Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis

The dipeptidyl peptidase IV gene family contains the four peptidases dipeptidyl peptidase IV, fibroblast activation protein, dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Dipeptidyl peptidase IV and fibroblast activation protein are involved in cell-extracellular matrix interactions and tissue remodeling. Fibroblast activation protein is upregulated and dipeptidyl peptidase IV is dysregulated in chronic liver disease. The effects of dipeptidyl peptidase 8 and dipeptidyl peptidase 9 on cell adhesion, cell migration, wound healing and apoptosis were measured by using green fluorescent protein fusion proteins to identify transfected cells. Dipeptidyl peptidase 9-overexpressing cells exhibited impaired cell adhesion, migration in transwells and monolayer wound healing on collagen I, fibronectin and Matrigel. Dipeptidyl peptidase 8-overexpressing cells exhibited impaired cell migration on collagen I and impaired wound healing on collagen I and fibronectin in comparison to the green fluorescent protein-transfected controls. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 enhanced induced apoptosis, and dipeptidyl peptidase 9 overexpression increased spontaneous apoptosis. Mechanistic investigations showed that neither the catalytic serine of dipeptidyl peptidase 8 or dipeptidyl peptidase 9 nor the Arg-Gly-Asp integrin-binding motif in dipeptidyl peptidase 9 were required for the impairment of cell survival, cell adhesion or wound healing. We have previously shown that the in vitro roles of dipeptidyl peptidase IV and fibroblast activation protein in cell-extracellular matrix interactions and apoptosis are similarly independent of catalytic activity. Dipeptidyl peptidase 9 overexpression reduced beta-catenin, tissue inhibitor of matrix metalloproteinases 2 and discoidin domain receptor 1 expression. This is the first demonstration that dipeptidyl peptidase 8 and dipeptidyl peptidase 9 influence cell-extracellular matrix interactions, and thus may regulate tissue remodeling.     

Cytochemistry of membrane proteases

Summary Membrane proteases that are detectable by cytochemical means are the classified exopeptidases, aminopeptidases A and M (or N), -glutamyl transpeptidase (which also acts as transferase), dipeptidyl peptidase IV and the endopeptidase, enteropeptidase (also known as enterokinase). Not yet classified are the possible expeptidase, tripeptidyl peptidase and endopeptidases I (Ala-endopeptidase) and II (Arg-endopeptidase). All these membrane proteases can be investigated with either chromogenic or fluorogenic procedures using synthetic peptide substrates. The most useful substrates are 4-methoxy-2-naphthylamine amino acids and peptides for cytochemical localizations at the light and electron microscope levels, for cytophotometric quantification and the study of membrane protease isoenzymes after analytical isoelectric focusing. Amino acid or peptide derivatives of naphthylamine AS can be recommended for light microscopical localization and cytofluorometric quantification, and 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin amino acids and peptides for the development of enzyme bands after isoelectric focusing. Cytochemistry reveals the heterogeneity in the distribution and species differences of membrane proteases in adult cells, tissues and organs and during development. It also reveals some common localizations, such as in small intestinal enterocytes and proximal tubule cells. The species and organ differences are substantiated and extended considerably by isoelectric focusing in combination with methods for the cytochemical detection of proteases. In addition, continuous cytophotometry or cytofluorometry (section and cultured cell biochemistry) allows the kinetic characteristics, initial reaction rates and maximum activities of all membrane proteases to be determined.The physiological functions of the endopeptidases and exopeptidases are still a matter of debate. However, from cytochemical inhibition studies with natural peptide substrates, e.g. peptide hormones, there is increasing evidence that the proteases detected with synthetic peptides play a decisive role in many physiological circumstances, e.g. in endocrine regulation mechanisms or the regulation of blood pressure. In this respect, capillary endothelium-linked surface membrane proteases may be especially important.     

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.     

Protease cytochemistry in the murine rodent, guinea-pig and marmoset placenta

Plasma membrane and lysosomal proteases, gamma-glutamyl transferase and extracellular matrix proteases were investigated by qualitative cytochemical means in the mature placenta of mice, rats, guinea-pigs and marmosets. These studies revealed similarities, which concerned primarily the lysosomal proteases in different structures of the placenta and all proteases and gamma-glutamyl transferases in the zone of placental shedding. However, species differences predominated. They were observed especially for amino-peptidase A and M, dipeptidyl peptidase IV and gamma-glutamyl transferase in the plasma membranes and extracellular matrix of the placental barrier and decidual cells of all species and the cells of the basal zone in rats and mice. Plasma membrane and extracellular matrix proteases in other parts of the placenta, e.g. the placenta stem of guinea-pigs and basal plate, amniotic and chorionic plate of marmosets occurred only in these species. Elastase substrates hydrolysing endopeptidase I and kallikrein-, thrombin-, plasmin-, plasminogen- and cathepsin B substrates hydrolysing endopeptidase II were not observed in any of these species. A general comparison of the species revealed similarities for the mouse, rat and guinea-pig placental barrier, but not for that of marmosets. The proteases of this zone in the marmoset placenta are more similar to the human situation, but do not correspond to it completely.     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

Protease cytochemistry in the murine rodent,guinea-pig and marmoset placenta

Summary Plasma membrane and lysosomal proteases, -glutamyl transferase and extracellular matrix proteases were investigated by qualitative cytochemical means in the mature placenta of mice, rats, guinea-pigs and marmosets. These studies revealed similarities, which concerned, primarily the lysosomal proteases in different structures of the placenta and all proteases and -glutamyl transferases in the zone of placental shedding. However, species differences predominated. They were observed especially for aminopeptidase A and M, dipeptidyl peptidase IV and -glutamyl transferase in the plasma membranes and extracellular matrix of the placental barrier and decidual cells of all species and the cells of the basal zone in rats and mice. Plasma membrane and extracellular matrix proteases in other parts of the placenta, e.g. the placenta stem of guinea-pigs and basel plate, amniotic and chorionic plate of marmosets occurred only in these species. Elastase substrates hydrolysing endopeptidase I and kallikrein-, thrombin-, plasmin-, plasminogen- and cathepsin B substrates hydrolysing endopeptidase II were not observed in any of these species. A general comparison of the species revealed similarities for the mouse, rat and guinea-pig placental barrier, but not for that of marmosets. The proteases of this zone in the marmoset placenta are more similar to the human situation, but do not correspond to it completely.In honour of Prof. P. van DuijnSupported by the BMFT (Project CMT 35) and Sfb 174In part presented at a Symposion on Progress in General, Applied and Diagnostic Histochemistry (Modra, Czechoslovakia on 12–15 April, 1984; abstracts published in Histochem J 17, No 5, 1985)     

Proteases in normal and diseased human skeletal muscle: a preliminary histochemical survey

Summary Seven proteases assumed to be aminopeptidases A, B and M, dipeptidyl peptidases II and IV, esteroproteinase and -glutamyltransferase were localized histochemically, using semipermeable membrane simultaneous coupling techniques, in unfixed cryostat sections of skeletal muscle removed from one healthy volunteer, six patients with disuse muscle atrophy, and 15 patients with some form of muscle disease. Normal muscle fibres showed weak reactions for aminopeptidases A and M and for the dipeptidyl peptidases, but no reactivity for -glutamyltransferase or esteroproteinase. No change was detected in diseased muscle fibres except that low -glutamyltransferase and esteroproteinase activities appeared in some cases. The activities of the seven enzymes were stronger in the intermysial connective tissue than in the muscle fibres, but were also unchanged in disease. The strongest reactions were found in some interstitial cells (mast cells and macrophages) and these were much increased in diseased muscle, particularly for dipeptidyl peptidases II and IV. The findings are interpreted in terms of the release of proteases from such cells and their subsequent involvement in the breakdown of myofibrillar proteins in muscle disease.     

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

The distribution of activities of membrane aminopeptidases (aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), gamma-glutamyltransferase (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 degrees C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4 degrees 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.     

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.     

Are diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV?

Dipeptidyl peptidase IV preferably hydrolyzes peptides and proteins with a penultimate proline residue. Umezawa and co-workers (Umezawa et al. (1984) J. Antibiotics 37, 422-425) reported that diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) are inhibitors for dipeptidyl peptidase IV. We could show that both compounds as well as other tripeptides with a penultimate proline residue are substrates for dipeptidyl peptidase IV. An apparent competitive inhibition by those compounds is a kinetic artifact due to the substrate-like structure of such tripeptides.     

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     

Peptidase activity of human colonic bacteria

Aminopeptidase activities of mixed faecal suspensions from four human donors and 12 of the most numerous species of human colonic bacteria were measured using alanine oligopeptides and various dipeptidyl- and amino acyl-arylamidase substrates. The pattern of hydrolysis of Ala(4)and Ala(5)in faecal suspensions, whereby Ala(2)was the first breakdown product, suggested that the main mechanism of peptide hydrolysis was dipeptidyl peptidase. Dipeptidyl p-nitroanilides and 4-methoxynaphthylamides were broken down more rapidly than amino acyl derivatives in three out of four individuals tested, consistent with this conclusion. The predominant Bacteroides spp. of the intestine also had greater dipeptidyl peptidase activity than amino acyl aminopeptidase activity, while Bifidobacterium, Clostridium, Enterococcus and Propionibacterium spp. had a more variable pattern of peptidase activities. Thus, peptide hydrolysis in the human intestine, as in the rumen, appears to be mainly a two-stage process which is initiated by dipeptidyl peptidases present in the most numerous Bacteriodes spp.     

Peptidases of the rumen bacterium, Prevotella ruminicola

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested.Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P. ruminicola. Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.     

Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar K(i) values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.     

A comparison of membrane enzymes of human and pig oesophagus; the pig oesophagus is a good model for studies of the gullet in man

Summary The distribution and relative catalytic activities of five plasma membrane enzymes (alkaline phosphatase, dipeptidyl peptidase IV, γ-glutamyl transpeptidase, microsomal alanyl aminopeptidase and glutamyl aminopeptidase) were examined in human and pig oesophagus. In both species, alkaline phosphatase activity occurred in basal and suprabasal cells of the epithelium and in capillaries. Stromal cells in the human submucosa were particularly reactive. Dipeptidyl peptidase IV was present in blood vessels and capillaries in man and pig and in submucous glands in the pig. The enzyme was also present in both species in the lamina propria cells immediately adjacent to the epithelial basal lamina. In the human, γ-glutamyl transpeptidase occurred in the epithelial basal cells and in isolated basal and lower prickle cells in the pig. Stromal cells in the human submucosa were strongly reactive and capillaries in the muscularis propria in both species moderately active. Microsomal alanyl aminopeptidase was detected in lamina propria cells adjacent to the epithelial basal cell layer in man and pig and at the apices of mucous cells in pig submucous glands. Weak glutamyl aminopeptidase activity was confined to capillaries in both species. The findings of this study, along with the ready availability of pig oesophagus, suggest that the pig may be a suitable model for studies of the gullet in man.     

Specific localization of membrane dipeptidase and dipeptidyl peptidase IV in secretion granules of two different pancreatic islet cells.

Endocrine cells require several protein convertases to process the precursors of hormonal peptides that they secrete. In addition to the convertases, which have a crucial role in the maturation of prohormones, many other proteases are present in endocrine cells, the roles of which are less well established. Two of these proteases, dipeptidyl peptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19), have been immunocytochemically localized in the endocrine pancreas of the pig. Membrane dipeptidase was present exclusively in cells of the islet of Langerhans that were positive for the pancreatic polypeptide, whereas dipeptidyl peptidase IV was restricted to cells positive for glucagon. Both enzymes were observed in the content of secretory granules and therefore would be released into the interstitial space as the granules undergo exocytosis. At this location they could act on secretions of other islet cells. The relative concentration of dipeptidyl peptidase IV was lower in dense glucagon granules, where the immunoreactivity to glucagon was higher, and vice versa for light granules. This suggests that, in A-cells, dipeptidyl peptidase IV could be sent for degradation in the endosomal/lysosomal compartment during the process of granule maturation or could be removed from granules for continuous release into the interstitial space. The intense proteolytic activity that takes place in the endocrine pancreas could produce many potential dipeptide substrates for membrane dipeptidase. (J Histochem Cytochem 47:489-497, 1999)     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed an increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and beta-N-acetyl-D-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and gamma-glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.     

Is there a tripeptidyl peptidase in the renal brush-border membrane?

A recent claim that the renal brush border contains a tripeptidyl peptidase [Andersen & McDonald (1987) Am. J. Physiol. 253, F649-F655] was examined. In a fluorescent assay, the hydrolysis of Gly-Pro-Met-2-naphthylamide (-NH-Nap) and Gly-Pro-Leu-NH-Nap by pig kidney microvilli was strongly inhibited by amastatin or di-isopropyl phosphorofluoridate (inhibitors of aminopeptidases and dipeptidyl peptidase IV). The products formed were shown to be Gly-Pro and Met-NH-Nap (or Leu-NH-Nap) and free 2-naphthylamine. Specific antibodies to pig and rat aminopeptidase N abolished the apparent tripeptidyl peptidase activity. We conclude that these substrates are hydrolysed by the sequential attack of dipeptidyl peptidase IV and aminopeptidase N and that pig and rat brush borders lack a detectable tripeptidyl peptidase.     

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-l-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-l-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.     

Immunofluorescence study of the extracellular matrix of the human placenta

Distribution of collagen types I, III, IV, V and fibronectin in human placental villi has been studied by indirect immunofluorescence. During 9-12 weeks of pregnancy the extracellular matrix of villi represents a network of filaments organized in bundles and aggregates that contain collagen types I and III and finer filaments of collagen types IV and V. Collagen type IV is regularly detected in basal membrane of capillaries and particularly in villous epithelium, collagen type V and fibronectin are occasionally detected there. Marked immunofluorescent reaction on collagen types IV and V and fibronectin, and weak reaction on collagen type III is observed in cellular islets around cytotrophoblasts. In the fetus born in term placental villi have uniform immunofluorescence in thick basal membranes of fetal capillaries and of chorionic epithelium. The immunofluorescent reaction specific for all collagen types is uniform in villous stroma. Distribution of different collagen types and fibronectin, including the unusual localization of membrane collagen type IV, in villous stroma and cellular islets of early and mature placenta is discussed.