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PubMed1996

Cultured giant fiber lobe of squid expresses three distinct potassium channel activities in selective combinations.

Neurons from the giant fiber lobe (GFL) of squid Loligo bleekeri were dissociated and cultured. The ionic currents were recorded using whole-cell patch clamp methods. The sodium current and the noninactivating potassium current like those elicited by the giant axon were among the currents expressed in axonal bulbs and bulblike structures upon dissociation. Meanwhile axonless cell bodies did not elicit such currents. Axonless cell bodies and some bulblike structures elicited two kinds of inactivating potassium currents, the slow- and the fast-inactivating current, which differed in their inactivation kinetics and pharmacology. Within 24 hr of plating, the current composition remained the same. While the noninactivating current was not sensitive to 4-aminopyridine, the two inactivating currents were sensitive, the slow-inactivating current being more sensitive. Selective combinations of the sodium current and the three potassium currents expressed in different structures of the acutely dissociated GFL could have resulted from cellular control of synthesis and transportation of the channel proteins to the somatic and the axonal membrane. The sodium current and the noninactivating potassium current could be recorded from some axonless cell bodies maintained in culture for over three days, indicating that the separation of the giant axon from its somata could result in the transportation of the channels normally expressed on the giant axon membrane to the somatic membrane.

4-Aminopyridine↗
PubMed2004

All roads lead to arginine: the squid protamine gene.

The protamine of squid is one of the most arginine-rich protamines (77%, mol/mol). It possesses a leading sequence that is posttranslationally removed during spermatogenesis in a manner that is analogous to that observed in some of its vertebrate protamine counterparts. In this paper we describe the gene sequence of the protamine of the squid Loligo opalescens. This represents the first complete gene sequence ever reported for an invertebrate protamine. Like those of vertebrate protamines, the messenger RNA is polyadenylated but the gene does not contain an intron. The promoter region contains the major transcriptional regulatory elements (CRE, TATA box, and CAP) that are also characteristic of the vertebrate protamine genes. It is unclear whether the similarities of protamines in species from both the deuterostome and the protostome branches represent the result of phylogenetic conservation or evolutionary convergence.

Amino Acid Sequence↗
PubMed2001

The evolution of bioluminescent oxygen consumption as an ancient oxygen detoxification mechanism.

Endogenous reductants such as hydrogen sulfide and alkylthiols provided free radical scavenging systems during the early evolution of life. The development of oxygenic photosynthesis spectacularly increased oxygen levels, and ancient life forms were obliged to develop additional antioxidative systems. We develop here the hypothesis of how "prototypical" bioluminescent reactions had a plausible role as an ancient defense against oxygen toxicity through their "futile" consumption of oxygen. As oxygen concentrations increased, sufficient light would have been emitted from such systems for detection by primitive photosensors, and evolutionary pressures could then act upon the light emitting characteristics of such systems independently of their use as futile consumers of oxygen. Finally, an example of survival of this ancient mechanism in present-day bioluminescent bacteria (in the Euprymna scolopes-Vibrio fischeri mutualism) is discussed. Once increasing ambient oxygen levels reached sufficiently high levels, the use of "futile" oxygen consumption became too bioenergetically costly, so that from this time the evolution of bioluminescence via this role was made impossible, and other mechanisms must be developed to account for the evolution of bioluminescence by a wide range of organisms that patently occurred after this (e.g., by insects).

Adaptation, Physiological↗
PubMed2001

Multi-elemental concentrations in tissues of Japanese common squid (Todarodes pacificus).

Forty-five elements were determined in mantle muscle and liver of juvenile Japanese common squid, Todarodes pacificus, collected from three locations in and near Japanese coasts, using a high-resolution inductively coupled plasma mass spectrometer (HR-ICP-MS) and an inductively coupled plasma atomic emission spectroscope (ICP-AES). Multivariate statistical techniques were applied to discriminate populations of squids from these three locations by treating absolute and relative concentrations of trace elements. Significant differences were found in the concentrations of elements, particularly for some alkaline and alkaline earth elements in the muscle and liver and for some 3d transition elements in the liver of three groups of squids. Squids from the Pacific Ocean could be distinguished from those of the Sea of Japan by discriminant function analysis of elemental concentrations. Based on the analysis, the squids collected from the Nemuro Strait in Japan were predicted to belong to those from the Sea of Japan. Elemental concentrations and discriminant function analysis can be used to identify subpopulations and migratory routes of squids.

Animals↗
PubMed1998

Highly processive motility is not a general feature of the kinesins.

Evidence is presented that the kinesin-related ncd protein is not as processive as kinesin. In low surface density motility experiments, a dimeric ncd fusion protein behaved mechanistically more similar to non-processive myosins than to the highly processive kinesin. First, there was a critical microtubule length for motility; only microtubules longer than this critical length moved in low density ncd surfaces, which suggested that multiple ncd proteins must cooperate to move microtubules in the surface assay. Under similar conditions, native kinesin demonstrated no critical microtubule length, consistent with the behavior of a highly processive motor. Second, addition of methylcellulose to decrease microtubule diffusion decreased the critical microtubule length for motility. Also, the rates of microtubule motility were microtubule length dependent in methylcellulose; short microtubules, that interacted with fewer ncd proteins, moved more slowly than long microtubules that interacted with more ncd proteins. In contrast, short microtubules, that interacted with one or a few kinesin proteins, moved on average slightly faster than long microtubules that interacted with multiple kinesins. We conclude that a degree of processivity as high as that of kinesin, where a single dimer can move over distances on the order of one micrometer, may not be a general mechanistic feature of the kinesin superfamily.

Adenosine Triphosphate↗
PubMed1997

Eyes absent: a gene family found in several metazoan phyla.

Genes related to the Drosophila eyes absent gene were identified in vertebrates (mouse and human), mollusks (squid), and nematodes (C. elegans). Proteins encoded by these genes consist of conserved C-terminal and variable N-terminal domains. In the conserved 271-amino acid C-terminal region, Drosophila and vertebrate proteins are 65-67% identical. A vertebrate homolog of eyes absent, designated Eya2, was mapped to Chromosome (Chr) 2 in the mouse and to Chr 20q13.1 in human. Eya2 shows a dynamic pattern of expression during development. In the mouse, expression of Eya2 was first detected in 8.5-day embryos in the region of head ectoderm fated to become the forebrain. At later stages of development, Eya2 is expressed in the olfactory placode and in a variety of neural crest derivatives. In the eye, expression of Eya2 was first detected after formation of the lens vesicle. At day 17.5, the highest level of Eya2 mRNA was observed in primary lens fibers. Low levels of Eya2 expression was detected in retina, sclera, and cornea. By postnatal day 10, Eya2 was expressed in secondary lens fibers, cornea, and retina. Although Eya2 is expressed relatively late in eye development, it belongs to the growing list of factors that may be essential for eye development across metazoan phyla. Like members of the Pax-6 gene family, eyes absent gene family members were probably first involved in functions not related to vision, with recruitment for visual system formation and function occurring later.

Age Factors↗
PubMed2000

Mixture interactions of glutamate and betaine in single squid olfactory neurons.

We used nystatin-patch techniques to characterize the responses of squid olfactory receptor neurons to the attractive odorant, L-glutamate, and to study mixture interactions between glutamate and the aversive odorant, betaine. We report that glutamate activates a cation-selective conductance that is permeable to Ca2+, K+, and Na+ and which would depolarize squid olfactory receptor neurons under physiological conditions. The responses to glutamate were concentration dependent. The EC50 of individual cells ranged from 0.3 mmol x l(-l) to 85.0 mmol x l(-l). We found that individual cells were capable of responding to both glutamate and betaine, and that the relative magnitudes of these responses varied from cell to cell. Finally, we report that current responses to binary mixtures of glutamate and betaine are suppressed relative to the sum of the responses to the individual odors in single squid olfactory receptor neurons.

Animals↗
PubMed1998

Betaine activates a hyperpolarizing chloride conductance in squid olfactory receptor neurons.

Isolated olfactory receptor neurons from the squid Lolliguncula brevis respond to betaine, a repellent odorant, with hyperpolarizing receptor potentials. Using perforated-patch techniques, we determined that the hyperpolarizing conductance was selective for Cl- and could be reversibly blocked by the Cl- channel blockers 4-acetamido-4'-isothio-cyanatistilbene-2,2'disulfonic acid and niflumic acid. Gramicidin-patch recordings revealed that [Cl-]i in squid olfactory receptor neurons is normally very low compared to vertebrate olfactory receptor neurons, and that activating a Cl- conductance would hyperpolarize the cell in vivo. The lack of dependence on internal or external K+ or Na+ ruled out the possibility that the Cl- conductance was generated by a cation-dependent cotransporter or pump. Common G-protein-dependent signalling pathways, including phospholipase C, arachidonic acid, and cyclic nucleotides, do not appear to be involved. Ca2+ imaging experiments showed that betaine did not affect [Ca2+]i, suggesting that the Cl- current is not Ca2+ dependent. Our findings represent the first report of an odorant-activated, hyperpolarizing chloride conductance in olfactory receptor neurons.

Animals↗
PubMed1999

Transient and persistent tetrodotoxin-sensitive sodium currents in squid olfactory receptor neurons.

Squid olfactory receptor neurons are primary bipolar sensory neurons capable of transducing water-born odorant signals into electrical impulses that are transmitted to the brain. In this study, we have identified and characterized the macroscopic properties of voltage-gated Na+ channels in olfactory receptor neurons from the squid Lolliguncula brevis. Using whole-cell voltage-clamp techniques, we found that the voltage-gated Na+ channels were tetrodotoxin sensitive and had current densities ranging from 5 to 169 pA pF-1. Analyses of the voltage dependence and kinetics revealed interesting differences from voltage-gated Na+ channels in olfactory receptor neurons from other species; the voltage of half-inactivation was shifted to the right and the voltage of half-activation was shifted to the left such that a "window-current" occurred, where 10-18% of the Na+ channels activated and did not inactivate at potentials near action potential threshold. Our findings suggest that in squid olfactory neurons, a subset of voltage-gated Na+ channels may play a role in generating a pacemaker-type current for setting the tonic levels of electrical activity required for transmission of hyperpolarizing odor responses to the brain.

Animals↗
PubMed2000

Ionic currents in cardiac myocytes of squid, Alloteuthis subulata.

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (IA), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of -40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (IK); and third, an outward, calcium-dependent, potassium current (IK(Ca)) sensitive to Co2+ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (INa), blocked by tetrodotoxin, inactivated at holding potentials more positive than -40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (ICa,L) with a sustained time course, suppressed by nifedipine or Co2+, and enhanced by substituting Ca2+ for Ba2+ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current.

4-Aminopyridine↗
PubMed2002

MgATP and phosphoinositides activate Na(+)/Ca(2+) exchange in bovine brain vesicles. Comparison with other Na(+)/Ca(2+) exchangers.

We investigated the metabolic modulation of the Na(+)/Ca(2+) exchanger in membrane vesicles obtained from bovine brain. The Na(+)/Ca(2+) exchanger was activated by MgATP with a K(0.5) of 336 micro M. Unlike the squid nerve Na(+)/Ca(2+) exchanger, this effect required no cytosolic component. Also, stimulation is the same in vesicles prepared and/or assayed at the ionic strength found in mammals (160 mM) or marine animals (300 mM). Other differences between squid and bovine nerve are that the bovine brain Na(+)/Ca(2+) exchanger is not stimulated by phosphagens, either phosphoarginine (molluscan source) or phosphocreatine (mammalian source); and that stimulation by MgATP in bovine brain is related to the production of polyphosphatidylinositides. In this regard bovine heart and brain Na(+)/Ca(2+) exchangers behave similarly. These results indicate that the mechanisms of metabolic regulation of the squid and mammalian nerve Na(+)/Ca(2+) exchangers are not alike and represent differences between species. Some differences found between bovine heart and brain exchangers, such as MgATP stimulation even at saturating [Ca(2+)] and the smaller degree of activation by adenosine 5'- O-(3-thiotriphosphate) (ATP-gamma-S) in the brain, may be related to the unequal isoform population in both tissues.

Adenosine Triphosphate↗
PubMed1998

Induction of apoptosis by cooperative bacteria in the morphogenesis of host epithelial tissues.

Associations with pathogenic bacteria have recently been shown to initiate apoptotic programs in the cells of their animal hosts, where host cell death is hypothesized to be a response of the immune system, either initiated as a mechanism of host defense or bacterial offense. In this study, we present evidence that bacterial initiation of apoptosis is neither restricted to pathogenesis nor to the initation of an immune response. In the cooperative association between the sepiolid squid Euprymna scolopes and the luminous bacterium Vibrio fischeri, the bacteria induce a dramatic morphogenesis of the host tissues during the first few days of interaction between these partners. The most striking change is the bacteria-triggered loss of an extensive superficial epithelium that potentiates the infection process. Our analyses of these tissues revealed that the bacteria induce apoptosis in the cells that comprise this epithelium within hours of the interaction with bacteria. Ultrastructural analysis revealed that after 24 h the integrity of the epithelium had been lost, i.e., the basement membrane had degenerated and the majority of the cells exhibited signs of apoptosis, most notably chromatin condensation. Analysis of these tissues with probes that reveal intracellular acidification showed that the cells first undergo an initial acidification beginning about 6-8 h after exposure to V. fischeri. As determined by end-labeling of DNA fragments, extensive endonuclease activity was detected at approximately 16-20 h post-infection. These data provide evidence that cooperative bacteria can participate in the remodeling of host tissues through the induction of host apoptotic programs.

Animals↗
PubMed2001

The unusual cartilaginous tissues of jawless craniates, cephalochordates and invertebrates.

A collagenous extracellular matrix was previously considered to be a requirement for classification of true cartilage. Data from the lamprey and hagfish now clearly indicate that both of these jawless craniates have extensive non-collagenous, yet cartilaginous endoskeletons. Non-collagenous cartilages are present in the cephalochordates (amphioxus) and in the invertebrates, although collagen-containing cartilages also are found in the invertebrates. This review summarizes current knowledge of the morphological, biochemical and molecular characteristics of the unusual non-collagenous cartilages in jawless craniates and the cartilaginous tissues in amphioxus and invertebrates. A least two types of non-collagenous cartilage matrix proteins are found in both the hagfishes and the lampreys, all of which are resistant to digestion by cyanogen bromide (CNBr). Although all four of these matrices show some similarities with each other, suggesting a family of non-collagenous, elastin-like proteins, it is clear that the major matrix proteins of each are different. New morphological and biochemical information on the cartilaginous tissues in squid, horseshoe crab and amphioxus reveals the presence of CNBr-insoluble, non-collagenous matrix proteins, potentially extending the jawless craniate family of cartilaginous proteins into the invertebrates. Details of the evolutionary relationships between these non-collagenous matrix proteins and the significance of the occurrence of these proteins as the major components of the cartilaginous tissues of jawless craniates, amphioxus, horseshoe crab and squid, all of which are capable of producing a variety of collagens in other tissues, remain to be investigated.

Amino Acids↗
PubMed2004

Inter-annual plasticity of squid life history and population structure: ecological and management implications.

Population size and structure, as well as individual growth rates, condition, and reproductive output, respond to environmental factors, particularly in short-lived and fast-growing squid species. We need to understand the mechanisms through which populations respond to environmental conditions, to predict when or if established relationships, used as management tools to forecast recruitment strength, might break down completely. Identifying characteristics of successful recruits who have grown under different environmental scenarios may improve our understanding of the mechanistic connections between environmental conditions and the temporal variation in life history characteristics that ultimately affect recruitment. This 5-year study sought to determine the association between key life history characteristics of southern calamary Sepioteuthis australis (growth rate, body size, and patterns of repro-somatic energy allocation) and the environmental conditions experienced by individuals on the east coast of Tasmania, Australia. Among years, all population and individual parameters examined were highly variable, despite the environmental regime during the study not encompassing the extremes that may occur in this dynamic region. Temperature was not clearly associated with any of the individual or population differences observed. Populations of apparently similar abundance were composed of individuals with strikingly different biological characteristics, therefore seeking relationships between abundance and environmental parameters at gross levels did not shed light on the mechanisms responsible for population size. Importantly, inter-annual differences in squid size, condition, reproductive investment, and possibly growth rate, were sex-specific, indicating that males and females responded differently to similar factors. Among years differences in body size were extreme, both among the male component of the population and between genders. The relative importance of many size-based processes that contribute to population size and structure (e.g. predation, starvation, competition, and reproductive success) will therefore vary inter-annually.

Age Factors↗
PubMed2006

Orientation in the cuttlefish Sepia officinalis: response versus place learning.

Several studies have demonstrated that mammals, birds and fish use comparable spatial learning strategies. Unfortunately, except in insects, few studies have investigated spatial learning mechanisms in invertebrates. Our study aimed to identify the strategies used by cuttlefish (Sepia officinalis) to solve a spatial task commonly used with vertebrates. A new spatial learning procedure using a T-maze was designed. In this maze, the cuttlefish learned how to enter a dark and sandy compartment. A preliminary test confirmed that individual cuttlefish showed an untrained side-turning preference (preference for turning right or left) in the T-maze. This preference could be reliably detected in a single probe trial. In the following two experiments, each individual was trained to enter the compartment opposite to its side-turning preference. In Experiment 1, distal visual cues were provided around the maze. In Experiment 2, the T-maze was surrounded by curtains and two proximal visual cues were provided above the apparatus. In both experiments, after acquisition, strategies used by cuttlefish to orient in the T-maze were tested by creating a conflict between the formerly rewarded algorithmic behaviour (turn, response learning) and the visual cues identifying the goal (place learning). Most cuttlefish relied on response learning in Experiment 1; the two strategies were used equally often in Experiment 2. In these experiments, the salience of cues provided during the experiment determined whether cuttlefish used response or place learning to solve this spatial task. Our study demonstrates for the first time the presence of multiple spatial strategies in cuttlefish that appear to closely parallel those described in vertebrates.

Animals↗
PubMed2005

Cultivation of microorganisms in the cultural medium made from squid internal organs and accumulation of polyunsaturated fatty acids in the cells.

The disposal and more efficient utilization of marine wastes is becoming increasingly serious. A culture media for microorganisms has been prepared from squid internal organs that are rich in polyunsaturated fatty acids (PUFAs). Both freshwater and marine bacteria grew well in this medium and some bacteria accumulated PUFAs in their lipids, suggesting uptake of exogenous PUFAs. Higher PUFA accumulations were observed in Escherichia coli mutant cells defective either in unsaturated fatty acid biosynthesis or fatty acid degradation, or both, compared to those without these mutations. Therefore, PUFA accumulation in cells can be improved by genetic modification of fatty acid metabolism in the bacteria. Squid internal organs would be a good source of medium, not only for marine bacteria but also for freshwater bacteria, and that this process may be advantageous to make efficient use of the fishery wastes and to produce PUFA-containing microbial cells and lipids.

Animals↗