Double-layer fluorescent zymography for processing protease detection.

We have developed a novel double-layer zymographic method for the detection of specific processing proteases of a target proprotease using a specific fluorescent substrate. The target processing proteases were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gel was subsequently incubated with the target proenzyme used as the substrate. A cellulose acetate membrane was immersed in 10% glycerol and then soaked in the fluorescent substrate solution. The slab gel of the processing protease was covered with the fluorescent substrate membrane, making a double layer. The double layer was incubated at 37 degrees C, and the released fluorescent band, in which the processing protease was located, was detected using UV light. The advantages of the double-layer fluorescent zymographic method are as follows: (i) the specific detection of target proprotease using a specific substrate, (ii) a relatively rapid and sensitive method, (iii) effective detection using small amounts of crude material, and (iv) wide applications that include the detection of processing proteases and activators for target proteases. Typical examples used for the detection of the processing proteases, such as plasminogen activator, chymotrypsinogen activator, procaspase-3 processing protease and caspase-3 activators, using this new method are described in this article.

High-resolution scanning electron microscopic evaluation of cell-membrane porosity by ultrasound.

The effect of low-intensity ultrasound on HL-60 cells (human promyelocytic leukemia cells) in the presence of the photo sensitizing drug merocyanine 540 (MC 540) was evaluated morphologically, using a high-resolution scanning electron microscope and a transmission electron microscope. Exposure of HL-60 cells to ultrasound without MC 540 resulted in a decrease of finger-like processes in the cells. The cells showed many undulating ruffles on the surface. Distinct pits or holes in the membrane were not observed in these cells. The surface of HL-60 cells treated only with MC 540 was relatively smooth compared with that of control cells. HL-60 cells exposed to ultrasound in the presence of MC 540 showed apparent surface deformation. Numerous crater-like depressions of heterogeneous dimensions were observed in many cells. In addition, various-sized pores were noted in the cell membranes of more damaged cells. These results indicate that cell degeneration was induced by a rapid change in cell membrane porosity during sonication in the presence of MC540.

Studies on 8-methoxyquinolones: synthesis and antibacterial activity of 7-(3-amino-4-substituted)pyrrolidinyl derivatives.

A series of 8-methoxyquinolones bearing 3-amino-4-methylpyrrolidines or 3-amino-4-fluoromethylpyrrolidines at the C-7 position was synthesized and their physicochemical and biological properties were evaluated. All of the compounds synthesized showed more potent activity than LVFX (3) against both gram-positive and negative bacteria. Increases in lipophilicity of these compounds had desirable effects on their potency of single intravenous toxicity and pharmacokinetic profiles in animals. Among the compounds synthesized, 1-fluorocyclopropyl derivatives 17 and 20, and 7-(cis-3-amino-4-fluoromethylpyrrolidinyl) derivative 19 (DC-756h) showed negative responses in the micronucleus test in mice while 1-cyclopropyl-7-(3-aminopyrrolidinyl) derivative 16 showed a positive response. These results suggested that the introduction of a fluorine atom into the 3-aminopyrrolidinyl substituent resulted in favorable influence on genetic toxicity as well as into the N-1 cyclopropyl substituent.

Human persistent left superior vena cava with doubled coronary sinus.

A heart with a persistent left superior vena cava was found in a 72 year old man during routine dissection. The right superior vena cava was absent. The right brachiocephalic vein crossed over the aortic arch and its branches to the left side of the heart. This variation reportedly occurs in about 0.16% of humans. The azygos vein was, as a result, also left sided with a hemiazygos vein on the right side. The left superior vena cava drained into a channel contained with the posterior wall of the left atrium of the heart above, and parallel with the coronary sinus. The channel of the left superior vena cava opened into the right atrium above the opening of the coronary sinus. The coronary sinus may have been doubled during embryonic development. The two vessels were joined by a small connecting vessel.

The true nature of the adductor brevis dually innervated by the anterior and posterior branches of the obturator nerve in humans.

To clarify the true nature and the mechanism of the human adductor brevis (specific adductor brevis, SAB) innervated dually by the anterior and posterior branches of the obturator nerve, we have carried out gross anatomical examination of 100 body halves of 50 adult Japanese cadavers. The SAB was found in 23 of the 100 thighs (23.0%), and its anterior and posterior surfaces received respectively the twigs from the anterior branch of the obturator nerve and the filament(s) from the posterior branch. The filament(s) was either indirectly derived from the medial stratum of the posterior branch through the formation of a common trunk with the twigs distributed in the obturator externus (14/23 thighs, 60.9%) or directly originating in the medial stratum of the posterior branch of the obturator nerve (9/23 thighs, 39.1%). In the close examination of the intramuscular distribution of the nerve to the SAB, the region innervated by the anterior branch of the obturator nerve could clearly be distinguished from that innervated by the posterior branch. The obturator nerve received fibers from L1234 (2/23 thighs) or from L234 (21/23 thighs), and the posterior branch of the obturator nerve ran through the obturator externus (18/23 thighs, 78.3%) or ran over the obturator externus (5/23 thighs, 21.7%), and finally emerged into the thigh. In view of the mode of origin of the filament(s), the structural element of the filament(s), and the pattern of entry of the filament(s) into the SAB, the fasciculus of the SAB, which is innervated by the posterior branch of the obturator nerve, was considered to originate in the obturator externus. Thus, the true nature of the SAB was concluded to be a complex product which was formed by a mechanism in which the fasciculus, which had separated from the obturator externus during the process of ontogeny, fused secondarily to the posterior surface of the regular adductor brevis. From findings in our series of studies, it was estimated that the maximum frequency of occurrence of the SAB could be 56%. Furthermore, from a statistical point of view, the segmental composition or course of the obturator nerve is not considered to be related to either the formation or the incidence of this muscle.

Musculus comitans nervi mediani (M. palmaris profundus).

Palmaris profundus muscles were found in two cadavers during routine dissection of the upper limb. This rare muscle was found in two forms. In the first case, the muscle resembled a diminutive palmaris longus with the belly arising from the common flexor tendon. In the second case however, a reversed muscle with the belly emerging from beneath the transverse carpal ligament and its long thin tendon extended to and inserted in the common flexor tendon. The similarity therefore of these muscles to variable forms of palmaris longus is remarkable but they differed in one very important aspect from palmaris longus. The muscles are of special interest because, in both cases, the muscles were found enclosed in a common fascial sheath with the median nerve. These unusual muscles, in spite of mimicking palmaris longus, may perhaps, be better named "musculus comitans nervi mediani" to denote their very important relationship to the median nerve, that of being the intimate traveling companion of the median nerve through the forearm and into the hand by way of the carpal canal beneath the transverse carpal ligament. In one case, a well developed median artery was also found which also entered the carpal canal along with the median nerve and its muscular companion.

Anatomic considerations of the peroneal nerve for division of the fibula during high tibial osteotomy.

Twenty legs in 10 cadavers were dissected to determine the course of the deep peroneal nerve from its origin to its termination. Particular attention was paid to defining: (1) its relationship to palpable landmarks, (2) the angle of the course of its proximal portion against the long axis of the fibula, (3) distribution of the proximal branch to the extensor hallucis longus muscle, and (4) safe areas of osteotomy in the proximal fibula during high tibial osteotomy. The extensor hallucis longus was often supplied by only one branch from the deep peroneal nerve at 99.8 mm (31.7%) distally from the apex of the fibula; this seems to explain why osteotomy of the fibula at its proximal one third often causes paralysis of this muscle. The findings suggest that safe areas for osteotomy in the proximal fibula during high tibial osteotomy are located up to 20.5 mm (6.5%) distal to the tip of the fibular head and that the safe angle of a periosteal incision against the fibular neck area is 64.1 degrees.

Staining of pancreatic centroacinar cells, liver bile canaliculi and testicular Leydig cells with a monoclonal antibody against adrenocortical cells.

The immunoreactivity of a monoclonal antibody against cell suspensions from guinea pig adrenal glands was examined at light- and electron-microscopic levels. In addition to the cell surface membrane of adrenocortical cells, the antibody labeled specific sites in the pancreas, liver and testis, but did not label any of the other tissues examined. In the pancreas, microvilli-like processes and the cell surface membrane of centroacinar cells were immunoreactive to the antibody. The microvilli of interlobular duct cells and pancreatic duct cells were also immunoreactive. In the liver, bile canalicular microvilli of hepatocytes were exclusively labeled. Membrane structures of cell organelles, mainly mitochondria, in testicular Leydig cells were also labeled. Immunoblot analysis showed that the monoclonal antibody bound to two common bands at molecular weights of approximately 62 kDa and 110 kDa in the pancreas, liver, testis, and adrenal gland. The two bands reacted with the digoxigenin-conjugated lectin, Sambucus nigra agglutinin (SNA), which recognizes sialic acid linked alpha (2-6) to galactose. Reaction patterns of SNA in the pancreas, liver and testis were similar to those of the monoclonal antibody; pancreatic centroacinar cells and interlobular duct cells, hepatocyte bile canaliculi and testicular Leydig cells were densely stained with SNA. Thus, the monoclonal antibody recognizes two common membrane glycoproteins containing sialic acids in the pancreas, liver, testis and adrenal cortex.

Structural organization of the initial lymphatics in the monkey mesentery and intestinal wall as revealed by an enzyme-histochemical method.

The structure and distribution of the initial lymphatics in whole mount preparations of the mesentery and intestinal walls of the Japanese monkey (Macaca fuscata) were studied using an enzyme-histochemical method (Kato et al., 1991, 1993). The lymphatic walls, colored dark brown by their positive 5'-nucleotidase (5'-Nase) activity, were clearly distinguished from the blood vessels (especially capillaries and arterioles) which were colored blue due to their positive alkaline phosphatase activity. The specificity and localization of both enzyme reactions were confirmed by comparative histochemical studies of the same specimen under a light microscope and scanning or transmission electron microscopes. Application of this staining method takes advantages of the overview preparation of flat membranous organs. The mesenterial area was generally lobulated in distribution with collecting lymphatics and blood vessels. At about the center of each lobule enclosed by the vessels, 5'-Nase-positive initial lymphatics assumed tubulo-saccular shapes, branching antler-like figure to form dense networks in the main lymph vascular pathway. Their apical parts revealed marked knob-like blind endings demarcated by a thin endothelial wall. No direct interconnection was recognizable between the lymphatic space and the tissue interstitium as a prelymphatic fluid pathway. In such preparations, 5'-Nase-positive lymphatic islands could be found isolated from the lymphatic network.

A supernumerary muscle between the adductors brevis and minimus in humans.

In order to elucidate the nerve supply of a supernumerary muscle observed between the adductors brevis and minimus in humans and to investigate its true nature and mechanism of formation, 100 body halves from 50 adult Japanese cadavers were subjected to gross anatomical examination. A supernumerary muscle was noted in 33 (33.0%) out of the 100 thighs. In each of these thighs, it arose from the upper part of the inferior ramus of the pubis and ran obliquely downwards and laterally. It was inserted into the anterior surface of the insertion aponeurosis of the adductor minimus (17/33 thighs, 51.5%), the upper part of the pectineal line (9/33 thighs, 27.3%), or the posterior side of the base of the lesser trochanter (7/33 thighs, 21.2%). It was supplied, from its posterior aspect, by a filament from the twig originating from the posterior branch of the obturator nerve and being distributed to the superficial fasciculus of the obturator externus (18/33 thighs, 54.5%) or by a twig directly originating from the posterior branch (15/33 thighs, 45.5%). The obturator nerve received fibers from L1234 (4/33 thighs), L234 (25/33 thighs), L2345 (2/33 thighs) or L34 (2/33 thighs) and, moreover, its posterior branch ran through (25/33 thighs, 75.8%) or over (8/33 thighs, 24.3%) the obturator externus to emerge into the thigh. Based on the topographical-anatomical relationships of this muscle to its nerve supply, it seems probable that it is formed by separation from the superficial layer of the obturator externus and changes into an independent structure during the process of ontogeny. Furthermore, from a statistical standpoint, the segmental composition or course of the obturator nerve is not related to either the formation or incidence of this muscle.

Enzyme-histochemical study on the fine distribution of the intramural lymphatics at the ileocecal junction of the monkey intestine.

The fine distribution of the intramural lymphatics at the ileocecal junction of the monkey intestine, especially in the lamina propria of the ileocecal valve, was examined by light and electron microscopy using enzyme-histochemical staining. The distinction between the lymphatics and the blood vessels was made by light microscopy on cold glycol methacrylate resin (JB-4) sections using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining. The lymphatics were found to show strong 5'-Nase activity and to comprise irregularly shaped vessels or spaces. The central lymphatic vessels (central lacteals) in low villi were seen to lie deep within the ALPase-positive subepithelial capillary network. In the ileum side of the ileocecal junction, the 5'-Nase-positive lymphatics were seen both in the superficial layer and the deep layer of the lamina propria. On the contrary, in the cecum side the mucosal lymphatics were less numerous in the superficial layer and were distributed mainly in the deep layer near the lamina muscularis mucosae. These lymphatics ran through the lamina muscularis and merged into the lymphatic network in the submucosa. The submucosal lymphatics communicated with each other at the ileocecal junction and formed a well-developed network. Collecting lymphatics with valves were also seen near the tunica muscularis (sphincter muscle) in the deep submucosa. These lymphatics traversed the muscle layer and drained into the subserosal lymphatics.

Enzyme-histochemical visualization of lymphatic capillaries in the mouse tongue: light and electron microscopic study.

The distinction between lymphatic capillaries and blood capillaries in the mouse tongue was studied enzyme-histochemically by light and electron microscopy. The lymphatic walls are characterized by a strong 5'-nucleotidase (5'-Nase) activity, whereas those of the blood capillaries reveal a significantly lower or no activity. The alkaline phosphatase (ALPase) activity, on the other hand, is markedly higher in the blood capillaries than in the lymphatic capillaries. The specific reaction of 5'-Nase activity in the lymphatic capillaries is obtained by simultaneous inhibition of ALPase on incubation in a medium (Wachstein and Meisel, 1957) with L-tetramisole for 5'-Nase histochemistry. The distribution and intensity of 5'-Nase activity in the lymphatic capillaries can be adequately visualized by comparison with serial cryostat sections for histochemical detection under light and backscattered imaging scanning electron microscopes. The reaction products of the 5'-Nase activity are localized on the outer surface of the cell membrane of the lymphatic endothelial cells, whereas those in the blood capillaries reveal a weak or no reaction. The present results demonstrates satisfactory isolated visualizations of 5'-Nase activity in the lymphatic capillaries and of ALPase activity in the blood capillaries.

Nerve supply to and the true nature of anterior supracostal muscle: three cases of the anterior supracostal muscle innervated by the external muscular branch of the first intercostal nerve having lateral cutaneous branch.

316 body-halves were examined in order to clarify nerve supply to human anterior supracostal muscle and to reveal the true nature of this particular muscle. 14 body-halves were found to have this muscle. It was innervated by the external muscular branch of the first intercostal nerve having lateral cutaneous branch in 3 out of 14 cases, and by the external muscular branch of the first intercostal nerve lacking lateral cutaneous branch in the remaining 11. As the former 3 cases have not been reported yet, we have examined the anatomy of these muscles in detail, and nerve supply to the anterior supracostal muscle and the true nature of this muscle were discussed in the present study. As a result, it was found that the anterior supracostal muscle is innervated essentially by an element of the components of the external muscular branch, and the muscle is derived from the first external intercostal muscle or rarely from the first and second external intercostal muscles. Moreover, it was inferred that the frequency of occurrence of the anterior supracostal muscle innervated by the intercostal nerve having lateral cutaneous branch is very low.

The human musculus obliquus externus abdominis innervated by ramus muscularis externus of intercostal nerves.

Each of the uppermost slips of the bilateral M. obliquus externus abdominis originated from the fifth rib and was innervated from its inner surface by the Ramus muscularis externus of the fifth intercostal nerve, in one corpse. Furthermore, the slip originating from the sixth rib of the right M. obliquus externus abdominis was innervated not only by the lateral cutaneous branches of the sixth and seventh intercostal nerves from its outer surface, but also by the Ramus muscularis externus of the sixth intercostal nerve from its inner surface. The slip originating from the fifth rib was given us an aspect in appearance as if it were the uppermost slip of origin of this muscle, but its true nature is considered to be the M. obliquus abdominis externus profundus from the viewpoint of its innervating nerve and pattern. In addition, the slip originating from the sixth rib is considered to be a substance formed by the dorsal adhesion of the M. obliquus abdominis externus profundus originating from the sixth rib to the slip of the M. obliquus externus abdominis originating from the same rib.