Antibodies in proteomics II: screening,high-throughput characterization and downstream applications

There are many ways in which the use of antibodies and antibody selection can be improved and developed for high-throughput characterization. Standard protocols, such as immunoprecipitation, western blotting and immunofluorescence, can be used with antibody fragments generated by display technologies. Together with novel approaches, such as antibody chips and intracellular immunization, these methods will yield useful proteomic data following adaptation of the protocols for increased reliability and robustness. To date, most work has focused on the use of standard, well-characterized commercial antibodies. Such protocols need to be adapted for broader use, for example, with antibody fragments or other binders generated by display technologies, because it is unlikely that traditional approaches will provide the required throughput.     

Myocardial interstitial glucose and lactate before,during, and after cardioplegic heart arrest

The interstitial fluid of the human myocardium was monitored in 13 patients undergoing aortic valve and/or bypass surgery before, during, and after hypothermic potassium cardioplegia. The regulation of glucose and lactate was studied after sampling with microdialysis. The following questions were addressed. 1). Is the rate of transcapillary diffusion the limiting step for myocardial uptake of glucose before or after cardioplegia? 2). Does cold potassium cardioplegia induce a critical deprivation of glucose and/or accumulation of lactate in the myocardium? Before cardioplegia, interstitial glucose was approximately 50% of the plasma level (P < 0.001). Interstitial glucose decreased significantly immediately after induction of cardioplegia and remained low (1.25 +/- 0.25 mM) throughout cardioplegia. It was restored to precardioplegic levels 1 h after release of the aortic clamp. Interstitial glucose then decreased again at 25 and 35 h postoperatively to the levels observed during cardioplegia. Interstitial lactate decreased immediately after induction of cardioplegia but returned to basal level during the clamping period. At 25 and 35 h, interstitial lactate was significantly lower than before and during cardioplegia. Glucose transport over the capillary endothelium is considered rate limiting for its uptake in the working heart but not during cold potassium cardioplegia despite the glucose deprivation following perfusion of glucose-free cardioplegic solution. Lactate accumulated during cardioplegia but never reached exceedingly high interstitial levels. We conclude that microdialysis provides information that may be relevant for myocardial protection during open-heart surgery.     

Antiendotoxin activity of protegrin analog IB-367 alone or in combination with piperacillin in different animal models of septic shock

The therapeutic efficacy of protegrin peptide IB-367 was investigated in three rat models of septic shock: (i) rats injected intraperitoneally with 1mg Escherichia coli 0111:B4 lipopolysaccharide, (ii) rats given an intraperitoneal injection of 2 X 10(10) CFU of E. coli ATCC 25922, and (iii) rats in which intra-abdominal sepsis was induced via cecal ligation and puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1mg/kg of IB-367, 60mg/kg piperacillin and 1mg/kg of IB-367 plus 60mg/kg piperacillin. The peptide demonstrated lower level of antimicrobial activity than piperacillin, nevertheless it exhibited the dual properties of antimicrobial and antiendotoxin agent. Finally IB-367 and piperacillin association showed to be the most effective therapeutic approach.     

A reversible abnormal form of myelin: an X-ray scattering study of human sural and rat sciatic nerves

A sural nerve dissected from a recently dead patient displayed an unusual X-ray diffraction pattern, suggesting that in situ and at the time of the patient's death the myelin sheaths were in a swollen state. Diffraction patterns of the swollen type were also recorded from: (1) a sural nerve from the corpse of a neurologically healthy person after soaking the nerve with Ringer solution at pH 5.5; (2) sciatic nerves dissected from rat cadavers at increasing time after death. In all the cases the swollen patterns reversed to the native type upon superfusion with Ringer solution at pH 7.3. The postmortem effect is to decrease the pH of the fluids surrounding the nerves in the cadavers. Our experiments show that the early postmortem processes have the effect of acidifying PNS nerves and that as a consequence of acidification the myelin sheaths swell.     

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (K_m) and V_max for α-naphthyl acetate were 1.54 mM and 360 μmol min⁻¹ mg of protein⁻¹, respectively.     

Ultrastructure of the female gonad of two temnocephalids (Platyhelminthes,Rhabdocoela)

The female gonad of Temnocephala dendyi and T. minor consists of a single germarium and two rows of vitellaria. It is enveloped by an extracellular lamina and accessory cells. Accessory cells are only peripherally located in the germarium while their cytoplasmic projections also fill the spaces between vitellocytes in the vitellarium. The main feature of oocyte maturation is the appearance of chromatoid bodies and the development of rough endoplasmic reticulum (R.E.R.) and Golgi complexes which appear to be correlated with the production of double-structured egg granules. The egg granules, which are localized in the cortical cytoplasm of mature oocytes, contain glycoproteins, are devoid of polyphenols and are similar in structure and composition to the cortical granules observed in some Digenea and Monogenea. Vitellocytes are typical secretory cells with well-developed R.E.R. and Golgi complexes which are involved in the production of shell globules and yolk. The multigranular pattern and the polyphenolic composition of the shell globules of the temnocephalids investigated are similar to those observed in other rhabdocoels, and in some Prolecithophora and Neodermata. This feature may represent a synapomorphy shared by these taxa. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and characterization of an acetyl xylan esterase from Bacillus pumilus

Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55 degrees C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis contant (Km) and Vmax for alpha-naphthyl acetate were 1.54 mM and 360 micromol min-1 mg of protein-1, respectively.