Morphological analysis of cat masseteric motoneurons after intracellular staining with horseradish peroxidase.

Intracellular injection of horseradish peroxidase (HRP) into 58 masseteric motoneurons identified by antidromic activation was performed in cats under pentobarbital anesthesia. Monosynaptic EPSPs were evoked by masseteric nerve stimuli in 52 cells, and were absent in the remaining six cells. The antidromic nature of the evoked spikes was confirmed by IS-SD separation observed at high frequency (50 Hz) stimulation. Motoneurons with monosynaptic excitation from masseter afferents showed IPSPs following stimulation of lingual and inferior alveolar nerves. Motoneurons which did not show monosynaptic excitation from masseter afferents showed no IPSPs from the above nerves. There were no differences in cell size or the number of stem dendrites between motoneurons with and without monosynaptic EPSPs. No recurrent collaterals were observed in any motor axons. Motoneurons with monosynaptic EPSPs were located at all rostrocaudal levels throughout the trigeminal motor nucleus, whereas motoneurons without such EPSPs were encountered only at the middle level. Dendrites of motoneurons with monosynaptic EPSPs did not extend into the medial portion of the nucleus where motoneurons innervating the anterior belly of the digastric muscle were located. In contrast, motoneurons without monosynaptic EPSPs had dendrite branches extending well into the medial part. The results show that there are two subpopulations of masseteric motoneurons that differ in peripheral inputs as well as dendritic morphology.

Adult T cell leukemia clinically manifested with ascites and pleural effusion.

A 62-year-old man with adult T cell leukemia (ATL) presented with complaints of anorexia and abdominal fullness. Evaluation revealed ascites and pleural effusion, but no lymph node swelling, hepatosplenomegaly, or skin involvement. The diagnosis of ATL was made by the detection of specific surface markers for T lymphocytes in pleural effusion and ascitic fluid, and by determination of human T cell leukemia virus type I (HTLV-I) proviral DNA integration in mononuclear cells of pleural effusion. This case was considered a rare type of ATL with infiltration of the pleura and peritoneum.

Regenerative process after experimental injury of hypoglossal nerves in guinea pigs.

The nerve regenerative process has been investigated by many studies. However, the quantification of the degree of crush of peripheral nerves has not yet been performed. The aim of this study was to determine and standardize the ligature intensity and crush level of the hypoglossal nerve of guinea pigs. The compound action potentials evoked by electric stimulation were used as an index of the degree of nerve crush. To demonstrate nerve regeneration after ligating and crushing of the right hypoglossal nerve, fluorescein isothiocynate conjugated cholera toxin-B subunit (CTb-FITC) was injected into the intact fiber of the left hypoglossal nerve, and the central side from the crushed region of the right hypoglossal nerve fiber. The total cross sectional area of the retrograde-labeled hypoglossal motoneurons was investigated under a confocal laser scanning microscope. The results of the evaluation using CTb-FITC indicated that the nerve regeneration occurred from two weeks after crush and recovered in six weeks.

[An approach of in vivo measurement for ultrasonic myocardial tissue characterization].

The purpose of this study is to characterize regional myocardial alternations of reflected ultrasound throughout a cardiac cycle in normal and ischemic myocardium. Integrated ultrasonic backscatter (2-5 MHz) gated R wave of ECG was measured at the base, middle and apex in 4 dogs, and the apex before and after ischemia in other 4 dogs. Quantitative ultrasonic backscatter (IB) was reflected to a steel reflector. At the apex and middle, where a cyclic pattern of IB was discernible, maximum values were recorded near end diastole and minimum near end systole. The amplitude of the variation of IB throughout a cardiac cycle for each region increase progressively from base to middle to apex. Time-averaged IB over a cardiac cycle (averaged IB) was similar for each area of the heart (base = -50.6 +/- 0.5 dB (mean +/- SE), middle -49.4 +/- 0.5 dB, apex -49.7 +/- 0.6 dB. p = NS for comparison of any two regions). After occluding left anterior descending coronary artery for 30 minutes, the variation of IB was markedly blunted and averaged IB increased significantly (-47.5 +/- 0.5 dB. p less than 0.01 compared with preocclusion (-50.8 +/- 0.5 dB]. These results suggested that IB and averaged IB may permit assessment of intrinsic geometrical changes throughout a cardiac cycle (contractile properties) and histological changes respectively.

[Analysis of factors affecting anaerobic threshold in healthy subjects].

The anaerobic threshold (AT) is used to determine the exercise capacity in patients with heart failure and healthy subjects. To determine the factors affecting AT, we determined the AT in healthy subjects, and examined the factors that determine AT in healthy subjects. One hundred and sixteen healthy subjects (79 men and 37 women) performed on a bicycle a stepwise increasing submaximal exercise. During the work test the parameters usually used in the detection of AT (Vo2, Vco2, VE), blood pressure, heart rate and oxygen saturation were recorded by a computerized system every minute. AT was determined from changes in ventilation and gas-exchange. The fat index was calculated from height and body weight measured at the beginning of the ventilatory function test. A significant correlation was obtained between AT and age, AT and fat index, AT and %VC, AT and maximum heart rate during exercise, AT and recovery rate of heart rate after exercise, and AT and Vo2 at rest. In addition, we examined the relationship among the parameters in 4 groups according to sex and age (30-49; younger, 50-69; older), because sex and age affected most parameters. We found a significant correlation between AT and fat index (older men and women), AT and %VC (younger and older women), AT and Vo2 at rest (younger and older men). We considered that the main factors that determined AT in healthy subjects were age, sex, fat index, %VC and Vo2 at rest.

The immature platelet fraction is a useful marker for predicting the timing of platelet recovery in patients with cancer after chemotherapy and hematopoietic stem cell transplantation.

Prediction of the timing of platelet recovery after chemotherapy and hematopoietic stem cell transplantation (HSCT) allows for optimal platelet transfusion. We assessed the clinical utility of the percentage value of the immature platelet fraction (IPF%) monitored using an XE-2100 automated hematology analyzer to predict the timing of platelet recovery after chemotherapy and HSCT. The IPF% was serially monitored in 31 patients with cancer who received 66 courses of chemotherapy and HSCT. In patients with cancer undergoing chemotherapy and HSCT, a transient increase in IPF% was observed 1-11 days prior to platelet recovery (>30 × 109 /l). In patients undergoing chemotherapy with a peak IPF% >10%, platelet recovery occurred significantly earlier than in those with IPF% peak values ≤10% (median periods were 2 and 5 days; P < 0.05). Platelet recovery appears to occur earlier in patients undergoing HSCT with a peak IPF% >10% than in those with IPF% peak values ≤10% (median periods were 2 and 6 days). Thus, the IPF% peak value is a useful parameter for predicting the timing of platelet recovery after chemotherapy and HSCT and has the potential to facilitate optimal platelet transfusion.

Immature platelet fraction measurement in patients with chronic liver disease: a convenient marker for evaluating cirrhotic change.

Platelet number is often used as an indicator of the severity of liver disease. Although inadequate thrombopoietin production and decreased platelet production have been proposed as major causes of cirrhotic thrombocytopenia, the underlying mechanism has not yet been fully clarified. We examined whether the measurement of the immature platelet fraction (IPF) in thrombocytopenic patients with liver dysfunction is useful as a rapid and noninvasive method for the differential diagnosis of chronic liver diseases. We examined 20 liver cirrhosis patients, 56 patients with chronic hepatitis, 9 patients with fatty liver, and 86 patients without liver disease. The percentage value of IPF (IPF%) was measured using an XE-2100 multiparameter automatic hematology analyzer. Using a receiver operating characteristic curve, we found diagnostic significance of the absolute platelet count and the absolute number of the IPF between cirrhotic patients and noncirrhotic patients, and developed a powerful multivariate discriminant analysis (MDA) function based on the platelet count and the IPF%. The diagnostic accuracy obtained by the MDA function was superior to that obtained by the absolute number of platelets and the IPF. We therefore propose our IPF% measurement for the diagnosis of liver cirrhosis.

Electron microscopic study of early formation of the tooth enameloid of a fish (Hoplognathus fasciatus). I. Odontoblasts and matrix fibers.

An electron microscope study was made on the tooth germs of Hoplognathus fasciatus in early developmental stage. Special attention was given to the odontoblasts, enameloid matrix fibers, calcification of enameloid and the hitherto controversial origin of the enameloid. 1. The ameloblasts and the odontoblasts are demarcated by a single layer of basement membrane which persists until immediately before the calcification of the enameloid matrix. 2. This histogenesis of the enameloid matrix begins with the formation of non-striated fibers 140-180 A thick. They are arranged in a direction vertical to the basement membrane and parallel to the sides of odontoblasts. Fibers with regular cross striations of 640 A periodicity later appear and the entire enameloid matrix is formed by these fibers. 3. Along with the formation of the enameloid, the odontoblasts assume a high columnar form, with a marked increase in cell organelles which show marked polarity suggesting active protein synthesis. Numerous odontoblastic processes are noted in the enameloid matrix. Granules, representing precurosors of enamleoid matrix fibers occur in the odontoblasts. Based on these findings, the enameloid matrix fibers must be of mesodermal origin. 4. Deposition of crystals of small size, needle and tube in shape, occurs in the circumference of the fiber bundles. As calcification progresses, crystals appear in the central portion of the bundles. Later, large crystals of rod and platelet shapes become intermingled. In addition to this, small crystals are fused, forming aggregates.

Phosphoprotein synthesis and secretion by odontoblasts in rat incisors as revealed by electron microscopic radioautography.

The secretory pathway of dentin phosphoproteins in rat incisors was studied by electron microscopic radioautography after the injection of 3H-serine, and the results were compared with those using 3H-proline as a tracer. Five min after injection of 3H-serine, radioactivity was found in the rough endoplasmic reticulum. At 10 min, silver grains were observed over the spherical portions of the cisface of the Golgi apparatus. At 20 min after injection, silver grains were seen over the cylindrical portions of the transface of the Golgi apparatus. The secretory granules showed the strongest reaction from 20 min to 1 hr. At 45 min, a significant labeled band appeared at the mineralization front. At 1 hr, the labeling at the mineralization front began to appear in the mineralized dentin, and after 12 hr this labeled band was located within the mineralized dentin. The pathway of 3H-proline was essentially the same as that of 3H-serine, but 3H-proline moved more slowly than 3H-serine, especially in transit from the rough endoplasmic reticulum to the Golgi apparatus. Secretory granules were heavily labeled from 30 min to 1 hr after injection of 3H-proline; no labeling was found at the mineralization front at 45 min. The labeling seen initially over the predentin was over the mineralized dentin no earlier than 6 hr after injection. The labeling pattern with 3H-serine is closely related to the localization of phosphoproteins, whereas the pattern with 3H-proline reflects the production of collagen rather than of phosphoproteins. The present radioautographic results indicate that dentin phosphoproteins are related to secretory granules and are secreted by odontoblasts at the mineralization front and also that phosphoproteins are involved in the process of mineralization of the circumpulpal dentin.

Gene expression of TGF-beta 1 and elaboration of extracellular matrix using in situ hybridization and EM radioautography during dentinogenesis.

BACKGROUND AND METHODS: The expressions of TGF-beta 1 and Type I collagen mRNA were studied by in situ hybridization and immunohistochemistry then the secretory pathway of dentin phosphoprotein was investigated electron microscopic radioautography in rat incisors. RESULTS AND CONCLUSIONS: Expression of TGF-beta 1 mRNA was observed in dental papilla cells before dentin formation. The signals were most intense in pre- and postodontoblasts and during dentinogenesis, but became weaker in the secretory region during the dentin formation. Type I collagen mRNA was expressed in essentially the same as that of TGF-beta 1. These results suggest that TGF-beta 1 plays an important role in the differentiation of, and collagen synthesis by odontoblasts. Radioautography showed radioactivity in the rough endoplasmic reticulum 5 min after injection of 3H-serine. Silver grains were observed over the cylindrical portions of the cis-face of the Golgi apparatus at 10 min and over the cylindrical portions of the transface at 20 min. The secretory granules showed the strongest reaction between 20 min and 1 h after injection. At 45 min, a significant labeled band appeared at the mineralization front. The pathway of 3H-proline was essentially the same as that of 3H-serine, but 3H-proline moved more slowly. Secretory granules were heavily labeled from 30 min; no labeling was found at the mineralization front at 45 min. The labeling pattern with 3H-serine appears to be closely related to the localization of phosphoproteins. Dentin phosphoproteins are related to secretory granules and are secreted by odontoblasts as the mineralization front, being involved in the process of dentin mineralization.

An immunocytochemical study of amelogenin proteins in the developing tooth enamel of the gar-pike, Lepisosteus oculatus (Holostei, Actinopterygii).

Previous studies have demonstrated the morphological similarity of the enamel-like layer found in the teeth of the coelacanth, lungfish and gar-pike to the enamel of tetrapods. In order to clarify the phylogenetic continuity between both structures, tooth germs of the gar-pike were immunocytochemically studied using an anti-bovine amelogenin polyclonal antibody. Intense immunoreaction was shown over the enamel-like matrix layer. Certain cell organelles associated with the secretory pathway of the ameloblasts were recognized as immunoreactive. These results indicate that the enamel-like layer of the gar-pike is a tissue homologous with the mammalian enamel because both possess a common, amelogenin-like substance.

Chlorophyll degradation in a Chlamydomonas reinhardtii mutant: an accumulation of pyropheophorbide a by anaerobiosis.

Chlorophyll degradation was investigated in cells of a chlorophyll b-less mutant of Chlamydomonas reinhardtii under aerobic and anaerobic conditions. During degradation of chlorophyll under anaerobic conditions, chlorophyll catabolite P535, an open-tetrapyrrole, was not excreted, but pyropheophorbide a was accumulated as the end product with a transient accumulation of chlorophyllide a and pheophorbide a in cells, in contrast to the breakdown under aerobic conditions. It is likely that in the absence of oxygen, degradation of chlorophyll a proceeds to pyropheophorbide a by three consecutive reactions, dephytylation, metal-releasing and demethoxycarbonylation, and then stops due to a limitation of the oxygen that the monooxygenase reaction requires for bilin formation. A novel enzyme catalyzing demethoxycarbonylation of pheophorbide a was partially purified. The enzyme activity increased dependent on the age of cells, and its increase was completely suppressed by cycloheximide. Production of P535 was also dependent on cytoplasmic protein synthesis.

Abortive secretion of an enamel matrix in the inner enamel epithelial cells during an enameloid formation in the gar-pike, Lepisosteus oculatus (Holostei, Actinopterygii).

The tooth in the gar-pike, Lepisosteus oculatus, an actinopterygian fish, is characterized by the occurrence of both enamel and enameloid, the former covering the tooth shaft and the latter, the tooth cap. Our previous research demonstrated that the enamel in this species was, as in the lungfish, immunoreactive for amelogenin, indicating its homologous nature with the mammalian tooth enamel, whereas the enameloid was completely immunonegative. The present study demonstrates that, during the early maturation stage of the enameloid formation, the inner enamel epithelial cells (IEECs) synthesize through a well-developed Golgi apparatus a fine-granular substance which is intensely immunoreactive for amelogenin. This substance was accumulated in a large saccule extended in a suprabasal zone of the cell; we were unable to find any morphological sign indicating a connection of the substance with the enameloid matrix. The abortive secretion of the enamel matrix-like substance in the IEEC during an enameloid formation was considered to be an instance of rudimental enamel formation. In the gar-pike, the synthesis of amelogenin in the IEEC has been demonstrated to occur independently from that of the enameloid matrix. The present findings demonstrate a prominent difference between the tooth enamel and enameloid.

Gene expression and localization of amelogenin in the rat incisor.

Gene expression and localization of amelogenin were studied in the developing rat incisor by the methods of in situ hybridization and immunohistochemistry. ISH revealed the first expression of amelogenin mRNA in the inner enamel epithelium of the cervical loop. The signals were clearly observed in pre-ameloblasts in the region bordering on predentin formation and became more intense toward the cells on the initial enamel matrix secretion. The maximal signals were found in the cytoplasm of secretory ameloblasts. From the terminal secretion zone, the signals then became gradually weaker toward the incisal edge but were still evident in the cytoplasm of shortening, transitional ameloblasts and those at the early maturation stage. No signals were found in the cells of the stratum intermedium and stellate reticulum throughout amelogenesis. Immunohistochemistry by means of an antibody against amelogenin C-telopeptide consisting of 12 amino acids revealed immunoreaction in the secretory ameloblasts reacting to the ISH. When a polyclonal antibody against amelogenin was used, immunoreaction was found in the distal ends of ruffle-ended ameloblasts (RA) in the maturation zone. Those results indicated that amelogenin is synthesized by ameloblastic cells from the inner enamel epithelium to the early maturation stage and is then resorbed by the RA.

Common epitopes of mammalian amelogenins at the C-terminus and possible functional roles of the corresponding domain in enamel mineralization.

The present studies were undertaken to investigate the presence of common epitopes of mammalian amelogenins at the C-terminus and the possible functional importance of the conserved C-terminal domain in enamel mineralization during mammalian amelogenesis. Enamel proteins, including the intact amelogenins and their degraded polypeptides, were isolated from the secretory enamel of pig, cow, rat, and rabbit incisors. Rabbit and rat antipeptide sera, as well as rat anti-25 kD and 20 kD pig amelogenin sera, were used to identify the amelogenins among the isolated matrix proteins of each of the animal species. The antipeptide sera were developed previously (Aoba et al. [19]) using as immunogens the two synthetic peptides, C13 and C25, which correspond to the last 12 (plus Cys for KLH-conjugation) and 25 amino acid residues of pig intact amelogenin, respectively. Reactivity of the enamel proteins with each antiserum was examined by Western blot analysis. The results of immunoblotting showed that a few enamel matrix proteins in each of the mammalian species were recognized by the anti-C13 serum, specifically, pig amelogenin at 25 kD (and trace components at 27, 22, and 18 kD), cow amelogenin at 28 kD (trace components at 26, 22, 19, and 14 kD), rat amelogenins at 28 and 26 kD (and a trace component at 20 kD), and rabbit amelogenins at 24 and 21 kD (and a trace at 13 kD). The anti-C25 serum reacted additionally with pig amelogenin at 23 kD, cow amelogenin at 27 kD (a major matrix constituent), and rabbit protein at 19 kD.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical demonstration of amelogenins and enamelins secreted by ameloblasts during the secretory and maturation stages.

Rabbit polyclonal antibodies against bovine amelogenins and enamelins which did not show any cross-reaction were raised, and ultrathin sections of rat incisors were examined by the protein A-gold and ABC methods. The immunoreactivity of amelogenins was found in dense granules in the intercellular spaces between preameloblasts, and later over the fine- and coarse-textured material. The immunoreactivity was present over the cell organelles associated with the secretory pathway, as well as pale and dark lysosomes of the presecretory and secretory ameloblasts. Here the enamel was immunolabeled in the intercrystal spaces. The immunoreactivity in multivesicular bodies was stronger in preameloblasts than in secretory ameloblasts. In the region of second ruffle-ended ameloblasts at the maturation stage, the immunolabeling was intense in the ruffled-border, but in the rough endoplasmic reticulum and Golgi apparatus, the immunolabeling was much weaker than at the secretory stage. The immunolabeling for enamelins showed essentially the same intracellular topographical pattern as that for amelogenins by the secretory stage, but was weaker. The immunoreactivity was found mainly attached to the enamel crystals. Double immunostaining of amelogenins and enamelins revealed that both immunoreactivities were present over the same cell organelles associated with secretion and lysosomal systems. It is suggested that the presecretory and secretory ameloblasts are actively involved in the secretion, degradation and resorption of enamel proteins, and that multivesicular bodies and lysosomes in the cells take part in these processes. Ameloblasts are considered to be related to the synthesis of enamelins.