Cellular expression of the monocarboxylate transporter (MCT) family in the placenta of mice.

Lactate plays an important role as an alternative energy substrate, especially in conditions with a decreased utility of glucose. Proton-coupled monocarboxylate transporters (MCTs) are essential for the transport of lactate, ketone bodies, and other monocarboxylates through the plasma membrane and may contribute to the net transport of lactate through the placental barrier. The present study examined the expression profile and subcellular localization of MCTs in the mouse placenta. An in situ hybridization survey of all MCT subtypes detected intense mRNA expressions of MCT1, MCT4, and MCT9 as well as GLUT1 in the placenta from gestational day 11.5. The expression of MCT mRNAs decreased in the intensity at the end of gestation in contrast to a consistently intense expression of GLUT1 mRNA. Immunohistochemically, MCT1 and MCT4 showed a polarized localization on the maternal side and fetal side of the two cell-layered syncytiotrophoblast, respectively. The membrane-oriented localization of MCTs was supported by the coexistence of CD147 which recruits MCT to the plasma membrane. However, the subcellular arrangement of MCT1 and MCT4 along the trophoblastic cell membrane was completely opposite of that in the human placenta. Although we cannot exactly explain the reversed localization of MCTs between human and murine placentas, it may be related to differences between humans and mice in the origin of lactate and its utilization by fetuses.

Functional mechanism of lung mosaic CT attenuation: assessment with deep-inspiration breath-hold perfusion SPECT-CT fusion imaging and non-breath-hold Technegas SPECT.

BACKGROUND: The functional mechanism of lung mosaic computed tomography attenuation (MCA) in pulmonary vascular disease (PVD) and obstructive airway disease (OAD) has not yet been fully clarified. PURPOSE: To clarify the mechanism of MCA in these diseases by assessing the relationship between regional lung function and CT attenuation change at MCA sites with the use of automated deep-inspiratory breath-hold (DIBrH) perfusion single-photon emission computed tomography (SPECT)-CT fusion images and non-breath-hold Technegas SPECT. MATERIAL AND METHODS: Subjects were 42 PVD patients (31 pulmonary thromboembolism, four primary/two secondary pulmonary hypertension, and five Takayasu arteritis), 12 OAD patients (five acute asthma, four obliterative bronchiolitis, and three bronchiectasis), and 12 normal controls, all of whom had MCA on DIBrH CT. The relationship between regional lung function and CT attenuation change at the lung slices with MCA was assessed using DIBrH perfusion SPECT-CT fusion images and non-breath-hold Technegas SPECT. The severity of perfusion defects with or without MCA was quantified by regions-of-interest analysis. RESULTS: On DIBrH CT and perfusion SPECT, in contrast to no noticeable CT attenuation abnormality and fairly uniform perfusion in controls, 60 MCA and 274 perfusion defects in PVD patients, and 18 MCA and 61 defects in OAD patients were identified, with a total of 77 ventilation defects on Technegas SPECT in all patients. SPECT-CT correlation showed that, throughout the 78 MCA sites of all patients, lung perfusion was persistently decreased at low CT attenuation and preserved at intervening high CT attenuation, while lung ventilation was poorly correlated with CT attenuation change. The radioactivity ratios of reduced perfusion and the intervening preserved perfusion at the 78 perfusion defects with MCA were significantly lower than those at the remaining 257 defects without MCA (P<0.0001). CONCLUSION: Although further validation is required, our results indicate that heterogeneous pulmonary arterial perfusion may be a dominant mechanism of MCA in PVD and OAD.

Calcium signaling in terminal Schwann cells associated with lanceolate sensory endings in rat vibrissae.

Transient elevations of the intracellular Ca2+ concentration ([Ca2+]i) in glia mediate various cell activities to regulate neuronal functions. The present study focuses on spatiotemporal dynamics of Ca2+ signaling in terminal Schwann cells, glial elements of the lanceolate sensory endings innervating the rat vibrissa. Arrays of lanceolate endings were enzymatically isolated from the vibrissal follicle, and subjected to [Ca2+]i image recording by time-lapse confocal microscopy. Each terminal Schwann cell displayed a round cell body, and extended long cytoplasmic stalks, which branched into two to five lamellae resting on different axon endings. Each axon ending, on the other hand, was covered on both flattened sides of the lancet by two Schwann lamellae of different cell origin. Thus the peripheral glia constituted an extensive network connecting the sensory endings. Image analyses of [Ca2+]i characterized the Schwann lamellae as functional compartments that can independently generate Ca2+ signals: these cell portions primarily responded to local mechanical stimuli with a [Ca2+]i spike, and individually initiated [Ca2+]i oscillations during bath application of the sensory modulator adenosine 5'-triphosphate (ATP). The stimulus-induced signals sometimes propagated along the glial network to activate neighboring lamellae after a delay of 2-4 sec. These findings suggest that the terminal Schwann cells contribute both to individual regulation and total coordination of the arrayed sensory endings.

Three-dimensional microanatomy of mechanoreceptors and their possible mechanism of sensory transduction.

The fine structure of sensory nerve endings and their topographical relationships with surrounding tissues were examined by a combination of scanning and transmission electron microscopy in order to analyze the mechanism of mechanoreception. Observations were reported on Ruffini endings in periodontal ligaments of rat incisors, and on longitudinal lanceolate endings surrounding rat sinus hairs. Both types of receptors exhibited the triplet structure known as the axon-Schwann cell complex; a flattened axon terminal was sandwiched between two Schwann cell lamellae. The two receptor types additionally revealed their specific modifications at each distal end, where fine tuft-like processes of Schwann cells projected into surrounding tissues with finger-like projections of an axon terminal attached to their bases. In the Ruffini endings of the periodontal ligament, the terminal tufts coiled about collagen bundles in favor of continuous transmission of tissue distortions to their accompanying axon fingers. In the lanceolate endings of sinus hair follicles, the Schwann cell tufts were suspended in an amorphous matrix with only their end feet anchored to rigid connective tissue elements. Terminal axon fingers associated with these Schwann cell processes were supposed to transiently deflect during acceleration and deceleration phases of a given hair movement because of inertia. The present study proposes the terminal tuft of Schwann cell processes and their accompanying axon fingers as a structural complex which potentially contributes to mechano-electric transduction.

Scanning electron microscopic observation of Merkel cells in the lamprey epidermis.

The Merkel cell in the epidermis has generally been regarded as a mechanoreceptor which detects tissue deformations with its microvilli, and subsequently releases certain transmitters to nerve endings. In order to analyze the mechanism of mechanoreception, the fine structure of lamprey Merkel cells and their relationships with surrounding tissue were examined by scanning electron microscopy (SEM) after exposure of the cells by NaOH maceration, as well as by transmission electron microscopy (TEM) according to a conventional method. By SEM, lamprey Merkel cells revealed small round cell bodies bearing numerous microvilli on the upper and lower poles in accord with previous TEM reports. Combined SEM and TEM observations showed that the Merkel cell bodies were tightly held in corresponding concavities of other epidermal cells, with some desmosomes connecting the cells with each other. On the other hand, microvilli of the Merkel cells extended freely in intercellular spaces bound with complex microplicae of epidermal cells. The regional difference in mechanical anchorage of the Merkel cells probably leads to transient deflection and subsequent recovery of their microvilli during a given mechanical stimulation, suggesting rapidly adapting mechanoreception by the cells.

Early anchoring collagen fibers at the bone-tendon interface are conducted by woven bone formation: light microscope and scanning electron microscope observation using a canine model.

To clarify the early process of recovery at the bone-tendon interface, we used light microscopy and SEM to examine the process of anchoring of collagen fibers to bone in a canine model. At two weeks, tendon, scar tissue, woven bone and lamellar bone were present at the insertion site. SEM revealed anchoring of collagen fibril bundles of the scar to the woven bone. By 4 weeks, the number of anchoring fibers had increased and a parallel arrangement of fibers was observed. SEM demonstrated deep penetration of fibers into the woven bone layer. In addition, the fibers were observed to project into and intermingle with the scar tissue. By 6 weeks, the anchoring fibers had developed fully and were distributed densely over the interface. SEM also revealed that the collagen fibril bundles in the scar tissue had connected with the collagen fibrils of the woven bone by way of the anchoring bundles. The woven bone was identifiable throughout the early stages of recovery as the interface between soft tissue and hard tissue. Throughout all experimental periods, no staining was observed at the interface of the tendon and bone by Saffranin-O. The formation of woven bone was important during early recovery of the tendon-bone interface prior to the completion of fibrocartilage-mediated insertion.

Development of a new calcium phosphate cement that contains sodium calcium phosphate.

A cement powder consisting of sodium calcium phosphate, Na3Ca6(PO4)5, in addition to tetracalcium phosphate and beta-tricalcium phosphate was prepared by pulverizing blocks of 4 wt% sodium-, 11 wt% carbonate-containing apatite samples that were heated at 1700 degrees C for 5 h. When mixed with 30 wt% malic acid or citric acid at a powder liquid ratio of 3:1, the cement set in 3 or 7 min at room temperature with compressive strength being around 52 or 27 MPa. In HeLa-cell cultures, the cement mixed with malic acid was less cytotoxic than the cement mixed with citric acid, which was far less cytotoxic than a commercial carboxylate cement used as a negative control, suggesting malic acid to be superior to citric acid as a liquid in this regard. Similar findings were also obtained with osteoclasts, of which culture experiments clearly suggested that the number of osteoclasts on the cement mixed with malic acid was significantly greater than that on the cement mixed with citric acid. Since osteoclastic response to substrates could be used as a maker in evaluating their bioresorbability associated with osteoclasts, the above finding may suggest that the cement that is to be mixed with malic acid would be more useful as bone substitutes.

[Lambert-Eaton myasthenic syndrome: diagnosis and treatment].

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of neuromuscular and autonomic transmission in which IgG autoantibodies lead to presynaptic voltage-gated calcium channel (VGCC) loss, or as a paraneoplastic disorder in association with small cell lung carcinoma (SCLC). Recent results strongly suggest that the antibodies to P/Q-type VGCC are the principal pathogenic factors in LEMS. Here, we present diagnosis and treatment of LEMS patients. Proximal weakness, depressed tendon reflexes, autonomic symptoms, and electrical posttetanic potentiation together are essential to accurately diagnose LEMS. The diagnosis is established immunologically by the presence of anti-P/Q-type VGCC antibodies, detected using the (125)I-omega-conotoxin MVIIC radioimmunoassay, which will be present in 85% of LEMS patients. The drug 3,4-diaminopyridine with anti-cholinesterase inhibitor is most effective in LEMS patients with or without SCLC. In LEMS with SCLC, specific tumor therapy will often improve the neurological disorder. In some cases plasmapheresis or intravenous immunoglobulin may be indicated.

Ultramicroanalysis of reducing carbohydrates by capillary electrophoresis with laser-induced fluorescence detection as 7-nitro-2,1,3-benzoxadiazole-tagged N-methylglycamine derivatives.

A method for ultramicroanalysis of carbohydrates using capillary electrophoresis with laser-induced fluorescence detection was developed, based on precapillary conversion to 7-nitro-2,1, 3-benzoxadiazole (NBD)-tagged N-methylglycamines. Although the derivatization involves two-step reactions, i.e., reductive N-methylamination followed by condensation with NBD-F, they can be carried out in a one-pot fashion and proceed quantitatively within ca. 50 min in total. Since the reaction conditions are mild, it does not cause desialylation. The derivatives can be well separated by capillary electrophoresis and sensitively detected by argon laser-induced fluorescence. It allowed detection of monosaccharides of down to nanomolar concentrations for analytical sample solution, which correspond to the attomole injected amounts, and good linearity was observed over a wide range. It was also successfully applied to analysis of N-glycans in a microgram quantity of a glycoprotein. Studies on the cleanup of derivatized product are also described in relation to N-glycan analysis.

Three-dimensional microanatomy of longitudinal lanceolate endings in rat vibrissae.

The longitudinal lanceolate endings are ubiquitous sensory terminals in the sinus and nonsinus hairs of mammals that form a palisade around the hair follicle. To analyze how the nerve endings detect hair movements, the present study re-examined their fine structure and relationships with surrounding connective tissue in rat vibrissae by using a combination of three methods: immunohistochemistry for S-100 protein, scanning electron microscopy of NaOH-macerated specimens, and transmission electron microscopy of serial sections. Observations showed the lanceolate endings to be represented by triplet units with a flattened axon terminal flanked on each side by a Schwann cell lamella, as reported previously. Two distinct parts were discriminated in the lanceolate ending: a principal portion in which the axon terminal protruded numerous fine fingers from between the Schwann cell coverings, and an apical cone that enclosed a large axon finger in an attenuated Schwann sheath. Long foot processes of Schwann cells fanned out distally from each apical cone. The principal portions of the lanceolate endings were firmly linked to the surrounding connective tissue by the narrow edges equipped with axon fingers, suggesting their continuous deformation by sustained hair deflections. In contrast, the apical cones were freely suspended in an amorphous matrix with only the end feet of the Schwann cell projections attached to rigid tissue elements. This part of the ending was proposed as a possible transducer site to generate rapidly adapting receptor potentials, both retreating and overshooting during the acceleration and deceleration phases of a given vibrissal movement.

Use of glass slides coated with apatite-collagen complexes for measurement of osteoclastic resorption activity.

This study was designed to evaluate the use of apatite-collagen complexes (ACC) coated onto glass slides for measurement of osteoclastic resorption activity. ACC-coated glass slides were prepared by immersion in beta-glycerophosphate solution for 7-14 days after glass slides coated with type I collagen had been treated with alkaline phosphatase and phosvitin. Osteoclast-containing cell suspensions were prepared from the long bones of 1-day-old rabbits and were seeded in medium 199 (containing 10% FBS) onto ACC-coated glass slides. After allowing the cells to attach for 1.5 h, the glass slides were incubated for periods of up to 96 h. The cells were observed by scanning electron microscopy and cytochemically for tartarate resistant acid phosphatase (TRAP) activity. Some slides were treated with FITC-phalloidin and anti-type I collagen antibody. TRAP-positive multinucleated cells were located in transparent spaces on the glass slides. These spaces did not stain immunohistochemically with anti-type I collagen antibody. Podosome formation was observed in the multinucleated cells facing the edge of the transparent spaces. The scanning electron microscopy demonstrated well-spread large cells located on the flattened surface on apatite particles covering the glass surface. Our results suggest that osteoclasts could resorb the apatite particles and coated collagen on the glass slide. The resorption lacunae appeared as transparent spaces, and the cytoskeleton of resorbing osteoclasts was observed in these spaces. ACC-coated glass slides could be useful for investigating the function and metabolic activities of osteoclasts.

Parasitic forms of a myxosporean in the kidney of the arctic lamprey, Lampetra japonica: an ultrastructural study.

We found frequent and unique parasitism by an unidentified myxosporean in the kidney of the arctic lamprey, Lampetra japonica, living in Japan. Trophozoites (pseudoplasmodia with or without sporoblasts) existed predominantly in the lumina of proximal urinary tubules, but were rarely found in any other regions of the kidney. Since no mature spores were produced in the trophozoites, exact identification of the species was impossible. Two parasitic forms were recognized in proximal urinary tubules: one adhering to the epithelial cells of renal tubules, and the other free-floating in the lumina of tubules. Ultrastructurally, the attaching trophozoites developed microvilli-like projections towards the apical surface of epithelial cells and vigorously interdigitated with microvilli of the brush border. In contrast, the whole surface of the floating trophozoite was smooth without any cell projections. The developed projections in the former type of trophozoite may contribute to their firm attachment to the epithelial cells and/or to absorption of nutrients via the epithelial cells. Against the myxosporean infection, the lamprey as the host exhibited a local immune reaction by disposition of numerous lymphocytes and macrophages into the epithelium of urinary tubules.

Three-dimensional ultrastructure of synoviocytes in the horse joint as revealed by the scanning electron microscope.

The synovial membrane displays a superficial cellular lining composed of two types of synoviocytes: "absorptive" macrophages (type A cells) and "secretory" fibroblast-like cells (type B cells). The types are intermingled and extend a variety of processes, rendering the cellular architecture of the synovial membrane difficult to visualize. Previous electron microscopic and histochemical studies failed to demonstrate the entire shape of synoviocytes, except our immunohistochemical study for protein gene product 9.5 in the horse joint. The present SEM study is the first to demonstrate the three-dimensional ultrastructure of synoviocytes as well as their distribution in the synovial membrane, using macerated samples from the horse carpal joints. The equine synovial membrane was largely covered by conspicuously developed synovial villi. Type A synoviocytes were closely similar to macrophages in regard to surface structure, and showed uneven distribution with the densest occurrence around the tips of the synovial villi. In the basal half of villi, type B synoviocytes, which were situated in close proximity to the synovial cavity, projected thick processes horizontally and intertwined to form a regular network of processes on the synovial surface. Those in the upper half of the villi were located in the abluminal layers and protruded an antenna-like process into the joint cavity with tips covered with long microvilli, in addition to forming the superficial plexus of processes. Type B cells were also provided with fine, membranous extensions that tended to cover the surface of synovial intima. The meshwork of horizontal processes, the antenna-like processes, and the membranous processes imply advantages in not only secretion but also sensation and regulation of the barrier function in the synovial membrane.

Osteoclastic responses to various calcium phosphates in cell cultures.

Disks made of hydroxyapatite, beta-tricalcium phosphate, carbonate apatite, tetracalcium phosphate, alpha-tricalcium phosphate, dicalcium phosphate dihydrate, and octacalcium phosphate were incubated in osteoclastic cell cultures for 2 days. The first five salts were sintered and the last two were compressed before incubation. Osteoclasts resorbed only the sintered carbonate apatite disks. However, osteoclasts were able to resorb octacalcium phosphate disks that were preincubated for 1 day in medium without cells, indicating that surface conditioning was important for osteoclastic resorption of this calcium phosphate. Although resorption did not occur, medium calcium and phosphorus changed to an appreciable extent after a 2-day incubation of beta-tricalcium phosphate, tetracalcium phosphate, alpha-tricalcium phosphate, and dicalcium phosphate dihydrate. These changes in the medium calcium and phosphate concentrations could explain why osteoclasts appeared to have lost their activity on these calcium phosphate disks and were not capable of resorbing them. With hydroxyapatite disks no changes were observed in the medium calcium and phosphorus before and after incubation. Moreover, the osteoclasts appeared to be essentially the same as with the sintered carbonate apatite disks and with bone slices used as a control. Nevertheless, no pits or lacunae were observed on the hydroxyapatite disks, indicating that sintered carbonate apatite should be superior to sintered hydroxyapatite as a bioresorbable bone substitute.

Porosity of the epithelial basement membrane as an indicator of macrophage-enterocyte interaction in the intestinal mucosa.

The epithelial basement membrane of intestinal villi is perforated with numerous small pores, through which free cells in the lamina propria communicate with the enterocytes. This study was a comparative analysis of the pores in the basement membrane by SEM after removal of the gut epithelium with OsO4 maceration. The porosity as represented by the area fraction of the pores varied along the baso-apical axis of villi in patterns specific for each animal species examined: consistent scantiness along the entire length of villi in mice, acute elevation in the second and third distal one-sixths of villi in rats, and gradual augmentation toward the villus tips in guinea pigs. Size distribution analyses of the pores indicated their heterogeneous enlargement in the regions of elevated porosity. Concomitant observation of lamina propria macrophages by histochemical labelings and by conventional TEM showed that the cells specifically clustered beneath the hyperporous basement membrane, with their thick processes penetrating it. The spatially-regulated patterns of perforation of the epithelial basement membrane indicate phase-specific interventions of lamina propria macrophages in the maturation or aging of enterocytes, which steadily proliferate in crypts and exfoliate at the villus tips.

Generation of intestinal T cells from progenitors residing in gut cryptopatches.

Cryptopatches (CPs) are part of the murine intestinal immune compartment. Cells isolated from CPs of the small intestine that were c-kit positive (c-kit+) but lineage markers negative (Lin-) gave rise to T cell receptor (TCR) alphabeta and TCR gammadelta intestinal intraepithelial T cells after in vivo transfer or tissue engraftment into severe combined immunodeficient mice. In contrast, cells from Peyer's patches and mesenteric lymph nodes, which belong in the same intestinal immune compartment but lack c-kit+Lin- cells, failed to do so. These findings and results of electron microscopic analysis provide evidence of a local intestinal T cell precursor that develops in the CPs.

Three-dimensional microanatomy of perineuronal proteoglycan nets enveloping motor neurons in the rat spinal cord.

Spinal motor neurons possess reticular coats of extracellular matrix proteoglycans on their somata and proximal dendrites. In order to define the anatomical background of the network, spatial relationships of the perineuronal proteoglycans with synaptic boutons and astrocyte processes were analyzed in rat motor neurons by TEM after histochemical detection of the substances with cationic iron colloid, and by SEM after exposure of the cytoarchitecture with NaOH maceration. Narrow intercellular channels filled with proteoglycan were found to extend along the surface of the neurons to form a homogeneous network of a mesh size of about 1 microm. The system of perineuronal channels consisted of two parts: a primary intervaricose net which meandered among synaptic boutons on the surface of the motor neuron, and secondary subvaricose nets which irrigated interfaces between larger boutons and the neuron. No elements in the perineuronal cytoarchitecture coincided with the meshwork of proteoglycan, indicating the involvement of postsynaptic factors in the distribution of the substance. Thin astrocyte processes surrounding the neurons formed a distinct network with heterogeneous meshes corresponding to boutons of various sizes. The perineuronal glial nets extended their surface area in contact with the intervaricose nets of proteoglycan by complex cellular interdigitations. The subvaricose nets of proteoglycan compartmentalized multiple synapses on large boutons, suggesting an involvement in the division of the synapses during development.

Neonatal granulocytosis is a postpartum event which is seen in the liver as well as in the blood.

In a recent series of studies, we demonstrated that stress in humans and animals, with resultant sympathetic nerve strain, induces severe granulocytosis, because granulocytes carry adrenergic receptors on the surface. Because activated granulocytes produce free radicals and superoxides, they sometimes induce tissue damage if the stress is too strong or continuous. Human neonates are also known to show high levels of granulocytes in the peripheral blood. In this study, we investigated whether such neonatal granulocytosis are a stress-associated response at birth. Both human and mouse materials, before and after birth, were used. The number of leukocytes in the blood, as well as some other factors in the serum, were measured. Although levels of granulocytes were found to be low in fetal humans and mice, they increased sharply after birth. In parallel with this postpartal granulocytosis, transaminases in sera increased transiently. In reference to results of a transient elevation in the levels of catecholamines at birth in mice, all these phenomena resemble stress-associated responses. Indeed, fatty liver and hematopoietic destruction in the liver were also observed in mice and humans. At this time, the production of inducible nitric oxide synthase (iNOS) by granulocytes in the liver was evident. These results suggest that neonatal granulocytosis is a postpartum event which results from various stresses (e.g., oxygen stress) at birth. This event may be responsible for such well-known neonatal phenomena as the termination of fetal hematopoiesis in the liver and as neonatal jaundice.

Scanning and transmission electron microscopy of Ruffini endings in the periodontal ligament of rat incisors.

The Ruffini organ is an arborized axon ending categorized as a low-threshold stretch receptor. We have previously shown that the lingual periodontal ligament of rat incisors is densely innervated with Ruffini endings. In the present study, fine structures in the surface of the periodontal Ruffini endings and their topographical relationship with the surrounding collagen fibers were observed by a combination of scanning and transmission electron microscopy to analyze the mechanism of the stretch reception. The entire length of the branches of the Ruffini endings, excepting their terminal portions, corresponded well with those depicted by previous investigators in the following points: (1) their cylindrical appearance covered by Schwann cell processes; (2) the presence of numerous axon fingers protruding through gaps in the Schwann sheath and; (3) their isolation from collagen fibers by multilayered basal lamina. On the other hand, tips of the axon branches-together with their Schwann sheaths-became attenuated and projected into tight bundles of collagen, indicating their susceptibility to mechanical deformations of the surrounding tissue. Margins of the axon terminals were conspicuously ruffled with long tongue-like projections of Schwann cells. The Schwann cell tongues twined around collagen bundles in their distal portions, and associated closely with fine axon projections in their proximal portions, suggesting their involvement in the mechanical transmission of stimuli to axon terminals.

Setting properties of alginate impression materials in dynamic viscoelasticity.

The viscoelastic properties of three alginate impression materials were investigated. A landmark of working time was calculated using a raw phase-time curve. A landmark of setting time was calculated from an inflection point of the divided difference of the first order in the share modulus-time curve. As a result, the working time was common at the point of delta = 45 degrees in variable frequencies. The mean value was obtained with less deviation. The setting times obtained were similar for variable frequencies. The mean values showed a distinct difference for each impression material.