Surgical excision of anal sac apocrine gland adenocarcinomas with and without adjunctive chemotherapy in dogs: 42 cases (2005-2011).

Objective-To identify variables associated with prognosis in dogs undergoing surgical excision of anal sac apocrine gland adenocarcinomas (ASACs) with and without adjunctive chemotherapy. Design-Retrospective case series. Animals-42 dogs with ASACs. Procedures-Information on signalment, clinical signs, diagnostic procedures, surgical procedures, adjunctive therapies, survival time, and disease-free interval was obtained from the medical records. Results-Survival time was significantly associated with the presence of sublumbar lymphadenopathy and sublumbar lymph node extirpation, with median survival time significantly shorter for dogs with sublumbar lymphadenopathy (hazard ratio, 2.31) than for those without and for dogs that underwent lymph node extirpation (hazard ratio, 2.31) than for those that did not. Disease-free interval was significantly associated with the presence of sublumbar lymphadenopathy, lymph node extirpation, and administration of platinum-containing chemotherapeutic agents, with median disease-free interval significantly shorter for dogs with sublumbar lymphadenopathy (hazard ratio, 2.47) than for those without, for dogs that underwent lymph node extirpation (hazard ratio, 2.47) than for those that did not, and for dogs that received platinum-containing chemotherapeutic agents (hazard ratio, 2.69) than for those that did not. Survival time and disease-free interval did not differ among groups when dogs were grouped on the basis of histopathologic margins (complete vs marginal vs incomplete excision). Conclusions and Clinical Relevance-Results suggested that in dogs with ASAC undergoing surgical excision, the presence of sublumbar lymphadenopathy and lymph node extirpation were both negative prognostic factors. However, completeness of surgical excision was not associated with survival time or disease-free interval.

Beta-diketones: peak shape challenges and the use of mixed-mode high-performance liquid chromatography methodology to obtain fast, high resolution chromatography.

Historically, indirect methods have been used for the HPLC analysis of beta-diketone compounds because of the very poor peak shapes and resolution obtained on conventional HPLC stationary phases. In this paper we demonstrate that it is possible to obtain good peak shapes for underivatised beta-diketone compounds, in a simulated reaction mixture, using only conventional mobile phases with mixed-mode stationary phase HPLC columns. Optimum conditions were obtained using the mixed-mode reversed-phase strong anion exchange column Primesep B, supplied by SIELC Technologies, with a 0.1% aq. TFA/MeOH gradient method and a column temperature of 55 degrees C.

Protein requirements in male adolescent soccer players.

Few investigations have studied protein metabolism in children and adolescent athletes which makes difficult the assessment of daily recommended dietary protein allowances in this population. The problematic in paediatric competitors is the determination of additional protein needs resulting from intensive physical training. The aim of this investigation was to determine protein requirement in 14-year-old male adolescent soccer players. Healthy male adolescent soccer players (N = 11, 13.8 +/- 0.1 year) participated in a short term repeated nitrogen balance study. Diets were designed to provide proteins at three levels: 1.4, 1.2 and 1.0 g protein per kg body weight (BW). Nutrient and energy intakes were assessed from 4 day food records corresponding to 4 day training periods during 3 weeks. Urine was collected during four consecutive days and analysed for nitrogen. The nitrogen balances were calculated from mean daily protein intake, mean urinary nitrogen excretion and estimated faecal and integumental nitrogen losses. Nitrogen balance increased with both protein intake and energy balance. At energy equilibrium, the daily protein intake needed to balance nitrogen losses was 1.04 g kg(-1) day(-1). This corresponds to an estimated average requirement (EAR) for protein of 1.20 g kg(-1) day(-1) and a recommended daily allowance (RDA) of 1.40 g kg(-1) day(-1) assuming a daily nitrogen deposition of 11 mg kg(-1). The results of the present study suggest that the protein requirements of 14-year-old male athletes are above the RDA for non-active male adolescents.

Single agent gemcitabine chemotherapy in dogs with spontaneously occurring lymphoma.

BACKGROUND: Gemcitabine has been shown to be effective as a single agent in a variety of tumors including nonHodgkin's lymphoma. Its use in veterinary medicine has been limited and to date this drug has not been used as a first-line therapy in dogs with lymphoma. HYPOTHESIS: Gemcitabine as a single agent may be efficacious in dogs presented for the first time with lymphoma. ANIMALS: Twenty-four dogs with spontaneously occurring lymphoma. METHODS: All dogs were clinically staged and given gemcitabine at 400 mg/m(2) over a 30-minute intravenous infusion weekly for 3 weeks and then given 1 week off treatment before starting a second cycle. RESULTS: A single dose of gemcitabine lowered both neutrophil count (decrease in mean neutrophil count from 10,640 cells/ microL to 3,140 cells/microL) and platelet count (decrease in mean platelet count from 201,290 cells/microL to 139,190 cells/microL) 7 days after administration. Consequently gemcitabine dosage was reduced at the second treatment in 8 of 21 dogs or a dose delay of 1-7 days and a reduction of dosage was used in 7 of 21 dogs. Seven dogs completed the assigned 4-week cycle. Two of these dogs had progressive disease and 5 had stable disease. No objective responses were seen in dogs treated with a second cycle of gemcitabine. CONCLUSIONS AND CLINICAL IMPORTANCE: Gemcitabine administration as a single agent resulted in hematologic toxicity and did not reduce lymphoma burden. If gemcitabine is to be used in veterinary medicine, additional prospective pharmacologic studies should be done to determine the appropriate dosage, regimen, and schedule of use before it can be recommended for use in the treatment of dogs with lymphoma as a single agent.

Peak leg muscle power, peak VO2 and its correlates with physical activity in 57 to 70-year-old women.

The two aims of this study were first to measure short-term muscle power (STMP) by means of a cycling force-velocity test (cycling peak power: CPP) and a vertical jump test (jumping peak performance: JPP) and second, to examine the relationships between physical activity (PA) level, peak oxygen uptake (peak VO2) and STMP in healthy elderly women. Twenty-three independent community-dwelling elderly women (mean age: 64+/-4.4) performed on separate days, a peak oxygen uptake test on cycle ergometer, a cycling force-velocity test and a vertical jump test. A questionnaire (QUANTAP) was used to assess lifespan exercise habits. Four indices expressed in kJ day(-1) kg(-1) were calculated. Two indices represented average past PA level: 1/quantity of habitual physical activity (QHPA), 2/quantity of sports activities (QSA). Two indices represented the actual PA level: 3/actual quantity of habitual physical activity (AQHPA), 4/actual quantity of sports activities (AQSA). CPP (6.3+/-1.2 W kg(-1)) was closely correlated to JPP (14.8+/-3.4 cm) (r=0.80, P<0.001). AQHPA and AQSA were only positively associated with peak VO2 (ml min(-1) kg(-1)) (r=0.49; r=0.50, P<0.05, respectively). Past PA level was not related to fitness measurements. Results show that in this population: (1) jumping peak performance was closely related to CPP measured in the laboratory; (2) the cardio-respiratory fitness was related to the actual habitual physical activity level; (3) only age and anthropometric variables explained the actual performances in multivariate analysis.

Validity of a V.O2 max prediction equation of the 2-km walk test in female seniors.

Walking is a useful exercise mode for most adults due to its general ease, acceptability, and safety. Therefore, many field tests based on performance in walking have been developed to predict V.O (2 max). Even if these tests are much easier to perform than laboratory tests, field tests have to be valid. The objective of the paper was to explore the accuracy and bias of a V.O (2 max) prediction equation of the 2-km Walk Test, in an active female senior group (n=18, mean age: 66.1+/-4.4). V.O (2 max) (l . min (-1)) was measured during cycle ergometry by direct gas analysis from a maximal test (step: 30 W, time: 2 min 30). V.O (2 max) related to body mass was then calculated (ml . min (-1) . kg (-1)). Subjects completed also the 2-km Walk Test (UKK Institute). V.O (2 max) (ml . min (-1) . kg (-1)) was then predicted from age, sex, body mass index, heart rate, and walking time measured during the 2-km Walk Test. Predicted V.O (2 max) and measured V.O (2 max) were highly correlated (r=0.63, p<0.01). Predicted V.O (2 max) (20.5+/-6.1 ml . min (-1) . kg (-1)) was not significantly different from measured V.O (2 max) (18.7+/-3.4 ml . min (-1) . kg (-1)). Prediction equation bias with its 95 % limits of agreement was - 1.8+/-4.8 ml . min (-1) . kg (-1) with a coefficient of variation of 24.2 %. In an active female senior population, the 2-km Walk Test offers a fairly accurate V.O (2 max) prediction. The training and learning effects can be neglected because when the test was repeated no significant bias was observed between the two trials.

Alendronate-induced chemical laryngitis.

Alendronate is a bisphosphonate used in treating osteoporosis. Its recognized side-effects include oesophageal irritation and ulceration. The authors describe a case of laryngitis induced by transient contact of this medication with the laryngeal mucosa. Successful management of this case is also detailed.

Carcinoma of the apocrine glands of the anal sac in dogs: 113 cases (1985-1995).

OBJECTIVE: To characterize the signalment, clinical signs, biological behavior, and response to treatment of carcinoma of the apocrine glands of the anal sac in dogs. DESIGN: Retrospective study. ANIMALS: 113 dogs with histologically confirmed carcinoma of the apocrine glands of the anal sac. PROCEDURE: Data on signalment, clinical signs, and staging were reviewed and analyzed along with treatment modality for potential association with survival time. RESULTS: Sex distribution was approximately equal (54% female, 46% male). One hundred four dogs underwent treatment consisting of surgery, radiation therapy, chemotherapy, or multimodal treatment. Median survival for treated dogs was 544 days (range, 0 to 1,873 days). Dogs treated with chemotherapy alone had significantly shorter survival (median, 212 days) than those receiving other treatments (median, 584 days). Dogs not treated with surgery had significantly shorter survival (median, 402 days) than those that underwent surgery as part of their treatment (median, 548 days). Dogs with tumors > or = 10 cm2 had significantly shorter survival (median, 292 days) than dogs with tumors < 10 cm2 (median, 584 days). Hypercalcemia was identified in 27% (n = 29) of dogs, and those dogs had significantly shorter survival (median, 256 days), compared with those that were normocalcemic (median, 584 days). Dogs with pulmonary metastasis had significantly shorter survival (median, 219 days) than dogs without evidence of pulmonary metastasis (median, 548 days). CONCLUSIONS AND CLINICAL RELEVANCE: Unlike most previous reports, this study revealed an approximately equal sex distribution, and results suggest a more favorable prognosis.

Regulatory domain conformational exchange and linker region flexibility in cardiac troponin C bound to cardiac troponin I.

Previously, we utilized (15)N transverse relaxation rates to demonstrate significant mobility in the linker region and conformational exchange in the regulatory domain of Ca(2+)-saturated cardiac troponin C bound to the isolated N-domain of cardiac troponin I (Gaponenko, V., Abusamhadneh, E., Abbott, M. B., Finley, N., Gasmi-Seabrook, G., Solaro, R.J., Rance, M., and Rosevear, P.R. (1999) J. Biol. Chem. 274, 16681-16684). Here we show a large decrease in cardiac troponin C linker flexibility, corresponding to residues 85-93, when bound to intact cardiac troponin I. The addition of 2 m urea to the intact cardiac troponin I-troponin C complex significantly increased linker flexibility. Conformational changes in the regulatory domain of cardiac troponin C were monitored in complexes with troponin I-(1-211), troponin I-(33-211), troponin I-(1-80) and bisphosphorylated troponin I-(1-80). The cardiac specific N terminus, residues 1-32, and the C-domain, residues 81-211, of troponin I are both capable of inducing conformational changes in the troponin C regulatory domain. Phosphorylation of the cardiac specific N terminus reversed its effects on the regulatory domain. These studies provide the first evidence that the cardiac specific N terminus can modulate the function of troponin C by altering the conformational equilibrium of the regulatory domain.

Site-site communication in the EF-hand Ca2+-binding protein calbindin D9k.

The cooperative binding of Ca2+ ions is an essential functional property of the EF-hand family of Ca2+-binding proteins. To understand how these proteins function, it is essential to characterize intermediate binding states in addition to the apo- and holo-proteins. The three-dimensional solution structure and fast time scale internal motional dynamics of the backbone have been determined for the half-saturated state of the N56A mutant of calbindin D9k with Ca2+ bound only in the N-terminal site. The extent of conformational reorganization and a loss of flexibility in the C-terminal EF-hand upon binding of an ion in the N-terminal EF-hand provide clear evidence of the importance of site-site interactions in this family of proteins, and demonstrates the strength of long-range effects in the cooperative EF-hand Ca2+-binding domain.

Characterization of the binding interface between the E-domain of Staphylococcal protein A and an antibody Fv-fragment.

Staphylococcal protein A (SpA) is a cell-surface component of Staphylococcus aureus. In addition to the well-characterized interaction between SpA and the Fc-region of human IgG, an alternative binding interaction between SpA and the Fab-region of immunoglobulin domains encoded by the V(H)3 gene family has been described. To characterize structurally the interface formed by SpA repeats and type-3 V(H)-domains, we have studied the 32-kDa complex formed between an E-domain mutant (EZ4) and the Fv-fragment of the humanized anti-HER2 antibody (Hu4D5-8) using heteronuclear NMR spectroscopy. Protocols were established for efficient incorporation of (15)N, (13)C, and (2)H into EZ4 and the V(H)- and V(L)-domains of the Fv, allowing backbone resonances to be assigned sequentially for EZ4 and the V(H)-domain in both free and complexed states. Broadening of certain V(H)-resonances in the free and bound Fv-fragment suggests microsecond to millisecond time-scale motion in CDR3. Residues experiencing significant chemical shift changes of backbone (1)H(N), (15)N, and (13)CO resonances upon complex formation delineate contiguous surfaces on EZ4 and the V(H)-domain that define the binding interfaces of the two proteins. The interaction surfaces identified by chemical shift mapping are comprised of predominantly hydrophilic residues. This is in contrast to the SpA-Fc interface which is predominantly hydrophobic in nature. Further analysis of the surface properties suggests a probable binding orientation for SpA- and V(H)3-domains.

Two-dimensional relayed-rotating-frame overhauser spectroscopy (1)H NMR experiments for the selective identification of 1,2-glycosidic linkages in polysaccharides.

This communication describes the use of two-dimensional relayed (TOCSY)-ROESY experiments for the rapid and selective identification of alpha/beta1,2-glycosidic linkages in polysaccharides. The method assists in the identification of cross-peaks in crowded regions of ROESY spectra by moving them to less congested areas. In addition, the appearance of the spectra provides information relating the location of the glycosidic linkage within the sequence of the glycan under study. Selection of solely the 1,2- linkages is achieved by appropriately tuning the duration of the TOCSY mixing period. The method is demonstrated both theoretically and experimentally for a variety of test case polysaccharides.

A TROSY CPMG sequence for characterizing chemical exchange in large proteins.

A new NMR spin relaxation experiment is described for measuring chemical exchange time constants from approximately 0.5 ms to 5 ms in 15N-labeled macromolecules. The pulse sequence is based on the Carr-Purcell-Meiboom-Gill technique [Carr and Purcell (1954) Phys. Rev., 94, 630-638; Meiboom and Gill (1958) Rev. Sci. Instrum., 29, 688-691; Loria et al. (1999) J. Am. Chem. Soc., 121, 2331-2332], but implements TROSY selection [Pervushin et al. (1997) Proc. Natl. Acad. Sci. USA, 94, 12366-12371] to permit measurement of exchange linebroadening contributions to the narrower component of the 1H-15N scalar-coupled doublet. This modification extends the size limitation imposed on relaxation measurements due to the fast decay of transverse magnetization in larger macromolecules. The new TROSY-CPMG experiment is demonstrated on a [U-98% 15N] labeled sample of basic pancreatic trypsin inhibitor and a [U-83% 2H, U-98% 15N] labeled sample of triosephosphate isomerase, a 54 kDa homodimeric protein.

Transverse-relaxation-optimized (TROSY) gradient-enhanced triple-resonance NMR spectroscopy.

Two modifications to sensitivity-enhanced gradient-selected TROSY-based triple-resonance NMR experiments are proposed that reduce the overall duration of the pulse sequences and minimize radiation damping effects on water-flipback solvent suppression. The modifications are illustrated for the HNCO-TROSY experiment, but are applicable to all triple-resonance experiments that detect proton magnetization after a reverse polarization transfer step from a (15)N spin. The methods are applied to yeast triosephosphate isomerase, a symmetric dimer with 248 amino acid residues per monomer.

NMR analysis of cardiac troponin C-troponin I complexes: effects of phosphorylation.

Phosphorylation of the cardiac specific amino-terminus of troponin I has been demonstrated to reduce the Ca2+ affinity of the cardiac troponin C regulatory site. Recombinant N-terminal cardiac troponin I proteins, cardiac troponin I(33-80), cardiac troponin I(1-80), cardiac troponin I(1-80)DD and cardiac troponin I(1-80)pp, phosphorylated by protein kinase A, were used to form stable binary complexes with recombinant cardiac troponin C. Cardiac troponin I(1-80)DD, having phosphorylated Ser residues mutated to Asp, provided a stable mimetic of the phosphorylated state. In all complexes, the N-terminal domain of cardiac troponin I primarily makes contact with the C-terminal domain of cardiac troponin C. The nonphosphorylated cardiac specific amino-terminus, cardiac troponin I(1-80), was found to make additional interactions with the N-terminal domain of cardiac troponin C.