Electrophoretic and immunocytochemical analysis of Hsp72 and Hsp73 expression in heat-stressed mouse testis and epididymis.

OBJECTIVE: One of the most profound events in stressed cells is the synthesis of a highly conserved family of proteins, the 'heat shock proteins' (Hsp). The Hsp70 family is the most diverse, and includes constitutive as well as stress-inducible proteins with overlapping or unique functions in different cell compartments. Elucidation of Hsp70 expression during different stages of spermatogenesis and maturation of germ cells is of particular interest due to their high sensitivity to heat treatment. STUDY DESIGN: Expression of the main isoforms of the Hsp70 family (constitutive Hsp73 and stress-inducible Hsp72) was determined in normal and heat-stressed mouse testes and epididymis from sexually mature (60-day-old) mice during spermatogenesis and maturation of germ cells. Immunocytochemical analysis and one- and two-dimensional gel electrophoresis were used to separate mouse testicular and epididymal proteins from saline extracts, followed by Western blotting. RESULTS: Using a polyclonal anti-Hsp70 antibody that recognizes both isoforms, inducible Hsp72 expression, was demonstrated immunocytochemically only in heat-stressed tissues, while a high level of constitutive Hsp73 isoform expression was found in both normal and heat-stressed mouse male reproductive tissues. Morphological studies have shown that round and elongated spermatids in the testes, as well as all segments of the epididymis, are most sensitive to heat stress. In the epididymis, the reaction was localized in different cell compartments. CONCLUSION: In heat stress conditions, Hsp73 is mobilized to prevent apoptosis in the testes and epididymis, and assists Hsp72 in the repair of stress-altered protein conformations.

Morphological characteristics of the canine and feline stomach mucosa.

The stomach mucosa structure in animals belonging to Order Carnivora indicates some specific characteristics in comparison with the other mammals. Between the bases of the mucosal glands and the lamina muscularis mucosae there is an additional plate which most of the morphologists have defined as lamina subglandularis. In currently used Nomina histologica this layer is indicated as stratum compactum in carnivorous stomach mucosa. The investigation aims were to study and compare canine and feline stomach tunica mucosa characteristics as well as to measure the thickness of stratum compactum and to specify some of the certain collagen types and fibronectin compounds. Conventional and differential histological and ultrastructural methods and immuno-histochemical approaches for investigation of the canine and feline stomach samples were used. The specific organization of the carnivorous stomach wall arrangement was established. In the structure of the canine stomach mucosa, no evidence of stratum compactum was observed. The presence of stratum compactum in feline stomach mucosa was ascertained and measured. Using an immunohistochemical method very high expression of collagen type IV and fibronectin, moderate positive reaction of collagen type III, and a comparatively weakest expression of collagen types I and V in the structure of stratum compactum from cat stomach mucosa was shown. The obtained results clarify the characteristics of the stomach mucosa morphology and could be used as a basis for distinguishing the stomach wall structure of the animal species belonging to Canidae and Felidae families although they are both carnivores.

Expression of alpha5beta1 integrin and fibronectin during early pregnancy in pigs.

The implantation in pig is superficial and non-invasive, involving phases of apposition, adhesion and attachment of conceptuses to endometrial surface epithelium. The role of integrins and ECM proteins is suggested. In the study, the expression of beta5beta1 integrin and FN on conceptus trophectoderm and endometrium during implantation and early pregnancy was investigated. The immunohistochemical localization of alpha5beta1 integrin and FN was performed on formalin-fixed, paraffin-embedded tissue sections using the ABC method. The results indicate that both conceptus and uterus expressed alpha5beta1 integrin and FN during early porcine pregnancy. The most intensive staining for alpha5beta1 integrin and FN was found in conceptus trophectoderm and endometrial surface epithelium in all investigated periods. During placentation the immunohistochemical staining for both alpha5beta1 integrin and FN was increased in trophectoderm and all endometrial structures. Since placenta in pigs is non-invasive, it can be suggested that both alpha5beta1 integrin and FN participate in molecular events leading to successful implantation and placentation in species with true epitheliochorial placenta.

Microprocessor controlled movement of solid colonic content using sequential neural electrical stimulation.

BACKGROUND AND AIMS: Invoked peristaltic contractions and movement of solid content have not been attempted in normal canine colon. The purpose of this study was to determine if movement of solid content through the colon could be produced by microprocessor controlled sequential stimulation. METHODS: The study was performed on six anaesthetised dogs. At laparotomy, a 15 cm segment of descending colon was selected, the proximal end closed with a purse string suture, and the distal end opened into a collecting container. Four sets of subserosal stimulating electrodes were implanted at 3 cm intervals. The segment of bowel was filled with a mixture of dog food and 50 plastic pellets before each of 2-5 random sessions of non-stimulated or stimulated emptying. Propagated contractions were generated using microprocessor controlled bipolar trains of 50 Hz rectangular voltage having 20 V (peak to peak) amplitude, 18 second stimulus duration, and a nine second phase lag between stimulation trains in sequential electrode sets. RESULTS: Electrical stimulation using the above mentioned parameters resulted in powerful phasic contractions that closed the lumen. By phase locking the stimulation voltage between adjacent sets of electrodes, propagated contractions could be produced in an aboral or orad direction. The number of evacuated pellets during the stimulation sessions was significantly higher than during the non-stimulated sessions (p<0.01). CONCLUSIONS: Microprocessor controlled electrical stimulation accelerated movement of colonic content suggesting the possibility of future implantable colonic stimulators.

Three-dimensional static parametric modelling of phasic colonic contractions for the purpose of microprocessor-controlled functional stimulation.

The study aimed at creating an integrated electromechanical model of invoked phasic contractions in canine colon during direct high frequency voltage stimulation. The model utilized data obtained from two large anaesthetized dogs that underwent laparotomy and serosal implantation of two circumferential electrode pairs into a distal segment of the left colon. The strength distribution of the stimulating electric field was analysed over a cylindrical mesh-surface grid modelling the interrogated colonic segment. Recordings of the stimulating current were utilized to model smooth muscle depolarization using linearized macroscopic tissue conductivity. The invoked contractile stress was related to the stimulating electric field strength using an exponential sigmoid function. Artificially produced occlusion of the lumen was derived for a pair of 5mm electrodes positioned on a cylindrical mesh-surface of 2 cm diameter and 15 cm length. The model simulated contractions invoked by stimuli of different amplitude (up to 12 V) with 98.6% accuracy of approximation. Macroscopic tissue conductivity was modelled as a combination of two first-order exponential terms involving a 3ms time constant. Real-time simulation of the current drawn by the smooth muscle during 10 V/50Hz bipolar voltage stimulation was performed. The integrated electromechanical model facilitates the quantification of microprocessor-controlled phasic colonic contractions.

Application of an object-oriented programming paradigm in three-dimensional computer modeling of mechanically active gastrointestinal tissues.

The aim of this study was to develop a novel three-dimensional (3-D) object-oriented modeling approach incorporating knowledge of the anatomy, electrophysiology, and mechanics of externally stimulated excitable gastrointestinal (GI) tissues and emphasizing the "stimulus-response" principle of extracting the modeling parameters. The modeling method used clusters of class hierarchies representing GI tissues from three perspectives: 1) anatomical; 2) electrophysiological; and 3) mechanical. We elaborated on the first four phases of the object-oriented system development life-cycle: 1) analysis; 2) design; 3) implementation; and 4) testing. Generalized cylinders were used for the implementation of 3-D tissue objects modeling the cecum, the descending colon, and the colonic circular smooth muscle tissue. The model was tested using external neural electrical tissue excitation of the descending colon with virtual implanted electrodes and the stimulating current density distributions over the modeled surfaces were calculated. Finally, the tissue deformations invoked by electrical stimulation were estimated and represented by a mesh-surface visualization technique.

Effects of the chromatographic fractions of the pig placental trophoblast on graft-versus-host reaction.

The trophoblast has a significant role in regulation of immune reactions at the materno-fetal interface by producing biologically active substances. In our previous studies five fractions with immunomodulatory activities were isolated by gel chromatography from trophoblast of pig placentas. To confirm the immunomodulatory effect of these trophoblast fractions on allogeneic in vivo systems and to obtain more evidence for the relevance of their activity on the maternofetal interface, their effect was studied on graft-versus-host reaction (GVHR). To assess the GVHR, the primary and secondary popliteal lymph nodes assay was used in mice. In the primary GVHR, 100 microg protein of Fraction 2-5, mixed with 5 x 10(6) allogeneic spleen cells (C57BL/6), were injected into one of the foot pads of recipient (BALB/c) mice. The secondary GVHR was induced in F1 (BALB/c x C57BL/6) mice by injection of spleen cells of BALB/c mice intraperitoneally preimmunized with allogeneic cells. The GVHR was measured by the weight of lymph nodes and by the lymphocyte proliferation. Flow cytometric analyses of the cells in the nodes with GVHR and under the influence of Fraction 4 or 5 were performed using monoclonal antibodies. In the primary GVHR, Fraction 4 or 5, injected simultaneously with allogeneic spleen cells, significantly suppressed the lymph nodes reactivity. Fractions 4 and 5 inhibited the ability of the spleen cells of mice intraperitoneally preimmunized with allogeneic cells to induce secondary GVHR in F1 mice. The Fraction 2 and 3 had no effect on GVHR. The results revealed that a group of proteins with Mr 37-7 kDa, isolated from trophoblast of pig placenta, strongly suppressed popliteal lymph node reactivity in the primary and secondary GVHR. The data provide convincing evidence for these fractions in vivo activity, for their effect across the species barrier and suggest the relevance of the same reactions on the materno-fetal interface.

Misinterpretation of human electrogastrograms related to inappropriate data conditioning and acquisition using digital computers.

Despite the fact that digital techniques for data acquisition and processing were widely used in electrogastrographic (EGG) research during the last decade, inappropriate signal conditioning and digitization are still potential pitfalls threatening the reliability of the experiments. The aim of this paper was to review: (1) the importance of the antialiasing low-pass filtering for reducing recording artifacts and interferences, (2) the advantages brought by the proper choice of filter cutoff frequency and the slope for the decrement of the minimal required sampling frequency, (3) the impact of incorrectly selected sampling frequency on data interpretations, with particular attention to the percent distribution ranges, and (4) the "leakage effect" related to the finite number of samples processed simultaneously in frequency domain representation of the recordings. A model of electrogastrographic (EGG) recording was mixed with a model of electrocardiographic (ECG) artifact. The resulting finite-duration signal was low-pass filtered and then digitized with a sampling frequency of 1 Hz. The cutoff frequency of the first-order low-pass filter was altered from 0.5 to 0.1 Hz. Amplitude frequency spectra of the digitized recordings were investigated. An example with a real human electrogastrogram in which an ECG artifact was present confirmed the simulation results. When a first-order anti-aliasing filter is utilized at least a fivefold difference between the filter cutoff frequency and the sampling frequency is recommended for compliance with the Nyquist theorem of digitization. Leakage effects associated with the finite-time duration of the recordings and the use of the discrete Fourier transform should be considered when frequency domain analysis is performed. Misinterpretation of the "bradygastric" and "tachygastric" ranges in the percent distribution of EGG frequency components is possible if inappropriate signal conditioning and digitization are employed.