No evidence of UV cone input to mono- and biphasic horizontal cells in the goldfish retina

Many animal species make use of ultraviolet (UV) light in a number of behaviors, such as feeding and mating. The goldfish (Carassius auratus) is among those with a UV photoreceptor and pronounced UV sensitivity. Little is known, however, about the retinal processing of this input. We addressed this issue by recording intracellularly from second-order neurons in the adult goldfish retina. In order to test whether cone-driven horizontal cells (HCs) receive UV cone inputs, we performed chromatic adaptation experiments with mono- and biphasic HCs. We found no functional evidence of a projection from the UV-sensitive cones to these neurons in adult animals. This suggests that goldfish UV receptors may contact preferentially triphasic HCs, which is at odds with the hypothesis that all cones contact all cone-driven HC types. However, we did find evidence of direct M-cone input to monophasic HCs, favoring the idea that cone–HC contacts are more promiscuous than originally proposed. Together, our results suggest that either UV cones have a more restricted set of post-synaptic partners than the other three cone types, or that the UV input to mono- and biphasic HCs is not very pronounced in adult animals.     

Basement membrane attachment is dispensable for radial glial cell fate and for proliferation, but affects positioning of neuronal subtypes

Radial glial cells have been shown to act as neuronal precursors in the developing cortex and to maintain their radial processes attached to the basement membrane (BM) during cell division. Here, we examined a potential role of direct signalling from the BM to radial glial cells in three mouse mutants where radial glia attachment to the BM is disrupted. This is the case if the nidogen-binding site of the laminin gamma1 chain is mutated, in the absence of alpha6 integrin or of perlecan, an essential BM component. Surprisingly, cortical radial glial cells lacking contact to the BM were not affected in their proliferation, interkinetic nuclear migration, orientation of cell division and neurogenesis. Only a small subset of precursors was located ectopically within the cortical parenchyma. Notably, however, neuronal subtype composition was severely disturbed at late developmental stages (E18) in the cortex of the laminin gamma1III4-/- mice. Thus, although BM attachment seems dispensable for precursor cells, an intact BM is required for adequate neuronal composition of the cerebral cortex.     

Subtype-specific expression of Fgf19 during horizontal cell development of the chicken retina

The mechanisms underlying retinal cell diversification are crucial to proper neural development. Fibroblast growth factor 19 (Fgf19) is expressed by developing horizontal cells (HCs) in the chicken retina. Although there are two major HC subtypes, axon-bearing and axon-less, the precise subtype expressing Fgf19 remains uncertain. Here we characterize Fgf19-expressing cells by co-labeling with antibodies against Lim1 (LIM homeodomain 1, or Lhx1), Islet1, and Prox1 (prospero-related homeobox 1) which are axon-bearing HC, axon-less HC, and pan-HC markers, respectively. We found that a subset of Fgf19-expressing cells was positive for Prox1 and Lim1 in the vitread neuroepithelium at embryonic day 4 (E4). By E9, the majority of Fgf19-expressing cells became positive for Prox1 and Lim1 prior to arrival at the prospective HC layer. In contrast, Fgf19-expressing cells did not overlap with the Islet1-positive population at any stage examined. These results suggest that Fgf19 is expressed by the early migratory horizontal precursors, and later by the presumptive axon-bearing HCs.     

Direct cell-to-cell contact between Kupffer cells and hepatocytes augments endotoxin-induced hepatic injury

This study focuses on the importance of direct contact between Kupffer cells (KCs) and hepatocytes (HCs) during the hepatic inflammatory response using an in vitro approach. The lipopolysaccharide (LPS)-induced inflammatory response in monocultures of porcine HCs and KCs were compared with cocultures prepared either with direct contact between KCs and HCs (DC cocultures) or without direct contact using cell culture membrane inserts. Our data show that DC cocultures exhibited the highest production of tumor necrosis factor (TNF)-alpha, interleukin-6, and nitric oxide (NO) compared with the other cultures. Immunohistochemical studies revealed that TNF-alpha was exclusively produced by KCs, whereas HCs were responsible for NO production after LPS stimulation. Biotransformation capacity, as determined by cytochrome P-450 and UDP glucuronosyl transferase enzyme activities, was most significantly decreased in DC cocultures. These results provide evidence that direct contact between KCs and HCs favors the extensive TNF-alpha production by KCs but in turn affects HC functionality and viability. These findings suggest that direct contact between KCs and HCs plays a key role in the development of a fulminating hepatic inflammatory response.     

Connexin hemichannel composition determines the FGF-1-induced membrane permeability and free [Ca2+]i responses

Cell surface hemichannels (HCs) composed of different connexin (Cx) types are present in diverse cells and their possible role on FGF-1-induced cellular responses remains unknown. Here, we show that FGF-1 transiently (4-14 h, maximal at 7 h) increases the membrane permeability through HCs in HeLa cells expressing Cx43 or Cx45 under physiological extracellular Ca(2+)/Mg(2+) concentrations. The effect does not occur in HeLa cells expressing HCs constituted of Cx26 or Cx43 with its C-terminus truncated at aa 257, or in parental nontransfected HeLa cells. The increase in membrane permeability is associated with a rise in HC levels at the cell surface and a proportional increase in HC unitary events. The response requires an early intracellular free Ca(2+) concentration increase, activation of a p38 MAP kinase-dependent pathway, and a regulatory site of Cx subunit C-terminus. The FGF-1-induced rise in membrane permeability is also associated with a late increase in intracellular free Ca(2+) concentration, suggesting that responsive HCs allow Ca(2+) influx. The cell density of Cx26 and Cx43 HeLa transfectants cultured in serum-free medium was differentially affected by FGF-1. Thus, the FGF-1-induced cell permeabilization and derived consequences depend on the Cx composition of HCs.     

Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan

The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.     

Chymotryptic digestion of Tetrahymena ciliary dynein. II. Pathway of the degradation of 22S dynein heavy chains

As shown in the preceding paper (Toyoshima, Y. Y., 1987, J. Cell Biol., 105:887-895) three-headed Tetrahymena 22S dynein consists of three heavy chains (HCs) and is decomposed into two-headed (H) and one-headed (L) fragments by chymotryptic digestion. To accurately determine the presence of multiple ATPases and ultimately the location of various domains, it is necessary to determine the identity of each HC fragment relative to the original HCs in 22S dynein. The degradation pathway of each HC was determined by peptide mapping and immunoblotting. The three HCs (A alpha, A beta, and A gamma) were immunologically different; although SDS-urea gel electrophoresis showed that A gamma HC was apparently resistant to the digestion, actually three distinct HCs contributed to the same band alternately. H fragment was derived from A beta and A gamma HCs, whereas L fragment originated from A alpha HC. Since both fragments were associated with ATPase activity, these results directly demonstrate the presence of multiple ATPase sites in Tetrahymena 22S dynein.     

The TSG-6/HC2-mediated Transfer Is a Dynamic Process Shuffling Heavy Chains between Glycosaminoglycans

The heavy chain (HC) subunits of the bikunin proteins are covalently attached to a single chondroitin sulfate (CS) chain originating from bikunin and can be transferred to different hyaluronan (HA) molecules by TSG-6/HC2. In the present study, we demonstrate that HCs transferred to HA may function as HC donors in subsequent transfer reactions, and we show that the CS of bikunin may serve as an HC acceptor, analogous to HA. Our data suggest that TSG-6/HC2 link HCs randomly on the CS chain of bikunin, in contrast to the ordered attachment observed during the biosynthesis. Moreover, the results show that the transfer activity is indifferent to the new HC position, and the relocated HCs are thus prone to further TSG-6/HC2-induced transfer reactions. The data suggest that HCs may be transferred directly from HA to HA without the involvement of the bikunin CS chain. The results demonstrate reversibility of the interactions between HCs and glycosaminoglycans and suggest that a dynamic shuffling of the HCs occur in vivo.     

Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism

Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.     

Dual role for Sox2 in specification of sensory competence and regulation of Atoh1 function

The formation of inner ear sensory epithelia is believed to occur in two steps, initial specification of sensory competent (prosensory) regions followed by determination of specific cell‐types, such as hair cells (HCs) and supporting cells. However, studies in which the HC determination factor Atoh1 was ectopically expressed in nonprosensory regions indicated that expression of Atoh1 alone is sufficient to induce HC formation suggesting that prosensory formation may not be a prerequisite for HC development. To test this hypothesis, interactions between Sox2 and Atoh1, which are required for prosensory and HC formation respectively, were examined. Forced expression of Atoh1 in nonprosensory cells resulted in transient expression of Sox2 prior to HC formation, suggesting that expression of Sox2 is required for formation of ectopic HCs. Moreover, Atoh1 overexpression failed to induce HC formation in Sox2 mutants, confirming that Sox2 is required for prosensory competence. To determine whether expression of Sox2 alone is sufficient to induce prosensory identity, Sox2 was transiently activated in a manner that mimicked endogenous expression. Following transient Sox2 activation, nonprosensory cells developed as HCs, a result that was never observed in response to persistent expression of Sox2. These results, suggest a dual role for Sox2 in inner ear formation. Initially, Sox2 is required to specify prosensory competence, but subsequent down‐regulation of Sox2 must occur to allow Atoh1 expression, most likely through a direct interaction with the Atoh1 promoter. These results implicate Sox2‐mediated changes in prosensory cells as an essential step in their ability to develop as HCs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 3–13, 2017     

Irreversible Heavy Chain Transfer to Hyaluronan Oligosaccharides by Tumor Necrosis Factor-stimulated Gene-6

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).     

Synthesis of nucleic acids and localization of atrial natriuretic peptide in the crayfish haemocytes

Three types of cells circulate in haemolymph of the crayfish Astacus astacus: agranular haemocytes (HCs I), small-granule haemocytes (HCs II) and large-granule haemocytes (HCs III). Their proliferation, differentiation and function remain poorly understood. By means of light and electron microscopic autoradiography using [3H]-thymidine, we have revealed that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To determine whether the HCs I are proliferating progenitor cells for the granular HCs, we have analyzed autographs of HC population in 1, 2, 7 and 21 days after a single [3H]-thymidine administration. Contrary to the expectation, we have failed to find labeled HCs II and HCs III. These findings raise doubts on the capacity of the HCs I to differentiate into two other types of HCs. By autoradiography using 3H-uridine, it has been detected that intensity of the RNA synthesis was the greatest in HCs I and less by a factor of two and four in HCs II and HCs III, respectively. Additionally, by EM immunocytochemistry, ANP-like immunoreactivity was revealed in the large granules of the HCs III. We assume that availability of ANP in secretory granules extends the possible functions of the crayfish HCs and suggests their participation in regulation of water-salt balance and immune responses.     

Diagnostic efficacy of immunocytochemistry on fine needle aspiration biopsies processed by thin-layer cytology

OBJECTIVE: To evaluate the efficacy of immunocytochemistry (ICC) performed on smears processed by thin-layer cytology (TLC). STUDY DESIGN: During the period January 2001-September 2003, 3,573 consecutive fine needle aspiration biopsies were processed with both conventional smears (CSs) and TLC diagnosed by a single pathologist; 113 required immunocytochemical study. CSs were fixed in ethanol whereas TLC slides were processed with the ThinPrep 2000 method (Cytyc Co., Marlborough, Massachusetts, U.S.A); both were stained with Papanicolaou stain. ICC staining was carried out on only TLC slides. RESULTS: The 113 cytologic cases were grouped as follows: 32 thyroid nodules with 16 histologic controls (HCs), 24 lymph nodes (regardless of location) with 15 HCs, 18 liver and pancreatic lesions (3 HCs), 11 lung nodules (6 HCs), 5 kidney and adrenal gland lesions (1 HC), 6 abdominal (2 HCs) and 4 mediastinal masses (1 HC), 6 salivary gland tumors (3 HCs), 4 bone masses (2 HCs) and 3 subcutaneous lesions (1 HC). ICC contributed to the diagnosis in 104 cases (92%), whereas it was inconclusive in 9. The cytologic diagnoses were histologically confirmed in 46 of 50 cases (92%). CONCLUSION: ICC can be successfully applied on TLC slides with better results than on CSs, and its yield can be useful in making the correct diagnosis on fine needle aspiration biopsy.     

Synthesis of nucleic acids and localization of atrial natriuretic peptide in crayfish haemocytes

Three types of cells circulate in the haemolymph of the crayfish Astacus astacus, i.e., agranular haemocytes (HCs I), small-granule haemocytes (HCs II), and large-granule haemocytes (HCs III). Their proliferation, differentiation, and function remain poorly understood. Using light and electron microscopic autoradiography with [³H]-thymidine, we found that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To verify whether HCs I are proliferating progenitor cells for granular HCs, we have analyzed autographs of the HC population 1, 2, 7, and 21 days after a single [3H]-thymidine administration. Contrary to our expectations, we have failed to find labeled HCs II and HCs III. These findings have raised doubts as to the capacity of HCs I to differentiate into two other types of HCs. With the use of ³H-uridine autoradiography, it was found that RNA synthesis was the most active in HCs I and 2 and 4 times lower in HCs II and HCs III, respectively. ANP-like immunoreactivity was revealed in large granules of the HCs III by electron microscopic immunocytochemistry. We assume that the presence of ANP in secretory granules extends the possible functions of crayfish HCs and suggests their participation in the regulation of the watersalt balance and immune response.     

ATPase sites in two-headed fragment of Tetrahymena 22S ciliary dynein.

Three-headed Tetrahymena 22S ciliary dynein was found to consist of three heavy chains (HCs) and decompose into two-headed and single-headed fragments upon chymotrypsin digestion. The three HCs (A alpha, A beta, and A gamma) were immunologically different, and presumed to be located on each of the head regions. The two-headed fragment contained A beta and A gamma HCs, while the A alpha HC originated in the single-headed fragment. Both fragments were associated with ATPase activity (Toyoshima, Y. (1987a) J. Cell Biol. 105, 887-895 and Toyoshima, Y. (1987b) J. Cell Biol. 105, 897-901). Using the two-headed dynein fragment, we attempted to determine the site of ATP hydrolysis in the fragment. After digestion of the fragment with 100 micrograms/ml thermolysin for 45 min, we noted eight thermolysin-digested polypeptides (TH 1, 2, 3, 4, 5 alpha, 5 beta, 6 alpha, and 6 beta). By precisely analyzing the degradation process and the products using peptide mapping, immunoblotting and high pressure liquid chromatography, it appeared that the two-headed fragment is dissociated as two separate fragments, each of which contained A beta or A gamma HC. Thermolysin digests, TH 1, 2, 5 alpha and 6 beta were found to be derived from A beta HC, while TH 3, 4, 5 beta and 6 alpha originated in the A gamma HC. Based on the measurements of ATPase activity of these polypeptides, we concluded that the ATPase site is located in the A beta and A gamma HCs, which may have their origins in each head of the two-headed fragment of Tetrahymena 22S ciliary dynein.     

A positive feedback synapse from retinal horizontal cells to cone photoreceptors

Cone photoreceptors and horizontal cells (HCs) have a reciprocal synapse that underlies lateral inhibition and establishes the antagonistic center-surround organization of the visual system. Cones transmit to HCs through an excitatory synapse and HCs feed back to cones through an inhibitory synapse. Here we report that HCs also transmit to cone terminals a positive feedback signal that elevates intracellular Ca(2+) and accelerates neurotransmitter release. Positive and negative feedback are both initiated by AMPA receptors on HCs, but positive feedback appears to be mediated by a change in HC Ca(2+), whereas negative feedback is mediated by a change in HC membrane potential. Local uncaging of AMPA receptor agonists suggests that positive feedback is spatially constrained to active HC-cone synapses, whereas the negative feedback signal spreads through HCs to affect release from surrounding cones. By locally offsetting the effects of negative feedback, positive feedback may amplify photoreceptor synaptic release without sacrificing HC-mediated contrast enhancement.     

Irreversible Heavy Chain Transfer to Chondroitin

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.     

The role of Wnt/β‐catenin signaling in proliferation and regeneration of the developing basilar papilla and lateral line

Canonical Wnt/β‐catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β‐catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β‐catenin signaling, we show that this pathway is active throughout the BP (E6‐E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β‐catenin activity in developing HCs, we performed gain‐ and loss‐of‐function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up‐regulated cell proliferation within the Sox2‐positive cells of the prosensory domains. Furthermore, Wnt/β‐catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside‐induced HC loss. Combined, our data suggest that Wnt/β‐catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β‐catenin signaling could play an important role in therapeutic HC regeneration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 438–456, 2014