A Study on Evaluation of Alkaline and Acid Phosphatase Isozymes During Different Developmental Stages of New Breeding Lines and Races of Bombyx mori L

It is interesting to note that different silkworm races reared in laboratory offer an important testing ground for the application of biochemical methods to taxonomic problems. Moreover, there is scarcity of knowledge on enzyme studies in new breeding lines and races of silkworm specially Bombyx mori L. Therefore, we designed the present study with the main goal to evaluate the activities of alkaline and acid phosphatases quantitatively during different developmental stages of new breeding lines and races viz. Kalimpong-A (KA), B18, Pure Mysore (PM), evolved R1 and R2 of Bombyx mori L. Quantitative estimations of in alkaline and acid phosphatases were expressed in terms of enzyme activity. Alkaline and acid phosphatase activities during the different developmental stages of KA, NB18, PM, evolved R1 & R2 races were determined using Sodium-1 naphthahyl phosphate as a substrate following the dye-coupling method. The assay mixture included 2 ml of substrate and 0.2 ml of enzyme extract and incubation was made for 30 minutes at 25°c. The reaction was stopped by adding 2 ml of post coupling solution (5 parts of 4% sodium dodecyl sulphate and 2 parts of 0.2% Fast red TR salt) and colorimetric determination were made at 540 nm. Results illustrated that the activity of phosphatases was found to be different and high activity was found in the larval stage, which is feeding stage followed by pupae.

Osseointegration Implant Post Coupling With External Prosthetic Limb – Continuation of Previous Case Reports “Stage III”

An ongoing update of the progress case report for the first patient treated with the Longitude™ osseointegration prosthesis implanted in an amputated residual femur is presented. The patient was given an intensive physical therapy program of strengthening and conditioning in anticipation of coupling to the external prosthesis. A custom prosthesis was fabricated based on the Plie’ 2.0 microprocessor knee system. The patient was then successfully trained on use and care of the prosthesis for ambulation without any complications.Keywords: Amputation, OsseointegrationAcknowledgement: Design concept by Concept Design & Development, LLC (CDD,LLC); Development and Manufacturing by Signature Orthopaedics, LTD; Centennial Hills Hospital Medical Center, Las Vegas, NV; and Institutional Review Board (IRB) by Joint Implant Surgery & Research Foundation.

Localization and cytophotometric analysis of cathepsin B activity in unfixed and undecalcified cryostat sections of whole rat knee joints.

Cathepsin B activity is demonstrated histochemically with a post-coupling method using Z-Arg-Arg-4-methoxy-2-naphthylamide as substrate and Fast Blue BB as coupling reagent in unfixed and undecalcified cryostat sections of whole rat knee joints. Sections were attached to transparent tape to keep the integrity of the tissue intact, such attachment being essential for precise precipitation of the final reaction product at sites of enzyme activity. Also essential was inclusion of polyvinyl alcohol in the enzyme incubation medium. High cathepsin B activity was found in osteoclasts, chondrocytes, fibroblasts, synovial cells, and bone marrow cells in knee joints after induction of arthritis. The final reaction product was precipitated as fine cytoplasmic granules probably corresponding to lysosomes. The reaction was specific because addition to the incubation medium of selective inhibitors of cathepsin B-like activity completely blocked the activity. The amount of final reaction product in synovium and in bone marrow cells was analyzed cytophotometrically. Specific formation of final reaction product was linear with incubation time up to 60 min at 37 degrees C and with section thickness up to 12 microns. Variation of the substrate concentration in the incubation medium revealed a KM value of 1.86 +/- 0.36 mM in synovial cells and 2.48 +/- 0.51 mM in bone marrow cells and Vmax values (expressed as mean integrated absorbance) of 1.18 +/- 0.10 in synovial cells and 1.02 +/- 0.11 in bone marrow cells. Both KM and Vmax values were significantly different in synovial cells and bone marrow cells (p less than 0.01) which could be owing to the presence of different isoenzymes in these tissues. We conclude that the described post-coupling method is sufficient to yield precise localization and that the method is valid for quantitative purposes.

Quantitative histochemical study of acid phosphatase activity in rat liver using a semipermeable membrane technique.

Acid phosphatase activity has been demonstrated in rat liver with the semipermeable membrane technique using naphthol AS-BI phosphate as substrate and hexazotized pararosaniline (HPRA) as simultaneous coupling agent. With this method the final reaction product (FRP) appeared in rat liver as intensely colored red granules in liver parenchymal cells and in Küpffer cells. The absorbance spectrum of the FRP peaks between 510 and 550 nm. A nonspecific reaction product, as has been found in skeletal muscle, did not occur in rat liver. A substrate concentration of 5 mM and a HPRA concentration of 10 mM result in optimum localization and activity. We concluded from the results with different enzyme inhibitors that lysosomal acid phosphatase was demonstrated. The mean absorbance of the FRP increased linearly with incubation time (15-60 min). Furthermore, we found a linear increase of the FRP with increasing section thickness (4-10 micron). When the simultaneous coupling method was replaced by a post-coupling technique, the colored reaction product was diffusely located throughout the cytoplasm. In conclusion, the simultaneous coupling technique in combination with the semipermeable membrane method is a valuable tool for detecting and quantifying lysosomal acid phosphatase activity in rat liver. We demonstrated that acid phosphatase activity is 1.2 times higher periportally than pericentrally in rat liver, and that 24 hr fasting before the experiments did not change the acid phosphatase activity.

Time-Resolved Infrared Spectroelectrochemistry

The generation of time-resolved infrared spectra of species formed at the electrode-solution interface is reported. The methodology involves electronic pre- or post-coupling of the electrochemical experiment to the interferometer retardation timing. By the sorting of the data points in each of the resulting sets of interferograms, which are accumulated over finite intervals of time after initiation of the electrochemical experiment, spectra representing very short integrated time periods are obtained. These time discretized spectra represent the time evolution of the vibrational structure at the electrode surface.

Ultrastructural localization of acid phosphatase activity in root tips by the p-(acetoxymercuric) aniline diazotate reagent and a comparison with a Gomori procedure

The localization of acid phosphatase was studied in root tip cells of pea and mung bean by use of a heavy metal azo-dye technique. Diazotized p-(acetoxymercuric) aniline in a post-coupling procedure, using naphthol AS-BI phosphate as substrate, yielded a fine particulate reaction product within vacuoles, intercellular spaces, multivesicular bodies and at various sites throughout the cytoplasm of pea root cells and differentiating mung bean protoxylem cells. An ultrastructural comparison with a modified Gomori lead-salt precipitation method revealed differences in the subcellular location of beta-glycerophosphatase and naphthol AS-BI phosphatase. The distribution of acid phosphatases within plant meristematic cells is discussed.