Chloride cells and pavement cells in gill epithelia of Acipenser naccarii: ultrastructural modifications in seawater‐acclimated specimens

Modifications in the chloride (mitochondria‐rich) and pavement cells of the gill epithelia of the Adriatic sturgeon Acipenser naccarii after their transfer under hypertonic environmental conditions (salinity 35) were examined by light and electron microscopy. In contrast to freshwater specimens, seawater‐acclimated fish showed a marked increase in the number and size of chloride cells. Ultrastructural modifications included: presence of a slightly invaginated apical crypt, a darker cytoplasm, a more compact tubular system, a major increase in cisternae from Golgi apparatus stacks and flattened‐out sacs with dilated ends that produced an increase in lateral and basal cell surfaces. All these changes indicated enhanced cellular activity. Pavement cells, which largely covered the chloride cells on the gill filament and lamella, exhibited a complex system of microridges on their apical surface. Typical features included numerous desmosomes that characterized the intercellular junction, and the presence in the apical cytoplasm of bundles of filaments and of electro‐dense vesicles in freshwater fish or clear vesicles in seawater‐acclimated animals.     

Fine structure of the skin cells of a stenohaline freshwater fish Cyprinus carpio exposed to diluted seawater

Seawater diluted to half (1.750% salinity) is lethal for adult carps after 3 h and 15 min. At lower salinities (0.350%-0.875%), the fish survived for longer periods, but only 0.175% salinity was innocuous. In carps, adapted to 0.175% salinity, the secretory activity of pavement cells was very high and their external ridges flattened or even disappeared. Mucus secretion was conspicuous, characterized by holocriny of old cells and apparition of young ones in large numbers. The intracellular mucus droplets often coalesced. Pavement cells and mucus cells were disconnected from their neighboring tissue fabric and were sloughing off. Mitotic figures of filament cells were frequent, suggesting high turnover. Club cells appeared near the epidermal surface. The number of pinocytotic vesicles of the basal cell layer markedly decreased, indicating a possible decrease in dermis-epidermis molecular transfers. Leucocytes, mainly lymphocytes penetrated into the epidermis, where also rodlet cells appeared. The low salinity tolerance of the carp might be related among other possible factors to the absence of chloride cells in the skin.     

Cellular responses in the skin of rainbow trout (Oncorhynchus mykiss) exposed to Rhine water

Trout, Oncorhynchus mykiss , were exposed to water from the Rhine for 24 days and their skin examined by light and electron microscopy. Relative to control fish mitotic figures were common and seen throughout the epidermis. Pavement cells in fish exposed to Rhine water contained significantly more secretory vesicles than control fish. Necrotic pavement cells were apparent after 24 h, and apoptotic cells from day 4 on. Mucous secretion was intense and the differentiation of mucous cells was stimulated. Some of these cells synthesized mucus of high electron density, probably of a serous composition. Leucocytes invaded the dermis and epidermis, and towards the end of the experiment many apoptotic and necrotic lymphocytes were found. In the dermis fibroblasts were abundant and actively producing collagen. Pigment containing cytoplasmic extensions of melanocytes penetrated into the epidermis. After 14 and 24 days of exposure many pigment cells, melanocytes, iridocytes and xanthocytes became apoptotic. Most of these changes are known from fish exposed to heavy metals, acid water or other stressful treatments, indicating that exposure to Rhine water is a stressful experience for trout.     

The source of lamellar mitochondria-rich cells in the air-breathing fish, Trichogaster leeri

Pavement cells and the mitochondria-rich cells (MRCs) are two of the main types of cells in fish gill epithelia. The pavement cells are generally responsible for gas exchange and MRCs for ion regulation. MRCs are found especially in the trailing edge and the interlamellar region of gill filament. In some species, MRCs are also observed in the gill lamellae. A previous study reported the likelihood of having lamellar MRCs in air-breathing fishes. Nevertheless, the source of lamellar MRCs is unclear. We used the air-breathing fish, Trichogaster leeri, to investigate the source of proliferated cells on the lamellae when 5-bromo-2-deoxyuridine (BrdU) was injected at different times before fish were sampled from deionized water. There were two major findings in this study. First, undifferentiated cells were found in the lamellae, as well as in the filaments. And, within 12-24 hr, a proliferated cell, identified as BrdU cell, could differentiate to an MRC in the gill lamellae. Second, the filaments and the lamellae in T. leeri responded to ionic stress differently but the proportion of the proliferated MRCs to the BrdU cells remained constant. Our results suggested that the lamellar MRCs were mainly differentiated from the cells that proliferated earlier from the lamellae.     

Molecular mechanisms controlling pavement cell shape in Arabidopsis leaves

Pavement cells have an interlocking jigsaw puzzle-shaped leaf surface pattern. Twenty-three genes involved in the pavement cell morphogenesis were discovered until now. The mutations of these genes through various means lead to pavement cell shape defects, such as loss or lack of interdigitation, the reduction of lobing, gaps between lobe and neck regions in pavement cells, and distorted trichomes. These phenotypes are affected by the organization of microtubules and microfilaments. Microtubule bands are considered corresponding with the neck regions of the cell, while lobe formation depends on patches of microfilaments. The pathway of Rho of plant (ROP) GTPase signaling cascades regulates overall activity of the cytoskeleton in pavement cells. Some other proteins, in addition to the ROPs, SCAR/WAVE, and ARP2/3 complexes, are also involved in the pavement cell morphogenesis.     

Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.     

Fine structure of the branchial epithelium in the teleost Oreochromis niloticus

We have studied the gill epithelium of Oreochromis niloticus using transmission electron microscopy with the particular interested relationship between cell morphology and osmotic, immunoregulatory, or other non‐regulatory functions of the gill. Pavement cells covered the filament epithelium and lamellae of gills, with filament pavement cells showing distinct features from lamellar pavement cells. The superficial layer of the filament epithelium was formed by osmoregulatory elements, the columnar mitochondria‐rich, mucous and support cells, as well as by their precursors. Light mitochondria‐rich cells were located next to lamellae. They exhibited an apical crypt with microvilli and horizontal small dense rod‐like vesicles, sealed by tight junctions to pavement cells. Dark mitochondria‐rich cells had long dense rod‐like vesicles and a small apical opening sealed by tight junctions to pavement cells. The deep layer of the filament epithelium was formed by a network of undifferentiated cells, containing neuroepithelial and myoepithelial cells, macrophage and eosinophil‐like cells and their precursors, as well as precursors of mucous cells. The lateral‐basal surface was coated by myoepithelial cells and a basal lamina. The lamellar blood lacunae was lined by pillar cells and surrounded by a basal lamina and pericytes. The data presented here support the existence of two distinct types of pavement cells, mitochondria‐rich cells, and mitochondria‐rich cells precursors, a structural role for support cells, a common origin for pavement cells and support cells, a paracrine function for neuroepithelial cells in the superficial layer, and the control of the lamellar capillary base by endocrine and contractile cells. Data further suggest that the filament superficial layer is involved in gill osmoregulation, that may interact, through pale mitochondria‐rich cells, with the deep layer and lamellae, whereas the deep layer, through immune and neuroendocrine systems, acts in the regeneration and defense of the tissue. J. Morphol. 2010. © 2010 Wiley‐Liss, Inc.     

Molecular characterization of water-selective AQP (EbAQP4) in hagfish: insight into ancestral origin of AQP4

Hagfish (Eptatretus burgeri) are agnathous and are the earliest vertebrates still in existence. Pavement cells adjacent to the mitochondria-rich cells show orthogonal arrays of particles (OAPs) in the gill of hagfish, a known ultrastructural morphology of aquaporin (AQP) in mammalian freeze-replica studies, suggesting that an AQP homolog exists in pavement cells. We therefore cloned water channels from hagfish gill and examined their molecular characteristics. The cloned AQP [E. burgeri AQP4 (EbAQP4)] encodes 288 amino acids, including two NPA motifs and six transmembrane regions. The deduced amino acid sequence of EbAQP4 showed high homology to mammalian and avian AQP4 (rat, 44%; quail, 43%) and clustered with AQP4 subsets by the molecular phylogenetic tree. The osmotic water permeability of Xenopus oocytes injected with EbAQP4 cRNA increased eightfold compared with water-injected controls and was not reversibly inhibited by 0.3 mM HgCl(2). EbAQP4 mRNA expression in the gill was demonstrated by the RNase protection assay; antibody raised against the COOH terminus of EbAQP4 also detected (by Western blot analysis) a major approximately 31-kDa band in the gill. Immunohistochemistry and immunoelectron microscopy showed EbAQP4 localized along the basolateral membranes of gill pavement cells. In freeze-replica studies, OAPs were detected on the protoplasmic face of the split membrane comprising particles 5-6 nm long on the basolateral side of the pavement cells. These observations suggest that EbAQP4 is an ancestral water channel of mammalian AQP4 and plays a role in basolateral water transport in the gill pavement cells.     

Re-Evaluation of the AASHTO-Flexible Pavement Design Equation with Neural Network Modeling

Here we establish that equivalent single-axle loads values can be estimated using artificial neural networks without the complex design equality of American Association of State Highway and Transportation Officials (AASHTO). More importantly, we find that the neural network model gives the coefficients to be able to obtain the actual load values using the AASHTO design values. Thus, those design traffic values that might result in deterioration can be better calculated using the neural networks model than with the AASHTO design equation. The artificial neural network method is used for this purpose. The existing AASHTO flexible pavement design equation does not currently predict the pavement performance of the strategic highway research program (Long Term Pavement Performance studies) test sections very accurately, and typically over-estimates the number of equivalent single axle loads needed to cause a measured loss of the present serviceability index. Here we aimed to demonstrate that the proposed neural network model can more accurately represent the loads values data, compared against the performance of the AASHTO formula. It is concluded that the neural network may be an appropriate tool for the development of databased-nonparametric models of pavement performance.     

Isolation and structural characterization of cap-binding proteins from poliovirus-infected HeLa cells.

In poliovirus-infected HeLa cells, poliovirus RNA is translated at times when cellular mRNA translation is strongly inhibited. It is thought that this translational control mechanism is mediated by inactivation of a cap-binding protein complex (comprising polypeptides of 24 [24-kilodalton cap-binding protein], 50, and approximately 220 kilodaltons). This complex can restore the translation of capped mRNAs in extracts from poliovirus-infected cells. We have previously shown that the virally induced defect prevents interaction between cap recognition factors and mRNA. Here, we show that the cap-binding protein complex (and not the 24-kilodalton cap-binding protein) has activity that restores the cap-specific mRNA-protein interaction when added to initiation factors from poliovirus-infected cells. Thus, the activity that restores the cap-specific mRNA-protein interaction and that which restores the translation of capped mRNAs in extracts from poliovirus-infected cells, copurify. The results also indicate, by an alternative assay, that the cap-binding protein complex is the only factor inactivated by poliovirus. We also purified cap-binding proteins from uninfected and poliovirus-infected HeLa cells. By various criteria, the 24-kilodalton cap-binding protein is not structurally modified as a result of infection. However, the 220-kilodalton polypeptide of the cap-binding protein complex is apparently cleaved by a putative viral (or induced) protease. By in vivo labeling and m7GDP affinity chromatography, we isolated a modified cap-binding protein complex from poliovirus-infected cells, containing proteolytic cleavage fragments of the 220-kilodalton polypeptide.     

Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells.

A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.     

Chloroplast division: squeezing the photosynthetic captive

Chloroplasts have evolved from a cyanobacterial endosymbiont and have been retained in eukaryotic cells for more than one billion years via chloroplast division and inheritance by daughter cells during cell division. Recent studies revealed that chloroplast division is performed by a large protein complex at the division site, encompassing both the inside and the outside of the two envelope membranes. The division complex has retained a few components of the cyanobacterial division complex to go along with other components supplied by the host cell. On the basis of the information about the division complex, we are beginning to understand how the division complex evolved, and how eukaryotic host cells regulate chloroplast division during proliferation and differentiation.     

Copper deprivation potentiates oxidative stress in HL-60 cell mitochondria.

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.     

Transformation in pneumococcus: protein content of eclipse complex.

A two-step purification of pneumococcal eclipse complex is described, which uses sucrose gradient sedimentation followed by agarose gel permeation chromatography. Purified complex contains, in addition to donor DNA single strands, macromolecular material that can be labeled with methionine or leucine during development of competence. This material co-chromatographed with eclipse complex DNA on hydroxylapatite, was dissociated from the DNA by sodium dodecyl sulfate, and was completely digested by Pronase. The sodium dodecyl sulfate-released material eluted as a single peak in sodium dodecyl sulfate chromatography. These properties were consistent with the noncovalent association with eclipse complex of a protein or class of proteins with a narrow range of polypeptide sizes. Evidence for the specific association of this protein with transforming DNA is eclipse was also obtained from parallel purification from 35S-labeled nontransformed cells; the amount of methionine label in the corresponding fractions in such cells was only 5% of that in transformed cells.     

Cytosolic chaperonin-containing t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycle.

The chaperonin-containing t-complex polypeptide 1 (CCT) is a cytosolic molecular chaperone composed of eight subunits that assists in the folding of actin, tubulin and other cytosolic proteins. We show here that the content of particular subunits of CCT within mammalian cells decreases concomitantly with the reduction of chaperone activity during cell cycle arrest at M phase. CCT recovers chaperone activity upon resumption of these subunits after release from M phase arrest or during arrest at S phase. The levels of alpha, delta and zeta-1 subunits decreased more rapidly than the other subunits during M phase arrest by colcemid treatment and recovered after release from the arrest. Gel filtration chromatography or native (nondenaturing) PAGE analysis followed by immunoblotting indicated that the alpha and delta subunit content in the 700- to 900-kDa CCT complex was appreciably lower in the M phase cells than in asynchronous cells. In vivo, the CCT complex of M-phase-arrested cells was found to bind lower amounts of tubulin than that of asynchronous cells. In vitro, the CCT complex of M phase-arrested cells was less active in binding and folding denatured actin than that of asynchronous cells. On the other hand, the CCT complex of asynchronous cells (a mixture of various phases of cell cycle) exhibited lower alpha and delta subunit content and lower chaperone activity than that of S-phase-arrested cells obtained by excess thymidine treatment. In addition, turnover (synthesis and degradation) rates of the alpha and delta subunits in vivo were more rapid than those of most other subunits. These results suggest that the content of alpha and delta subunits of CCT reduces from the complete active complex in S phase cells to incomplete inactive complex in M phase cells.     

Learning receptive field properties of complex cells in V1

There are two distinct classes of cells in the primary visual cortex (V1): simple cells and complex cells. One defining feature of complex cells is their spatial phase invariance; they respond strongly to oriented grating stimuli with a preferred orientation but with a wide range of spatial phases. A classical model of complete spatial phase invariance in complex cells is the energy model, in which the responses are the sum of the squared outputs of two linear spatially phase-shifted filters. However, recent experimental studies have shown that complex cells have a diverse range of spatial phase invariance and only a subset can be characterized by the energy model. While several models have been proposed to explain how complex cells could learn to be selective to orientation but invariant to spatial phase, most existing models overlook many biologically important details. We propose a biologically plausible model for complex cells that learns to pool inputs from simple cells based on the presentation of natural scene stimuli. The model is a three-layer network with rate-based neurons that describes the activities of LGN cells (layer 1), V1 simple cells (layer 2), and V1 complex cells (layer 3). The first two layers implement a recently proposed simple cell model that is biologically plausible and accounts for many experimental phenomena. The neural dynamics of the complex cells is modeled as the integration of simple cells inputs along with response normalization. Connections between LGN and simple cells are learned using Hebbian and anti-Hebbian plasticity. Connections between simple and complex cells are learned using a modified version of the Bienenstock, Cooper, and Munro (BCM) rule. Our results demonstrate that the learning rule can describe a diversity of complex cells, similar to those observed experimentally.     

Human ETS1 oncoprotein. Purification, isoforms, -SH modification, and DNA sequence-specific binding.

The human ETS1 proto-oncogene proteins have been isolated from the T-cell leukemia line, CEM, by immunoaffinity chromatography and their identity confirmed by NH2-terminal amino acid sequencing. Incubation of CEM cells with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) indicates that ETS proteins can be modified in their cellular context and that pretreatment of the cells with N-ethylmaleimide (NEM) protects ETS1 proteins from TLCK modification. These data show that ETS1 proteins can exist in at least two different states, -SH-available and -SH-protected. Renatured human ETS1 has DNA sequence-specific binding to the PEA3 (CAGGAAGT) motif. The ETS1.PEA3 complex can be observed by electrophoretic mobility shift assays (EMSA). Purified ETS1 retards a band which is exactly the same size as a complex that is retarded from nuclear extracts prepared from CEM cells. Reduced ETS1 is required to form the ETS1.PEA3 complex, however; modification of the ETS1 -SH groups by either NEM or by TLCk does not inhibit formation of the complex. The ETS1.PEA3 complex formed with TLCK-modified ETS1 has a slower mobility than the complex formed with unmodified ETS1. Zone sedimentation analysis of purified ETS1 indicates that it is the monomer of ETS1 which binds to the PEA3 oligonucleotide.     

Latent forms of transforming growth factor-beta (TGF beta) derived from bone cultures: identification of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF beta

Transforming growth factor-beta (TGF beta) is produced by most tissues, including bone, as a complex that is biologically inert. Release of TGF beta homodimer from this latent complex is necessary for TGF beta to exert effects on target cells. Thus, the nature of the latent complex and the mechanisms responsible for TGF beta release are the key to understanding TGF beta actions. We have found that murine calvarial bone cultures secrete multiple latent forms of TGF beta. Using analytical chromatography and Western blot analysis, we have compared bone latent TGF beta with the previously characterized latent complex present in platelets and with simian TGF beta precursor, which is stably expressed in a latent form by Chinese hamster ovarian (CHO) cells. A major component of the bone material appears to be a latent complex of 100 kDa, consisting of mature TGF beta (25-kDa homodimer). Like the recombinant TGF beta precursor, it elutes from a Mono-Q fast pressure liquid chromatography anion exchange column at 0.2 M NaCl and shows a very similar banding pattern on Western blots. Thus, this bone complex closely resembles recombinant TGF beta precursor expressed in a latent form by CHO cells and differs from the naturally occurring platelet complex, which has an additional 135-kDa binding protein that is bound through disulfide bonds to the precursor proregion. Western blot analysis also indicates that, like CHO cells, which express recombinant TGF beta precursor, but unlike other cell types, the bone cultures secrete detectable amounts of uncleaved TGF beta precursor. The bone calvarial culture is the first example of a naturally occurring system that expresses the 100-kDa latent TGF beta complex.     

Purification of aspartate transcarbamylase from Drosophila melanogaster.

A purification procedure is described by which aspartate transcarbamylase was obtained from cultured cells of Drosophila melanogaster as part of a high-molecular-weight enzyme complex. The complex is shown to contain several polypeptides. An antiserum directed against the complex enzyme inhibited in vitro the activity of aspartate transcarbamylase, carbamylphosphate synthetase and dihydro-orotase which were shown to copurify on a sucrose gradient and by gel electrophoresis. A fast preparation procedure using this antiserum yielded a 220 000-molecular-weight protein in addition to the polypeptides present in the complex. A purification procedure is also described to obtain aspartate transcarbamylase from second instar larvae of Drosophila. At this stage, the enzyme is not complexed with carbamylphosphate synthetase and dihydro-orotase but exhibits the same molecular weight as the aspartate transcarbamylase moiety found in the high-molecular-weight complex of cultured cells.