Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer.

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations.

Ultralow-frequency Raman system down to 10 cm(-1) with longpass edge filters and its application to the interface coupling in t(2+2)LGs.

Ultralow-frequency (ULF) Raman spectroscopy becomes increasingly important in the area of two-dimensional (2D) layered materials; however, such measurement usually requires expensive and nonstandard equipment. Here, the measurement of ULF Raman signal down to 10 cm(-1) has been realized with high throughput by combining a kind of longpass edge filters with a single monochromator, which are verified by the Raman spectrum of L-cystine using three laser excitations. Fine adjustment of the angle of incident laser beam from normal of the longpass edge filters and selection of polarization geometry are demonstrated how to probe ULF Raman signal with high signal-to-noise. Davydov splitting of the shear mode in twisted (2+2) layer graphenes (t(2+2)LG) has been observed by such system in both exfoliated and transferred samples. We provide a direct evidence of twist-angle dependent softening of the shear coupling in t(2+2)LG, while the layer-breathing coupling at twisted interfaces is found to be almost identical to that in bulk graphite. This suggests that the exfoliation and transferring techniques are enough good to make a good 2D heterostructures to demonstrate potential device application. This Raman system will be potentially applied to the research field of ULF Raman spectroscopy.

Central administrations of hemopressin and related peptides inhibit gastrointestinal motility in mice.

BACKGROUND: Hemopressin was identified as an endogenous inverse agonist/antagonist of CB1 receptor, whereas VD-hemopressin(α) [VD-Hpα] and VD-hemopressin(ß) [VD-Hpß] were found as the novel endogenous peptidic agonists of cannabinoid receptors. As cannabinoids are potent modulators of gastrointestinal (GI) motility, our aim was to characterize the effects of hemopressin and related peptides on GI motility in vivo. METHODS: The responses of intracerebroventricular (i.c.v.) administration of the reference compound WIN55,212-2, hemopressin, and related peptides to GI motility were investigated by measuring upper GI transit, colonic bead expulsion, and whole gut transit in mice. KEY RESULTS: Central administration of the classical cannabinoid receptor agonist WIN55,212-2 dose-dependently slowed upper GI transit, colonic expulsion, and whole gut transit via CB1 receptor. Similarly, Hpα, VD-Hpα, and VD-Hpß delayed upper GI transit and colonic expulsion after i.c.v. administration. At the high doses, Hpα and VD-Hpß inhibited whole gut transit, whereas VD-Hpα had no effect on whole gut transit. In addition, the effects of these three peptides on GI transit were antagonized by the CB1 receptor selective antagonist AM251, but not by the CB2 receptor selective antagonist AM630. CONCLUSION & INFERENCES: The endogenous cannabinoid peptide ligands hemopressin, VD-Hpα, and VD-Hpß inhibited GI transit through the activation of CB1 , but not CB2 cannabinoid receptors. The lower potencies of the hemopressin and related peptides in GI transit assays may be important for the future development of cannabinoid peptides as the therapeutic analgesics with limited GI side effects.

Pulsed radiofrequency inhibited activation of spinal mitogen-activated protein kinases and ameliorated early neuropathic pain in rats.

Background: Pulsed radiofrequency (PRF) has been widely used to treat chronic pain, but the effectiveness and mechanisms in preventing early neuropathic pain have not been well explored. Even fewer knowledge is available in its impact on glia-mediated nociceptive sensitization. This study aims to elucidate the modulation of PRF on nerve injury-induced pain development and activation of spinal mitogen-activated protein kinases (MAPKs). Methods: In a rat spinal nerve ligation (SNL) model, a low-volt PRF treatment was applied to the L5 dorsal root ganglion after nerve injury. Nociceptive behaviours were measured by von Frey and heat withdrawal tests at multiple time points. MAPK activations, including p-ERK and p-p38, as well as TNF-á level in the spinal dorsal horn were assessed and the cell types that expressed MAPK activation were identified by double immuno fluorescence staining.Results: We found that SNL promptly induced neuropathic pain in the affected hind limb for over 1 week as well as increased p-ERK and p-p38 in the spinal dorsal horn. PRF significantly attenuated SNL-induced mechanical allodynia and thermal hyperalgesia for 5­7 days. PRF also inhibited ERK and p38 activations, which were found majorly located within neurons and microglia, respectively. Besides, PRF significantly suppressed expression of TNF-á in the spinal dorsal horn throughout the course. Conclusions: Low-volt PRF significantly ameliorated SNL-induced acute pain. Inferentially, PRF may inhibit spinal sensitization by down-regulating spinal MAPK activations and activation-mediated cytokine release.We demonstrated that early PRF treatment in acute nerve injury helps to ameliorate neuropathic pain development.

Involvement of elevated expression of multiple cell-cycle regulator, DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein), in the growth of breast cancer cells.

To investigate the detailed molecular mechanism of mammary carcinogenesis and discover novel therapeutic targets, we previously analysed gene expression profiles of breast cancers. We here report characterization of a significant role of DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein) in mammary carcinogenesis. Semiquantitative RT-PCR and northern blot analyses confirmed upregulation of DTL/RAMP in the majority of breast cancer cases and all of breast cancer cell lines examined. Immunocytochemical and western blot analyses using anti-DTL/RAMP polyclonal antibody revealed cell-cycle-dependent localization of endogenous DTL/RAMP protein in breast cancer cells; nuclear localization was observed in cells at interphase and the protein was concentrated at the contractile ring in cytokinesis process. The expression level of DTL/RAMP protein became highest at G(1)/S phases, whereas its phosphorylation level was enhanced during mitotic phase. Treatment of breast cancer cells, T47D and HBC4, with small-interfering RNAs against DTL/RAMP effectively suppressed its expression and caused accumulation of G(2)/M cells, resulting in growth inhibition of cancer cells. We further demonstrate the in vitro phosphorylation of DTL/RAMP through an interaction with the mitotic kinase, Aurora kinase-B (AURKB). Interestingly, depletion of AURKB expression with siRNA in breast cancer cells reduced the phosphorylation of DTL/RAMP and decreased the stability of DTL/RAMP protein. These findings imply important roles of DTL/RAMP in growth of breast cancer cells and suggest that DTL/RAMP might be a promising molecular target for treatment of breast cancer.

Radiotherapy for low-grade gastric marginal zone lymphoma: a retrospective study.

BACKGROUND: We evaluated the efficacy and toxicity of radiotherapy (RT) in patients with low-grade gastric marginal zone lymphoma. METHODS: A retrospective review of consecutive cases of gastric marginal zone lymphoma treated by radical RT at the Peter MacCallum Cancer Centre and Radiation Oncology Victoria between January 1980 and September 2003 was carried out. RESULTS: Eighteen patients (11 men and 7 women) were identified. The median age at commencement of RT was 65 years (range 42-84 years). Prior treatment included Helicobacter pylori eradication in 12 patients, chemotherapy in 7 and surgery in 2, whereas 2 patients had no prior therapy. The median time to progression after commencement of last treatment before RT was 4.8 months (range 0-129.4 months). The radiation fields included the stomach plus perigastric and coeliac nodes in 15 patients (83%), stomach plus spleen in 2 patients (11%) and stomach plus para-aortic nodes in 1 patient (6%). The median RT dose was 30 Gy (range 30-36 Gy) in a median 20 fractions (range 17-24 fractions). One patient required treatment interruption for acute toxicity. A complete response on post-RT biopsies was achieved in 17 of 18 patients (94%). With a median follow up of 4.5 years after RT, 3 of these 17 patients (18%) have had a recurrence. At the last follow up, 11 patients were alive in continuous complete histological remission. No late renal toxicity was identified. CONCLUSION: Radiotherapy is an effective, well-tolerated treatment for patients with low-grade gastric marginal zone lymphoma, including those who have had prior therapy.

Monoclonal antibodies against human ribosomal P proteins penetrate into living cells and cause apoptosis of Jurkat T cells in culture.

OBJECTIVE: This study was designed to determine the role of autoantibodies to the ribosomal P protein (anti-P Abs) in the pathogenesis of systemic lupus erythematosus (SLE) using monoclonal anti-P antibodies (anti-P mAbs). METHODS: Anti-P mAbs were prepared by a standard hybridoma procedure using recombinant human P1 and P2 proteins as immunogens. We studied the reactivities of these mAbs to P proteins, their binding and penetration capabilities in different cell lines and their apoptotic effects on Jurkat T cells. RESULTS: In addition to recognizing human P0, P1 and P2 proteins, the anti-P mAb 9B6-4 bound to 20-40% and penetrated 50-90% of astrocytes, Jurkat T cells and lung cancer cells via the P0 surface protein. Treatment with the mAb 9B6-4 also caused increases in the percentages of Jurkat T cells in the sub-G1 phase of the cell cycle (14.8%) and undergoing apoptosis (21.3%). CONCLUSION: Anti-P autoantibodies may play a role in the pathogenesis of lymphopenia or lymphocyte dysfunction in SLE.

p55Cdc/cdc20 overexpression promotes early g1/s transition in myeloid cells.

p55Cdc/Cdc20 is expressed in cycling mammalian cells and has been shown to be an activator of the mitotic spindle assembly checkpoint. We previously showed that overexpression of p55Cdc/Cdc20 in myeloid cells resulted in accelerated apoptosis and inhibition of granulocyte differentiation in the murine myeloid cell line 32Dcl3. p55Cdc/Cdc20 protein expression is detected in cells at late G1 phase of the cell cycle but is maximal during G2 phase. We report in this paper that inducible expression of p55Cdc/Cdc20 in 32Dcl3 cells results in premature transition from G1 to S phase. To characterize the mechanism of this early transition, we examined the expression of critical regulatory proteins during the cell cycle. Although expression of cyclin D, cyclin E, cdk2, and cdc2 did not change significantly between p55Cdc/Cdc20-overexpressing and control cells, p27Kip1 protein levels were lower and cdk2 activity higher during G1 to S transition in p55Cdc/Cdc20-overexpressing cells compared to control cells. Cyclin B1 levels were lower at early G1 phase in cells overexpressing p55Cdc/Cdc20. Our results suggest that p55Cdc/Cdc20 may play an important role in G1 to S transition during myelopoiesis.

In vitro activity of quinupristin/dalfopristin against gram-positive bacteria Haemophilus influenzae and Branhamella catarrhalis in Taiwan.

BACKGROUND: Over the past decade, resistance of Gram-positive cocci to common antibiotics has steadily increased. New antibacterial agents that are active against multidrug-resistant pathogens are urgently needed for the treatment of these pathogens. We conducted an in vitro study on the activity of quinupristin/dalfopristin and other antibiotics against common clinical isolates of the gram-positive cocci, Haemophilus influenzae and Branhamella catarrhalis. METHODS: The agar dilution method described by the National Committee for Clinical Laboratory Standards was used to determine the minimum inhibitory concentrations (MICs) of bacterial isolates from clinical specimens obtained from patients in a medical center. RESULTS: All Staphylococcus aureus isolates were inhibited by quinupristin/dalfopristin (< or = 2 micrograms/ml). The MIC90s were 1 mg/ml for both methicillin-sensitive and -resistant S aureus. Quinupristin/dalfopristin inhibited streptococci at a concentration of 1 microgram/ml or less. The MIC90s were 1 microgram/ml for Streptococcus pneumoniae, S pyogenes and viridans streptococci. Ampicillin-resistant Enterococcus faecium was inhibited by quinupristin/dalfopristin at 0.5 to 4 micrograms/ml, with an MIC90 of 1 microgram/ml. H influenzae was inhibited by quinupristin/dalfopristin at 0.25 to 8 micrograms/ml, with an MIC90 of 4 micrograms/ml. B catarrhalis was inhibited by quinupristin/dalfopristin at 0.25 to 1 microgram/ml, with an MIC90 of 1 microgram/ml. CONCLUSIONS: We found that quinupristin/dalfopristin showed good in vitro activity against staphylococci, streptococci and B catarrhalis but less in vitro activity against H influenzae.

Molecular diagnosis of fragile X syndrome and distribution of CGG repeats in the FMR-1 gene in Taiwanese.

BACKGROUND AND PURPOSE: Fragile X syndrome, the most frequent form of inherited mental retardation, is caused by abnormal expansion of the CGG trinucleotide repeats in the 5' untranslated region of the FMR-1 gene. In this study, we describe the prenatal diagnosis of fragile X syndrome and the distribution of CGG repeat numbers in the FMR-1 gene, which has not been previously reported in Taiwanese. METHODS: Using polymerase chain reaction (PCR), we determined the range of the CGG repeats in the FMR-1 gene in 316 normal individuals (350 X chromosomes) and 349 mentally retarded patients (429 X chromosomes). For prenatal diagnosis of fragile X syndrome, DNA extracted from amniotic fluid cells was used for PCR determination of CGG repeats. RESULTS: Because there were no significant differences between the distribution of the (CGG)n alleles between the mentally retarded and normal subjects, the data were pooled. Among the 779 X chromosomes studied, 24 different alleles were identified with a low of 16 and a high of 45 CGG repeats. The 29 repeat allele was the most common, followed by the 30 and the 28 repeat alleles. We effectively amplified slightly expanded premutation alleles of up to about 90 CGG repeats. In the prenatally diagnosed fetus, a normal 29 repeat allele was found. CONCLUSIONS: Determination of the distribution of the CGG repeats in the FMR-1 gene in Taiwanese is useful in genetic counseling regarding fragile X syndrome. Prenatal molecular diagnosis of the syndrome can be successfully performed using amniotic fluid cells.

A non-radioactive polymerase chain reaction method for diagnosis of Machado-Joseph disease.

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is caused by unstable CAG trinucleotide repeat expansion in the coding region of the MJD gene. In this study, we describe a non-radioactive polymerase chain reaction (PCR) method to detect the CAG repeat range of the MJD gene. This technique allows direct visualization of the PCR products on ethidium bromide-stained agarose gels within hours. In this study, genomic DNA samples isolated from peripheral lymphocytes, amniotic fluid cells, and chorionic villi were tested with two sets of commonly used MJD primers. PCR conditions were optimized, which resulted in clear visualization of both the primer sets on 3% agarose gels. Ten out of 25 candidate MJD patients have been identified with this method to date, with no false-positive or false-negative diagnoses. This simple, reliable, and cost-effective method can be used for patient diagnosis, pre-symptomatic diagnosis, and prenatal diagnosis.

Integrin-mediated migration of murine B82L fibroblasts is dependent on the expression of an intact epidermal growth factor receptor.

To evaluate the mechanisms by which epidermal growth factor (EGF) regulates actin-based cellular processes such as cell migration, we first examined the effects of EGF on cell adhesion, which is essential for cell migration. In mouse B82L fibroblasts transfected with the full-length EGF receptor, EGF promotes cell rounding and attenuates cell spreading on fibronectin, laminin, and vitronectin, and thus appears to reduce the strength of cell adhesion. Moreover, EGF synergizes with multiple extracellular matrix (ECM) components in the promotion of integrin-mediated cell migration of several different cell types, including fibroblasts and various carcinoma and osteosarcoma cell lines. Interestingly, co-presentation (co-positioning) of EGF with laminin or fibronectin is essential for EGF-stimulated migration. When EGF is mixed with the cells instead of the ECM components, it has little effect on cell migration. These results suggest that co-presentation of EGF with ECM components can enhance the polarization events required for directional cell movement. To identify the EGF receptor elements critical for the EGF stimulation of cell migration, B82L fibroblasts were transfected with either mutated or wild-type EGF receptors. Surprisingly, we found that B82L-Parental cells that lack the EGF receptor are not able to migrate to fibronectin, even though they can adhere to fibronectin. However, the introduction of wild-type EGF receptors into these fibroblasts enables them to migrate toward fibronectin even in the absence of EGF. The requirement of the EGF receptor for cell migration does not appear to result from the secretion of EGF or TGF-alpha by the cells transfected with the EGF receptor. Furthermore, cells expressing EGF receptors that are kinase-inactive, or C-terminally truncated, exhibit little migration toward fibronectin, indicating that an intact EGF receptor kinase is required for fibronectin-induced cell migration. In addition, neutralizing anti-EGF receptor antibodies attenuate cell migration in the presence of EGF, and inhibit migration to fibronectin or laminin alone. These results further suggest that the EGF receptor is downstream of integrin activation in the signal transduction pathways leading to fibroblast migration.

Application of spinal pain mapping in the diagnosis of low back pain--analysis of 104 cases.

BACKGROUND: Low back pain is probably the most common pain problem seen in a general pain clinic and the cause of low back pain can be enigmatic at times. Often the pain sources are difficult to identify with the conventional diagnostic modalities. Spinal pain mapping is a sequence of well organized nerve block procedures. We undertook this study to evaluate the usefulness of this modality in diagnosing low back pain of uncertain etiology. METHODS: In this prospective study, 104 consecutive adult patients who underwent spinal pain mapping were examined and analyzed. All patients had intractable low back pain of undetermined etiology after medical history, physical examination and 4-view roentgenographic evaluation of the lumbar spine had been undertaken to locate it. In addition, 41 patients (39%) had one or more of the following tests done, which included CT, MRI, EMG/NC but all failed to delineate the causes of the pain. All patients failed to respond to the conservative therapies. RESULTS: With pain mapping the source of pain was found to be caused by sacro-iliac joint in 6%, lumbar nerve root in 20%, facet joint in 24%, combined lumbar nerve root and facet disease in 24%, internal disc disorder in 7%, combined facet and sacro-iliac joint in 4% and lumbar sympathetic dystrophy in 2% of patients. Pain mapping failed to demonstrate the causes of the pain in the remaining 13% of the patients. CONCLUSIONS: Considering the difficult nature of this group of patients, spinal pain mapping provided a useful functional approach to the diagnosis of low back pain with obscure etiology in 87% of patients in our series.

Agreement between a frequency-weighted filter for continuous biomechanical measurements of repetitive wrist flexion against a load and published psychophysical data.

A previous pilot study demonstrated that a force and frequency-weighted filter network could be developed for processing continuous biomechanical measures of repetitive wrist motions and exertions. The current study achieves the objective by modelling subjective discomfort for repetitive wrist flexion using controlled posture, pace and force. A three-level fractional factorial experiment was conducted involving repetitive wrist flexion (2 s/motion, 6 s/motion, 10 s/motion) from a neutral posture to a given angle (10 degrees, 28 degrees, 45 degrees) against a controlled resistance (5 N, 25 N, 50 N) using a Box Behnken design. Ten subjects participated. Discomfort was reported on a 10 cm visual analogue scale. Results of response surface regression analysis revealed that main effects of force, wrist flexion angle, and repetition were all significant (p < 0.05) and that no second-order effects were observed. Linear regression analysis on these factors established a discomfort model on which the filter characteristics were based. The pure error test model revealed no significant lack of fit (p > 0.05). The continuous model was compared and agreed with discrete psychophysical data from other published studies. The model was used for generating parameters for a force and frequency-weighted digital filter that weighs continuous wrist postural signals with corresponding force in proportion to the equal discomfort function as a function of frequency of repetition. These filters will enable integration of large quantities of biomechanical data in field studies.

Validation of a frequency-weighted filter for continuous biomechanical stress in repetitive wrist flexion tasks against a load.

This experiment validates a frequency-weighted filter for continuous measurements of force, posture and repetition using a stimulated industrial task. A peg transfer task was used requiring subjects to repetitively insert pegs into holes with controlled resistance. Ten subjects performed the task for six conditions. All wrist flexion angular data were recorded continually using an electrogoniometer and processed through the filter. Subjective discomfort was reported after performing the task for 1 h using a 10 cm visual analogue scale. Results from linear regression analysis showed that the instrument reliably estimated subjective discomfort (r2 = 0.873). Applications and limitations of this instrument are explored.

Efficacy of a stress management program for patients with hepatocellular carcinoma receiving transcatheter arterial embolization.

Transcatheter arterial embolization (TAE), a common treatment for patients with unresectable hepatocellular carcinoma (HCC), can provoke severe physical discomfort and psychologic stress. The purpose of this study was to investigate the effect of a combination of health education, muscle relaxation, and back massage on reducing physical and psychologic stress in HCC patients receiving TAE. A quasi-experimental design was used. Forty patients with HCC (30 men and 10 women) with a mean age of 57 +/- 12 years were recruited and randomly assigned to the control or experimental group. The effectiveness of the stress management program was evaluated using a knowledge questionnaire, a worry inventory, a state-trait anxiety inventory, and a physical distress scale. After completing the stress management program, the experimental group had a greater mean increase in knowledge score than the control group (5.1 vs 0.8, p < 0.0001) and a greater mean decrease in worry score (-8.2 vs 1.1, p < 0.0001). The mean decrease in the anxiety score in the experimental group was also significantly greater than in the control group before TAE (-5.8 vs 3.2, p < 0.001) and 2, 4, 6, and 7 days after TAE (-8.2 vs 7.1, p < 0.001; -8.7 vs 3.2, p < 0.001; -9.8 vs -2.1, p < 0.05; -11 vs -0.9, p < 0.05). The patients in the experimental group had a smaller mean increase in physical distress score than the control group at 2, 4, 6, and 7 days after TAE (34.7 vs 50.2, 20.9 vs 29.6, 10.6 vs 18.2, 3.9 vs 11.2, all p < 0.05). This stress management program effectively reduces the stress of HCC patients undergoing TAE.

A single metric for quantifying biomechanical stress in repetitive motions and exertions.

The relative effects of repetition, force and posture were studied in order to investigate how continuous biomechanical measurements can be combined into a single metric corresponding to subjective discomfort. A full factorial experiment was conducted involving repetitive wrist flexion from a neutral posture to a given angle against a controlled force. Seven subjects performed the task using two paces (20 and 4 motions/min), two force levels (15 and 45 N) and two angles (15 and 45 degrees) for 1 h each. Discomfort was reported on a 10 cm visual analogue scale anchored between 'no discomfort' and 'very high discomfort'. Repeated measures analysis of variance showed that all main effects were statistically significant (p < 0.05) and no significant interactions were observed. A linear regression model was fitted to the data and used for generating frequency weighted digital filters that shape continuous recordings of repetitive motions and exertions into an output proportional to relative discomfort. The resulting high-pass digital filter had a 22 dB/decade attenuation slope. A simulated industrial task used for validating the model involved repetitively transferring pegs across a horizontal bar and inserting them into holes against a controlled resistance. Angular wrist data were recorded using an electrogoniometer and filtered. Six subjects performed the task of the three conditions consisting of (1) 15 wrist flexion, 15 N resistance and 6 motions min, (2) 15 wrist flexion. 45 N resistance and 12 motions/min, and (3) 45 degrees wrist flexion, 45 N resistance and 15 motions/min. Subjective discomfort was reported after performing the task for 1 h. Pearson correlations between subjective discomfort ratings and the integrated filtered biomechanical data for individual subjects ranged from 0.90 to 1.00. The pooled correlation across subjects was 0.67. This approach may be useful for physical stress exposure assessment and for design of tasks involving repetitive motions and exertions.

Novel point mutation within intron 10 of FMR-1 gene causing fragile X syndrome.

The majority of cases involving fragile X syndrome are due to expansion of a (CGG)n trinucleotide repeat at the 5' untranslated region of the FMR-1 gene. Deletion and intragenic loss of function mutations of the FMR-1 gene also have been reported. Here, we report a C to T point mutation at the 14th nucleotide in intron 10 of the FMR-1 gene in three unrelated fragile X patients. However, the (CGG)n repeat of FMR-1 in those patients does not expand. To determine the effect of this mutation on the patients' FMR-1 transcripts, total RNA from peripheral blood cells was reverse transcribed and amplified by polymerase chain reaction (RT-PCR). Direct and subcloned sequencing of the RT-PCR products revealed that the transcripts from the allele with C to T mutation skip exon 10 entirely, resulting in a joining of exons 9 and 11. Deletion of exon 10 results in frame-shift and premature termination of translation, which removes the highly conserved region that encoding the KH2 and RGG box domains of FMRP. Interestingly, a male of the three patients has another G to A substitution in exon 15. However, the intron 10 mutation is sufficient for development of fragile X syndrome.

Laminin responsiveness is associated with changes in fibroblast morphology, motility, and anchorage-independent growth: cell system for examining the interaction between laminin and EGF signaling pathways.

Laminin can influence the adhesion, differentiation, and motility of several cell types, including epithelial and neural cells. In addition, laminin, which contains an epidermal growth factor (EGF)-like motif, can stimulate DNA synthesis in fibroblasts possessing the EGF receptor, but laminin does not compete for EGF binding. To further investigate laminin action in fibroblasts, and the relationship between laminin and EGF receptor function, we have developed a system wherein cells containing laminin-binding activity were cloned from a mouse fibroblast cell line (B82L-wt) that cannot adhere to laminin but that have been transfected with the wild-type human EGF receptor. Although only the isolated clones can efficiently attach to laminin-coated plates, all the cells can adhere to plastic, fibronectin, and collagen l, and all exhibit comparable levels of cell surface-associated laminin. Ligand-binding assays showed that the cells with laminin attachment activity possess high-affinity EGF binding (Kd approximately 0.4 nM), and all express a similar level of the human EGF receptor. However, when compared to the B82L-wt cells, the cells with laminin-binding activity exhibit altered morphology, anchorage-independent growth, and motility. Specifically, the morphology of the fibroblasts possessing laminin binding activity appears more elongated and they spread more extensively on plastic plates. Analysis of their growth in soft agar revealed that the clones have a 2-5-fold increase in colony formation in comparison to the B82L-wt cells. The cells possessing laminin attachment ability also exhibit laminin-induced motility, and this movement is directional (chemotaxis) rather than random (chemokinesis), indicating functional laminin receptors and signaling pathways. To examine the specific laminin receptors involved in these effects, the influence of anti-integrin subunit antibodies on cell adhesion and migration was evaluated. These studies showed that an anti-alpha 6 integrin antibody can completely inhibit the clonal cells' attachment and migration to laminin, and anti-alpha 6 immunoblots revealed that only the clones express measurable levels of alpha 6. These data indicate that alpha 6-containing integrins contribute to the laminin-mediated attachment and motility of these clones and that this system may also influence the morphology and anchorage-independent growth of these fibroblasts. In addition, these cells provide a unique system for examining the interaction between EGF and laminin receptor action.

Exposure assessment of biomechanical stress in repetitive manual work using frequency-weighted filters.

A quantitative exposure assessment strategy for physical stress associated with repetitive manual tasks is proposed using continuous biomechanical data measured directly from electrogoniometers or force sensors. This paper describes an efficient method for reducing large quantities of biomechanical data into a quantifiable metric that accounts for recognized musculoskeletal exposure factors, including repetitiveness, postural or forceful exertion stress, and duration. A frequency domain approach is used for averaging elemental data recorded for repetitive cycles. Parameters for frequency-weighted filters are developed using psychophysical data for equivalent discomfort levels resulting from repetitive movements of different amplitudes and frequencies. These filters enable continuous biomechanical data to be filtered and integrated, resulting in a single quantity corresponding to psychophysical response characteristics for repetitive motion stress. It is anticipated that a similar approach may be used for epidemiological response characteristics. Applications of this theory may make it possible for assessing exposure to physical stress in a manner analogous to the way in which sound level meters are used for measuring exposure to acoustic noise. Repetitive wrist flexion and localized discomfort was used for demonstrating the feasibility of this approach. Suitable data reduction techniques are necessary for evaluating work methods, job designs, and for conducting large scale detailed epidemiological investigations of cumulative trauma disorder risk factors. Frequency-weighted filters based on human response to physical stress at different frequencies can greatly simplify exposure analysis and ultimately may make it possible for quantitative exposure limits to be established.