ATP-mediated cell–cell signaling in the organ of Corti: the role of connexin channels

Connexin 26 (Cx26) and connexin 30 (Cx30) form hemichannels that release ATP from the endolymphatic surface of cochlear supporting and epithelial cells and also form gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca²⁺ mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y₂ and P2Y₄ receptors, PLC-dependent generation of IP₃, release of Ca²⁺ from intracellular stores, instigating the regenerative propagation of intercellular Ca²⁺ signals (ICS). The range of ICS propagation is sensitive to the concentration of extracellular divalent cations and activity of ectonucleotidases. Here, the expression patterns of Cx26 and Cx30 were characterized in postnatal cochlear tissues obtained from mice aged between P5 and P6. The expression gradient along the longitudinal axis of the cochlea, decreasing from the basal to the apical cochlear turn (CT), was more pronounced in outer sulcus (OS) cells than in inner sulcus (IS) cells. GJ-mediated dye coupling was maximal in OS cells of the basal CT, inhibited by the nonselective connexin channel blocker carbenoxolone (CBX) and absent in hair cells. Photostimulating OS cells with caged inositol (3,4,5) tri-phosphate (IP₃) resulted in transfer of ICS in the lateral direction, from OS cells to IS cells across the hair cell region (HCR) of medial and basal CTs. ICS transfer in the opposite (medial) direction, from IS cells photostimulated with caged IP₃ to OS cells, occurred mostly in the basal CT. In addition, OS cells displayed impressive rhythmic activity with oscillations of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) coordinated by the propagation of Ca²⁺ wavefronts sweeping repeatedly through the same tissue area along the coiling axis of the cochlea. Oscillations evoked by uncaging IP₃ or by applying ATP differed greatly, by as much as one order of magnitude, in frequency and waveform rise time. ICS evoked by direct application of ATP propagated along convoluted cellular paths in the OS, which often branched and changed dynamically over time. Potential implications of these findings are discussed in the context of developmental regulation and cochlear pathophysiology.     

Variation in the cell cycle time in the crypts of lieberkühn of the mouse

The durations of the phases of the cell cycle were measured at different levels in the jejunal crypts of male Balb/c mice. A mean cell cycle time of 12.3 h was found for the whole crypt. In cell positions 1 and 2, the cell cycle time was 16.7 h, and this time steadily decreased to a value of between 10 and 11 h for cell positions above 11. It is concluded that basally situated crypt cells in the mouse are cycling relatively slowly, and that they form the functional stem cell pool for the crypt. These cells may also compose the potential stem cell pool which repopulates the crypt after death of proliferative cells.     

Ultrastructural analysis of the granulosa — Luteal cell transition in the ovary of the dog

The transition from ovarian granulosa to lutein cell during the estrus cycle of 60 pregnant and non-pregnant beagle bitches was analyzed by light and electron microscopy (both 100 and 1000 KV). Early proestrus was characterized by a gradual rise in serum estrogen levels, hyperplasia of the granulosa cells, the accumulation of follicular fluid, and the development of tortuous intercellular channels. During the second half of proestrus, serum estrogen levels continued to rise, but growth, division, and differentiation of the granulosa cells was minimal. Estrus was marked by the first acceptance of the male and a well-defined LH peak In the subsequent 24 hour period, the granulosa-lutein cells hypertrophy rapidly and develop a large Golgi apparatus, small profiles of granular endoplasmic reticulum, numerous microfilaments, and large gap junctions between the cells. Mitochondria also proliferate, enlarge, and elongate, but retain lamelliform cristae. Luteinization of the cells and progesterone secretion begin just after ovulation which in turn occurs about 24 hours after the LH peak. On the third and fourth day of estrus, numerous small vesicles of agranular endoplasmic reticulum fill the extoplasm and the mitochondria swell up and round off. The vesicles rapidly fuse into whorled and flattened cisternae or anastomosing tubules of agranular endoplasmic reticulum, while the mitochondria develop tubulovesicular cristae. These structures gradually become organized with respect to the basal lamina. The Golgi apparatus is centered over the pole of the nucleus that faces the pericapillary space. Stacked and whorled cisternae of agranular ER develop in the lateral margins and avascular end of the cell while mitochondria and tubular elements of agranular ER predominate in the central medial and most basal portions of the cytoplasm. Microfilaments are ubiquitous and appear to be instrumental in this orientation process. The cell surface develops three distinct regional specializations that coincide with the underlying cellular compartments: interconnecting pleomorphic folds fill the pericapillary space; long tenous microvilli project from the lateral cell surface and form tortuous intercellular channels and canaliculi; and large gap junctions form along the margins of the cell furthest removed from the basal lamina. By the sixth day of estrus, the granulosa-luteal cell transition is nearly complete and serum progesterone levels are on the rise.     

Genotypical differences in aluminum resistance of maize are expressed in the distal part of the transition zone. Is reduced basipetal auxin flow involved in inhibition of root elongation by aluminum?

Short-term Al treatment (90 microM Al at pH 4.5 for 1 h) of the distal transition zone (DTZ; 1-2 mm from the root tip), which does not contribute significantly to root elongation, inhibited root elongation in the main elongation zone (EZ; 2.5-5 mm from the root tip) to the same extent as treatment of the entire maize (Zea mays) root apex. Application of Al to the EZ had no effect on root elongation. Higher genotypical resistance to Al applied to the entire root apex, and specifically to the DTZ, was expressed by less inhibition of root elongation, Al accumulation, and Al-induced callose formation, primarily in the DTZ. A characteristic pH profile along the surface of the root apex with a maximum of pH 5.3 in the DTZ was demonstrated. Al application induced a substantial flattening of the pH profile moreso in the Al-sensitive than in the Al-resistant cultivar. Application of indole-3-acetic acid to the EZ but not to the meristematic zone significantly alleviated the inhibition of root elongation induced by the application of Al to the DTZ. Basipetal transport of exogenously applied [(3)H]indole-3-acetic acid to the meristematic zone was significantly inhibited by Al application to the DTZ in the Al-sensitive maize cv Lixis. Our results provide evidence that the primary mechanisms of genotypical differences in Al resistance are located within the DTZ, and suggest a signaling pathway in the root apex mediating the Al signal between the DTZ and the EZ through basipetal auxin transport.     

Identification of cell adhesive sequences in the N-terminal region of the laminin α2 chain

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.     

Plasma levels of TGF-β1 in homeostasis of the inflammation in sickle cell disease

Sickle cell disease (SCD) represents a chronic inflammatory condition with complications triggered by the polymerization of hemoglobin S (Hb S), resulting in a series of cellular interactions mediated by inflammatory cytokines, as the transforming growth factor beta (TGF-β), which plays an important role in inflammation resolution. This study assessed the relation between SCD inflammation and the plasma concentration of TGF-β1, and also checked the influence of the presence of −509C/T polymorphism in TGFB1 gene on TGF-β1 plasma values. The plasma levels of TGF-β1 were quantified by ELISA in 115 patients with SCD (genotypes SS, SD-Los Angeles, Sβ-thalassemia and SC) and in 58 individuals with no hemoglobinopathies (Hb AA), as the control group. The −509C/T polymorphism in TGFB1 gene was screened by PCR-RFLP. The correlation between TGF-β1 plasma levels and the inflammation was based on its association with the count of platelets, total white blood cells (WBC) and neutrophils in the peripheral blood. Patients with SCD showed plasma levels of TGF-β1 higher than the control group, especially the Hb SS genotype, followed by the group with Hb SD. Polymorphism investigation showed no interference in the values obtained for the cytokine in the groups evaluated. All SCD groups showed TGF-β1 levels positively correlated to the platelets and WBC counts. The original data obtained in this study for SCD support the involvement of TGF-β1 in regulating of the inflammatory response and suggest that this marker possibly may become a potential therapeutic target in the treatment of the disease.     

Effect of aluminum on the binding properties of α-chymotrypsin

 The effect of aluminum ions on the binding properties of α-chymotrypsin has been studied. The results show that aluminum does not affect the catalytic rate constant k _cat, but it acts as an enzyme activator favoring the binding of the substrate to the catalytic site (i.e. decreasing K _m). Furthermore, aluminum binding to α-chymotrypsin displays about a threefold decrease in its affinity for the macromolecular inhibitor bovine pancreatic trypsin inhibitor (BPTI). Altogether, the different effect of aluminum on the binding of synthetic substrates (e.g. N-α-benzoyl-l-tyrosine ethyl ester, BTEE) and macromolecular inhibitors (e.g. BPTI) to α-chymotrypsin suggests the occurrence of an aluminum-linked conformational change in the enzyme molecule which brings about a marked structural change at the primary and secondary recognition sites for substrates and inhibitors. The modulative effect exerted by aluminum on the enzyme hydrolytic activity has been investigated also as a function of pH. The ion-linked effect appears to be dependent on the pH in a complex fashion, which suggests that aluminum binding is controlled by the protonation of at least two classes of residues on the enzyme molecule. Received: 5 December 1996 / Accepted: 11 March 1997     

Regulated expression of the integrin α9β1 in the epithelium of the developing human gut and in intestinal cell lines: Relation with cell proliferation

The integrin α9β1 is one of the recently identified integrins whose expression is restricted to specialized tissues. Its exact function is still unknown. In the present study, we have analyzed the expression of the α9 subunit in human fetal and adult small intestinal and colonic epithelia as well as in intestinal cell lines by indirect immunofluorescence, immunoprecipitation, Western blot, and Northern blot. In intact tissues, the antigen was restricted to the basolateral domain of epithelial cells in intestinal crypts at the fetal stage and was absent in the adult. The α9β1 integrin was also detected in the intestinal cell lines HIEC-6 and Caco-2/15. The presence of α9β1 in HIEC-6 was found to be consistent with their proliferative crypt-like status. In Caco-2/15 cells, the integrin was present at high levels in proliferating cells but was downregulated when cells cease to grow and undertake their differentiation. EGF treatment, which is known to maintain Caco-2/15 cells in a proliferative state, resulted in higher levels of α9 as compared to control cells. Taken together, these observations suggest a relation between integrin α9β1 expression and proliferation in human intestinal cells. J. Cell. Biochem. 71:536–545, 1998. © 1998 Wiley-Liss, Inc.     

Do we underestimate the importance of water in cell biology?

Liquid water is a highly versatile material. Although it is formed from the tiniest of molecules, it can shape and control biomolecules. The hydrogen-bonding properties of water are crucial to this versatility, as they allow water to execute an intricate three-dimensional 'ballet', exchanging partners while retaining complex order and enduring effects. Water can generate small active clusters and macroscopic assemblies, which can both transmit information on different scales.     

What is the role of cell division in leaf development?

An idea underlying a great deal of research and discussion in plant cell and developmental biology is that the spatial regulation of cell division plays a key role in plant development. In this article, the role of cell division in two aspects of leaf development is analysed: morphogenesis (leaf initiation, growth, and the generation of leaf shape) and histogenesis (the differentiation of leaf cells to form the various cell types that make up a functional leaf). The point of view that emerges from this analysis is that the rate and pattern of cell division is important for leaf development, but does not dictate leaf size, shape, or cell fate.     

Effect of ionophores on the processing of theβ-amyloid precursor protein in different cell lines

Summary 1. Alzheimer's disease is characterized by the deposition in the brain of extracellular amyloid plaques and vascular deposits consisting mostly of amyloid-peptide (A). A, a polypeptide of 39–43 amino acids (M _r, 4 kDa), is derived proteolytically from a family of proteins of 695–770 amino acids (M _r, 110–140 kDa) called-amyloid precursor protein (APP).2.APP, an integral membrane glycoprotein, is extensively posttranslationally modified within the endoplasmic reticulum (ER) and various Golgi compartments.APP is cleaved by proteases in either the trans-Golgi network or the post-Golgi apparatus and then secreted as a truncated soluble form into the conditioned media of cultured cells and cerebrospinal fluid samples from human subjects.APP can be processed either by an antiamyloidogenic secretory pathway or by an endosomal/lysosomal pathway.3. I studied the effect of two ionophores on the processing ofAPP in cultured cells. Monensin and, in some cases, ammonium chloride increase the intracellular accumulation ofAPP in several cell lines and may alter its processing. Monensin, which had the most consistent effects, also inhibited secretion ofAPP in a differentiated (growth factor mediated) cell line. Nigericin, with greater K⁺ selectivity, was less able to alter the accumulation and possible processing of the protein.4. These results suggest that the increase in the accumulation of intracellularAPP observed after treating cells with ionophores has some specificity. The selective effect of these ionophores on the metabolism ofAPP may provide a model system to analyze the pathways for studying maturation, secretion, and degradation ofAPP.     

Do changes in the cell membrane structure induce the generation of lipid peroxidation products which serve as first signalling molecules in cell to cell communication?

Evidence is presented that mammalian and plant cells respond equally to any event which changes their cell membrane structure. Proliferation, wounding or aging induces generation of lipidhydroperoxides from cell wall phospholipids. These are transformed to signalling compounds, some of these induce apoptosis. If the exerted impact exceeds a certain level, the original enzymic reaction switches to a non-enzymic one which produces peroxylradicals. The latter are not liberated enzymically. Peroxylradicals generate a second set of signalling compounds, but cause also severe damage: they epoxidize double bonds, and oxidize proteins, sugars and nucleic acids. Such reactions occur in all inflammatory diseases. Lipidhydoperoxides and their degradation products are incorporated in fat. Apparently, these compounds are transferred partly to LDL. Such LDL is still recognized by the cell LDL receptor. Toxic lipid peroxidation products are therefore introduced into cells and might be able to damage cells from inside long before the typical signs of atherosclerosis and other chronic diseases become visible.     

PH-dependent cell–cell interactions in the green alga Chara

Protoplasma - Characean internodal cells develop alternating patterns of acid and alkaline zones along their surface in order to facilitate uptake of carbon required for photosynthesis. In this...     

A cyto-biochemical study of the ‘canal’ formation in the yeast cell wall

Summary A distinction between the chemical composition of ultrastructurally modified regions and the rest of the cell wall (canals) of the yeast Schwanniomyces occidentalis was shown by cytochemical staining of cell wall polysaccharides. The formation of canals was induced by cultivation of yeasts on hydrocarbons and was parallelled by the enhancement of -glucosidase, -glucanase and -mannosidase activities which were all capable of degrading cell wall polysaccharides. The presence of cycloheximide prevented canal formation. We assume that these hydrolases modified definite cell wall regions transforming them into canals.     

Is immune cell activation the missing link in the pathogenesis of post-diarrhoeal HUS?

Haemolytic uraemic syndrome (HUS), which is caused by Shiga toxin (Stx)-producing Escherichia coli, is the commonest cause of acute renal failure in childhood. It is widely believed that HUS develops following the release of Stx, an AB5 toxin that inhibits protein synthesis and has a direct toxic effect on the kidney endothelium. There remains, however, a mismatch between the current understanding of the pathogenesis of HUS and the evolution of the clinical signs of the disease. Our hypothesis is that Stx-mediated immune cell activation in the gut is the missing link in the pathogenesis of this condition, initiating the characteristic renal pathology of HUS either alone or in synergy with Stx. Validation of this hypothesis could lead to a targeted anti-inflammatory approach aimed at modulating immune cell function in HUS.     

Down-regulation of Pkd2 by siRNAs suppresses cell–cell adhesion in the mouse melanoma cells

The Pkd2 gene encodes an integral protein (~130 kDa), named polycystin-2 (PC-2). PC-2 is mainly involved in autosomal dominant polycystic kidney disease. Recently, polycystin-1/polycystin-2 complex has been shown to act as an adhesion complex mediating or regulating cell–cell or cell–matrix adhesion, suggesting that PC-2 may play a role in cell–cell/cell–matrix interactions. Here, we knocked down the expression of Pkd2 gene with small interfering RNAs (siRNAs) in the mouse melanoma cells (B16 cells), indicating that the cells transfected with the targeted siRNAs significantly suppressed cell–cell adhesion, but not cell–matrix adhesion, compared to the cells transfected with non-targeted control (NC) siRNA. This study provides the first directly functional evidence that PC-2 mediates cell–cell adhesion. Furthermore, we demonstrated that PC-2 modulated cell–cell adhesion may be, at least partially, associated with E-cadherin. Collectively, these findings for the first time showed that PC-2 may mediate cell–cell adhesion, at least partially, through E-cadherin.