Biochemical and Immunological Characterization of Collagenase in Tissues of Metamorphosing Bullfrog Tadpoles

Tissue inhibitor of metalloproteinases (TIMP, a specific inhibitor of collagenase) was found to inhibit thyroid hormone-induced tail regression, suggesting the important role of collagenase in this process. Collagenase was purified from culture media of back skin of tadpole of bullfrog, Rana catesbeiana . Anti-tadpole collagenase polyclonal antisera were obtained against the purified enzyme. The antibody inhibited the activity of tadpole collagenase. The antisera reacted to tissues of adult bullfrogs, tadpoles of african clawed frog, Xenopus laevis , and adult newts, Cynopus pyrrhogaster , and also reacted to human fibroblast collagenase. Immunoblot analyses suggested that tadpole collagenase lacks the procollagenase which is generally found in mammalian collagenases. Intense immunological stains were observed for the tissues of thyroid hormone-treated tadpoles as compared to those of untreated animals. Thyroid hormone increased amounts of collagenase not only in epidermal layer but also in mesenchymal tissues including fibroblastic cells.     

Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4-dihy-droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage-dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O₂ in N₂) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.     

Evidence for resegmentation in the formation of the vertebral column using the novel approach of retroviral-mediated gene transfer

We have examined the somitic cell contribution to the vertebral column of the chick by genetic labeling of sclerotomal cells in early development. Single somites of embryonic Day 2 embryos were filled with retroviral particles containing the lacZ transducing vector BAG. After a further 14 or 17 days of incubation the embryos were fixed and the vertebral column was sectioned and stained histochemically for the lacZ gene product beta-galactosidase. Cells staining for the enzyme were found exclusively on the injected side of two vertebral segments; the staining was largely restricted, however, to the caudal half of the more rostral segment and the rostral half of the next more caudal segment. No embryos were observed with labeling in less than two vertebral segments. Moreover, labeled cells were not uniformly distributed within the labeled region of each vertebra; the neural arch, for example, usually contained a higher proportion of labeled cells than did the centrum. These observations support the concept of resegmentation, whereby a vertebra forms from sclerotomal cells derived from two consecutive somites resulting in a vertebral column shifted by one half segment with respect to the segmented boundaries of the somites. The quantitative distribution of labeled cells in the vertebrae also suggests that sclerotomal cells populate the region of a future vertebral segment in an orderly fashion dependent on when the cells migrate from the somite.     

Potassium-selective amphotericin B channels are predominant in vesicles regardless of sidedness.

Amphotericin B (AmB) is a membrane-active antibiotic which has been shown to increase ion and small molecule permeability in a variety of model and biological membrane systems. A major mechanistic model, based on BLM systems, proposes that amphotericin forms barrellike pores with cholesterol which are cation selective when added to one side of the membrane and anion selective when added to both sides. We have tested this hypothesis on small and reverse-phase large unilamellar vesicles (SUV and REV) with and without cholesterol. The method used to measure K+, Cl-, and net ion currents is based on ion/H+ exchange detected by the entrapped pH probe pyranine. We find that AmB forms channels which have net selectivity for K+ over Cl- regardless of sidedness or sterol content in SUV. REV with 10% cholesterol also show net K+ selectivity with double-sided addition. Differences are noted between cholesterol- and non-sterol-containing vesicles consistent with at least two separate modes of action: (1) cholesterol-containing SUV form some larger diameter pores which allow the passage of larger ions especially when added to both sides; (2) SUV without sterol form pores which are still K+ over Cl- selective, but larger ions do not pass. The latter mode of action precludes a sterol/pore type of model but not necessarily a barrellike model consisting only of amphotericin molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stage-specific cuticular proteins of Ostertagia circumcincta and Ostertagia ostertagi

In this study we have shown that NHS-biotin and I¹²⁵-streptavidin can detect cuticular polypeptides of Ostertagia spp. The labelled polypeptide profile of intact nematodes is simple compared to the profile obtained by labelling homogenates. None of the major internal polypeptides are labelled and the subset of proteins labelled in intact nematodes appears to be mainly surface associated. The results presented here demonstrate that NHS-biotin may be used as a reagent for the analysis of surface polypeptides. The surface polypeptide profiles of the five major developmental stages (L1, L2, L3, L4 and adult) of Ostertagia circumcincta show a series of stage-specific molecules with no polypeptides common to all stages, indicating that the cuticle is a dynamic structure which changes throughout the life cycle. Similarity comparison of Ostertagia ostertagi L3 and L4 stage surface profiles showed that each stage is clearly distinct; comparison of these stages between the two species shows an overall similarity.     

Electrophoretically estimated genetic distance and divergence time between chimpanzee and man

Amount of genetic differentiation between chimpanzee and man was estimated from the result of comparative electrophoretic screening of blood protein variations at 32 independent genetic loci. TheNei's genetic distance (D) was calculated as 0.4514, and from this value the divergence time between the two species was estimated as 2.26 million years; considering the variation among amino-acid substitution rate in different proteins, the corrected figures were given as genetic distance of 0.5706 and divergence time of 2.85 million years. This genetic difference is considered too small the two species to be allocated in different families, in accordance with the results of the similar kind of analyses byKing andWilson (1975) and Bruce andAyala (1979). Discussions were made for a discrepancy between the divergence times estimated by using and not by using the splitting time recognized by paleoprimatologists as a reference, and for the difference in the estimations made in different laboratories.     

Population genetics of Japanese monkeys: II. Blood protein polymorphisms and population structure

Genetic variability in individual troops of the Japanese macaque (Macaca fuscata fuscata) was quantified by the proportion of polymorphic loci and the average heterozygosity per individual from the results of starch-gel electrophoreses of blood proteins controlled by 32 independent genetic loci. The former averaged 9.2% and the latter 1.3%, the values being remarkably lower than those estimated for other animal populations. Geographical distribution of the genetic variations was not uniform in the whole species but the variants occurred only in limited areas. Assuming the selective neutrality of segregating alleles and the two-dimensional stepping-stone model of population structure, the genetic migration rate between the local demes per generation could be estimated to average less than inverse of average effective deme size. Here, the local deme is not a troop itself, but it consists of several troops tightly connected with each other by frequent exchanges of reproductive males. Analyses of correlation between geographic and genetic distances between troops revealed that the gene constitutions of two troops apart more than 100 km on an island could be regarded as practically independent of each other. These results suggest that the population structure of the Japanese macaque species has a tendency to split into a number of local subpopulations in which the effect of random genetic drift is prevailing.     

Factors Contributing to the Poor Myelination in the Brain of the Snell Dwarf Mouse

Abstract: Conventional histological examination of the pituitary does not distinguish Snell dwarf mutants (dw/dw) from their normal littermates (+/?) in the neonatal stage. However, immunohistochemical examination of pituitaries of litters born to heterozygous Snell parents revealed that in approximately 25% of the glands examined, the number of positive cells was very low in the neonatal stage. We attempted to delineate the events resulting in the poor myelination in the brain of the Snell dwarf mouse, and to devise an immunohistochemical method for identifying the mutant neonate. Differences in the brain weights of the dw/dw and +/? mice first became apparent on the 10th day of age, and from this time on no further increase in the weight of the dwarf mouse brain was recorded. Increase in CNPase activity was found to be suppressed in the cerebrum and brain stem throughout the developmental stage, but not in the other parts of the brain. The yield of isolated myelin decreased by 58% in the mutant mouse, but CNPase activity was equivalent to that of control myelin. Differences in DNA content per cerebrum from the dw/dw and +/? mice first became apparent on the 10th day of age. Henceforth, the dw/dw mice showed no further increase, although the +/? mice continued to increase. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the cerebrum is most active, was suppressed to about 50% of the control level in all parts of the dwarf brain. These findings indicate that the poor myelination found in the mutant cerebrum is a hypomyelination due to reduced oligodendroglial proliferation caused by lack of circulating growth hormone.     

Neurochemical Studies on the Cerebellar Hypoplasia of Gunn Rat (Hereditary Hyperbilirubinemic Rat)

The cerebellar hypoplasia induced by hereditary hyperbilirubinemia in the Gunn rat was analyzed neurochemically and immunohistochemically. The antiserum against myelin basic protein was used to visualize the arborization of the fibers in the cerebellum. Arborization was very scarce in the affected lobes of the homozygous (jj) cerebellum. Na,K-ATPase activity did not show significant differences between the jj and the control (Jj) cerebellum. The concentration of norepinephrine in the jj cerebellum was about 1.5 times that of the control. However, the activation ratio of the Na,K-ATPase by norepinephrine and other catecholamines such as dopamine and isoproterenol was about twice as high as the basal activity, and no significant difference was observed between the jj and the Jj cerebella. The glutamic acid decarboxylase activity of the jj cerebellum did not differ significantly from that of the control.     

2-Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice

Abstract: The 2-deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2-deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2-deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2-deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.