Phase I dose-escalation trial of tucatinib in combination with trastuzumab in patients with HER2-positive breast cancer brain metastases.

BACKGROUND: Brain metastases are frequent in HER2-positive breast cancer. ONT-380 (tucatinib) is a potent selective inhibitor of HER2 with intracranial activity in preclinical models. PATIENTS AND METHODS: This was a phase I study of tucatinib with trastuzumab, without chemotherapy, in patients with progressive, measurable HER2-positive brain metastases. The study tested two schedules of tucatinib: cohort A was twice daily and cohort B was once daily. The primary objective was determination of the maximum tolerated dose (MTD). Secondary end points included objective response (intracranial and extracranial) using modified RECIST and clinical benefit rate (CBR). RESULTS: Overall, 41 patients were enrolled (cohort A, n = 22; cohort B, n = 19). Patients had a median of two prior treatments for metastatic breast cancer and 83% had progressed after prior brain radiation. The MTD of tucatinib for cohort A was 300 mg twice daily and for cohort B was 750 mg once daily. The most common dose-limiting toxicities included thrombocytopenia and aspartate transaminase/alanine aminotransferase elevation. Grade 3/4 aspartate transaminase/alanine aminotransferase elevation occurred in nine of 41 patients (22%). Intracranial responses were observed in two of 17 (12%) patients in cohort A and one of 17 (6%) patients in cohort B treated at the MTD. In cohort A, CBR at 16 weeks was 35% (n = 6). In cohort B, CBR at 16 weeks was 53% (n = 9). Of 15 patients overall who experienced clinical benefit, 12 (80%) had received prior neratinib and/or lapatinib. Median progression-free survival for cohorts A and B was 3.4 and 4.1 months, respectively. CONCLUSION: The combination of tucatinib and trastuzumab is tolerable and demonstrated preliminary evidence of efficacy in patients with HER2-positive brain metastases. CLINICAL TRIAL REGISTRATION: NCT01921335.

Achilles tendinitis in systemic lupus erythematosus: search for an associated inflammatory disease.

OBJECTIVES: Except for traumatic and iatrogenic causes, Achilles tendinitis (AT) is mostly encountered in the context of inflammatory rheumatic diseases. This study aimed to describe AT in systemic lupus erythematosus (SLE). PATIENTS AND METHODS: Among 158 SLE patients who fulfilled the SLE criteria of the ACR classification followed between 1980 and 2013, we selected those who experienced at least one episode of AT not caused by traumatic or toxicity factors. RESULTS: Eight patients (one male, seven females), median age 52 years (range: 35-68), presented with 11 episodes of AT within an average of 10.5 (0-21) years after SLE diagnosis. Clinical presentation of SLE was mainly cutaneous (eight of eight), and articular (seven of eight). Axial symptoms were reported in six patients, two of whom had HLA-B27-positive status, and fulfilled the Amor and European Spondylarthropathy Study Group criteria. Resolution of AT was good with nonsteroidal anti-inflammatory topical or systemic drug therapies, which kept SLE quiescent and avoided any increase of specific treatment. CONCLUSION: Although the association is rare, when AT occurs in SLE patients, physicians should look for associated spondylarthritis.

[Whipple disease revealed by anti-TNFα therapy]. / Maladie de Whipple révélée par les traitements anti-TNFα

INTRODUCTION: Whipple disease is a rare infectious disease with protean clinical manifestations. This infection may mimic chronic inflammatory rheumatisms such as rheumatoid arthritis or spondylarthritis. In this context, introduction of a biotherapy after a diagnostic hesitation does not always lead to early complications. Sometimes, the clinical degradation follows an initial improvement, encouraging continuation of the immunosuppressive treatment and leading consequently to a greater diagnostic delay. CASE REPORTS: We report two cases of Whipple disease diagnosed in the context of an inflammatory disease with anti-TNFα failure. The first patient was a 53-year-old man who presented with an axial and peripheral spondylarthritis who was treated with etanercept and adalimumab. The second was a 42-year-old man who received adalimumab and then etanercept for a peripheral spondylarthritis. CONCLUSION: Whipple disease should be suspected in all patients who present with a chronic inflammatory rheumatism that is partially or not controlled with anti-TNFα therapy and who had persisting elevated acute phase reactants.

Visual acuity testability and comparability in Australian preschool children: the Sydney Paediatric Eye Disease Study.

PURPOSE: To establish standardised protocols for vision screening, testability and comparability of three different vision tests were examined in a population-based, cross-sectional sample of preschool children (Sydney Paediatric Eye Disease Study). METHODS: Measurement of presenting monocular distance visual acuity (VA) using the Amblyopia Treatment Study (ATS) HOTV protocol, was attempted by all (1774) children aged≥24 months. In addition, in children aged≥60 months (576), VA was also tested using the logMAR retro-illuminated HOTV or Early Treatment Diabetic Retinopathy Study (ETDRS) linear charts (CSV 1000). Children able to have both eyes tested monocularly were considered. RESULTS: Testability significantly increased with age for all VA tests. The ATS HOTV with an overall testability of 80% (females: 82%, males: 78%) was the most testable of the VA tests (P<0.0001). In children aged <3 years testability was low (≤47%) rising to≥80% in children aged≥3. In children≥60 months, testability was higher for the HOTV (94%) than the ETDRS (59%) chart. In those that did two VA tests, mean difference of the ATS HOTV compared with the HOTV(CSV) was -0.1, and compared with ETDRS was -0.12 (P<0.0001). CONCLUSIONS: Children aged <3 years had poor VA testability, whereas those 3 years and above were highly testable using the ATS HOTV. The HOTV (CSV) retro-illuminated test was appropriate for children aged >5 years, and may be possible in younger children with early educational exposure. When comparing VA measures using these tests, the higher VA attained using the ATS HOTV, needs to be taken into account.

Efficacy of rituximab in primary Sjogren's syndrome with peripheral nervous system involvement: results from the AIR registry.

OBJECTIVE: To evaluate rituximab (RTX) in primary Sjögren's syndrome (pSS) with peripheral nervous system (PNS) involvement. METHODS: Patients with pSS and PNS involvement who were included in the French AIR registry were analysed. RESULTS: 17 patients (age 60 years (44-78 years); 14 were female) were analysed. Neurological improvement was noted in 11 patients (65%) at 3 months. Rankin scale decreased from 3 (1-5) to 2 (1-5), 2 (1-5) and 2 (1-6) after 3, 6 and 9 months (p=0.02). European Sjögren's Syndrome Disease Activity Index decreased from 18 (10-44) to 11 (5-20), 11 (5-29) and 12 (5-30) after 3, 6 and 9 months (p<0.05). RTX was effective in neurological involvement in 9/10 patients with vasculitis or cryoglobulinaemia (90%) (group 1) at 3 months and in 2/7 cases (29%) without cryoglobulinaemia and vasculitis (p=0.03). Rankin and European Sjögren's Syndrome Disease Activity Index scales decreased significantly in group 1. CONCLUSION: RTX seems effective in cryoglobulinaemia or vasculitis-related PNS involvement in pSS.

Carcinoma de Células Escamosas de Tiroides: A Propósito de un Caso / Primary squamous cell thyroid carcinoma

El carcinoma de células escamosas de tiroides (CCET) es un tumor infrecuente y agresivo. Su etiología es incierta. Ante la presencia de carcinoma escamoso en la glándula tiroides debe excluirse la posibilidad de infiltración de un tumor originado en una estructura adyacente o de metástasis de otros carcinomas. El tratamiento de elección es la cirugía radical. La mayoría de los pacientes fallecen antes del año debido a progresión local de la enfermedad. Presentamos el caso de un paciente con una masa tiroidea, con diagnóstico histológico de carcinoma escamoso (con inmunohistoquímca negativa para tiroglobulina, TTF1 y calcitonina y positiva para p63 y citok5). Los estudios clínicos, endoscópicos y radiológicos excluyeron otros sitios de origen de carcinoma escamoso. Fue tratado con quimio y radioterapia, falleciendo por progresión local luego de 9 meses.

Primary squamous cell thyroid carcinoma (PSCTC) is a rare and aggressive tumor of uncertain origin. When squamous carcinoma is diagnosed, it is mandatory to exclude the possibility of primary tumor arising from an adjacent structure or representing metastases from a primary growth elsewhere. Aggressive surgical resection is the treatment of choice. However, the prognosis is poor, with a median survival of less than a year. Death is usually secondary to progression of local disease. We report a case of a patient presenting with a thyroid mass; biopsy was consistent with squamous cell carcinoma. On immunohistochemistry tumor cells were negative for TTF1, thyroglobulin and calcitonin. Cancer cells were positive for p63 and citok5. Extensive workup excluded the possibility of extrathyroid origin. The patient was treated with chemoradiotherapy; he died 9 months later due to local progression.

The ABRF Edman Sequencing Research Group 2008 Study: investigation into homopolymeric amino acid N-terminal sequence tags and their effects on automated Edman degradation.

The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.

Large scale refolding and purification of the catalytic domain of human BACE-2 produced in E. coli.

Evidence for a key role of beta-amyloid (Abeta) in Alzheimer's disease has led to considerable interest in potential therapeutic strategies targeting enzymes involved in processing the amyloid precursor protein (APP). Beta-site APP Cleaving Enzyme (BACE or beta-secretase) is a membrane bound aspartyl protease that has been shown to be directly involved in Abeta production and, therefore, is at the forefront of therapeutic targets in the treatment of Alzheimer's disease. BACE-2, an enzyme closely related to BACE, regulates Abeta production in a manner antagonistic to BACE, suggesting that non-selective inhibition of BACE-2 by BACE inhibitors might impair the lowering of Abeta. The design of BACE inhibitors that do not inhibit BACE-2 would be enhanced by structural and kinetic studies, efforts that typically demand considerable amounts of both enzymes. A BACE-2 construct containing 19 residues of the BACE prosegment followed by the BACE-2 catalytic domain sequence, Asp36-Trp447, was produced in E. coli inclusion bodies (IB) at 110-140 mg/L cell culture. Exploration of a variety of refolding conditions resulted in an efficient method for refolding the resulting pro-BACE-2 construct, and this protein undergoes facile autocatalytic cleavage, optimal at pH 4, at the Leu40- downward arrow-Ala41 bond. Refolded BACE-2 was purified by anion exchange, molecular sieve, and affinity chromatographies, yielding 105 mg of homogeneous enzyme (kcat/ Km = 1.2 x 10(4) x M(-1) x sec(-1)) from 8 liters of E. coli cell culture.

Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction.

BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)(6) and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (His)(6) derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 A resolution.

High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms.

BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.

ABRF ESRG 2006 study: Edman sequencing as a method for polypeptide quantitation.

The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% each of peptide C and its acetylated form, peptide C*. Participating laboratories were provided with two tubes, each containing 100 picomoles of the peptide mixture (as determined by quantitative amino acid analysis) and were asked to provide amino acid assignments, peak areas, retention times at each cycle, as well as initial and repetitive yield estimates for each peptide in the mixture. Details about instruments and parameters used in the analysis were also collected. Participants in the study with access to a mass spectrometer (MALDI-TOF or ESI) were asked to provide information about the relative peak areas of the peptides in the mixture as a comparison with the peptide quantitation results from Edman sequencing. Positive amino acid assignments were 88% correct for peptide C and 93% correct for peptide B. The absolute initial sequencing yields were an average of 67% for peptide (C+C*) and 65.6 % for peptide B. The relative molar ratios determined by Edman sequencing were an average of 4.27 (expected ratio of 4) for peptides (C+C*)/B, and 1.49 for peptide C*/C (expected ratio of 1); the seemingly high 49% error in quantification of Lys(Ac) in peptide C* can be attributed to commercial unavailability of its PTH standard. These values compare very favorably with the values obtained by mass spectrometry.

ABRF ESRG 2005 study: identification of seven modified amino acids by Edman sequencing.

Identification of modified amino acids can be a challenging part for Edman degradation sequence analysis, largely because they are not included among the commonly used phenylthiohydantion amino acid standards. Yet many can have unique retention times and can be assigned by an experienced researcher or through the use of a guide showing their typical chromatography characteristics. The Edman Sequencing Research Group (ESRG) 2005 study is a continuation of the 2004 study, in which the participating laboratories were provided a synthetic peptide and asked to identify the modified amino acids present in the sequence. The study sample provided an opportunity to sequence a peptide containing a variety of modified amino acids and note their retention times relative to the common amino acids. It also allowed the ESRG to compile the chromatographic properties and intensities from multiple instruments and tabulate an average elution position for these modified amino acids on commonly used instruments. Participating laboratories were given 2000 pmoles of a synthetic peptide, 18 amino acids long, containing the following modified amino acids: dimethyl- and trimethyl-lysine, 3-methyl-histidine, N-carbamyl-lysine, cystine, N-methyl-alanine, and isoaspartic acid. The modified amino acids were interspersed with standard amino acids to help in the assessment of initial and repetitive yields. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the ESRG Web site. The ABRF ESRG 2005 sample is the seventeenth in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.

Long-term results of en bloc transplantation of pediatric kidneys into adults using a vicryl mesh envelope technique.

En bloc transplantation of pediatric kidneys into adults is a suitable measure to help correct the shortage of available kidneys. This practice, however, is not widespread because of the high incidence of vascular complications. Our institution has previously described a vicryl mesh technique for en bloc kidneys, with an attempt to reduce the incidence of vascular complications. The purpose of this study was to evaluate the long-term results of recipients with en bloc kidneys stabilized with this technique. The charts of 644 adult renal transplants performed between July 1987 and July 1999 were reviewed. During this period, 14 adult patients have received 14 pairs of en bloc pediatric kidneys using the vicryl mesh technique. All patients received OKT3 as an induction immunosuppression with cyclosporine started 10-14 d after the transplant. The median donor age was 24 months (range 14-84 months), and the median recipient age was 49 yr (range 23-68 yr). The mean recipient weight was 79 kg (range 60-114 kg). The mean cold ischemia time was 14.2 hr. None of the patients developed vascular or urological complications. Delayed graft function and moderate acute rejection occurred in one patient each. At a mean follow-up of 51 months (range 7-96 months), all 14 patients maintained excellent renal function with a mean creatinine of 1.01 mg/dL. Renal measurements pre-operatively and at follow-up ultrasound examinations were available in 9 patients, and the mean length of the kidneys had grown approximately 5.0 cm. These data demonstrate that minimal vascular and immunological complication rates can be achieved with pediatric en bloc kidneys using the vicryl mesh envelope technique.

The development and validation of a dysphagia-specific quality-of-life questionnaire for patients with head and neck cancer: the M. D. Anderson dysphagia inventory.

OBJECTIVE: To design a reliable and validated self-administered questionnaire whose purpose is to assess dysphagia's effects on the quality of life (QOL) of patients with head and neck cancer. DESIGN: Cross-sectional survey study. METHODS: Focus groups were convened for questionnaire development and design. The M. D. Anderson Dysphagia Inventory (MDADI) included global, emotional, functional, and physical subscales. One hundred consecutive adult patients with a neoplasm of the upper aerodigestive tract who underwent evaluation by our Speech Pathology team completed the MDADI and the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36). Speech pathologists completed the Performance Status Scale for each patient. Validity and reliability properties were calculated. Analysis of variance was used to assess how well the MDADI discriminated between groups of patients. RESULTS: The internal consistency reliability of the MDADI was calculated using the Cronbach alpha coefficient. The Cronbach alpha coefficients of the MDADI subscales ranged from 0.85 to 0.93. Test-retest reliability coefficients of the subscales ranged from 0.69 to 0.88. Spearman correlation coefficients between the MDADI subscales and the SF-36 subscales demonstrated construct validity. Patients with primary tumors of the oral cavity and oropharynx had significantly greater swallowing disability with an adverse impact on their QOL compared with patients with primary tumors of the larynx and hypopharynx (P<.001). Patients with a malignant lesion also had significantly greater disability than patients with a benign lesion (P<.001). CONCLUSIONS: The MDADI is the first validated and reliable self-administered questionnaire designed specifically for evaluating the impact of dysphagia on the QOL of patients with head and neck cancer. Standardized questionnaires that measure patients' QOL offer a means for demonstrating treatment impact and improving medical care. The development and validation of the MDADI and its use in prospective clinical trials allow for better understanding of the impact of treatment of head and neck cancer on swallowing and of swallowing difficulty on patients' QOL.

[W206R]-procaspase 3: an inactivatable substrate for caspase 8.

We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser(24)-H(277)) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extracts of recombinant Escherichia coli by immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive. Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment. The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysis.

Anterior intramuscular transposition with ulnar nerve decompression at the elbow.

The effectiveness of decompression and anterior intramuscular transposition of the ulnar nerve for treatment of severe cubital tunnel syndrome was evaluated. A consecutive series of 39 anterior intramuscular transpositions were reviewed. One surgeon performed the transpositions between 1993 and 1997 in 34 patients who presented with clinically severe cubital tunnel syndrome. Clinical outcome and satisfaction with surgery were assessed. The results showed early clinical improvement of 77% of patients (mean followup, 3.34 months). With repeated assessments later, the same group of patients had clinical improvement of 62% (mean followup, 30.9 months). Patients younger than 50 years, individuals who underwent external neurolysis, or patients who had a previously failed subcutaneous transposition had fewer satisfactory results.

Late adolescents' perspectives on marital rape: the impact of gender and fraternity/sorority membership.

Although recent studies of marital rape have examined both victims' and perpetrators' social and psychological characteristics, little attention has been directed to the attitudes of others toward marital rape. Using a systematic sample of college students, this study examined attitudes toward marital rape--in particular, the impact of gender and fraternity/sorority membership on respondents' (1) views regarding marital rape compared to rape by a stranger; (2) feelings about possible actions a woman who is a victim of marital rape can take; and (3) attitudes toward legislation pertaining to marital rape. It was found that college women were significantly more likely than college men to say they strongly agree that marital rape and stranger rape should be treated as similar crimes. In addition, nonfraternity men were significantly more likely than fraternity men to indicate that they strongly approve of marital rape legislation and that husbands who perpetrate marital rape should be prosecuted. Sorority membership had little impact on women's responses.

Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor.

Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k(cat) and a 15-fold-lower K(m ATP) although K(m peptide) was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.

Further experience with Botox injection for tracheoesophageal speech failure.

BACKGROUND: Some patients fail to acquire tracheoesophageal (TE) speech after laryngectomy because of pharyngeal constrictor hypertonicity. Botox injection relieves hypertonicity, but there are little objective data regarding outcomes, duration of effect, and reinjection rates. METHODS: Hypertonicity was identified by means of insufflation testing and confirmed videofluoroscopically in 23 unsuccessful TE speakers. Each patient received an EMG-guided Botox injection. Additional injections were offered if the first injection failed to produce fluent speech. RESULTS: Overall, 20 of 23 patients (87%) achieved fluent TE speech production after Botox injections; 5 after additional injections. Two patients declined further intervention, and 1 failed to achieve fluent TE speech production even after 3 Botox injections. The longest sustained effect was 37 months, the shortest was 5 months for 1 patient who required reinjection of Botox to maintain her TE speech production. CONCLUSIONS: Botox injection relieves constrictor hypertonicity in selected cases of TE speech failure with little need for reinjection to maintain long-term speech success.