Lamina, a novel multicellular form of Methanosarcina mazei S-6.

A novel multicellular form of Methanosarcina mazei S-6 is described. It was termed lamina, and it formed during the exponential growth phase when packets or single cells were grown in 40 mM trimethylamine and a total concentration of 8.3 to 15.6 mM Ca2+ and/or Mg2+, in cultures that were not shaken. A distinct molecular event represented by the increment in expression and a spatial redistribution of an antigen during lamina formation is documented.     

Interconversion of large packets and small groups of cells of Micrococcus rubens: dependence upon magnesium and phosphate.

Micrococcus rubens, a gram-positive occus, usually forms large, cubic packets of more than 500 cells that are regularly arranged in three-dimensional cell groups. In medium with extremely low concentration of Mg2+ and phosphate, in which the cells can only grow on a agar surface, it formed small groups of 2 to 20 cells. Irregularly arraged cell groups of intermediated size were obtained in culture media containing intermediated concentrations of Mg2+ and phosphate. Mutants that formed irregular cell groups of intermediate size under normal culture conditions were also obtained.     

The fine structure of blood follicles in the earthworm genera Amynthas and Lumbricus (Annelida: Oligochaeta)

Blood follicles of the earthworm Amynthas are hemoglobin-containing, sac-like dilatations of blood vessels which connect to the general circulation. Grape-like clusters of follicles are found posterior to the pharynx, among tufts of micronephridia, and single follicles are located among cells of the pharyngeal gland. In Lumbricus, follicles take the form of simple swellings and irregular-shaped diverticula of nephridial capillaries. The fundamental structure of the wall of follicles and of vessels in both genera is the same and consists of two layers: an extracellular vascular lamina and an outer (coelomic) covering of smooth muscle-like myoperithelial cells. Hemocytes may be free and circulating or they may facultatively attach to the vascular lamina as littoral cells, constituting an incomplete endothelium-like surface. Hemocytes that appear to be in the process of attaching or detaching are rounded, while adherent cells are flattened and elongate. Free and littoral hemocytes actively endocytose packets of circulating extracellular hemoglobin. Hemocytes within follicles possess radiating cell processes which also endocytose hemoglobin. Although these cells were presumed to secrete hemoglobin, staining with 3,3′-diaminobenzidine confirms the presence of hemoglobin only within pinosomes and not within protein-synthesizing or packaging organelles. The presence of hemosiderin-like bodies suggests that follicular hemocytes catabolize hemoglobin. Blood follicles apparently provide a means of significantly increasing cell-surface area for hemoglobin processing, without substantially increasing the volume and pumping load of the circulatory system.     

Growth of linear spicules in cultured primary mesenchymal cells of sea urchin embryos is bidirectional

The growth of spicules of the sea urchin embryo was studied in a simple in vitro system in which primary mesenchymal cells attach to the substrata, migrate and fuse via filopodia, and subsequently deposit CaCO3, which in most cases is in the form of linear rods. The use of autoradiographs following 45Ca2+ pulse-chase labeling revealed that in such a system linear spicules that formed had two focal points of growth. Elongation occurred by addition of approximately equivalent amounts of Ca2+ to both ends of each rod. Multiform spicules having variable numbers of elongation sites (tips) also showed a similar pattern of Ca2+ deposition. Thus, the growth of both linear and relatively complex skeletal forms is apparently accomplished by the same basic mechanism.     

Hemocytes contribute to both the formation and breakdown of the basal lamina in developing wings of Manduca sexta

Monoclonal antibodies (MAbs) raised against wing tissues of Manduca sexta recognize epitopes shared by both hemocytes and basal laminae. During the last larval stadium, the basal lamina of moth wing epithelium forms after hemocytes have migrated into the space adjacent to basal surfaces of epithelial cells. As adult development commences, hemocytes participate in phagocytosis of the same basal lamina; and as dissolution of the basal lamina proceeds (day 2-day 5 post-pupation), wing epithelial cells send forth long basal processes and rearrange within the plane of the epithelium. During this period of cell rearrangement, the immunoreactivity of the basal lamina decreases in concert with an increase in immunoreactive vesicles within hemocytes; and at the ultrastructural level, hemocytes have been observed to engulf fragments of basal lamina. The distribution of immunolabel in the developing moth wing suggests that hemocytes contribute not only to the formation of the wing's basal lamina but also to its breakdown. Since basal laminae are probably important determinants of epithelial form and pattern, hemocytes also contribute to the shaping of epithelial populations.     

The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans

Three known genes guide circumferential migrations of pioneer axons and mesodermal cells on the nematode body wall. unc-5 affects dorsal migrations, unc-40 primarily affects ventral migrations, and unc-6 affects migrations in both directions. Circumferential movements still occur, but are misdirected whereas longitudinal movements are normal in these mutants. Pioneer growth cones migrating directly on the epidermis are affected; growth cones migrating along established axon fascicles are normal. Thus these genes affect cell guidance and not cell motility per se. We propose that two opposite, adhesive gradients guide circumferential migrations on the epidermis. unc-5, unc-6, and unc-40 may encode these adhesion molecules or their cellular receptors. Neurons have access to the basal lamina and the basolateral surfaces of the epidermis, but mesodermal cells contact only the basal lamina. These genes probably identify molecular cues on the basal lamina that guide mesodermal migrations. The same basal lamina cues, or perhaps related molecules on the epidermal cell surfaces, guide pioneer neurons.     

Removal of insect basal laminae using elastase

We have used the enzyme elastase to remove the basal lamina of epithelia from two insects: the upper Malpighian tubules of Rhodnius prolixus and imaginai discs of Drosophila melanogaster. Removal of the basal lamina was confirmed using scanning and transmission electron microscopy. Use of the technique on the Malphighian tubules of Rhodnius reveals for the first time the three-dimensional organization of the circumferential folds of the basal plasma membrane. Elastase is much more effective in removing the basal lamina than are the enzymes hyaluronidase, collagenase, and chymotrypsin, either alone or in combination. Following elastase treatment, cells of the Malpighian tubules dissociate with only mild mechanical agitation into single, viable cells. Treatment with elastase removes the basal laminae of imaginai discs of Drosophila and accelerates evagination as has been previously described for trypsin. To obtain single cell preparations from elastase-treated imaginai discs, mechanical stirring in Ringer low in Ca²⁺ was required. In addition to its usefulness in cell isolation, elastase treatment allows examination of the effect of removal of basal laminae on the physiology and development of insect epithelia.     

Ca2+ signaling in identified T-lymphocytes from human intestinal mucosa. Relation to hyporeactivity, proliferation, and inflammatory bowel disease

Ca2+ entry across the plasma membrane is necessary for the activation and proliferation of T-lymphocytes. Human intestinal lamina propria lymphocytes physiologically exhibit minimal proliferation in response to antigen receptor stimulation when compared with peripheral blood T-lymphocytes. This hyporeactivity is partially abolished in inflammatory bowel disease. We hypothesized that differences in Ca2+ signaling could be related to the disease. To test this possibility, we measured Ca2+ signals in identified lymphocytes from human blood and human intestinal mucosa. Ca2+ signals in lamina propria T-lymphocytes from non-inflamed tissue were drastically reduced when compared with Ca2+ signals of blood T-lymphocytes from the same persons. However, Ca2+ signals in T-lymphocytes from inflamed intestinal mucosa were much higher than the ones from non-inflamed mucosa and almost reached levels of Ca2+ signals in peripheral blood T-cells. Furthermore, Ca2+ influx was closely linked to cell proliferation in both peripheral blood T-lymphocytes and lamina propria lymphocytes cells. We conclude that differences in Ca2+ signaling can explain the differences of T-lymphocyte reactivity in blood versus lamina propria and, importantly, also between T-lymphocytes from inflamed and non-inflamed intestinal mucosa. Ca2+ channels in the plasma membrane of T-lymphocytes might thus prove an excellent target to screen for immunosuppressiva to potentially treat the symptoms of inflammatory bowel disease.     

The inhibitory effects of synthetic short peptides, mimicking MICA and targeting at NKG2D receptors, on function of NK cells

NKG2D is an activating receptor expressed on most of human NK cells, one of whose ligands is MICA. Based on the crystal structure of NKG2D-MICA complex, we synthesized three short peptides (P1, P2 and P3), mimicking functional alpha1 and alpha2 domain of MICA. The inhibitory effects of three peptides on NK-92 cells, a human NK cell line against Hela cells were observed and the inhibitory percentage was 38% at maximum for P1+P2+P3 in concentration of 1nM. The same peptides had no effect on NK-92 cell against target cells lacking MICA (K562 cells line). The unrelated peptides as controls had no effect on the system. Two peptides (P2 and P3) were prolonged at one or both ends, and the longer forms of peptides exerted stronger inhibitory effects than their shorter forms. Each combination of two peptides exerted a stronger function than single peptide (P1, P2, P3), indicating that shedding of longer amino acid sequence of alpha1 domain or more domain sites of MICA are better than shorter sequence and fewer sites. P1+P2+P3 revealed the almost same inhibitory rate as the soluble MICA (sMICA). P1+P2+P3 were also able to alleviate the concanavalin A-induced murine autoimmune hepatitis in vivo, conforming the similarity of NKG2D between human and mice. The results demonstrate that MICA-mimicking peptides will be useful to search the specific functional sites for NKG2D-MICA interaction, but also promising in explaining NKG2D-related autoimmunity.     

不同耐旱性大麦品种叶片发育的研究

选用耐旱性不同的两个大麦品种作为研究对象,分析其叶片结构的异同。结果表明:两个大麦品种的叶片发育可以分为幼叶萌发期、幼叶抽出期、幼叶生长期和叶片成熟期四个阶段,其中在幼叶萌发期,叶片结构无明显差异。经PAS染色,从幼叶生长期开始,耐旱性弱的Moroc 9-75,含淀粉粒的叶肉细胞少,淀粉粒颗粒小; 耐旱性强的HS 41-1,含淀粉粒的叶肉细胞多,淀粉粒颗粒大。遭受干旱胁迫后,两个品种的植株长势明显较弱,叶片短而窄; 表皮细胞角质层变厚,叶片中叶肉细胞变小,叶肉细胞胞间隙变大,叶肉细胞破裂现象增多; PAS染色反应显示,含淀粉粒的叶肉细胞减少,淀粉粒颗粒变小或基本没有; HS 41-1解体的细胞不如Moroc 9-75多。因此,在光镜下,叶片结构的差异,特别是细胞含有的淀粉粒大小与数量的区别,是植物对水分胁迫的一种适应; 同时叶脉对植物刚性的影响较大。     

The localization of macrophage subsets and dendritic cells in the gastrointestinal tract of the mouse with special reference to the presence of high endothelial venules

Summary This study concerns the distribution of macrophages and dendritic cells (DC) in the gastrointestinal tract of the mouse. Heterogeneity of macrophage population was found by using the MOMA-1, MOMA-2, ERTR-9, Mac-1 and F4/80 monoclonal antibodies. MOMA-1, ERTR-9, Mac-1 and F4/80+ cells were detected mostly at the villous cores in the lamina propria of the villi, whereas MOMA-2+ cells were primarily found around the crypts at the base of the villi. These MOMA-2+ cells revealed a granular appearance throughout the cytoplasm and displayed a strong acid phosphatase (AcPh) activity. Few MOMA-2+ cells were seen at the top of the villi in the epithelium. Although MOMA-1 and ERTR-9+ cells have similar morphology and the same distribution patterns in the lamina propria, they are likely different populations, because in Peyer's patches (PP), MOMA-1+ cells were present, whereas ERTR-9+ cells could not be detected. Both populations displayed AcPh activity. Strongly stained Mac-1+ cells were abundantly seen in the lamina propria of the small intestine. F4/80+ cells were rare. NLDC-145+ cells with AcPh activity and weak Ia staining were also found. In the PP-associated villi and in the T-dependent area of PP, dendritic NLDC-145+ cells, which were strongly Ia positive, were detected. MIDC-8+ cells were found only in the T-dependent area. Few NLDC-145+ cells (dendritic cells) were found in the upper part of the oesophagus. These cells were also stained with the MIDC-8 antibody. The MECA-325 monoclonal antibody recognized high endothelial venules (HEV) in PP and blood vessels at the base of the villi of the jejunumileum and caecum. Unlike in PP, the endothelium of the venules in the villi was flat.     

Basal lamina of ovarian follicle regulates an inward Cl- current in differentiated granulosa cells

Patch-clampexperiments were conducted to study the effects of basal lamina(basement membrane) of preovulatory chicken ovarian follicle onmembrane currents in differentiated chicken granulosa cells in ahomologous system. The membrane capacitance (measure of total membranearea) was smaller in cells cultured on intact basal lamina than that ofcontrol cells. The granulosa cells expressed outward and two inwardcurrents. A small fraction of the cells (3%) expressed only atransient fast-activating and -inactivating inward current carried byCa²⁺. The majority of the cells, however, expressed aslowly activating and inactivating inward current (carried byCl) that was superimposed on the transientCa²⁺ current. All cells expressed an outward currentcharacteristic of the delayed-rectifier K⁺ current. Theremoval of extracellular Ca²⁺ led to elimination of theslow inward Cl current, indicating that it is aCa²⁺-dependent Cl current. Both peakamplitude and current density of the inward Cl currentwere significantly lower in cells cultured on freshly isolated intactbasal lamina (or basal lamina stored at 4°C for 12 mo) than those ofcontrol cells; however, basal lamina had no significant effect on thedensity of the outward current. Similar to the observations made forintact basal lamina, solubilized basal lamina suppressed the inwardCl current in differentiated granulosa cells. These datashow that homologous basal lamina modulates aCa²⁺-dependent Cl current in differentiatedgranulosa cells. These findings provide a partial explanation for themechanisms that subserve the reported effects of basal lamina (basementmembrane) on the metabolic functions of differentiated granulosa cells.

     

Structure of the body cavity of the sea spider <Emphasis Type="Italic">Nymphon brevirostre</Emphasis> Hodge, 1863 (Arthropoda: Pycnogonida)

The microscopic anatomy and ultrastructure of the body cavity and adjacent organs in the sea spider Nymphon brevirostre Hodge, 1863 (Pycnogonida, Nymphonidae) were examined by transmission electron microscopy. The longitudinal septa subdividing the body cavity are described: (1) Dohrn’s horizontal septum, (2) lateral heart walls, and (3) paired ventral septa consisting of separate cellular bands. The body cavity is a hemocoel, it has no epithelial lining and is only bordered by a basal lamina. The epidermis, heart, and Dohrn’s septum are not separated from each other by basal laminae and may have a common origin. The cellular bands forming the longitudinal ventral septa are not covered with the basal lamina and presumably derive from cells belonging to the hemocoel. The roles of the morphological structures studied for the circulation of hemolymph are discussed. The gonad lies inside Dohrn’s septum, it is covered with its own basal lamina and surrounded by numerous lacunae of the hemocoel entering the septum. The gonad wall is formed with a single layer of epithelium. The same epithelial cells form the gonad stroma. The gonad cavity is not lined with the basal lamina; muscle cells are present in the gonad wall epithelium, thus rendering the lumen similar to a coelomic cavity. Freely circulating cells of two types are found in the hemocoel: small amebocytes containing electronic-dense granules that are similar to granulocytes of other arthropods, as well as hemocytes with large vacuoles of varying structure that are comparable with plasmatocytes; however some of these may be activated granulocytes.     

FLEXIBILITY IN ONTOGENY AS SHOWN BY THE CONTRIBUTION OF THE SHOOT APICAL LAYERS TO LEAVES OF PERICLINAL CHIMERAS

The development of leaves on apically stable, periclinal chimeras was studied in a number of dicot genera. The mutant cell layers of the shoot apex and the tissues derived from them were as active developmentally as the normal layers. Ontogeny was the same in these chimeras as in nonchimeras, and growth of their leaves can be outlined as follows. Formation of the buttress, the axis, and the lamina of simple dicot leaves were independent events. In each the first growth included derivatives of the apical layers, usually three in number, found in the apex of the shoot and the lateral buds. Most cell divisions in the outer layers (L-I and L-II) were anticlinal relative to the new structures. Therefore, in the proximal regions of the buttress, axis (petiole and midrib), and lamina, the derivative cells of L-I and L-II were usually present in single layers. The rest of the internal tissue was from L-III. As formation of the axis and the lamina proceeded, derivatives of L-II replaced L-III internally in the distal and marginal regions leaving cells of L-III behind. Both the determinate growth of leaves and the pattern of cell divisions at and near the leading edges of growth meant that no cells in the leaf were comparable to the initial cells of the shoot apex. As the lamina extended, there were extensive intercalary cell divisions, both anticlinal and periclinal, so that in any given region of a leaf the layers of internal cells were from either L-II or L-III. At any point along the axis, L-III participated or did not participate in laminar extension. At any given stage in laminar growth either of two sister cells in any internal layer divided either a few times or extensively. The extreme variability in direction and frequency of cell division during leaf development was under an overriding genetic control, which resulted in the normal or typical size, shape and thickness of leaves.     

Extracellular Matrix Components Regulating Glandular Differentiation and the Formation of Basal Lamina of a Human Pancreatic Cancer Cell Line in Vitro

The interaction between the extracellular matrix and human tumor-cell clones S2-013 and S2-020, derived from a pancreatic cancer cell line (SUIT-2), was examined in vitro, using various cell differentiation-promoting matrices in two- and three-dimensional cultures. S2-013 cells (well-differentiated tubular adenocarcinoma in xenografts in nude mice) cultured in Matrigel formed glandular structures. Ultrastructural observation revealed a morphological polarity of cells and a distinct basal lamina. On the other hand, S2-020 cells (poorly differentiated tubular adenocarcinoma in xenografts) cultured in Matrigel formed neither glandular structures nor a basal lamina, but only cell aggregates. The morphology of these two sublines cultured in Matrigel expressed the histological degree of differentiation which they presented in nude mice. In contrast, in type I collagen gel, S2-013 cells formed glandular structures without a basal lamina, and in soft agar, they were able to form neither glandular structures nor a basal lamina. S2-020 cells cultured in type I collagen gel or soft agar formed the same simple cell aggregates as in Matrigel. Matrices used in a three-dimensional culture influenced the degree of differentiation in S2-013 cells but had no effect on the morphological differentiation in S2-020 cells. To detect the factors which induce basal lamina formation, S2-013 cells were cultured on a microporous membrane coated with extracellular matrix components such as laminin, type IV collagen, and fibronectin. S2-013 cells formed a basal lamina only on the laminin. These cell lines may be useful in investigating the mechanisms regulating the formation of glandular structures and basal lamina.     

The clock input to the first optic neuropil of Drosophila melanogaster expressing neuronal circadian plasticity

In the first optic neuropil (lamina) of the fly's visual system, two interneurons, L1 and L2 monopolar cells, and epithelial glial cells show circadian rhythms in morphological plasticity. These rhythms depend on clock gene period (per) and cryptochrome (cry) expression. In the present study, we found that rhythms in the lamina of Drosophila melanogaster may be regulated by circadian clock neurons in the brain since the lamina is invaded by one neurite extending from ventral lateral neurons; the so-called pacemaker neurons. These neurons and the projection to the lamina were visualized by green fluorescent protein (GFP). GFP reporter gene expression was driven by the cry promotor in cry-GAL4/UAS-GFP transgenic lines. We observed that the neuron projecting to the lamina forms arborizations of varicose fibers in the distal lamina. These varicose fibers do not form synaptic contacts with the lamina cells and are immunoreactive to the antisera raised against a specific region of Schistocerca gregaria ion transport peptide (ITP). ITP released in a paracrine way in the lamina cortex, may regulate the swelling and shrinking rhythms of the lamina monopolar cells and the glia by controlling the transport of ions and fluids across cell membranes at particular times of the day.     

Pairwise knockdowns of cdc2-related kinases (CRKs) in Trypanosoma brucei identified the CRKs for G1/S and G2/M transitions and demonstrated distinctive cytokinetic regulations between two developmental stages of the organism

Expression of the cdc2-related kinase 3 (CRK3) together with expression of CRK1, -2, -4, or -6, were knocked down in pairs in the procyclic and bloodstream forms of Trypanosoma brucei, using the RNA interference technique. Double knockdowns of CRK3 and CRK2, CRK4, or CRK6 exerted significant growth inhibition and enriched the cells in G2/M phase, whereas a CRK3 plus CRK1 (CRK3 + CRK1) knockdown arrested cells in both G1/S and G2/M transitions. Thus, CRK1 and CRK3 are apparently the kinases regulating the G1/S and G2/M checkpoint passages, respectively, whereas the other CRKs are probably playing only minor roles in cell cycle regulation. A CRK1 + CRK2 knockdown in the procyclic form was found to cause aberrant posterior cytoskeletal morphogenesis (X. M. Tu and C. C. Wang, Mol. Biol. Cell 16:97-105, 2005). A CRK3 + CRK2 knockdown, however, did not lead to such a change, suggesting that CRK2 depletion can lead to the abnormal morphogenesis only when procyclic-form cells are arrested in the G1 phase. The G2/M-arrested procyclic form produces up to 20% stumpy anucleated cells (zoids) in the population, suggesting that cytokinesis and cell division are not blocked by mitotic arrest but are apparently driven to completion by the kinetoplast cycle. In the bloodstream form, however, G2/M arrest resulted in little zoid formation but, instead, enriched a population of cells each containing multiple kinetoplasts, basal bodies, and flagella and an aggregate of multiple nuclei, indicating failure in entering cytokinesis. The two different cytokinetic regulations between two distinct stage-specific forms of the same organism may provide an interesting and useful model for further understanding the evolution of cytokinetic control among eukaryotes.     

Distribution of transition metal ions in multiple forms of Methanosarcina hydrogenase

There were significant levels of in vitro hydrogenase activity in Methanosarcina strains. The multiple forms of hydrogenase were observed in cell free extracts of cells grown on methanol. Strains having poor growth on H₂ : CO₂ had four forms while strains having normal growth on all substrates contained two forms of hydrogenase. These multiple forms differ in their charges as well as in their composition of transition metal ions. The strain having normal growth showed higher incorporation of ⁶³Ni²⁺ and ⁶⁵Zn²⁺. Both hydrogenases, A and D, of strain P₃ had methylviologen and F₄₂₀-reducing activity and contained Zn²⁺ and Co²⁺ respectively. Hydrogenases A and D of strains P₁ and P₄ also had similar characteristics whereas hydrogenases B and C had only methylviologen reducing activity.     

独叶草的根和节部及叶的解剖学研究

报道了独叶草根、节部和叶的剖解学特征。这些器官在解剖学上表现出的突出点是：根具２个以上的根毛区（与星叶草机相同）,中有少量的次生生长,皮层细胞中具内生真菌;变态叶的叶迹或为单迹单维管组织束,或为单迹２维管组织束,或２迹在向皮层外部延伸过程中合并为具２条维管组织束的单迹;叶柄维管束不存在厚壁的维管束鞘,且在由基部向顶部延伸的过程中常发生复杂的分枝及汇合;叶片具有同形的叶肉植物,叶脉维管束鞘具２层细胞