End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that abut 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.     

Analysis of linkage between beta-lactoglobulin and J blood group system loci in cattle

Linkage between loci controlling variants of beta-lactoglobulin and blood groups of the J system in cattle was studied by means of stochastic genetic methods reported earlier. The studies were conducted on a herd of Black Pied cattle improved with Holstein sires; population genetic data were analyzed. A plot for lod score was constructed, and point (r - 0.28) and interval estimations of the coefficient of recombination were obtained. The results are in good agreement with earlier reported data on other subjects.     

Quantitative structure/activity relationship for the rate of conversion of C4-substituted catechols by catechol-1,2-dioxygenase from Pseudomonas putida (arvilla) C1

Dot-immunoblotting assay (DIA) using five monoclonal antibodies (MAbs) to infectious bronchitis virus (IBV) was used to detect and classify the viruses propagated in embryonated chicken eggs. Using a group-specific MAb 3F5, 10 reference strains and 12 Korean isolates of IBV were successfully detected by DIA, and the lowest virus titer of IBV detected by DIA was approximately less than 10(3.8) mean embryo infective dose/ml. For evaluating the diagnostic efficiency, DIA was compared with the conventional infectious bronchitis (IB) diagnostic method. IBV antigens in allantoic fluid from embryonated eggs inoculated with IB-suspected field samples were specifically detected by DIA within only one or two egg passages, whereas the conventional embryonated egg inoculation method required four to seven egg passages for confirming IBV infection. These results indicated that DIA could significantly reduce time and cost for IB diagnosis. For examining the possibility of classifying IBV by DIA, four strain-specific MAbs, 3A4, 2A3, 6F7, and 2C6, were used. According to the MAb reacting patterns to the IBV antigens, the 10 IBV reference strains were classified into six groups; seven strains belonged to three different groups, and the other three strains each belonged to an individual group. In the case of 12 Korean isolates of IBV, they were classified in six groups. Among the six groups, the MAb reacting patterns of three groups matched those of the IBV reference strains, but the others did not. These data suggest that at least three variant serotypes of IBV exist in Korea.     

A rare and little known variant of hypospadias

Hypospadia is a congenital defect presenting as different variants of dysplasia of the distal urethra with ectopy of the urethral external orifice and distortion of the penis. A variant of hypospadia not yet described in Russian medical literature was observed in a newborn boy. He had normal penis and scrotum, but on the under-side of the penis there was a site of thin skin protruding as a resonance bag in urination. At the site of the thin skin the back urethral wall was absent with a cavity covered by the thin skin. In urination urine filled the cavity blowing up the skin above it to the form of the resonance bag. This variant of hypospadia was designated as segmental urethral hypoplasia.     

Cocaine-induced activation of striatal neurons during focused stereotypy in rats

As psychomotor stimulants, both amphetamine and cocaine elicit episodes of repetitive motor activation (focused stereotypy) known to involve the mesostriatal dopamine system. During amphetamine-induced focused stereotypy, motor-related neurons in the striatum respond with either an excitation or inhibition, depending on dose and behavioral pattern, whereas nonmotor-related units are inhibited. To assess striatal activity during the focused stereotypy induced by cocaine, both types of striatal units were recorded in ambulant rats. Either 20 or 40 mg/kg cocaine caused highly focused sniffing and head bobbing, which occurred in conjunction with activation of both motor- and nonmotor-related neurons. The activation of motor-related units was evident even when firing rate was compared during periods of matched pre- and post-drug behavior, arguing against movement as the sole basis for the drug-induced neuronal excitation. Subsequent administration of haloperidol (1.0 mg/kg) reversed but did not completely block the neuronal activation, while the behavioral response shifted away from focused stereotypy toward an increase in ambulation. Thus, the level of activation of both motor- and nonmotor-related striatal neurons may play a critical role in the behavioral response pattern induced by cocaine.