The histochemical pattern of mechanically or chemically injured rabbit cornea after aprotinin treatment: relationships with the plasmin concentration of the tear fluid

Summary Plasmin, a serine protease, was recently found to be involved in corneal ulcerative processes in humans and rabbits. In our experiments, plasmin activity was found in the tear fluid after mechanical and chemical damage of the rabbit cornea, such as de-epithelization and burning with alkali. The plasmin concentrations in the tear fluid were dependent on the severity of injury. The highest plasmin activity (2.0–3.0 g ml^–1) occurred after severe alkali damage to large areas of the cornea, and the lowest activity (0.4–1.0 g ml^–1) after mechanical injury (de-epithelization).Plasmin concentrations up to 1.0 ml^–1 were associated with increased activities of lysosomal hydrolases in epithelial cells and keratocytes beneath the epithelium. Plasmin activities increased as the inflammatory reaction developed. When plasmin activity in the tear fluid was higher than 1.0 g ml^–1, inflammatory cells were found in the corneal stroma. Levels of 1.5–2.0 g ml^–1 were connected with higher numbers of inflammatory cells (particularly polymorphonuclear leukocytes) with increased activities of lysosomal hydrolases. Very high plasmin activities (2.5–3.0 g ml^–1) accompanied corneal ulcerative processes.The local application of aprotinin (Trasylol, Bayer), an inhibitor of plasmin, and also of some other proteases, was found to be necessary for the healing of severe corneal injuries in which highly elevated plasmin activity in the tear fluid and inflammatory cellulization of the cornea occurred (severe damage). It was beneficial in cases in which medium plasmin activity occurred in the tear fluid and inflammatory changes in the cornea were not too extensive. If used very early after injury, aprotinin prevents the appearance of high plasmin activity in the tear fluid, reduces the invasion of inflammatory cells into the corneal stroma, and accelerates the healing. Even the corneal transparency is restored in many cases.     

Dipeptidyl peptidase IV (DPPIV) activity in the tear fluid as an indicator of the severity of corneal injury: a histochemical and biochemical study

Comparative histochemical and biochemical studies on the catalytically active protease Dipeptidyl peptidase IV (DPPIV), have been performed in the rabbit cornea and the tear fluid using a sensitive fluorogenic substrate, Gly-Pro-7-amino-4-Trifluoromethyl Coumarine (AFC). In both normal and experimentally injured corneas, DPPIV activity was detected histochemically and in the tear fluid biochemically. In contrast to the normal cornea where DPPIV activity was absent and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPPIV, which was associated with high DPPIV activity in the tear fluid. In conclusion, Gly-Pro-AFC was found to be useful for comparative histochemical and biochemical studies on DPPIV activity in the experimentally injured rabbit eye. Using the method of the tear film collection by a short touch of substrate punches to the respective site of the cornea or conjunctiva we can show that in experimental injuries (wearing of contact lenses, irradiation of the cornea with UVB rays), the damaged corneal cells were the main source for DPPIV activity in the tear fluid. It is suggested that the activity of DPPIV measured in the tear fluid might serve as an indicator of early corneal disorders, e.g. corneal vascularization related to contact lens wear.     

Histochemical changes in the rabbit cornea and plasmin activity in the tear fluid during contact lens wear. Favourable influence of protease inhibitors (aprotinin,PC5, elastatinal)

Summary Plasmin activity in the tear fluid of the rabbit eye was examined during the wearing of soft contact lenses (SCL) and compared with the occurrence of corneal disturbances assessed in cryostat sections. Plasmin activity was determined with a semiquantitative method using dry punches of filter paper previously soaked in 0.1 M Tris-HCl buffer solution containing mmol/l d-Val-Leu-Lys-FCA (trifluoromethylaminocoumarine), pH 7.2. Punches were applied to the corneal surface for 5 s (tear collection) and incubated in wet chamber. The time of appearance of the bright yellow fluorescence in UV light was recorded and taken as a measure of plasmin activity. For calibration punches soaked in solutions containing plasmin in various concentrations, and processed in the same manner were used. Changes in the cornea were examined histochemically using methods of choice for acid glycosidases, proteases, dehydrogenases, and Na⁺-K⁺-ATPase. SCL with high and low water content were worn in rabbits in 1, 2, 4, 7, 14, 21 and 28 days.Decreased activity of Na⁺-K⁺-ATPase, GGT, and SDH in the corneal endothelium and epithelium were not accompanied by detectable plasmin activity in the tear fluid. Pronounced damage of the corneal epithelium (increased activities of acid glycosidases, acid proteases, LDH, markedly decreased activity of SDH) was accompanied by low concentration of plasmin (0.4–1.0 g/ml) in the tear fluid. Middle activity of plasmin (1.0–2.0 g/ml) was detectable when PMNs were present in the corneal stroma. High plasmin activity (2.0–3.0 g/ml) correlated with corneal ulceration and vascularization. The occurrence of both — plasmin activity and corneal disturbances was highly dependent on the water content of SCL (which goes parallel with oxygen permeability), duration of SCL wear, mechanical stress, and bacterial contamination. Mechanical irritation is considered to be the main factor leading to the appearance of plasmin activity in the tear fluid. The local application of aprotinin which inhibits plasmin and some other serine proteases, enables us to prolong the harmless wear of SCLH (approximately one week). The combination of aprotin-in with leukocyte elastase inhibitors (elastatinal and particularly PC5), prevents ulceration of the cornea and inhibits corneal vascularization after SCLL wear. Vascularization of the cornea does not occur if protease inhibitors are combined with flurbiprofen, an anti-inflammatory drug of cyclooxygenase pathway of arachidonic acid. Protease inhibitors also improved the course of bacterial keratitis.     

Comparative biochemical studies of the murine fatty acid transport proteins (FATP) expressed in yeast

The fatty acid transport protein (FATP) family is a group of proteins that are predicted to be components of specific fatty acid trafficking pathways. In mammalian systems, six different isoforms have been identified, which function in the import of exogenous fatty acids or in the activation of very long-chain fatty acids. This has led to controversy as to whether these proteins function as membrane-bound fatty acid transporters or as acyl-CoA synthetases, which activate long-chain fatty acids concomitant with transport. The yeast FATP orthologue, Fat1p, is a dual functional protein and is required for both the import of long-chain fatty acids and the activation of very long-chain fatty acids; these activities intrinsic to Fat1p are separable functions. To more precisely define the roles of the different mammalian isoforms in fatty acid trafficking, the six murine proteins (mmFATP1-6) were expressed and characterized in a genetically defined yeast strain, which cannot transport long-chain fatty acids and has reduced long-chain acyl-CoA synthetase activity (fat1Delta faa1Delta). Each isoform was evaluated for fatty acid transport, fatty acid activation (using C18:1, C20:4, and C24:0 as substrates), and accumulation of very long-chain fatty acids. Murine FATP1, -2, and -4 complemented the defects in fatty acid transport and very long-chain fatty acid activation associated with a deletion of the yeast FAT1 gene; mmFATP3, -5, and -6 did not complement the transport function even though each was localized to the yeast plasma membrane. Both mmFATP3 and -6 activated C20:4 and C20:4, while the expression of mmFATP5 did not substantially increase acyl-CoA synthetases activities using the substrates tested. These data support the conclusion that the different mmFATP isoforms play unique roles in fatty acid trafficking, including the transport of exogenous long-chain fatty acids.     

Histochemical changes in the rabbit cornea and plasmin activity in the tear fluid during contact lens wear. Favourable influence of protease inhibitors (aprotinin, PC5, elastatinal).

Plasmin activity in the tear fluid of the rabbit eye was examined during the wearing of soft contact lenses (SCL) and compared with the occurrence of corneal disturbances assessed in cryostat sections. Plasmin activity was determined with a semiquantitative method using dry punches of filter paper previously soaked in 0.1 M Tris-HCl buffer solution containing mmol/l D-Val-Leu-Lys-FCA (trifluoromethylaminocoumarine), pH 7.2. Punches were applied to the corneal surface for 5 s (tear collection) and incubated in wet chamber. The time of appearance of the bright yellow fluorescence in UV light was recorded and taken as a measure of plasmin activity. For calibration punches soaked in solutions containing plasmin in various concentrations, and processed in the same manner were used. Changes in the cornea were examined histochemically using methods of choice for acid glycosidases, proteases, dehydrogenases, and Na(+)-K(+)-ATPase. SCL with high and low water content were worn in rabbits in 1, 2, 4, 7, 14, 21 and 28 days. Decreased activity of Na(+)-K(+)-ATPase, GGT, and SDH in the corneal endothelium and epithelium were not accompanied by detectable plasmin activity in the tear fluid. Pronounced damage of the corneal epithelium (increased activities of acid glycosidases, acid proteases, LDH, markedly decreased activity of SDH) was accompanied by low concentration of plasmin (0.4-1.0 micrograms/ml) in the tear fluid. Middle activity of plasmin (1.0-2.0 micrograms/ml) was detectable when PMNs were present in the corneal stroma. High plasmin activity (2.0-3.0 micrograms/ml) correlated with corneal ulceration and vascularization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical and biochemical studies on the red and white muscle in rabbit

1. The total amount of triglyceride was 6.00 +/- 0.14 mg/g wet tissue in soleus, 1.50 +/- 0.52 in extensor digitorum longus and 1.83 +/- 0.88 in gastrocnemius muscle. 2. The amounts of triglycerides in the individual types were calculated to be very large, moderate and very small in type 1, 2A and 2B, respectively, when compared with histochemical studies. 3. Differences in fatty acid composition of triglycerides were seen between the soleus and extensor digitorum longus, and gastrocnemius showed intermediate values. 4. These results might be important corresponding to differences in energy metabolism in different fiber types.     

Comparative enzyme histochemical studies on the development of teeth in man and mouse

Summary On the basis of a material consisting of primordia of deciduous teeth from 17 human foetuses and tooth germs from 20 newborn mice and 4 mouse foetuses, the histochemical distribution of succinic dehydrogenase, LDH, BDH, glucose-6-phosphate-dehydrogenase, NADH₂ cytochrome C reductase, and NADPH₂ diaphorase was studied.The investigation comprised partly mapping of the above-mentioned oxidative enzymes in human tooth germs during the period from bud stage to bell stage with incipient hard-tissue formation, and partly a comparative enzyme histochemical investigation of the dental development in man and in the mouse.In both materials there was an increase of all the demonstrated enzyme systems in the ameloblasts in early amelogenesis. On the other hand, the ameloblasts of the two materials showed differences during the period of matrix production: In the human ameloblasts there was activity of succinic dehydrogenase basally as well as apically, while the non-human ameloblasts showed such activity only basally.The odontoblasts of both materials showed a definite reaction for glucose-6-phosphatedehydrogenase, LDH, and BDH, but slight activity of succinic dehydrogenase. This indicates the existence of the pentose shunt as the source of energy in dentinogenesis. On the basis of the enzyme reactions studied, neither material showed a subodontoblastic layer or any reaction in the hard tissues.Material supplied by the Association for the Aid of the Crippled Children, New York. Aided by grants from the Danish State Research Foundation and the Rask-Ørsted Foundation.     

2-alpha-(N-dansyl-4-aminophenylthio)-N-acetyl-9-O-acetylneuraminic acid. A new specific and highly sensitive substrate in sialate-O-acetylesterase assay.

2-alpha-(N-Dansyl-4-aminophenylthio)-N-acetyl-9-O-acetylneuraminic acid (10) was prepared as a new specific and highly sensitively detectable sialate-9-O-acetyl-esterase substrate. It is built up from a sialidase-stable aminophenyl-alpha-thioketoside of N-acetylneuraminic acid. By labeling this thioketoside with dansyl chloride a fluorescent neuraminic acid derivative was prepared which allows determinations down to the picomol range. Regioselective acetylation with trimethylorthoacetate results in the corresponding 9-O-acetyl derivative. After incubation with esterase from bovine brain the hydrolysis products were separated on a HPLC column and fluorimetrically detected at 334 nm excitation and 564 nm emission. The Km value of 2.5 mM was in the range between the values of the completely unspecific methylumbelliferyl acetate and the less sensitively detectable N-acetyl-9-O-acetylneuraminic acid which have been used up to now as standard substrates.     

Comparative studies of the phage T2 and T4 DNA (N6-adenine)methyltransferases: amino acid changes that affect catalytic activity.

The bacteriophage T2 and T4 dam genes code for a DNA (N6-adenine)methyltransferase (MTase). Nonglucosylated, hydroxymethylcytosine-containing T2gt- virion DNA has a higher level of methylation than T4gt- virion DNA does. To investigate the basis for this difference, we compared the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases. Results from Western blotting (immunoblotting) showed that the same amounts of MTase protein were produced after infection with T2 and T4. Kinetic analyses with purified homogeneous enzymes showed that the two MTases had similar Km values for the methyl donor, S-adenosyl-L-methionine, and for substrate DNA. In contrast, they had different k(cat) values (twofold higher for T2 Dam MTase). We suggest that this difference can account for the ability of T2 Dam to methylate viral DNA in vivo to a higher level than does T4 Dam. Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 [T4, Ser; T2, Pro], 26 [T4, Asn; T2, Asp], and 188 [T4, Asp; T2, Glu]), we have produced hybrid proteins to determine which residue(s) is responsible for increased catalytic activity. The results of these analyses showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and noncanonical sites. Moreover, a single substitution of either residue 20 or 26 was sufficient to increase the k(cat) of T4 Dam.     

Comparative studies in biochemical composition of benthic invertebrates (bivalves, ophiuroids) from the Northeast Water (NEW) Polynya

Two species of ophiuroids (Ophiopleura borealis and Ophiacanthabidentata) and bivalves (Astarte crenata and Arctinulagroenlandica) were collected within and adjacent to the Northeast Water Polynya off the northeast coast of Greenland, and analyzed for total moisture, protein, lipid, and ash content. Body composition varied significantly among stations within species. Lipid and protein contents of Astarte crenata were higher in clams collected from the eastern Westwind Trough stations compared to those collected from the western Westwind Trough stations. However, the mean lipid and protein contents were significantly higher in Ophiopleura borealis sampled from the western Westwind Trough compared to the eastern Westwind Trough. Ophiopleura borealis are mobile deposit feeders and scavengers and may not be as directly dependent on sedimentary biogenic material as Astarte crenata and Ophiacanthabidentata. Samples of both Ophiopleura borealis and Ophiacantha bidentata from the Belgica Trough had lower protein and lipid contents compared to those sampled in the western Westwind Trough. Samples of Arctinula groenlandica from two locations on the Ob Bank had significantly different protein levels from each other. These data support previous work, indicating that biogenic materials resulting from primary production in the ice-free western area of the Northeast Water Polynya are advected eastward down the western Westwind Trough and that the Belgica Trough has lower organic input compared to the Westwind Trough. Received: 15 May 1997 / Accepted: 1 October 1997     

Morphological and histochemical studies on the parotid gland of Praomys (Mastomys) natalensis

The parotid gland of Praomys (Mastomys) natalensis follows the same anatomical pattern of Rodentia since no peculiar morphological and histochemical features were found in the present study that differentiate it from homologous glands in other rodents. It consists of serous secreting components and intralobular ducts that demonstrate no sexual dimorphism. Histochemical studies reveal that the glandular cells of the Praomys' parotid gland contain no acid mucosubstance, but that material of a neutral glycoprotein nature can be found in the secreting cells of acini, epithelium of the ducts, and the ductal lumina.