Kinematic characteristics of canine hindlimb movement during sit-to-stand and stand-to-sit motions.

Sit-to-stand and stand-to-sit motions are basic motions for daily animal life, and these motions are used as therapeutic exercises for dogs with functional impairments. The sit-to-stand motion is divided into several phases for kinesiological assessment in human rehabilitation and physical therapy. However, these motions in dogs have not been characterized in detail. We examined canine hindlimb kinematic characteristics during sit-to-stand/stand-to-sit motions and compared the characteristics with those during walking. In addition, we tried to classify phases of the movements based on kinematic characteristics of the transition of the range of motion of the hindlimb. We used a three-dimensional motion analysis system to evaluate the motions of eight clinically healthy beagles. During the sit-to-stand motion, the total range of motion (ROM) in the hip joint flexion/extension was half of that of during walking, but the total ROM of the hindlimb external/internal rotation relative to the pelvis and flexion/extension of the stifle and the tarsal joints were significantly larger than those of walking, suggesting that sit-to-stand exercise causes movements of hindlimb joints without marked changes in hip joint flexion/extension movement. Both sit-to-stand and stand-to-sit motions could not be divided into multiple phases only by the transition of the range of motion of the hindlimb.

Functional assessment of the gluteus medius, cranial part of the biceps femoris, and vastus lateralis in Beagle dogs based on a novel gait phase classification.

In humans, walking analysis based on the gait phase classification has been used for interpretation of functional roles of different movements occurring at individual joints, and it is useful for establishing a rehabilitation plan. However, there have been few reports on canine gait phase classification, and this is one of the reasons for preventing progress in canine rehabilitation. In this study, we determined phases of the canine gait cycle (GC) on the basis of the phase classification for human gait. The canine GC was able to be divided into initial contact (IC) and the following 5 phases: loading response (LR), middle stance (MidSt), pre-swing (PSw), early swing (ESw), and late swing (LSw). Next, the hind limb joint angles of the hip, stifle and tarsal joints and results of surface electromyography of the gluteus medius (GM), cranial part of the biceps femoris (CBF) and vastus lateralis (VL) muscles in relation to the gait phases were analyzed. The activities of three muscles showed similar changes during walking. The muscle activities were high in the LR phase and then declined and reached a minimum in the PSw phase, but they increased and reached a peak in the LSw phase, which was followed by the LR phase. In conclusion, the multiphasic canine GC was developed by modification of the human model, and the GC phase-related changes in the muscle activity and joint angles suggested the functions of GM, CBF and VL muscles in walking.

A case of primary (AL) amyloidosis with predominantly vascular amyloid deposition in the kidney.

We report a 70-year-old man with primary (AL) amyloidosis with predominantly vascular deposition of amyloid diagnosed by renal biopsy, who was successfully treated using two chemotherapy regimens. There was rapid elevation of the serum creatinine level without remarkable proteinuria or hematuria. Renal histological examination showed some thickened arterial walls with amyloid fibril accumulation, and only a small amount of amyloid deposition in the glomeruli. Immunohistochemical examination was positive for anti-kappa staining. Serum immunoelectrophoresis and immunofixation testing did not show monoclonal proteins, and urine immunoelectrophoresis did not show Bence-Jones proteins. Serum free light chain (FLC) analysis showed that the serum FLC level and FLC kappa/lambda ratio were abnormally high for his renal function. He received two courses of VAD (vincristine, doxorubicin, and dexamethasone), followed by BD (bortezomib and dexamethasone), resulting in a hematologic partial response. Renal amyloidosis with vascular-limited amyloid deposition has few urinary findings. Early diagnosis of this condition is challenging, because kidney biopsies are not usually performed in patients without significant urinary findings. We suggest several currently available methods of achieving earlier detection of this condition.

Stent failure in the management of malignant extrinsic ureteral obstruction: risk factors.

There is still controversy surrounding the indications for performing either a retrograde ureteral stent or percutaneous nephrostomy to manage malignant extrinsic ureteral obstruction (MEUO). We retrospectively analyzed 53 patients who underwent a decompression of MEUO using retrograde ureteral stent. Ureteral stent failure occurred in 18 of 53 patients (34%). Multivariate analysis showed that gastrointestinal cancer as the primary disease, poor preoperative performance status and severe preoperative hydronephrosis were independent predictors of stent failure. Based on the present data, we propose an algorithm for the management of MEUO.

Wide Range of Biotin (Vitamin H) Content in Foodstuffs and Powdered Milks as Assessed by High-performance Affinity Chromatography.

The biotin (vitamin H) contents of various foodstuffs were determined by using a newly developed high-performance affinity chromatography with a trypsin-treated avidin-bound column. Biotin was derivatized with 9-anthryldiazomethane (ADAM) to fluorescent biotin-ADAM ester. A wide range of biotin contents were found in various foodstuffs depending upon the species (strain), season, organ (of plants and animals), geography, freshness, preparation method and storage method. Among the foodstuffs and fermented foods tested, it was found that wide distributions of biotin content were observed in powdered milk, natto, sake (rice wine), beer, edible oil and sea weed. Since powdered milk is important for child health and development, 14 kinds of powdered and special milks for use in children's diseases were intensively measured. We found that several special milk powders for children with allergies contained low levels of free biotin. Use of these powdered milks caused skin diseases and alopecia in some patients possessing thermolabile serum biotinidase, and administration of free biotin improved their symptoms dramatically. Therefore, it is essential to estimate the total and free biotin contents on each foodstuff in order to improve effective biotin intake and support better health and quality of life for people.

Determination of biotin (vitamin H) by the high-performance affinity chromatography with a trypsin-treated avidin-bound column.

A method for measuring biotin by affinity-chromatography was developed using a trypsin-treated avidin silica gel column. Elution was by a linear gradient of propan-2-ol in an acidic phosphate buffer system containing 0.7 M NaCl (pH 2.4). Biotin was derivatized with 9-anthryldiazomethane (ADAM) to the fluorescent biotin-ADAM ester and a linear calibration line was obtained from 0 to 1.39 pmol with a detection limit of 69.5 fmol. Biotin was measured after hydrolysis in 2.25 M sulphuric acid for 1h at 120 degrees C and the recovery for biocytin was 65.7+/-2.53%, and hence a correction factor of 1.52 was used for the total biotin analysis. The recovery of added biotin from the serum was more than 98% using this correction factor of 1.52. Biotin was also measured in nutritional supplemental foods and foodstuffs, and we found that chicken egg yolk, "natto", rice bran, royal jelly, and dried yeast contained highest levels of biotin. Biotin was also found in ferments by Bacillus natto, yeast, and some acetic acid bacterium. Storage foods such as beans, nuts and eggs also contained abundant biotin. Biotin was also determined and replacement monitored in the serum of suspected biotinidase deficiency patients. This affinity-chromatographic method for biotin determination was shown to be a robust and reliable and is well suited for biochemical and nutritional research.

Determination of specific activities and kinetic constants of biotinidase and lipoamidase in LEW rat and Lactobacillus casei (Shirota).

Enzyme kinetic parameters, such as K(m), V(max) (or V), k(cat)/K(m), and K(i) (by biotin or lipoic acid) for biotinidase and lipoamidase were determined in Lewis (LEW) rat and Lactobacillus casei (Shirota) using fluorimetric high-performance liquid chromatography (HPLC). It was found that the final protein concentration below 0.1mg/ml is sufficient to obtain linear hydrolytic reaction and to determine the Michaelis-Menten type kinetic parameters (K(m), V, K(i)). We applied this HPLC enzyme assay method onto the rat and some bacteria. The highest specific activities (Vs) for biotinidase were found in Lactobacillus casei (Shirota) and rat kidney. It was also found that the largest K(i) by product for biotinidase and lipoamidase were present in the Lactobacillus casei (Shirota). There has been found specie (between rat and mouse) differences and tissue (organ) differences, together with tissue region differences and sex differences in some tissues. Summary of the distributions of both enzymes in LEW rat was also presented. Therefore, this HPLC determination method for the enzyme kinetic parameters in tissues is expected to be an indispensable tool for the investigation of the various diseases in humans.

Predictive value of N-acetylglucosaminyltransferase-V for superficial bladder cancer recurrence.

PURPOSE: GnT-V is an enzyme that catalyzes beta1-6 branching of N-acetylglucosamine on asparagine (N)-linked oligosaccharides of cell proteins. GnT-V expression has been closely related to malignant potentials in colon cancer, brain cancer and hepatocellular carcinoma. We determined whether GnT-V expression is predictive of superficial bladder cancer recurrence. MATERIALS AND METHODS: The cohort comprised 60 consecutive patients with first time superficial bladder cancer treated with transurethral resection. None of the patients received prophylactic intravesical therapy until recurrence. Paraffin embedded tumor specimens were immunohistochemically examined by the avidin-biotin peroxidase method using monoclonal antibody against GnT-V. Kaplan-Meier survival curves were generated to determine disease-free survival. Univariate and multivariate analyses were done to compare GnT-V expression to other clinical and pathological variables. RESULTS: GnT-V expression correlated inversely with tumor grade and stage. The positive incidence of GnT-V in G1 to G3 tumors was 7 of 9 (78%), 21 of 43 (49%) and 3 of 8 (38%), respectively. GnT-V was positive in 26 of 44 cases of pTa (60%) and in 5 of 16 of pT1 (31%) disease. The 31 patients with positive GnT-V expression had significantly higher disease-free survival than the 29 with negative GnT-V expression (log rank test p = 0.0034). Multivariate analysis revealed that patient age, pT, grade and negative GnT-V expression were independent predictors of recurrence (p = 0.015, 0.001, 0.019 and 0.011, respectively). CONCLUSIONS: Immunohistochemical detection of GnT-V is an independent predictor of superficial bladder cancer recurrence.

Carbohydrate structure and differential binding of prostate specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy.

Serum prostate-specific antigen (PSA) assay is widely used for detection of prostate cancer. Because PSA is also synthesized from normal prostate, false positive diagnosis cannot be avoided by the conventional serum PSA test. To apply the cancer-associated carbohydrate alteration to the improvement of PSA assay, we first elucidated the structures of PSA purified from human seminal fluid. The predominant core structure of N-glycans of seminal fluid PSA was a complex type biantennary oligosaccharide and was consistent with the structure reported previously. However, we found the sialic acid alpha2-3 galactose linkage as an additional terminal carbohydrate structure on seminal fluid PSA. We then analyzed the carbohydrate moiety of serum PSA from the patients with prostate cancer and benign prostate hypertrophy using lectin affinity chromatography. Lectin binding was assessed by lectin affinity column chromatography followed by determining the amount of total and free PSA. Concanavalin A, Lens culinaris, Aleuria aurantia, Sambucus nigra, and Maackia amurensis lectins were tested for their binding to the carbohydrates on PSA. Among the lectins examined, the M. amurensis agglutinin-bound fraction of free serum PSA is increased in prostate cancer patients compared to benign prostate hypertrophy patients. The binding of PSA to M. amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, was also confirmed by surface plasmon resonance analysis. These results suggest that the differential binding of free serum PSA to M. amurensis agglutinin lectin between prostate cancer and benign prostate hypertrophy could be a potential measure for diagnosis of prostate cancer.

Size-exclusion chromatography of biological samples which contain extremely alkaline proteins.

An improved size-exclusion chromatography (SEC) was developed to isolate extremely basic (alkaline) proteins, such as trypsin (pI=10.5), lysozyme (pI=11), and histone (pI=10.8). Develosil 300 Diol-5 (300 x 8 mm I.D., 30 nm average pore diameter) column was used with an eluent of 0.1 M sodium phosphate, 1.5 M sodium chloride, glycerol (40%, v/v), 2-propanol (10%, v/v), and Brij-58 (1%, v/v). Under these conditions, the final apparent pH becomes to 4.0, and pH adjustment is not necessary. Column temperature and flow rate were 15 degrees C and 0.2 ml/min, respectively. This elution system is stable and reliable, and applications onto human pancreatic juice, human bile, and tissue homogenates were successfully achieved. Since this system is convenient for protein analysis, it is expected to be generally applicable to clinical and biochemical research for identifying protein components in combination with microsequencing.