Study of elastase-type activity in blister fluids of recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized clinically by blister formation due to minor trauma and ultrastructurally by a progressive disappearance of anchoring fibrils at the dermoepidermal junction and of the oxytalan-type fibers which belong to the elastic fiber system. In this study, we determined the elastase-type activity in blister fluid obtained from 8 patients suffering from RDEB as compared to the suction fluid of experimental blisters in a healthy person and to the blister fluid of a patient suffering from epidermolysis bullosa simplex. One patient with dominant dystrophic epidermolysis of the albopapuloid type was also studied. Seven of the eight children with RDEB showed highly elevated values. The eighth child, treated with etretinate, as well as the patient suffering from dominant epidermolysis bullosa had moderately increased values. The determination of elastase-type activity in the blister fluid could therefore be useful to establish the differential diagnosis of recessive dystrophic epidermolysis bullosa.     

Effect of physical environment on the conformation of ricin. Influence of low pH.

The molecular properties of ricin (the toxic lectin from Ricinus communis seeds, RCA II or RCA 60) were evaluated by analytical ultracentrifugation, viscosimetry, c.d., fluorescence and equilibrium dialysis. Measurements of sedimentation (S0(20,W) = 4.60 S) and viscosity (eta = 2.96 X 10(-2) dl/g) indicated that, at neutral pH, the ricin molecule is very compact. Various transitions were explored, and a pH-triggered change in the ricin conformation was observed between pH 7 and 4. In this range, the sedimentation coefficient, far-u.v. c.d. and fluorescence altered simultaneously without unfolding. Below pH 7 the change in the ricin conformation was accompanied by a decrease in the affinity of ricin for galactosides, and at pH 4.0 by an alteration in its binding capacity. These effects of low pH are discussed in relation to the physical conditions encountered by ricin molecules during their entry into living cells.     

A chromatin core particle obtained by selective cleavage of histones by clostripain.

Rat liver chromatin core particles digested with clostripain yield a structurally well-defined nucleoprotein particle with an octameric core made up of fragmented histone species (designated H'2A, H'2B, H'3 and H'4, respectively) after selective loss of a sequence segment located in the N-terminal region of each core histone. Sequential Edman degradation and carboxypeptidase digestion unambiguously establish that histones H2A, H2B, H3 and H4 are selectively cleaved at the carboxyl side of Arg 11, Lys 20, Arg 26 and Arg 19 respectively and that the C-terminal sequences remain unaffected. Despite the loss of the highly basic N-terminal regions, including approximately 17% of the total amino acids, the characteristic structural organization of the nucleosome core particle appears to be fully retained in the proteolyzed core particle, as judged by physicochemical and biochemical evidence. Binding of spermidine to native and proteolyzed core particles shows that DNA accessibility differs markedly in both structures. As expected the proteolyzed particle, which has lost all the in vivo acetylation sites, is not enzymatically acetylated, in contrast to the native particle. However, proteolyzed histones act as substrates of the acetyltransferase in the absence of DNA, as a consequence of the occurrence of potential acetylation sites in the core histones thus rendered accessible. The possible role of the histone N-terminal regions on chromatin structure and function is discussed in the light of the present observations with the new core particle obtained by clostripain proteolysis.     

Discrimination of distinct proteinases at the four structural levels of rat liver mitochondria.

Rat liver mitochondrial fractions corresponding to four morphological structures (matrix, inner membrane, intermembrane space and outer membrane) contain proteinases that cleave casein components at different rates. Proteinases of the intermembrane space preferentially cleave kappa-casein, whereas the proteinases of the outer membrane, inner membrane and matrix fractions degrade alpha S1-casein more rapidly. Electrophoretic separation of the degradation products of alpha S1-casein and kappa-casein in polyacrylamide gels shows that different polypeptides are produced when the substrate is degraded by the matrix, by both membranes and by the intermembrane-space fraction. Some of the degradation products resulting from incubation of the caseins with the mitochondrial fractions are probably the result of digestion by contaminating lysosomal proteinase(s). The matrix has a high peptidase activity, since glucagon, a small peptide, is very rapidly degraded by this fraction. These observations strongly suggest that distinct proteinases, with different specificities, are associated respectively with the intermembrane space and with both membrane fractions.     

Streptomyces K15 DD-peptidase-catalysed reactions with suicide beta-lactam carbonyl donors.

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency.     

Urinary catecholamine responses to basic types of physical activity

Urinary adrenaline, noradrenaline, heart rate, and subjective ratings were obtained from 9 healthy males during six different physical activities, ranging in intensity from lying down to running. Heart rate, subjective ratings and noradrenaline excretion reflected the work load in the different conditions. Adrenaline, on the other hand, failed to show this relationship. There was no significant increase in adrenaline excretion even at the highest work load (corresponding to a heart rate of 160 bpm). It was concluded that urinary adrenaline may safely be used as an indicator of mental factors even in situations with different levels of physical activity.     

Ontogenetic development of corticotropin-releasing factor (CRF)-containing neural elements in the brain of the chicken during incubation and after hatching

Summary In chicken embryos of different ages and in young chickens after hatching, neural elements reacting with antibodies generated against synthetic ovine corticotropin-releasing factor (CRF) were studied by means of the peroxidase-anti-peroxidase (PAP) technique at the lightmicroscopic level. CRF-immunoreactivity was first observed in perikarya located in the periventricular part of the hypothalamus on the 14th day of the incubation period. CRF-containing neural elements were detected on the same day of incubation in the external zone of the median eminence, but not in all investigated animals. In extrahypothalamic sites, immunoreactive perikarya were demonstrable in the central gray of the mesencephalon on the 15th day of incubation. Furthermore, immunoreactive cells appeared in other brain regions such as nucleus accumbens and dorsomedial nucleus of the thalamus after hatching. The present observations provide information regarding the functional development of the hypothalamo-hypophyseal-adrenal axis in the chick embryo.     

Oxygen consumption during embryonic development of the annual fish Nothobranchius korthausae with special reference to diapause

The oxygen consumption of Nothobranchius korthausae eggs in different developmental stages, including diapause II and III, was measured. Oxygen consumption increases exponentially during embryonic development. In diapause II and III there is a drop in oxygen consumption, which attains a minimal level in diapause II after 3 weeks and in diapause III after 2 weeks. During early development the embryos can escape from hypoxic stress by entering diapause I and II. During late embryogenesis embryos in diapause III can escape from hypoxic stress by hatching. We conclude that survival of annual fish embryos is enhanced during conditions of low oxygen concentration by reduced oxygen consumption rates during diapause.     

Formation of partially phosphorylated phosphorylase in isoproterenol stimulated rat hearts

Summary Phosphorylase ab hybrid was demonstrated in perfused rat hearts and during the in vitro conversion of purified rat heart phosphorylase b. Phosphorylase ab hybrid was determined in rat heart extracts by the activating effect of AMP in the presence of caffeine. These results were confirmed by the quantitative determination of incorporated ³²P in vitro and through the characteristic inhibition of ab hybrid by glucose-6-phosphate.As shown by our results, in aerobically perfused control hearts only the ab hybrid represents the active form of phosphorylase, its activity reaching about 20% of the total. In response to isoproterenol (5–1000 ng), the amount of ab hybrid rose to about 30–40%, preceding the rise of the a form, which increased in a dose-dependent manner up to 45% of the total.The great sensitivity of the ab form to AMP activation and glucose-6-phosphate inhibition supports its physiological significance in heart under in vivo conditions as well. Our results strongly suggest that the activity ratio -AMP/+AMP reflects rather the percentage ratio of phosphorylated subunits than that of the activated (partially or totally phosphorylated) phosphorylase molecules.