Histochemistry of some proteases in the normal rabbit,pig and ox corneas

Summary The distribution of activities of membrane aminopeptides (aminopeptidases M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), -gluamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4°C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4°C) for the demonstration of lysosomal enzymes.In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity.Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Hydroxylysine glycosides in the collagen of normal and scarred rabbit corneas

Rabbit corneal scar collagen contains a lower content of glucosylgalactosylhydroxylysine than normal corneal collagen. Although the ratio of lysine to total hydroxylysine in normal and scar collagen is approximately the same, the relative proportion of glycosylated hydroxylysine to non-glycosylated hydroxylysine is different. These observations relate to the disposition of glycoside residues in scar and normal collagen and to the ultrastructural characteristics of the collagen fibrils.     

Histochemistry of proteases in ependyma, choroid plexus and leptomeninges

Aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV) and gamma-glutamyl transferase (GGT) were demonstrated histochemically in cryostat sections of the rat brain to show the reaction pattern of ependyma, choroid plexus and leptomeninges. GGT was only demonstrable in the cell membranes of ependymal cells and in the leptomeninges; however, APA, APM and DAP IV showed a variable degree of activity in the capillary endothelium of the choroid plexus as well as in the leptomeninges. On the basis of these results, it is postulated that peptides in the cerebrospinal fluid can be cleaved extraventricularly by the enzymes demonstrated in the leptomeninges.     

Histochemistry of proteases in ependyma,choroid plexus and leptomeninges

Summary Aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV) and -glutamyl transferase (GGT) were demonstrated histochemically in cryostat sections of the rat brain to show the reaction pattern of ependyma, choroid plexus and leptomeninges. GGT was only demonstrable in the cell membranes of ependymal cells and in the leptomeninges; however, APA, APM and DAP IV showed a variable degree of activity in the capillary endothelium of the choroid plexus as well as in the leptomeninges. On the basis of these results, it is postulated that peptides in the cerebrospinal fluid can be cleaved extraventricularly by the enzymes demonstrated in the leptomeninges.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Adrenal medullary basophilia in ox,pig and sheep

Summary In ox, pig and sheep the adrenaline storing cells are intensely basophilic compared with the noradrenaline storing cells when aldehyde fixed tissue is stained with toluidine blue at pH 5.0 and above. This has been shown to be due to carboxyl groups from the glutamate rich chromaffin granule soluble protein. In isolated chromaffin granules adenosine nucleotides also bind the dye. Fixation of adrenal medulla in agents not containing aldehydes, or the use of cryostat sections results in equal basophilia in the adrenaline and noradrenaline storing cells. The probable mechanism of the differential basophilia of the two sorts of medullary cells following aldehyde fixation is discussed.     

Histochemistry of implantation in the rabbit

Summary The distribution of glycogen, acid and neutral mucopolysaccharides, hydrolases alkaline phosphatases, and carbohydrate dehydrogenases is described in the rabbit blastocyst and its surroundings over the implantation period from 5 to 9 days.Glycogen is found mainly in the embryonic tissues, where peaks of concentration coincide with differentiation, some is also seen in the uterine epithelium and secretion, and large quantities accumulate in the developing decidua.Neutral mucopolysaccharide is found in the yolk-sac and embryonic endoderm, and as secretion droplets in the uterine epithelium and secretion.Acid mucopolysaccharide occurs in the embryonic coverings and uterine secretion.RNA is associated in the embryo with developing tissues, and accumulates in the developing decidual cells.Hydrolases (acid phosphatase, and B esterase) increase their activity in the trophoblast knobs, in developing syncytiotrophoblast, and in the embryonic endoderm. Degeneration of the uterine epithelium is associated with maximal hydrolase activity.Trophoblastic alkaline phosphatase activity (non-specific and specific) decreases from 5 to 7 days of gestation, then increases markedly in the developing syncytiotrophoblast. AMPase appears in the embryonic mesoderm. In the uterine epithelium intense brush border staining is seen, and TPPase and UDPase become visible for a short period in the Golgi region. Phosphatases increase their activity in the decidua to 8 days and then decrease.Carbohydrate dehydrogenases (except -glycero-phosphate and -hydroxy-butyrate dehydrogenases) increase their activity in embryonic tissues, particularly in the developing syncytiotrophoblast and endoderm. Symplasma formation in the uterine epithelium is also associated with increase in enzyme activity, and a similar increase, up to 8 days of gestation, is seen in the decidua with isocitrate, malate, glucose-6-phosphate, lactate, succinate, and furfuryl alcohol dehydrogenases.Some correlation is found between the histochemical findings and the phenomena of epithelial removal, uterine secretion, decidual formation and function, giant cell function, morphogenesis, and histiotrophic nutrition, and the results are compared with previous findings for the rat in which implantation is morphologically, and probably physiologically a very different process.     

Histochemistry of connective tissue cells in subcutaneous adipose tissue of normal and decapitated pig fetuses

Connective tissue cells that are histochemically and morphologically distinct from 'fibroblasts' are localized around developing hair follicles in the pig and rat. Immature adipose tissue is limited to small areas immediately around fully descended hair follicles in the rat hypodermis. In the present study, connective tissue around large nerves and blood vessels in fetal pig subcutaneous tissue was examined for the presence of enzymes typical of adipocytes. Samples from decapitated pig fetuses were studied so that the effects of an altered hormonal profile could be examined. Samples of dorsal subcutaneous adipose tissue were obtained from fetuses at 65, 70, 85, 90, 110, and 112 days of gestation. Fetuses were decapitated in utero at 45 days of gestation, and adipose tissue samples were obtained from these fetuses at 110 days of gestation. A close spatial relationship was observed between the growth of large blood vessels and nerves and fat cell cluster development in the older (greater than 70 days) fetuses. Connective tissue cells that were contiguous with fat cell clusters were histochemically identical to adipocytes. The lipid histochemistry of the reactive connective tissue cells (histochemically identical to adipocytes) was variable in young fetuses. In all 110-and 112-day-old fetuses, the reactive cells contained lipid, whereas the reactive cells in decapitated fetuses were devoid of lipid. The reactive connective tissue cells were not associated with capillaries and did not contain basement membranes. The histochemistry of these cells suggests that they respond to a particular hormonal or metabolic profile as do adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemistry of the placenta of the pig

Summary The distribution of glycogen, RNA, lipid, acid mucopolysaccharides, diastase-resistant PAS-positive material, hydrolytic enzymes, specific and non-specific alkaline phosphatases, and carbohydrate dehydrogenases is described in the foetal and maternal placenta of the pig throughout gestation. The results are compared with the findings in similar and other placental types, and some deductions regarding the functional significance of the enzymes in the structures in which they are observed are made.     

Biliary excretion of some diquaternary ammonium cations in the rat, guinea pig and rabbit

1. The extent of the excretion in the bile and urine of the (14)C-labelled dications, diquat, paraquat, morfamquat, decamethonium and dimethyltubocurarine in bile-duct-cannulated rats, guinea pigs and rabbits was examined. 2. These compounds were excreted unchanged in bile and urine, except diquat, which was metabolized to a significant extent (18% of the dose) in the rabbit only. 3. The extent of the biliary excretion of diquat (mol wt. of ion 184), paraquat (186), decamethonium (258) and morfamquat (469) was less than 10% of the dose in the three species, whereas that of dimethlytubocurarine (653) was greater than 10% in the rat and rabbit but not in the guinea pig. 4. These results together with data from the literature suggest that the molecular weight at which the excretion of dications in the bile exceeds 10% of the dose is in the region of 500-600, which differs from the values for monocations (Hughes et al., 1973) and anions (Millburn et al., 1967; Hirom et al., 1972).     

Proteins of the hard keratins of echidna, hedgehog, rabbit, ox and man

In the accompanying paper it has been shown that two major groups of proteins (low-sulphur and high-sulphur) of ovine wool, horn, and hoof contain similar components although the overall proportions of the groups of proteins and the relative proportions of components within the groups may show significant differences. In the present paper it has been shown for five other species (echidna, hedgehog, rabbit, ox and man) that the hard keratins produced by one animal contain the same groups of protein components but in different relative proportions. The wide apparent differences in the type and relative proportions of the the low-sulphur components which comprise the major constituent proteins of the microfibrils suggest that microfibrils can tolerate a considerable variation in the constituent proteins and still produce functional structures. The low-sulphur protein components are sufficiently well resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis to make this procedure potentially useful for animal identification and classification.     

Minor proteases in the stomach of the pig

1. Pepsins C and D and probably pepsin B as well as the major enzyme pepsin have been detected on starch-gel electrophoretograms of concentrates of the gastric contents of four pigs. 2. The presence of pepsin B is further indicated by a high ratio of peptidase activity to haemoglobin-digesting activity after treatment at pH6.9.     

Laminins in normal,keratoconus, bullous keratopathy and scarred human corneas

The laminin composition (LMalpha1-alpha5, beta1-beta3, gamma1 and gamma2 chains) of normal corneas and corneal buttons from keratoconus, bullous keratopathy (BKP), Fuchs' dystrophy + BKP, Fuchs' dystrophy without BKP and scar after deep lamellar keratoplasty (DLKP) was investigated with immunohistochemistry. The epithelial basement membranes (BMs) of both normal and diseased corneas contained LMalpha3, alpha5, beta1, beta3, gamma1 and gamma2 chains. The epithelial BM morphology was altered in the different diseases. Scarring was associated with irregular BM and ectopic stromal localization of different laminin chains. The Descemet's membrane (DM) contained LMalpha5, beta1 and gamma1 chains in all cases and additionally LMbeta3 and gamma2 chains in the majority of keratoconus corneas. The interface in the DLKP cornea had patches of LMalpha3, alpha4, alpha5, beta1 and beta2 chains, and an extra BM-like structure under the Bowman's membrane. These results suggest that laminin chains participate in the process of corneal scarring and in the pathogenesis of some corneal diseases. The novel finding of LMalpha3, beta3 and gamma2 in the DM of keratoconus buttons indicates that this membrane is also involved in the disease and that some cases of keratoconus may have a congenital origin, without normal downregulation of the LMbeta3 chain.     

Effect of vitamin E-deficiency on the activity of some lysosomal and non-lysosomal proteases in rabbit muscles.

The activity of different cathepsins and neutral proteinases was measured in normal and vitamin E-deficient rabbit muscles using specific substrates. Among the changes of enzyme activities in dystrophy caused by vitamin E-deficiency the increase in the activity of cathepsin B is the most striking. The activity of cathepsin H, both in the fast and slow muscles and that of MMP-ase in the slow muscle remains practically unchanged. Activities of other proteases significantly increase. The change in the activity of proteolytic enzymes in striated muscle of vitamin E-deficient rabbits seems to be selective. As a rule the increase in the activity is higher in fast than in slow muscles.     

Characterization of pulmonary surfactant from ox, rabbit, rat and sheep.

1. Pulmonary surfactants from ox, rabbit, rat and sheep were isolated and analysed. 2. All preparations had a high anenoic phosphatidylcholine content and would produce stable surface tensions of 0.01 Nm-1 or less. 3. Protein content was 8-18% of the dry weights. A number of proteins were observed; their overall composition were high in hydrophobic amino acid residues. 4. Lipid content varied from 79% (ox) to 90% (rabbit) with phosphatidylcholine representing from 58% (sheep) to 83% (rabbit) of the total lipid. The surfactant preparations were rather similar in lipid composition except that sheep surfactant contained about 10% lysophosphatidylcholine. 5. Hexadecanoic acid was the principal fatty acid. It was particularly high in phosphatidylcholine. 6. Phosphatidylglycerol was a minor constituent of all surfactants but phosphatidyldimethylethanolamine was not detected.