; body mass index (BMI): 31+/-6 kg m(-2); WOMAC: 41.5+/-16.1; Kellgren-Lawrence score mode: 4), reduced trabecular spacing (Tb.Sp) and increased free ends (FE) were correlated with decreased JSW after accounting for BMI, gender and knee malalignment. These relationships were dependent on site of JSW measurement. CONCLUSION: High reproducibility in quantifying bone sclerosis using Tb.Sp and its significant relationship with JSW demonstrated potential for assessing OA progression. Increased trabecular FE and reduced porosity observed with smaller JSW suggest collapsing subchondral bone or trabecular plate perforation in advanced knee OA.

DOGSET: pre-designed primer sets for fine-scale mapping and DNA sequence interrogation in the dog.

DOGSET is an online resource that provides access to primer sequences that have been computationally mined from the reference genome using heuristic algorithms. The electronic repository includes PCR primers corresponding to 32,135 markers for genetic mapping and 334,657 sequence-tagged gene elements for targeted re-sequencing and mutation discovery. A customized report that tailors primer design to wet bench protocols can be exported for a region of interest by specifying genome coordinates in a graphical user interface.

Novel high-affinity PPARgamma agonist alone and in combination with paclitaxel inhibits human anaplastic thyroid carcinoma tumor growth via p21WAF1/CIP1.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists demonstrate antitumor activity likely through transactivating genes that regulate cell proliferation, apoptosis, and differentiation. The PAX8/PPARgamma fusion oncogene, which is common in human follicular thyroid carcinomas appears to act via dominant negative suppression of wild-type PPARgamma, suggesting that it may be a tumor suppressor gene in thyroid cells. We have identified a novel high-affinity PPARgamma agonist (RS5444) that is dependent upon PPARgamma for its biological activity. This is the first report of this molecule and its antitumor activity. In vitro, the IC50 for growth inhibition is approximately 0.8 nM while anaplastic thyroid carcinoma (ATC) tumor growth was inhibited three- to fourfold in nude mice. siRNA against PPARgamma and a pharmacological antagonist demonstrated that functional PPARgamma was required for growth inhibitory activity of RS5444. RS5444 upregulated the cell cycle kinase inhibitor, p21WAF1/CIP1. Silencing p21WAF1/CIP1 rendered cells insensitive to RS5444. RS5444 plus paclitaxel demonstrated additive antiproliferative activity in cell culture and minimal ATC tumor growth in vivo. RS5444 did not induce apoptosis but combined with paclitaxel, doubled the apoptotic index compared to that of paclitaxel. Our data indicate that functional PPARgamma is a molecular target for therapy in ATC. We demonstrated that RS5444, a thiazolidinedione (Tzd) derivative, alone or in combination with paclitaxel, may provide therapeutic benefit to patients diagnosed with ATC.

Relationship between myocardial metabolites and contractile abnormalities during graded regional ischemia. Phosphorus-31 nuclear magnetic resonance studies of porcine myocardium in vivo.

The mechanisms responsible for changes in myocardial contractility during regional ischemia are unknown. Since changes in high-energy phosphates during ischemia are sensitive to reductions in myocardial blood flow, it was hypothesized that myocardial function under steady-state conditions of graded regional ischemia is closely related to changes in myocardial high-energy phosphates. Therefore, phosphorus-31 nuclear magnetic resonance spectroscopy was employed in an in vivo porcine model of graded coronary stenosis. Simultaneous measurements of regional subendocardial blood flow, high-energy phosphates, pH, and myocardial segment shortening were made during various degrees of regional ischemia in which subendocardial blood flow was reduced by 16-94%. During mild reductions in myocardial blood flow (subendocardial blood flow = 83% of nonischemic myocardium), only the ratio of phosphocreatine to inorganic phosphate (PCr/Pi), Pi, and [H+] were significantly changed from control. PCr, ATP, and PCr/ATP were not significantly reduced from control with mild reductions in blood flow. Changes in myocardial segment shortening were most closely associated with changes in PCr/Pi (r = 0.94). Pi and [H+] were negatively correlated with segment shortening (r = -0.64 and -0.58, respectively) and increased over twofold when blood flow was reduced by 62%. Thus, these data demonstrate that PCr/Pi is sensitive to reductions in myocardial blood flow and closely correlates with changes in myocardial function. These data are also consistent with a role for Pi or H+ as inhibitors of myocardial contractility during ischemia.

BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines.

Human BRG1 is a component of the evolutionarily conserved SWI-SNF chromatin remodeling complex. BRG1 has been implicated in growth control through its interaction with the tumor suppressor pRb and may consequently serve as a negative regulator of proliferation. Postulating that BRG1 may itself be a tumor suppressor gene, we screened a panel of tumor cell lines to determine whether the gene is targeted for mutation. We report that the COOH-terminal region of BRG1 is homozygously deleted in two carcinoma cell lines, prostate TSU-Pr1 and lung A-427. In addition, biallelic inactivations of BRG1 were observed in four other cell lines derived from carcinomas of the breast, lung, pancreas, and prostate; their mutations in BRG1 included three frameshift lesions and one nonsense lesion. Point mutations were also discovered in a number of other cell lines, however in most cases any effect of these mutations on BRG1 function remains to be established. A variety of different mutations within BRG1, in several cell lines, suggest that BRG1 may be targeted for disruption in human tumors. Significantly, reintroduction of BRG1 into cells lacking BRG1 expression was sufficient to reverse their transformed phenotype inducing growth arrest and a flattened morphology. These data strongly support the model that BRG1 may function as a tumor suppressor and strengthen the hypothesis that the regulation of gene expression through chromatin remodeling is critical for cancer progression. It will be important to confirm these observations in primary tumors.

Human mitogen-activated protein kinase kinase 4 as a candidate tumor suppressor.

Mitogen-activated protein kinases function in signal transduction pathways that are involved in controlling key cellular processes in many organisms. A mammalian member of this kinase family, MKK4/JNKK1/SEK1, has been reported to link upstream MEKK1 to downstream stress-activated protein kinase/JNK1 and p38 mitogen-activated protein kinase. This mitogen-activated protein kinase pathway has been implicated in the signal transduction of cytokine- and stress-induced apoptosis in a variety of cell types. Here, we report that two human tumor cell lines, derived from pancreatic carcinoma and lung carcinoma, harbor homozygous deletions that eliminate coding portions of the MKK4 locus at 17p, located approximately 10 cM centromeric of p53. In addition, in a set of 88 human cancer cell lines prescreened for loss of heterozygosity, we detected two nonsense and three missense sequence variants of MKK4 in cancer cell lines derived from human pancreatic, breast, colon, and testis cells. In vitro biochemical assays revealed that, when stimulated by MEKK1, four of the five altered MKK4 proteins lacked the ability to phosphorylate stress-activated protein kinase. Thus, the incidence of coding mutations of MKK4 in the set of cell lines is 6 of 213 (approximately 3%). These findings suggest that MKK4 may function as a suppressor of tumorigenesis or metastasis in certain types of cells.

Characterization of a carboxy-terminal BRCA1 interacting protein.

There are several lines of evidence indicating that the carboxy-terminal region of the tumor suppressor protein BRCA1 is a functionally significant domain. Using the yeast two-hybrid and in vitro biochemical assays, we show that a protein, CtIP, interacts specifically with the carboxy-terminal segment of human BRCA1 from residues 1602-1863. A germ line truncation mutation, Y1853ter, that removes the last 11 amino acids from the carboxy-terminus of BRCA1, abolishes not only its transcriptional activation function, but also binding to CtIP. The function of CtIP is unknown, but its reported association with a transcriptional repressor CtBP lends further support that it may have a role in transcription. A sequence based screen of a panel of 89 tumor cell line cDNAs for mutations in the CtIP coding region identified five missense variants. In the pancreatic carcinoma cell line, BxPC3, the non-conservative lysine to glutamic acid change at codon 337 is accompanied with apparent loss of heterozygosity or non-expression of the wild type allele. Thus it is plausible that CtIP may itself be a tumor suppressor acting in the same pathway as BRCA1.