Proteases of human brain

Growing appreciation of the multiple functions of proteolytic enzymes in intracellular protein degradation and post-translational modification, in the release of biologically active macromolecules and peptides from precursors and in cellular protein regulation and quality control has stimulated interest in proteases in neurobiology and neuropathology. In this article, the proteinases and peptidases thus far studied in the human central nervous system are reviewed with respect to their enzymology, anatomical and cytological distributions and contributions to neurological and psychiatric disease states. Though information concerning brain proteases in man is fragmentary, it suffices to establish the importance of these complex systems for advancing knowledge of human cerebral function in health and disease.The authors are privileged to submit this contribution for the Special Issue of Neurochemical Research honoring Professor K. A. C. Elliott—distinguished pioneer in neurochemistry and a cherished mentor and former colleague of one of us (AP).     

Catecholamines in fetal pig plasma and the response to acute hypoxia and chronic fetal decapitation

Summary Dopamine, norepinephrine and epinephrine were measured by radioenzymatic assay in blood plasma samples drawn from the umbilical arteries of 30 anaesthetised Landrace pig fetuses. Just prior to term, the concentrations of dopamine (0.46±0.14 ng·ml^–1) and norepinephrine (1.74±0.60 ng·mg^–1) were lower than earlier in gestation, whereas epinephrine concentrations at term (0.80±0.31 ng·ml^–1) were similar to those at mid-gestation, intervening stages of gestation having higher levels of plasma epinephrine. Fetal hypoxia was induced by clamping the umbilical cord for 2 min and the catecholamines determined in arterial blood samples immediately thereafter, then again 3 min after removal of the clamp. Inconsistent effects of cord clamping on catecholamine levels were seen at 55 days, but thereafter, in all but one instance, the hormone levels were increased. Fetuses near term tended to respond less than fetuses at 75 and 96 days gestation (term=114±1 day). Catecholamines were also present in the circulation of fetuses decapitated at 42 days gestation and studied at 109±1 days. The average concentrations of dopamine (1.12±0.27 ng·ml^–1) and norepinephrine (8.23±3.04 ng·ml^–1) were greater than in intact fetuses, the plasma epinephrine levels being comparable to, or slightly higher than, those in intact fetuses. The results demonstrate that catecholamines are present in the circulation of the intact and decapitated pig fetus and that the actual concentrations and the type of response to umbilical cord clamping are dependent on gestation age.     

Temperature-dependent morphological changes in membranes of bacillus stearothermophilus

Bacillus stearothermophilus cells vary the lipid fatty acid composition of cytoplasmic membranes with growth temperature. Spin label studies of such membranes have been interpreted to indicate lateral lipid phase separations at the growth temperature. We have now used freeze-fracture electron microscopy to confirm the spin label studies. Freeze-fracture faces of protoplasts indicate slight but distinct protein aggregation at the growth temperature. Aggregation increases rapidly with decreasing quench temperature in wild-type cells. In contrast we were unable to demonstrate extended protein segregation in membranes of a temperature-sensitive mutant that contains more than 58% branched fatty acids. Storage of protoplasts for prolonged times below the lipid phase transition results in the appearance of corrugated fracture faces with 300- to 500-Å repeat patterns, although this organism does not synthesize lecithins.     

The carbonyl reactivity of 3-bromopyruvate and related compounds

The covalent hydration of β-halopyruvic acids and β-halopyruvamides has been investigated in the uv region by stopped-flow techniques. In aqueous solutions of β-bromopyruvate and related compounds, the geminal diol is the predominant form at all pH values. Kinetic investigations have also been carried out on these hydrations. General bases enhance the rate of approaching the equilibrium. Specific acid catalysis was not detected. In the water-catalyzed reaction of pyruvamides, the relationship between the equilibrium constants K and the rate constants of the forward reaction shows that the transition state is more productlike.     

Development of projections between areas of the nervous system

A theory of projections in the nervous system (such as the retino-tectal projection) is proposed. Components of axonal growth cones and target tissue interact and cooperate, within the area of contact, to generate a guiding parameter, in the simplest case a guiding substance of distributionp. The components which are involved in this production are assumed to have graded distributions with respect to position in the projecting and target area, respectively. The distributionp thus produced guides the growth cone in the direction of maximal slope until the minimal value ofp is reached. In this way, each growth cone can be guided to a position on the target tissue which depends on the origin of the fiber in such a manner that a projection results. Adhesive forces could but need not be involved in the guiding mechanism. The slope ofp may interfere with an intracellular pattern forming mechanism within the growth cone, determining the polarity of activation (as modelled previously on the basis of autocatalysis and lateral inhibition) and thus the direction of growth. For the generation of a distribution ofp leading to a reliable projection, simple graded distributions in the projecting and target area suffice, involving one or two components in each dimension. Their effect on the generation ofp may be activatory as well as inhibitory. Exponential gradients give rise to particularly simple mapping functions. The following is an example of this general type of model: Growth cones as well as target tissue contribute to the production of a guiding substance. For each dimension, there is, in the target tissue, an exponentially graded component exerting (directly or indirectly) two functions: it actively produces guiding substancep and it interacts, in an inhibitory fashion, with the production ofp by a component of the growth cone (which is, in turn, graded with respect to position of origin in the projecting area). While the theory is proposed as a fair approximation of the primary events in neural projections, superimposed regulatory effects can also be incorporated. These include fiber-fiber interactions, mechanisms smoothing out unequal density distributions of axon terminals and effects of time of arrival of fibers on the projection, which have been proposed previously as primary mechanisms generating projections. A further extension of the model is to assume that crude and more refined positional specificity is determined in a combinatorial fashion, allowing the possibility of interchanges and transformations of parameters.     

Catechol oxidase from green olives: Properties and partial purification

Catechol oxidase was extracted from an acetone powder prepared from green olive. The enzyme was purified 240-fold by ammonium sulphate fractionation followed by ion exchange chromatography and gel filtration. The enzyme was characterized by substrate specificity and response to inhibitors. Between 7 and 9 bands having catechol oxidase activity could be detected by gel electrophoresis and electrofocusing. The purified enzyme had an estimated MW of 42 000. The enzyme was strongly inhibited by diethyldithiocarbamate. Inhibition by chloride was strongly dependent on pH. The enzyme did not oxidise monophenols.     

Instability of plasmid DNA sequences: Macro and micro evolution of the antibiotic resistance plasmid R6-5

Summary Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possibly other replicons occur more frequently than has hitherto been appreciated. The sequence changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existed in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous,and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.     

The influence of ovulation number and ovarian dimensions on embryo production in superovulated cattle

Twenty-one cycling Angus heifers and five Holstein cows received a subcutaneous (SC) injection of 50 mg of progesterone (P) in oil for 14 consecutive days. On day 6 of (P) treatment, animals were injected intramuscularly (IM) with 6 mg of estradiol valerate, and on day 13, received an IM injection of 2,000 IU of Pregnant Mare Serum Gonadotropin. Three additional Angus heifers were used as non-hormone treated controls. Seventeen of 21 heifers and 4 of 5 cows (81%) exhibited estrus within 48 to 132 hr following P treatment. Two of the five animals in which estrus was not observed were palpated as pregnant and discarded from the study. Treatment animals showing estrus were randomly assigned either to Group I, animals bred by natural service, or Group II, animals artificially inseminated with two straws of frozen semen at 12-hr intervals for a total of four breedings. Twenty-one animals were slaughtered 2 to 6 days after the onset of estrus, and those animals in which estrus was not detected were slaughtered 10 days after the last P injection. Two of the 24 treated animals had no ovulations. A total of 397 ovulation points ($\text{397}\text{22}$) were counted for a mean ovulation rate of 18 ovulations per animal. One hundred and fifty-six ova were recovered ($\text{156}\text{397}$) for a collection rate of 39%. Group I animals had 44 of 66 (67%) of their ova fertilized while 23 of 71 (32%) of the ova in Group II were fertilized. Nineteen unfertilized eggs were collected from the three animals not observed in estrus. No differences in fertilization rates between the Group I and Group II animals were found. Mean ovarian width, length and weight in the treated animals was measured and found to be 3.5 ± 1.1 cm, 4.8 ± 1.4 cm, and 21.7 ± 21.2 gm, respectively. Ovarian width, length and weight were all positively correlated with the number of ovulations per ovary r=.74, r=.74, and r=.55, respectively. No significant correlation existed between ovarian width (r=.16), lenght (r=.21), or weight (r=.13) when compared to ova recovery rate. This result suggests that ovarian size or weight may not be the limiting factor involved in embryo recovery.     

Analysis of early stages of budding inHydra by means of an endogenous inhibitor

Summary Buds originate inHydra attenuata at a position 1/3 of the body length from the basal disc. The position with respect to the vertical axes is determined first and the position of the bud on the circumference of this budding region is specified later.Bud formation in hydra is reversibly prevented by pre-treatment with an inhibitor purified from hydra tissue (Berking, 1977). Some hours after the end of the treatment with the inhibitor, bud formation is resumed. From the starting or restarting point of development after the inhibitory treatment to the visible beginning of bud formation, 4 intermediary stages were distinguished on the basis of different responses to a second treatment with inhibitor. The pre0treatment is followed immediately by a period of maximal sensitivity to the inhibitor, which varies in length. At the conclusion of this phase the time interval required for the visible appearance of buds is fixed (12 h). In this and the following phase another application of inhibitor can cancel the entire preparatory process from the pre-treatment onwards. A transition to near complete resistance to inhibitor is the basis for defining a third phase. In a fourth phase, immediately before the evagination of the bud starts, the proesence of the inhibitor will again hinder the development. Upon removal of the inhibitor the suppressed buds will appear.