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Convergent evolution of crystallin gene regulation in squid and chicken: the AP-1/ARE connection.

Previous experiments have shown that the minimal promoters required for function of the squid SL20-1 and SL11 crystallin genes in transfected rabbit lens epithelial cells contain an overlapping AP-1/antioxidant responsive element (ARE) upstream of the TATA box. This region resembles the PL-1 and PL-2 elements of the chicken beta B1-crystallin promoter which are essential for promoter function in transfected primary chicken lens epithelial cells. Here we demonstrate by site-directed mutagenesis that the AP-1/ARE sequence is essential for activity of the squid SL20-1 and SL11 promoters in transfected embryonic chicken lens cells and fibroblasts. Promoter activity was higher in transfected lens cells than in fibroblasts. Electrophoretic mobility shift and DNase protection experiments demonstrated the formation of numerous complexes between nuclear proteins of the embryonic chicken lens and the AP-1/ARE sequences of the squid SL20-1 and SL11 crystallin promoters. One of these complexes comigrated and cross-competed with that formed with the PL-1 element of the chicken beta B1-crystallin promoter. This complex formed with nuclear extracts from the lens, heart, brain, and skeletal muscle of embryonic chickens and was eliminated by competition with a consensus AP-1 sequence. The nonfunctional mutant AP-1/ARE sequences did not compete for complex formation. These data raise the intriguing possibility that entirely different, nonhomologous crystallin genes of the chicken and squid have convergently evolved a similar cis-acting regulatory element (AP-1/ARE) for high expression in the lens.

Animals↗

Glutathione S-transferase and S-crystallins of cephalopods: evolution from active enzyme to lens-refractive proteins.

Our previous studies have shown that the S-crystallins of cephalopod (Ommastrephes sloani pacificus) eye lenses comprise a family of at least ten members which are evolutionarily related to glutathione S-transferase (GST, EC 2.5.1.18). Here we show by cDNA cloning that there are at least 24 different S-crystallins that are 46-99% identical to each other by amino acid sequence in the squid Loligo opalescens. In each species, all but one S-crystallin (SL11 in O. pacificus and Lops4 in L. opalescens) examined has an inserted central peptide of variable length and sequence. cDNA expression studies conducted in Escherichia coli showed that squid GST (which is expressed little in the lens) has very high enzymatic activity using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate; by contrast, SL20-1 of O. pacificus and Lops12 of L. opalescens (which are encoded by abundant lens mRNAs) have no GST activity. Interestingly, SL11 and Lops4 have some enzymatic activity with the CDNB substrate. Site-specific mutations at Y7 or W38, both residues essential for activity of vertebrate GSTs, or insertion of the central peptide present in the inactive SL20-1, reduced the specific activity of squid GST by 30- to 100-fold. These data indicate that the S-crystallins consist of a family of enzymatically inactive proteins (when using CDNB as a substrate) which is considerably larger than previously believed and that GST activity was lost by gradual drift in sequence as well as by insertion of an extra peptide by exon shuffling. The results are also consistent with the idea that SL11 and Lops4 are orthologous crystallins representing the first descendants of the ancestral GST gene in the pathway which gave rise to the extensive S-crystallin family of lens proteins.

Amino Acid Sequence↗

Quantitative analysis of sodium and potassium activation delays in fresh axons of the squid: Loligo forbesi.

Activation kinetics of the sodium and potassium conductances were re-examined in fresh axons of Loligo forbesi exhibiting very little if any potassium accumulation and a very small leak conductance, special attention being paid to the initial lag phase which precedes the turning-on of the conductances. The axons were kept intact and voltage-clamped at 2-3 degrees C. In all cases, the rising phase of the currents could be fitted with very good accuracy using the Hodgkin-Huxley (1952) equations although, in most cases, the turning-on of the conductance did not coincide with the beginning of the depolarizing test pulse. The delay which separates the change in potential and the turning-on of current (the activation delay) was analyzed quantitatively for different prepulse and pulse potentials. The measured activation delay differed significantly from the delay predicted by the original HH equations. This difference (the 'non-HH delay') varied with prepulse and pulse potentials. For the potassium current, the relationship between the non-HH delay and pulse potential for a constant prepulse was bell shaped, the maximum value (0.7 ms for a prepulse to -80 mV) being reached for about 0 mV. For this same current, the relationship between the non-HH delay and the prepulse potential for a constant pulse potential was sigmoidal, starting from a minimum value of around 0.5 ms at -100 mV and rising to 5 ms at -15 mV. Essentially similar results were obtained for the sodium current although the non-HH delay was three to five times smaller and the dependency upon prepulse potential not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The early phase of sodium channel gating current in the squid giant axon. Characteristics of a fast component of displacement charge movement.

A fast component of displacement current which accompanies the sodium channel gating current has been recorded from the membrane of the giant axon of the squid Loligo forbesii. This component is characterized by relaxation time constants typically shorter than 25 microseconds. The charge displaced accounts for about 10% (or 2 nC/cm2) of the total displacement charge attributed to voltage-dependent sodium channels. Using a low noise, wide-band voltage clamp system and specially designed voltage step protocols we could demonstrate that this component: (i) is not a recording artifact; (ii) is kinetically independent from the sodium channel activation and inactivation processes; (iii) can account for a significant fraction of the initial amplitude of recorded displacement current and (iv) has a steady state charge transfer which saturates for membrane potentials above +20 mV and below -100 mV. This component can be modelled as a single step transition using the Eyring-Boltzmann formalism with a quantal charge of 1 e- and an asymmetrical energy barrier. Furthermore, if it were associated with the squid sodium channel, our data would suggest one fast transition per channel. A possible role as a sodium channel activation trigger, which would still be consistent with kinetic independence, is discussed. Despite uncertainties about its origin, the property of kinetic independence allows subtraction of this component from the total displacement current to reveal a rising phase in the early time course of the remaining current. This will have to be taken into account when modelling the voltage-dependent sodium channel.

Animals↗

Phloretin affects the fast potassium channels in frog nerve fibres.

The effect of phloretin (20-100 microM), a dipolar organic compound, on the voltage clamp currents of the frog node of Ranvier has been investigated. The Na currents are simply reduced in size but not otherwise affected. Phloretin has no effect on the slow 4-aminopyridine-resistant K channels. However, the voltage dependence and time course of the fast K conductance (gK) is markedly altered. The gK (E) curve, determined by measuring fast tail currents at different pulse potentials, normally exhibits a bend at -50 mV, indicating the existence of two types of fast K channels. Phloretin shifts the gK (E) curve to more positive potentials, reduces its slope and its maximum and abolishes the distinction between the two types of fast K channels. The effect becomes more pronounced with time. Phloretin also markedly slows the opening of the fast K channels, but has much less effect on the closing. Opening can be accelerated again by a long depolarizing prepulse which presumably removes part of the phloretin block. It is concluded that phloretin selectively affects the fast K channels of the nodal membrane. The results are compared with similar observations on the squid giant axon.

4-Aminopyridine↗

Functional states of an excitable membrane and their dependence on its parameter values.

The property of an excitable membrane of a nerve cell to change the type of electrical activity has been examined with the change of the value of applied current (I). The dependence of this property on the values of the membrane parameters is determined. Two different functional states depending on the values of the membrane parameters are considered. For one of the states a change in the value of I is accompanied by a change in the type of activity (damped periodic oscillations jump to undamped periodic oscillations or vice versa). For the other state the type of activity remains phasic (damped periodic oscillations) for each value of I. For the mathematical model of a membrane we have considered the problem of obtaining the boundary, partitioning the parameter space into the regions to which these functional states correspond. We suggest a mathematical set of this problem and give its algorithm. These boundaries have been constructed for two different variable parameters of the model. A good agreement between the boundaries and the experimental values of sodium and potassium conductances for different excitable membranes has been obtained.

Algorithms↗

Two and three dimensional reductions of the Hodgkin-Huxley system: separation of time scales and bifurcation schemes.

We study two different two-dimensional reductions of the Hodgkin-Huxley equations. We show that they display the same qualitative bifurcation scheme as the original equations but overestimate the current range where periodic emission occurs. This is essentially due to the assumption that the evolution of the sodium activation variable m is instantaneous with respect to the dynamics of the variables h and n, an hypothesis that breaks down at high values of the injected current. To prove this point we compare the current-amplitude relation, the current-frequency relation, and the shapes of individual spikes for the two reduced models to the results obtained for the original Hodgkin-Huxley model and for a three-dimensional model with instantaneous sodium activation. We show that a more satisfying agreement with the original Hodgkin-Huxley equations is obtained if we modify the evolution equation for the potential by incorporating the prominent features of the dynamics of m.

Animals↗

On the relationship between the number of negative slope regions in the voltage-current curve of the Hodgkin-Huxley model and its parameter values.

We consider the problem of the existence of a negative slope region (NSR) in the voltage-current curve of the neuronal membrane and the relationship between this phenomenon and the membrane parameters. For the Hodgkin-Huxley model it is proposed to determine the dependence of the number of NSR on the values of the maximal sodium (gNa) and potassium (gk) conductances. The method is suggested for constructing the boundaries on the (gNa, gk) plane, in passing through which the number of NSR changes to 1. Using the method we partition the (gNa, gk) plane into the regions corresponding to the curves with the different number of NSR. This number can be changed from 0 to 2 in changing the values of gNa and gk over the physiologically possible range.

Animals↗

Neurofilamentous network and filamentous matrix preserved and isolated by different techniques from squid giant axon.

The contribution of the neurofilamentous network to the structure of the squid giant axon was analyzed electron-microscopically. Axial 10-nm filaments cross-linked by radial 5-nm bridges form a network that is present in preparations prepared by a variety of techniques. The axoplasm is differentiated into dense and less dense regions. In the presence of Co(II) ions, the neurofilamentous network was remarkably well preserved and appeared to be associated with a dense web of fine filament matrix, which also was identified in extracted axoplasm and in fractions enriched with neurofilament protein complex. In the presence of La(III) ions, the neurofilamentous network had a coarse and open appearance. The stereo images of extracted and critical-point dried axoplasm suggested that the neurofilamentous network contains ordered lattice-like regions. Extracted preparations of extruded axoplasm and fractions enriched with neurofilament protein complex suggested that the properties of the network are determined by the neurofilament protein complex. It is proposed that the neurofilamentous network is the essential determinant of the form of the axon, and that the order within the network is determined by the radial components of the network. The structures observed in the different preparations are not artifacts, but rather are related closely to their native state in the axon.

Animals↗

In vitro polymorphism and phase transitions of the neurofilamentous network isolated from the giant axon of the squid (Loligo pealei L.).

Using electron microscopy (EM), optical diffraction and image reconstruction techniques, we have demonstrated polymorphism of neurofilamentous network (NFN) in vitro based on phase transitions of the protein assemblies. The specific polymorphic appearances depended upon a number of factors, such as K+, Mg2+, Ca2+ ions, as well as the charge and hydration state of the molecules. Furthermore, modifications initiated by the state of phosphorylation of the sidearm proteins played an important role, especially in determining the sidearm disposition of the NFN. The Ca2+-activated protease removed the sidearms. Other enzymes activated by Ca2+ may initiate new association patterns of the peptide remnants and the intercoiling of two smooth neurofilaments (NFs) into paired helical filament-like (PHF-like) strands. Prolonged storage of the isolated NFs in Rubinson-Baker solution resulted in autocrosslinking and intercoiling of modified NFN components. The in vitro polymorphism and phase transitions of squid NFN induced under controlled conditions have been compared to modifications of cytoskeleton observed by EM in frontal lobe biopsies of Alzheimer patients. We conclude that similar processes, as induced in vitro, do occur in neurons of Alzheimer patients.

Alzheimer Disease↗

Removal of the Schwann sheath from the giant nerve fiber of the squid: an electron-microscopic study of the axolemma and associated axoplasmic structures.

The axolemma is associated structurally and functionally with the axoplasm, forming an axolemma-ectoplasm complex. To study the structure of this complex, a new technique was developed for removing the Schwann sheath from a portion of the giant nerve fiber. An isolated fiber was treated, without loss of excitability, with trypsin dissolved in natural seawater. Next, the fiber was treated with a mild fixative and then was placed in a hypertonic solution of sucrose in seawater. The elevated sheath was transected and everted, thus exposing the surface of the axon. Desheathed axons were examined by scanning and transmission electron microscopy. The surface of the axon has a ridge-and-groove pattern, reflecting an underlying helical arrangement of filaments which bundle and unbundle. Both left and right axons of the squid possess right-handed helical twists with a tilt angle of 10 degrees. Hemispherical protuberances about 1.5 microns at their base are observed along the ridges. Thin sections of the desheathed axons reveal that the desheathing procedure leaves the axolemma intact. Desheathed axons display electron-dense bodies associated with the axolemma and with the filaments of the ectoplasm similar to the dense bodies observed in whole fibers fixed in the presence of 10 mM Co(II) ions. Axons perfused for 40 min with a solution containing 2 mM Co(II) ions retain their excitability and display a smooth inner ectoplasmic face. A portion of the axolemma, together with adhering ectoplasm, was removed from desheathed axons, mounted between folding double grids, stained, and critical-point dried. Through this novel method a network of 10 nm filaments spaced 40 nm apart and cross-linked by filaments 5 to 7 nm in diameter was demonstrated.

Animals↗

Immunocytochemical localization of a rhodopsin-like protein in the lipochondria in photosensitive neurons of Aplysia californica.

Polyclonal antibodies directed against squid opsin were used in immunocytochemical and immunoblot experiments to identify a rhodopsin-like protein in photosensitive neurons of Aplysia. Aldehyde-fixed abdominal and cerebral ganglia were embedded in paraffin for peroxidase anti-peroxidase analysis or used whole for immunofluorescence studies. Ganglia were embedded in Lowicryl K4M for electron-microscope immunocytochemistry. In both the cerebral and abdominal ganglia, light-microscope immunocytochemical results showed reaction product deposited around the neuronal cell periphery corresponding in position to the lipochondria. In the abdominal ganglion, the giant cell R2, located in the right rostral quarter, and neurons in the right caudal quarter were consistently labeled with anti-opsin. Electron-microscopic studies demonstrated ferritin-labeling of the lipochondria in R2 and other immunoreactive neurons. Immunoblot analysis of R2 and cerebral neuron extracts was used to identify two prominent immunoreactive protein bands at 85 000 and 67 500 molecular weight.

Animals↗

Cell junctions in early embryos of squid (Loligo pealei).

Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7-8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12-13). During germinal layer establishment (stages 12-13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavage-stage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.

Animals↗

The cells that secrete the beaks in octopods and squids (Mollusca, Cephalopoda).

A single layer of cell secrets the hard cephalopod beaks. The beccublasts are tall columnar cells that separate the beak from the surrounding buccal muscles, and must serve to attach these muscles to the beak. Within the cell layer there are three types of cells. The first, and most frequently found contain cell-long fibrils. These fibrils may have contractile and tensile properties. Complex trabeculae extend from the beccublasts into the matrix of the beak. The fibrils are attached to these trabeculae and at the other end of the cells they are anchored near to the beccublast-muscle cell interface, closely associated with the muscles that move the beak. The second group of cells contain masses of endoplasmic reticulum the cysternae of which are arranged along the long axis of the cell. These cells also contain dense granules and are probably the major source of beak hard tissue. It is probable that each cell secretes its own column of beak hard tissue. The third group of cells cells contains a mixture of fibrils and secretory tissue. In the beccublast layer there are changes in the proportion of the three types of cells depending upon the region sampled. In the region where growth is most active there are mostly secretory cells, whereas near the biting and wearing tip there are mainly anchoring type cells.

Animals↗

Ciliated sensory neurons in the lip of the squid Lolliguncula brevis Blainville.

The lip of Lolliguncula brevis is a muscular fold covered by a simple columnar epithelium and overlayed medially and distally by a non-cellular cuticle. Bipolar sensory cells in the epithelium have a shallow pocket with cilia and microvilli at the free end of their dendrite. The cilia project out onto the surface through pores in the cuticle. Cells with intracellular cilia were observed below the epithelium. These cells send a process toward the surface of the lip. These two receptors are compared to the receptors previously described from the lip of Sepia.

Animals↗

Potassium channels in squid neuron cell bodies: comparison to axonal channels.

The squid giant axon is formed from the fusion of many axons whose cell bodies are located in the giant fiber lobe (GFL) of the stellate ganglion. We measured macroscopic potassium channel currents in both squid giant axons and in the cell bodies. These currents appear similar in many ways, but were differently affected by the amino group modifying reagent, trinitrobenzene sulfonic acid (TNBS). TNBS increased the steady-state amplitude of the axonal currents but decreased the somal currents. We also studied single K channel currents in the giant fiber lobe cells, for comparison with single K channel currents measured previously in the axons. We found two common classes of K channels, distinguished by their conductances (11 and 25 pS) and kinetic properties. The two channel types resemble the 10 and 20 pS channels that have been described in squid giant axons. These results suggest that the cell bodies and axons express fundamentally similar K channel types. The different effects of amino group modification on macroscopic K currents in the two regions may arise from small (perhaps posttranslational) modifications of the core proteins.

Amines↗