Further characterization of cysteine proteinase inhibitors purified from rat and human epidermis.

Cysteine proteinase inhibitors isolated from rat and human epidermis were purified to homogeneity and had isoelectric points of pH 4.31 and pH 5.10, respectively, Both inhibitors caused noncompetitive inhibition to the same degree against papain (EC 3.4.22.2), but the activity of human inhibitor against rat liver cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), and L (EC 3.422.-) was more effective than that of rat inhibitor. Dependency on pH was observed with rat inhibitor for cathepsins B and H, and with human inhibitor for cathepsin L. The reaction of the inhibitors with papain and cathepsins H and L occurred immediately, while the inhibition reaction of cathepsin B increased progressively during a preincubation time up to 40 min. Incubation at pH 7.0 maximized the progressive inhibitory activity. These findings demonstrate that cysteine proteinase inhibitors from rat and human epidermis inhibited a variety of cysteine proteinases. However, the inhibitor and enzyme interaction depends upon the enzyme, inhibitor source, and experimental conditions such as pH and preincubation time.

Biochemical properties of thiol proteinase inhibitor purified from psoriatic scales.

Epidermal thiol proteinase inhibitor (EPI) was extracted from normal and psoriatic cornified cells with 10 mM Tris-HCl, pH 8.0, and purified by papain-Sepharose affinity chromatography and gel filtration. Both EPIs showed a single band and the same mobility in gel electrophoresis with and without sodium dodecyl sulfate. Their immunological identity also was seen by agar diffusion. The inhibitor activity of EPIs to papain and rat liver lysosomal enzymes which caused a local inflammatory reaction after intradermal injection was determined on alpha-N-benzoyl-DL-arginine-2-naphthylamide and azocasein. Their activities to papain were the same at pH 8.0, but EPI of psoriasis showed only 50% of the activity of normal cells at pH 5.0 and 6.0. EPI of normal cells was heat stable, while that of psoriasis was reduced in activity after heating at 90 degrees C. Inhibitor activity of EPI from psoriatic cells toward the lysosomal enzymes, cathepsin B and/or cathepsin H and cathepsin L, was also inferior to EPI from normal cells at all pHs studied. We suggest the possibility that the inflammatory response associated with psoriasis seems in part to result from epidermal cells producing a less effective EPI, which may be a natural anti-inflammatory substance.

Light and electron microscopic study of cholinergic and noradrenergic elements in the basolateral nucleus of the rat amygdala: evidence for interactions between the two systems.

Pharmacological studies have suggested that the cholinergic (ACh) and noradrenergic (NA) systems in the amygdala (AM) play an important role in learning and memory storage and that the two systems interact to modulate memory storage. To obtain anatomical evidence for the interaction, the organization of the ACh and NA fibers in rat AM was investigated by immunocytochemistry for choline acetyltransferase (ChAT) and dopamine-beta-hydroxylase (DBH) in conjunction with light, confocal laser scanning, and electron microscopy (LM, CLSM, and TEM, respectively). LM showed that the ChAT immunoreactivity was densest in the basolateral nucleus (BL), whereas the DBH immunoreactivity was densest in the posterior BL. CLSM demonstrated that the ChAT-immunoreactive profiles in the BL were frequently located in juxtaposition to the DBH-immunoreactive axons. The TEM observations were as follows: The majority of the synapses formed by ChAT-immunoreactive terminals were symmetric, but DBH-immunoreactive axons formed both asymmetric and symmetric synapses. The ChAT-immunoreactive terminals usually established the symmetric synaptic contacts with the DBH-immunoreactive terminals and varicosities. The DBH-immunoreactive terminals formed the asymmetric synapses with the ChAT-immunoreactive dendrites of the intrinsic neurons within the AM. The results provide anatomical substrates for mnemonic functions of the ACh and NA systems and for the interactions between the two systems in the AM.

Synthesis and antiinflammatory and hypnotic activity of 5-alkoxy-3-(N-substituted carbamoyl)-1-phenylpyrazoles.

5-Alkoxy-3-(N-substituted carbamoly)-1-phenylpyrazoles were prepared and tested for antiinflammatory and hypnotic activity. Four compounds showed antiinflammatory activity and three possessed hypnotic properties.

Three-dimensional microvasculature of the hair follicle.

The three-dimensional microvasculature of the hair follicle of the adult Wistar rat was demonstrated by scanning electron microscopy of vascular corrosion casts. The anagen hair follicle was surrounded by the basket-like capillary network which was supplied by the branches of the subcutaneous artery and drained into the veins continuous with the subcutaneous vein. The capillary network surrounding the anagen hair follicle was most dense at its bottom, and became sparse at its upper part. The telogen hair follicle was surrounded by only a few capillaries. Transmission electron microscopy showed that the capillaries around the hair bulb possessed fenestrations. Our results indicate that the microvasculature of the anagen hair follicle is so organised as to supply the hair bulb with abundant blood, which is the most important area for hair growth.

A micro-anatomical model of the distribution of myocardial endomysial collagen.

Myocardial connective tissue probably provides passive support for regulating heart tensile strength and stiffness and ultimately for controlling heart mechanics through its endomysial part. However, endomysial collagen micro-arrangement is still a matter of debate. In order to define the fine distribution of left ventricle endomysial collagen, we applied the NaOH-scanning electron microscopy (SEM) maceration method (one of the techniques of choice for studying collagen micro-arrangement) to rabbit heart. Gomori-reticulum staining was used for correlated light microscopy (LM) observations. The SEM-NaOH method allowed isolation of collagen by removing other extracellular matrix components and cells and preserved collagen structure and position. Endomysial collagen appeared arranged in laminae that delimited the lacunae that were left empty by macerated myocytes and small vessels (mostly capillaries). These laminae were formed by reticular fibers, as confirmed by LM observations of Gomorireticulum-stained samples, and were organized in irregularly meshed networks made of thin (single) and thick (composed) filaments. In longitudinal views, collagen laminae extended the entire length of lacunae. In transversal views, the cut surface of the laminae appeared to be made of collagen bundles. These observations provide an updated microanatomical view of endomysial collagen distribution, which integrates previous studies. This model is based on the evidence that collagen laminae enveloped the surface of small vessels and myocytes. Thus, a type of myocyte-myocyte or capillary-myocyte "laminar connection" anchored to the entire cell length here is emphasized, rather than a type of "strut connection" anchored to defined loci, as usually described. This structure explains better how endomysium may provide the necessary support for heart compliance and protection against overstretch.

Comparative analysis of insulo-acinar portal system in rats, guinea pigs, and dogs.

The insulo-acinar portal system in the rat, guinea pig, and dog was comparatively analyzed using corrosion casting method in scanning electron microscopy and confocal laser scanning microscopy. In all animals examined, there were three types of arterioles according to their destination: 1) the arteriole which supplied the capillary glomerulus of the islet, 2) the arterioles which directly branched out into capillaries around the acini, and 3) the arterioles which supplied the duct system. In the rat, the afferent vessel usually ended in the cortical layer of the islet and its main branches ran along this layer before giving secondary capillary branches into the deeper regions, while in the dog and guinea pig, the region where the afferent arterioles branched out into secondary capillary branches varied among individual islets. There were three types of efferent vessels of the islet: 1) the insulo-acinar portal vessels that radiated from the islet to join the capillary network in the exocrine pancreas, 2) the emissary venules of the islet, leading directly into the systemic circulation, and 3) the insulo-ductal portal vessels which drained into the peri-ductal capillary network. In the rat and guinea pig, the intralobular islets possessed both the insulo-acinar portal vessels and the emissary venules, while the interlobular islets possessed emissary venules with occasionally occurring insulo-acinar portal vessels. In the dog, most of the islets were located within the lobule and possessed preferentially the insulo-acinar portal vessels. In this animal, the lobule was supplied by several microvascular units, in the center of which was located the capillary glomerulus of the islet. The peri-insular zone of the unit was mainly supplied by the insulo-acinar portal vessels, while the periphery, the tele-insular zone, was directly supplied by arterioles as well. The venules originated at the periphery of the unit. The islet in the dog had virtually no emissary venules. Confocal laser scanning microscopy of the rat islets showed that B cells occupied the core of all islets. The microvascular architecture within the rat islet appeared to be organized as to drain blood from the A and D cell area to the B cell area of the islet.

Organization of the blood and lymphatic microvasculature of the gallbladder in the guinea pig: a scanning electron microscopic study.

The organization of the blood and lymphatic microvessels of the gallbladder in the guinea pig is demonstrated by scanning electron microscopy (SEM) of vascular corrosion casts, and SEM of KOH-macerated tissues. In the lamina propria of the gallbladder, there is a dense network of subepithelial capillaries. The network is supplied by the arterioles that come off the arterial plexus located deep in the lamina propria. The network gathers into the postcapillary venules continuous with the collecting venular plexus located immediately below the subepithelial capillary network. The precapillary arterioles are sparsely surrounded by a single layer of circularly oriented extensions of smooth muscle cells. The terminal arterioles are endowed with circularly oriented fusiform smooth muscle cells. The nervous plexus is also noticed along the terminal arterioles. The capillaries are embraced by flat prolongations of pericytes. The postcapillary venules are sparsely surrounded by stellate pericytes and the collecting venules are sparsely surrounded by elongated or branched spindle-shaped, primitive smooth muscle cells which extend their long process in various directions along the vascular wall. The lymphatics are mostly located in the subserosal layer. The tips of the initial lymphatics are closed by endothelial cells, although there are frequently some gaps between them. The thin flaps of the lymphatic endothelial cells overlap or interdigitate with each other. The luminar surfaces of the lymphatics show oval nuclear protrusions, while the abluminal surfaces showed numerous microfolds except for the oval and flat nuclear portions. The lymphatics possess neither smooth muscle cells nor pericytes.

Correlation of glutamate-induced apoptosis with caspase activities in cultured rat cerebral cortical neurons.

In cultured rat cortical neurons lactate dehydrogenase (LDH) activity in the medium, a cell-death marker, increased gradually after exposure to glutamate (100 microM to 1 mM) for 60 min and reached a plateau at 24 to 30 h. Neuronal death was mainly apoptotic as suggested by typical electron microscopic findings, fluorescent double staining with membrane-permeating and nonpermeating chromatin dyes, nick end labeling, and assessment of DNA fragmentation by agarose gel electrophoresis. After 1 mM glutamate exposure, a rise of interleukin-1beta converting enzyme (ICE)-like protease activity in neurons was parallel to cysteine protease p32 (CPP32)-like protease activity and declined before CPP32-like protease activity reached the peak (at 6 h). LDH activity in the medium of glutamate-exposed neurons was decreased by specific ICE and/or CPP32 inhibitors, acetyl-L-tyrosyl-L-valyl-L-alanyl-L-aspart-1-al (Ac-YVAD-CHO) and acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspart-1-al (Ac-DEVD-CHO), respectively, in a dose-dependent manner. Fluorescent double staining of nuclei also demonstrated that at 100 microM each inhibitor prevented neuronal apoptosis and that this effect was additive. Among agonists corresponding to various glutamate receptor subtypes, N-methyl-D-aspartate (NMDA) and kainate induced apoptosis in cortical neuronal cultures while alpha-amino-3-hydroxy-5-methylisoxazole-4-propinate (AMPA) did not. The metabotropic glutamate receptor agonist, 1-aminocyclopentane-1S, 3R-dicarboxylate (ACPD) prevented apoptosis. Interestingly, apoptosis at 24 h after agonist or antagonist exposure correlated closely with caspase activity 6 h after exposure.

Measurements of the auricle in the human fetus.

The auricular length, auricular base length and auricular width were measured in 94 human fetuses with crown-rump (CR) lengths ranging from 49 mm (approximately 11 weeks of gestational age) to 250 mm (approximately 31 weeks of gestational age). The three measurement values showed linear increases as the CR length increased, suggesting that they are useful parameters to indicate intrauterine growth. The measurement values also suggested that the mandibular and hyoid derivatives did not grow independently, but did grow with maintaining a certain relationship.

Differential expression of Tenomodulin and Chondromodulin-1 at the insertion site of the tendon reflects a phenotypic transition of the resident cells.

The insertion site of the tendon to the skeletal element is hypovascular and is one of the most common sites of dysfunction in the musculoskeletal system. However, the resident cells have been poorly defined due to a lack of a specific marker for tenocytes. We previously reported that Tenomodulin (Tnmd) and Chondromodulin-1 (Chm1) are homologous angiogenesis inhibitors and predominantly expressed in the avascular region of tendons and cartilage, respectively. In this study, we analyzed the expression of Tnmd, Chm1, alpha 1 chain of the type I collagen (Col1a1) and alpha 1 chain of the type II collagen (Col2a1) at the insertion site of the Achilles, patellar, or rotator cuff tendons of 1-week-old rabbits by in situ hybridization analysis. Tnmd was co-expressed with Col1a1 in tenocytes of these tendons, while Chm1 and Col2a1 were detected in chondrocytes of the hyaline cartilage. Interestingly, the cell population between Tnmd/Col1a1 positive tenocytes and Chm1/Col2a1 positive chondrocytes expressed Col1a1 but none of the other markers (Tnmd, Chm1, and Col2a1). Red blood cells were exclusively present at the interface between the tendon substance and cartilage in the insertion site of the Achilles tendon. Lack of Tnmd and Chm1 in this newly characterized cell population may allow the transitional zone between the poorly vascularized tendon and cartilage to establish the unique vascular pattern for blood supply.

Three-dimensional organization of lymphatics and its relationship to blood vessels in rat small intestine.

The lymphatic organization and its relationship to the vascular system in the rat small intestine was studied by scanning electron microscopy of corrosion casts and freeze-fractured tissues, and by light microscopy of injected preparations. The villus possessed 3-10 or more central lacteals depending upon the villous width. The lacteals in each villus possessed interconnections between adjacent ones and were surrounded externally by the villous capillary network. At the villous base, the lacteals fused and formed a wide sinus, from which 2 or 3 lymphatics descended and led into the submucosal ones. In the muscularis externa there was a coarse lymphatic network which, together with the submucosal one, drained into collecting lymphatics continuous with the mesenteric ones. The central lacteals and the sinus were lined with thin endothelial cells with cytoplasmic leaves interdigitating with those of adjacent ones. There were tissue channels in the villous interstitial space, which opened through the gaps between the lymphatic endothelial cells into the central lacteals. The voluminous lacteals in the villi suggest their great potential for lymph formation. The existence of collecting lymphatics with valves in the muscularis externa suggests that contraction of the layer is involved in transporting lymph towards the efferent lymphatics.

Three-dimensional organization of the collagen fibrillar framework of the human and rat livers.

The collagen fibrillar framework in the human and rat liver was demonstrated by a cell-maceration/scanning electron microscope (SEM) method. Maceration of fixed tissues with alkali plus water successfully removed the cellular elements, exposing collagen fibrils which measured about 60 nm in diameter and were identified as such by transmission electron microscopy (TEM). The normal human liver contained 12.4 mg of collagen fibrils/g of wet tissue, while rat livers contained 1.3 mg of collagen fibrils/g of wet tissue. In the Glisson's sheaths were condensations of collagen fibrils which extended to the hepatic lobules. In the spaces of Disse collagen fibrils ran either solitarily or in bundles and formed sheaths for housing the sinusoids. The central veins and the sublobular veins were also surrounded by the collagen fibrillar sheaths which were continuous with those in the spaces of Disse. Between adjacent sheaths of sinusoids frequently stretched collagen fibrillar bundles which were confirmed by TEM to occur in inter-hepatocellular spaces continuous with the spaces of Disse. The collagen fibrillar layer of the human liver capsule was much thicker (70-100 microns in thickness) than that of the rat liver (less than 5 microns in thickness). The collagen fibrils of the capsule were also continuous with those in the spaces of Disse. The collagen fibrillar framework of the liver is presumed not only to mechanically support the tissue, but also to form a microenvironment for hepatocytes and cells in the Disse's space.

Structure of lymphatics in rat cecum with special reference to submucosal collecting lymphatics endowed with smooth muscle cells and valves. I. A scanning electron microscopic study.

The three-dimensional structure of lymphatic vessels in the rat cecum was studied by KOH-collagenase digestion/scanning electron microscopy (SEM), and corrosion casting/SEM. Abluminal surfaces of the lymphatic vessels show flat elliptical nuclear regions and flat cytoplasmic processes interdigitated with adjacent ones. The lymphatic capillaries closed by interdigitations of flat endothelial processes at their initial portion begin at the various levels of the mucosa. They descend and pass through the muscularis mucosa to connect with the lymphatic vessels in the submucosa. They form polygonal meshwork, the distances between intersections being about 0.2-0.5 mm. They also have valves, the distances between adjacent valves being about 0.1-0.6 mm. Most of the submucosal lymphatic vessels are surrounded by either periendothelial cells or typical smooth muscle cells. The polygonal meshworks made up of stellate periendothelial cells with many irregular processes embrace the initial segment of the collecting lymphatics. As they proceed proximally, the periendothelial cells become elongated and branch out by threes or fours, thus presenting the appearance of smooth muscle cells. These branches are connected side by side and run obliquely along the vessels, thus forming polygonal meshworks around the vessels. The more proximal collecting lymphatic vessels are surrounded by circularly oriented smooth muscle cells. Our results indicate that most of the lymphatic vessels in the submucosa are collecting ones and possess smooth muscle cells as well as valves. This suggests that the lymphatic vessels in the submucosa actively contract and propel the lymph towards the mesenteric lymphatic vessels.

The maceration technique in scanning electron microscopy of collagen fiber frameworks: its application in the study of human livers.

This paper reviews the cell-maceration/scanning electron microscopic (SEM) technique and its application in the study of human livers. The maceration of glutaraldehyde-fixed tissues with 2N-NaOH and water at room temperature effectively and consistently removes all the cells, thus exposing collagen fiber networks. SEM of the macerated tissues shows three-dimensional arrangements of collagen fibers more clearly than previously reported methods. High resolution SEM observations of macerated and non-macerated collagen fibrils of the rat tail tendon have revealed that both show similar cross-striated bandings that are determined by an alternate succession of elevated and depressed segments along the collagen fibrils, with a period of approximately 65 nm. Three ridges have been observed in the nonmacerated collagen fibrils: two on the margins of the elevated segments and one at an intermediate point of the depressed segment. The macerated collagen fibrils show a straight arrangement with slightly wavy microfibrils. The subendothelial spaces of Disse in the human liver contain abundant collagen fibers. There are some collagen fibers that stretch between adjacent collagen fiber sheaths in the subendothelial spaces of Disse, either forming a mono-layered network or coursing individually. The collagen fibers in the spaces of Disse are continuous with those in the liver capsule and in the Glisson's sheaths and with those around the central and sublobular veins. The collagen fibers in human livers form a network of the liver as a whole, thus constituting a hepatoskeletal system.

Three-dimensional organization of the connective tissue fibers of the human pancreas: a scanning electron microscopic study of NaOH treated-tissues.

A method for scanning electron microscope (SEM) study of reticular fibers in their original shapes and locations is described. This technique was employed to demonstrate the three-dimensional organization of the reticular fibers of the human pancreas. The cellular elements were effectively removed by treatment of the tissue pieces with a 10% aqueous solution of NaOH for 3-4 days at room temperature. Thin layers of the reticular fibers surrounding the acini and ducts formed a three-dimensional interstitial compartment. The reticular fiber sheaths for the blood vessels coursed through the compartment. In the lobule, there were scattered round or oval capsules for the islets of Langerhans. The capsule also consisted of reticular fibers. Within the capsule, reticular fiber sheaths for accommodating islet capillaries, representing the pericapillary spaces, formed a three-dimensionally anastomosing network. The channels for the capillaries ensheathed by the reticular fibers in the islet were continuous with those in the surrounding exocrine pancreas; thus, the insulo-acinar portal system was confirmed to exist in the human pancreas. This study also maintains that the present method is useful for examining the microvascular organization of the islet.