Immunolocalization of an inwardly rectifying K+ channel, K(AB)-2 (Kir4.1), in the basolateral membrane of renal distal tubular epithelia.

Immunolocalization of K(AB)-2 (Kir4.1), an inwardly rectifying K+ channel with a putative ATP-binding domain, was examined in rat kidney where expression of K(AB)-2 mRNA was previously shown. Anti-K(AB)-2 antibody was raised in rabbit and then affinity-purified. An immunohistochemical study revealed that K(AB)-2 immunoreactivity was detected specifically in the basolateral membrane of distal tubular epithelia. Therefore, K(AB)-2 is the first K+ channel shown to be localized in the basolateral membrane of renal epithelia. The finding suggests that K(AB)-2 may contribute to supplying K+ to the Na(+)-K+ pump, which is abundant in the basolateral membrane of distal tubular epithelia, as well as to maintenance of the deep negative membrane potential of these cells.

Electron microscopic observation on phase transition of purple membrane.

Structural changes during the thermal phase transition of purple membrane were observed by freeze-fracture electron microscopy. Native Halobacterium halobium cells contain broad purple membrane areas about 1 micron in diameter. The boundary separating purple and red membranes is obvious. On warming at 80 degrees C, particles of red membrane spread out beyond the boundary. Then a purple membrane area is eventually divided into small areolae of similar size, about 0.1 micron in diameter. By cooling down slowly, the purple membrane area is reformed and the crystalline arrangement also restored.

Photoreceptor disk membranes of Lampetra japonica.

With the aim of characterizing photoreceptor outer segments and obtaining in situ observation of macromolecular variations due to cell types as well as adaption, we counted the number of outer segment disk membranes using electron micrographs of ultrathin sections as well as intramembrane particles on the complementary replicas of the retina of Lampetra japonica. Long photoreceptor cells (LPCs, cone-type cells) numbered 10,000/mm2 in the central as well as peripheral regions, while short ones (SPCs, rod-type cells) numbered 30,000/mm2 in the same regions. The LPC outer segment exhibited 306 disks on average during the light cycle versus 364 during the dark cycle. 12.0% of the LPC disks during the light cycle versus 13.4% during the dark cycle represented the "open" disks. The SPC outer segment exhibited 470 disks on average during the light cycle versus 507 during the dark cycle. 11.1% of the SPC disks during the light cycle versus 13.6% during the dark cycle represented the "open" disks. The LPC disk membrane contained 44.3 particles/0.01 microns 2 during the light cycle versus 39.5 particles during the dark cycle, 95% of which were derived from the protoplasmic fracture (PF) face. The SPCs contained 36.0 particles/0.01 micron 2 during the light cycle versus 43.6 during the dark cycle, 90% of which were derived from the PF-face. The present findings contradict the frequently cited hypothesis that an "open" disk, retaining continuity with the plasmalemma, is preserved characteristically into later stages by the cone outer segment. The significance of the intramembrane particles for the activity of the photoreceptor membrane is discussed.

Three-dimensional and ultrastructural ICAM-1 distribution in the choroid plexus, arachnoid membrane and dural sinus of inflammatory rats induced by LPS injection in the lateral ventricles.

To investigate immunological environment in the cerebrospinal fluid (CSF) system, ultrastructural and three-dimensional localization of intercellular adhesion molecule-1 (ICAM-1) was studied in the choroid plexus, arachnoid membrane and dural sinus of LPS-stimulated rats with immuno-SEM and TEM. The choroid plexus epithelial cells expressed rich ICAM-1 along the microvilli. The arachnoid trabeculae fibroblast-like cells demonstrated ICAM-1 expression on both sides facing the subarachnoid space moderately. The dural sinus endothelial cells, however, showed only few ICAM-1 expression and no specific localization. These results suggest that the choroid plexus and arachnoid membrane may play an important mutual role for leukocyte migration in the CSF system, and that the CSF system may function in immunoreaction independently of the vascular system with the aid of up-regulated ICAM-1 expression.

Localization of mRNAs for CDP-diacylglycerol synthase and phosphatidylinositol synthase in the brain and retina of developing and adult rats.

CDP-diacylglycerol (CDP-DAG) synthase (CDS) is known as one of the key enzymes in the lipid synthesis including phosphoinositides (PIs) production. Phosphatidylinositol (PtdIns) synthase (PIS) catalyzes a formation of PtdIns from CDP-DAG in the PI cycle which produces several second messengers. We compared the gene expression for a presumably PI cycle specific-CDS molecule and a PIS using Northern blot analysis and in situ hybridization histochemistry in the central nervous system (CNS) and retina of developing and mature rats. Whereas no significant expression for CDS was detected during the prenatal stage in any CNS regions, PIS mRNA had already expressed on the prenatal day 15 throughout the neuroaxis including the spinal cord. During the postnatal stages, the gene expression for both CDS and PIS was detected widely in the gray matters throughout the entire brain. The expression for CDS was at higher levels in the olfactory mitral cells, the occipital cortex, the subiculum and hippocampal CA1 pyramidal cells, and the cerebellar Purkinje cells. On the other hand, the expression for PIS was at high levels in the olfactory mitral cells, the cerebral cortex, the hippocampal and dentate neuronal layer and the cerebellar Purkinje and granule cells. No significant expression for CDS or PIS was detected in the ventricular germinal zone, the cerebellar external granular layers, the mature ependyma or entire white matters. The expression for CDS and PIS decreased slightly throughout the CNS on P49. The significance of the parallel and discrepant expression patterns in terms of relative intensity between the two enzyme molecules was discussed in relation to the membrane turnover and signal transduction.

Anatomical and neurochemical peculiarities of the pika retina: basis for lack of circadian rhythm of core temperature.

We have previously reported a complete lack of circadian rhythm in the body temperature of pikas in contrast to other lagomorphs. In this present study, the anatomical and neurochemical findings by immunohistochemical, photo and electron microscopic methods reveal that the photoreceptor system of this animal is poorly developed. This probably explains their stable core body temperature which help them survive in cold temperatures.

Characterization of lamprey rhodopsin by isolation from lamprey retina and expression in mammalian cells.

A visual pigment was extracted from lamprey retina and was expressed in cultured mammalian cells (293S) using a cDNA fragment isolated from lamprey retina. The extracted pigment, a putative lamprey rhodopsin, had an absorption maximum at 503 nm. The recombinant lamprey rhodopsin, reconstituted with 11-cis-retinal, showed an absorption maximum at about 500 nm. Both pigments reacted with an anti-bovine rhodopsin antibody (Rh29), which recognizes the short photoreceptor cells in lamprey retina. Unlike rhodopsins of higher vertebrates, the lamprey rhodopsin bleached gradually in the presence of 100 mM hydroxylamine even in the dark. Our results suggest that, despite its high similarities with other vertebrate rhodopsins, lamprey rhodopsin has a character different from those of higher vertebrates.

Preference of peanut agglutinin labeling for long-wavelength-sensitive cone photoreceptors in the dace retina.

Peanut agglutinin (PNA) was known for its selective binding to cone cells. In the present study, we investigated whether there was any difference in PNA binding among various subtypes of cone photoreceptor cells in the dace retina. The outer segments of the long-double- and long-single-cone cells were preferentially labeled with PNA. Ultrastructural pre-embedding labeling revealed that the binding sites of PNA were confined to the calycal processes of these cells. By contrast, only slight labeling was discerned on the corresponding regions of other types of cone cells. The results indicate that PNA can distinguish the long-wavelength-sensitive cone from the short-to-middle-wavelength-sensitive cone cells.

Lectin cytochemical analysis of glycoconjugates in photoreceptor cell membranes of Lampetra japonica.

Seven types of ferritinized lectin were used to examine the distribution of glycoconjugates on the outer segment membranes of lamprey photoreceptor cells. Ultrastructural pre-embedding labeling revealed that peanut agglutinin, soybean agglutinin and Ricinus communis agglutinin I were preferentially bound to the proximal, lateral and luminal surfaces of the long cell outer segments, whereas Griffonia simplicifolia agglutinin II and concanavalin A agglutinin were bound to the corresponding surfaces of the short cell outer segments. The results indicate that there is marked difference in the composition of glycoconjugates over the outer segment membranes between long and short photoreceptors.

Development of apical-surface structures of mouse otic placode.

In 8.0 gestation-day embryos, a central cilium and microvilli were present on the apical surface of the cells of the presumptive otic placode and other ectoderm regions as well. The cilium at this stage lacked a central pair of fibres and was regarded as a primitive cilium with 9+0 composition. The microvilli gained in length and density of distribution as soon as the otic placode began to invaginate in 8.5-d. embryos. With further development in 9.5-11.0-d. embryos, they took on a regular distribution pattern. Narrowing and concomitant bulging out appeared in the cells along the neck of the otic invagination in 8.5-d. embryos. Marked regional differences also occurred in the apical-surface structures of the cells, particularly in the relatively more advanced form of the embryos. Kinocilia of the adult vestibular sensory cells were found to lack a continuous central pair of fibres, resembling the apical cilia of the otic placode. This finding seems to suggest that a vestibular kinocilium represents a direct descendant from the primordial cilium, which generally emerges from the primordial ectodermal cells and is entirely lost from the keratinizing ectoderm in the later development.

Fine structure of guinea pig vestibular kinocilium.

The fine structure of the utricular kinocilium of the guinea pig was examined with transmission electron microscopy after treatment with tannic acid to enhance resolution of internal morphology. The utricular kinocilium was devoid of inner dynein arms and a central pair of microtubules, while a set of outer dynein arms and radial spokes was found. This supports the hypothesis that the vestibular kinocilium is non-motile. Internal electron-dense particles at the attachment sites of the stereo-kinociliar bonds were situated in the immediate periphery of the outer dynein arms, although no visible connection existed between these structures. Findings obtained in the present study seem to give insight on the mechanism of mechanosensory transduction in the vestibular sensory cells.

Microtubules of guinea pig cochlear epithelial cells.

By tannic acid staining, the 13-protofilament composition of cochlear hair cell microtubules, an impressive contrast against the 15-protofilament microtubules in cochlear pillar cells, was verified. The 15-protofilament microtubules formed a large and stiff cytoskeletal bundle in pillar cell bodies involving abundant actin filaments. The bundles were always situated vertically, i.e., longitudinally to the cell body axis, and were most numerous in the outer as well as the inner pillar cells in the basal turn, decreasing gradually toward the apex. Such gradient architecture of the pillar cell cytoskeleton can be correlated with the tuning mechanism for traveling waves of sound containing variable frequencies.

Fine structure of the lamina basilaris of guinea pig cochlea.

The lamina basilaris of guinea pig cochlea was studied with SEM after trypsin treatment, and with TEM of resin sections and deep-etching replicas. The lamina consists of radial, evenly compacted filaments in the zona arcuata, and radial, discretely bundled filaments in the zona pectinata. In both zones, elementary filaments measured about 12 nm in thickness on the replica. The filaments formed more or less irregular passing bridges with each other and, eventually, a three-dimensional network which was continuous with the basement membrane under the supporting cells.

Early expression of intercellular adhesion molecule-1 in the corneal endothelium stimulated by endotoxin: an immuno-scanning electron microscopical study.

Three-dimensional localization of the intercellular adhesion molecule-1 (ICAM-1) in the corneal endothelium stimulated by Salmonella typhimurium endotoxin was investigated using immuno-scanning electron microscopy (SEM). Samonella typhimurium endotoxin, 100 micrograms, was injected in Lewis rats weighing 200 grams. The animals, including the controls, were sacrificed and both eyes enucleated at 0, 3, 12 and 24 hours after injection (n = 3 each time). After resection, the corneas were immersed in hypothermic University of Wisconsin solution with monoclonal mouse-anti-rat ICAM-1 IgG and then goat-anti-mouse IgG coupled to 15 nm gold particles. Then the corneas were prepared conventionally for scanning electron microscopy. Histotopographical examination with immuno-SEM revealed that ICAM-1 antigen increased on the corneal endothelium by 3 hours postinjection. The particles were arranged along the cytoplasmic processes, especially at the summits. The number of particles was 3.3 +/- 0.8/microns2 in the control, 3.6 +/- 0.8/microns2 at 0 hour, 14.4 +/- 0.9/microns2 at 3 hours, 25.4 +/- 1.4/microns2 at 12 hours, and 22.7 +/- 2.6/microns2 at 24 hours postinjection. ANOVA indicated that the time-course was an important factor (P < 0.01). Our results showed that ICAM-1 could be augmented in the corneal endothelium by endotoxin. The interrelationship between ICAM-1 expression and cytoplasmic processes seems to be important for the neutrophil-binding mechanism.

Development and distribution of the major pollen allergen (Cry j I) in male flower buds of Japanese cedar (Cryptomeria japonica).

We investigated the production of the major pollen allergen [Cry j I] of Japanese cedar (Cj) in the course of male flower bud development. We found that most of the pollen was at the tetrad stage in early September, and they developed to the mature stage in mid-October (1987) or early November (1988). Large amounts of Cry j I seemed to be extractable to ABS solution from the mature stage pollen but not from the pollen at other stages, that is, the tetrad and immature stages. Mature pollen could be ruptured with ammonium bicarbonate buffer, but tetrad and immature pollen could not. By immunofluorescent technique, antigen Cry j I was detected in the pollen only at the mature stage of Cj pollen development. Therefore, we think that Cry j I is produced at the time of pollen maturation.

Vincula tendinum of human hands with special reference to vascular patency.

As persisting parts of the mesotendon, Vinculum breve and Vinculum longum are known to serve as transport and conduction pathways to the intravaginal segments of flexor tendons. For morphological evaluation of their constancy, they were dissected out of, measured, and histologically examined on the 2nd-5th fingers in 53 hands of donated cadavers. The Vincula brevia existed in all cases more or less in association with the insertion of the superficialis as well as profundus flexor tendon, meanwhile the Vincula longa varied in number from 0 to 3 per tendon from one finger to the others, even 4 in the case of the index superficialis tendon. Light microscopic investigation of vascular patency was carried out on semithin resin sections of 137 Vincula longa obtained from the flexor superficialis and profundus tendons of 53 index fingers. Up to 5 or more arterial and venous vessels were counted per Vinculum longum. The complete absence of a patent artery was recognized in 6 out of 62 Vincula longa of the flexor superficialis and in 22 out of 75 of the profundus flexor. Most frequently, the Vincula longa had a single artery and a single vein. Considering the constant presence of vessels on the cross section, the Vinculum breve is regarded as essential for maintaining the tendons at work. In contrast, the significance of Vinculum longum will be variable for the microcirculation of intravaginal segments of the flexor tendons individually and also in the course of aging.

[Effects of high dose steroid on the experimentally induced facial palsy].

As a conservative treatment for Bell's palsy, the high dose steroid therapy introduced by Stennert in 1982 are highly evaluated clinically from many investigators. In the present study, model animals with experimentally-induced facial paralysis were prepared using guinea pigs in which the effect of administration of high dose steroid on the recovery process of the palsy was investigated electrophysiologically and morphologically. The main trunk of the facial nerve was exposed under Nembutal anesthesia, and compressed for 10 sec. using a needle holder. The model animals were divided into three groups, a high dose steroid group (Group A) and a low dose steroid group (Group B) and control group (Group C). The evoked EMGs of orbicularis oris muscle were recorded before operation and on the third, seventh and 14th days after operation, and each time course of the amplitude changes in Groups A, B and C was compared. Recovery patterns of amplitude in Group A was much more rapid than in Groups B and C. Light microscopy revealed extensive destruction and existence of only a small number of normal myelinated fibers on the 14th day after operation in Group C. In Group A, however, these changes were slight. And the numbers of normal myelinated fibers decreased to a minimum in Group C, while the number of fibers in Group A already increased on the 14th day. Electron microscopy, in Group C, disclosed distortion of the sheath of myelinated fibers and abnormal changes in the axon. But in Group A, dense lamellar structure of myelin sheath and no axonal degeneration were disclosed. These experimental results support the efficacy of high dose steroid therapy upon Bell's palsy.

"SIF" cells in the sympathetic ganglia of the bullfrog, Rana catesbeiana: variety in population and innervation.

"Small intensely fluorescent" (SIF) cells appeared singly or, more frequently, in variably-sized clusters in the sacroccygeal 8th and 9th sympathetic ganglia of the bullfrog. Smaller clusters containing only two to nine SIF cells accounted for 61% of 1773 clusters examined. The largest cluster contained 283 cells. The number of cells in individual ganglia also varied from 21 to 3332. SIF cells, solitary as well as in smaller clusters, received no distinct form of the synaptic contact. In contrast, the cells in larger clusters were frequently innervated by nerve endings that were similar in vesicular constitution to the nerve endings on principal ganglion (PG) cells. No synaptic contact was found between SIF cells and PG cells. SIF cells were also characterized by their location in the vicinity of blood capillaries with a continuous endothelium. Our observation seems to suggest that larger clusters of SIF cells receiving nerve endings are linked to a paracrine and/or endocrine system. Chemical influence via the blood stream and intraganglionic milieu for non-innervated SIF cells in the solitary or smaller clusters is a subject for speculation. An interneuronal role of SIF cells to relay stimuli to PG cells seems unlikely. The possible functions here assigned to SIF cells could be variable in efficiency depending on their population and density.

Gap junction and its cytoskeletal undercoats as involved in invagination-endocytosis.

Plasmamembrane and its cytoskeletal undercoat were characterized by electron microscopy in gap junctions (GJs) of steroidogenic cells of the guinea pig and bullfrog adrenal glands. In both species GJs varied in shape considerably and measured 0.1-4 microns in diameter. Planar GJs were not provided with any distinct form of the undercoat. In contrast, variably invaginating GJs had a network of actin-containing microfilaments located in the protruding cytoplasm arising from either one of adjoining cells. In the deeper invaginations, on the contrary, parallel arrays of actin-containing microfilaments formed a submembranous sheath in the withdrawing cytoplasm. The microfilaments were arranged at right angles for the long axis of the invagination. In completely internalized GJs, the network and sheath became less organized or obscured. A mechanical force driving the invagination-endocytosis involving GJ areas is presumably generated by the microfilament network and sheath, organized differently in forms, but working in concert together. It is also likely that there is another dissolving process for GJs via clathrin-coated vesicles.