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.     

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.     

Developmental acquisition of a rapid calcium-regulated vesicle supply allows sustained high rates of exocytosis in auditory hair cells

Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca(2+) currents during various voltage stimulations. Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca(2+) efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca(2+) sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca(2+)-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane.     

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.     

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.     

Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia

Hairy cells (HCs) are mature malignant B cells that contain a number of constitutively active signaling molecules including GTP-bound Rac1, protein kinase C, and Src family kinases. Because Rac1 is a component of the reactive oxidant species (ROS)-generating NADPH oxidase system, we investigated the role of this GTPase in ROS production in HCs. In this study, we show that ROS production in HCs involves a flavin-containing oxidase dependent on Ca2+, but not on GTP-Rac1 or protein kinase C. This suggests the involvement of the nonphagocytic NADPH oxidase NOX5, an enzyme found in lymphoid tissues, but not in circulating lymphocytes. By using RT-PCR and Southern and Western blotting and by measuring superoxide anion production in membrane fractions in the absence of cytosolic components, we demonstrate for the first time that HCs (but not circulating normal B cells or some other lymphoid cell types) express NOX5. We also demonstrate that inhibition of NADPH oxidase in HCs results in a selective increase in the activity of Src homology region 2 domain-containing phosphatase 1 (SHP-1). Furthermore, SHP-1 in HCs coimmunoprecipitates with tyrosine phosphorylated CD22 and localizes in the same cellular compartment as NOX5. This allows the inactivation of SHP-1 by NOX5-generated ROS and contributes to the maintenance of the constitutive activation of HCs.     

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.     

Robust innate immune responses at the placenta during early gestation may limit in utero HIV transmission

In 2019, >90% of new HIV infections in infants globally occurred vertically. Studies suggest intrauterine transmission most often occurs in the third trimester; however, there are no mechanistic studies to support these observations. We therefore obtained early/mid-gestation and term placentae from 20 HIV/Hepatitis B/CMV negative women. Isolated primary placental macrophages (Hofbauer cells [HCs]) were exposed to HIV-1_BaL and/or interferon (IFN)-α, IFN-β, IFN-λ1, and RIG-I-like receptor (RLR) agonists. qRT-PCR, FACS, ELISA, Luminex, and Western blot analyses determined expression of activation markers, co-receptors, viral antigen, cytokines, antiviral genes, and host proteins. Early gestation HCs express higher levels of CCR5 and exhibit a more activated phenotype. Despite downregulation of CCR5, term HCs were more susceptible to HIV replication. Early gestation HCs displayed a more activated phenotype than term HCs and HIV exposure lead to the further up-regulation of T-cell co-stimulatory and MHC molecules. Limited HIV replication in early/mid gestation HCs was associated with increased secretion of anti-inflammatory cytokines, chemokines, and a more robust antiviral immune response. In contrast, term HCs were more susceptible to HIV replication, associated with dampening of IFN-induced STAT1 and STAT2 protein activation. Treatment of early/mid gestation and term HCs, with type I IFNs or RLR agonists reduced HIV replication, underscoring the importance of IFN and RLR signaling in inducing an antiviral state. Viral recognition and antiviral immunity in early gestation HCs may prevent in utero HIV infection, whereas diminished antiviral responses at term can facilitate transmission. Defining mechanisms and specific timing of vertical transmission are critical for the development of specific vaccines and antiviral therapeutics to prevent new HIV infections in children globally.     

Overactivation of Notch1 signaling induces ectopic hair cells in the mouse inner ear in an age-dependent manner

Background

During mouse inner ear development, Notch1 signaling first specifies sensory progenitors, and subsequently controls progenitors to further differentiate into either hair cells (HCs) or supporting cells (SCs). Overactivation of NICD (Notch1 intracellular domain) at early embryonic stages leads to ectopic HC formation. However, it remains unclear whether such an effect can be elicited at later embryonic or postnatal stages, which has important implications in mouse HC regeneration by reactivation of Notch1 signaling.

Methodology/Principal Findings

We performed comprehensive in vivo inducible overactivation of NICD at various developmental stages. In CAG^CreER+; Rosa26-NICD^loxp/+ mice, tamoxifen treatment at embryonic day 10.5 (E10.5) generated ectopic HCs in the non-sensory regions in both utricle and cochlea, whereas ectopic HCs only appeared in the utricle when tamoxifen was given at E13. When tamoxifen was injected at postnatal day 0 (P0) and P1, no ectopic HCs were observed in either utricle or cochlea. Interestingly, Notch1 signaling induced new HCs in a non-cell-autonomous manner, because the new HCs did not express NICD. Adjacent to the new HCs were cells expressing the SC marker Sox10 (either NICD+ or NICD-negative).

Conclusions/Significance

Our data demonstrate that the developmental stage determines responsiveness of embryonic otic precursors and neonatal non-sensory epithelial cells to NICD overactivation, and that Notch 1 signaling in the wild type, postnatal inner ear is not sufficient for generating new HCs. Thus, our genetic mouse model is suitable to test additional pathways that could synergistically interact with Notch1 pathway to produce HCs at postnatal ages.     

Functional hemichannels formed by human connexin 26 expressed in bacteria

Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure–function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs.     

Differentiation of embryonic stem cells into inner ear vestibular hair cells using vestibular cell derived-conditioned medium

Vestibular hair cells (V–HCs) in the inner ear have important roles and various functions. When V–HCs are damaged, crippling symptoms, such as vertigo, visual field oscillation, and imbalance, are often seen. Recently, several studies have reported differentiation of embryonic stem (ES) cells, as pluripotent stem cells, to HCs, though a method for producing V–HCs has yet to be established. In the present study, we used vestibular cell conditioned medium (V-CM) and effectively induced ES cells to differentiate into V–HCs. Expressions of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5) were significantly increased in ES cells cultured in V-CM for 2 weeks, while those were not observed in ES cells cultured without V-CM. On the other hand, the cochlear HC-related marker Lmod3 was either not detected or detected only faintly in those cells when cultured in V-CM. Our results demonstrate that V-CM has an ability to specifically induce differentiation of ES cells into V–HCs.     

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.     

Delayed mitochondrial dysfunction in apoptotic hair cells in chinchilla cochleae following exposure to impulse noise

Apoptotic death of hair cells (HCs) in the cochlea has been found following exposure to intense noise. The current study was designed to examine the mitochondrial energetic function of HCs during the course of noise-induced apoptosis. Two aspects of the mitochondrial energetic function, succinate dehydrogenase (SDH) activity and mitochondrial membrane potential (MMP), were examined in HCs of chinchilla cochleae following exposure to a series of 75 pairs of impulse noises at 155 dB pSPL. The results showed that nuclear condensation and uptake of propidium iodide or trypan blue appeared at 10 min after the noise exposure, indicating a rapid progression of HC apoptosis. However, SDH activity was preserved at this time point. As the time elapsed (1 hr or 24 hrs) after the noise exposure, all newly-generated apoptotic HCs showed strong SDH activity, indicating the preservation of SDH activity during the course of apoptosis. Examination of MMP with rhodamine 123 staining revealed that MMP was sustained in the apoptotic HCs having mild nuclear condensation, even after the occurrence of cell membrane leakage. MMP was reduced with further progression of nuclear condensation. These results suggest the presence of a delayed mitochondrial dysfunction in apoptotic HCs following exposure to intense noise. Research was supported by the Grant NIDCD 1R03 DC006181-01A1.     

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.     

Carbon Monoxide (CO) Is a Novel Inhibitor of Connexin Hemichannels

Hemichannels (HCs) are hexamers of connexins that can form gap-junction channels at points of cell contacts or “free HCs” at non-contacting regions. HCs are involved in paracrine and autocrine cell signaling, and under pathological conditions may induce and/or accelerate cell death. Therefore, studies of HC regulation are of great significance. Nitric oxide affects the activity of Cx43 and Cx46 HCs, whereas carbon monoxide (CO), another gaseous transmitter, modulates the activity of several ion channels, but its effect on HCs has not been explored. We studied the effect of CO donors (CORMs) on Cx46 HCs expressed in Xenopus laevis oocytes using two-electrode voltage clamp and on Cx43 and Cx46 expressed in HeLa cells using a dye-uptake technique. CORM-2 inhibited Cx46 HC currents in a concentration-dependent manner. The C-terminal domain and intracellular Cys were not necessary for the inhibition. The effect of CORM-2 was not prevented by guanylyl-cyclase, protein kinase G, or thioredoxin inhibitors, and was not due to endocytosis of HCs. However, the effect of CORM-2 was reversed by reducing agents that act extracellularly. Additionally, CO inhibited dye uptake of HeLa cells expressing Cx43 or Cx46, and MCF-7 cells, which endogenously express Cx43 and Cx46. Because CORM-2 carbonylates Cx46 in vitro and induces conformational changes, a direct effect of that CO on Cx46 is possible. The inhibition of HCs could help to understand some of the biological actions of CO in physiological and pathological conditions.     

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).     

Elucidation of the Sodium‐Storage Mechanism in Hard Carbons

Hard carbons (HCs) are the most promising candidate anode materials for emerging Na‐ion batteries (NIBs). HCs are composed of misaligned graphene sheets with plentiful nanopores and defects, imparting a complex correlation between its structure and sodium‐storage behavior. The currently debated mechanism of Na⁺‐ion insertion in HCs hinders the development of high‐performance NIBs. In this article, ingenious and reliable strategies are used to elaborate the correlation between the structure and electrochemical performance and further illuminate the sodium‐storage mechanism in HCs. First, filling sulfur into the micropores of HCs can remove the low‐voltage plateau, providing solid evidence for its association with the pore‐filling mechanism. Along with the decreased concentration of defects/heteroatoms at higher treatment temperature, the reduced sloping capacity confirms the adsorption mechanism in the sloping region. Finally, the similar sodium‐insertion behaviors of HCs with ether‐based and ester‐based electrolytes indicate that no Na⁺ ions intercalate between the graphene layers. The determined adsorption‐pore‐filling mechanism encourages the design of more efficient HC anode materials with high capacity for high‐energy NIBs.     

pH changes in the invaginating synaptic cleft mediate feedback from horizontal cells to cone photoreceptors by modulating Ca2+ channels

Feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the center-surround-receptive field organization of retinal neurons. Recordings from cone photoreceptors in newt retinal slices were obtained by the whole-cell patch-clamp technique, using a superfusate containing a GABA antagonist (100 microM picrotoxin). Surround illumination of the receptive field increased the voltage-dependent calcium current (ICa) in the cones, and shifted the activation voltage of ICa to negative voltages. External alkalinization also increased cone ICa and shifted its activation voltage toward negative voltages. Enrichment of the pH buffering capacity of the extracellular solution increased cone ICa, and blocked any additional increase in cone ICa by surround illumination. Hyperpolarization of the HCs by a glutamate receptor antagonist-augmented cone ICa, whereas depolarization of the HCs by kainate suppressed cone ICa. From these results, we propose the hypothesis that pH changes in the synaptic clefts, which are intimately related to the membrane voltage of the HCs, mediate the feedback from the HCs to cone photoreceptors. The feedback mediated by pH changes in the synaptic cleft may serve as an additional mechanism for the center-surround organization of the receptive field in the outer retina.