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Ion-induced conformational and stability changes in Nereis sarcoplasmic calcium binding protein: evidence that the APO state is a molten globule.

Nereis sarcoplasmic Ca(2+)-binding protein (NSCP) is a calcium buffer protein that binds Ca(2+) ions with high affinity but is also able to bind Mg(2+) ions with high positive cooperativity. We investigated the conformational and stability changes induced by the two metal ions. The thermal reversible unfolding, monitored by circular dichroism spectroscopy, shows that the thermal stability is maximum at neutral pH and increases in the order apo < Mg(2+) < Ca(2+). The stability against chemical denaturation (urea, guanidinium chloride) studied by circular dichroism or intrinsic fluorescence was found to have a similar ion dependence. To explore in more detail the structural basis of stability, we used the fluorescent probes to evaluate the hydrophobic surface exposure in the different ligation states. The apo-NSCP exhibits accessible hydrophobic surfaces, able to bind fluorescent probes, in clear contrast with denatured or Ca(2+)/Mg(2+)-bound states. Gel filtration experiments showed that, although the metal-bound NSCP has a hydrodynamic volume in agreement with the molecular mass, the volume of the apo form is considerably larger. The present results demonstrate that the apo state has many properties in common with the molten globule. The possible factors of the metal-dependent structural changes and stability are discussed.

Animals↗

Eggs and embryos from the Cambrian.

The early evolution of metazoans is a major focus of biological attention, but is the historical record revealed in the Cambrian "explosion" an accurate reflection of original events? The key questions concern the nature of the earliest animals and when they originated. One widely-mooted suggestion is that planktotrophic larvae, typified by the annelidan trochophore and echinoid pluteus, existed long before the metazoan radiations evident in the Cambrian fossil record. This idea could be consistent for recent evidence of divergence times, based on molecular "clocks," of phyla appearing well before the Cambrian. Now a surprising new discovery of eggs with blastomeres and embryos with well-defined anatomy from the Cambrian (c.530 Myr ago) of China and Siberia promises to extend the arena of debate. In one case a convincing ontogeny can be traced from eggs to adult tube-dwelling cnidarians. In the other example a possible protostome, unhatched and wrapped around the egg, shows segmentation and possibly nascent sclerites. In both, these cases development is direct, i.e., there is no evidence for any planktotrophic larval stage. The implications for our perceptions of both the Cambrian 'explosion' and metazoan phylogeny could be considerable.

Animals↗

Micrognathozoa: a new class with complicated jaws like those of Rotifera and Gnathostomulida.

A new microscopic aschelminth-like animal, Limnognathia maerski nov. gen. et sp., is described from a cold spring at Disko Island, West Greenland, and assigned to Micrognathozoa nov. class. It has a complex of jaws in its pharynx, and the ultrastructure of the main jaws is similar to that of the jaws of advanced scleroperalian gnathostomulids. However, other jaw elements appear also to have characteristics of the trophi of Rotifera. Jaw-like structures are found in other protostome taxa as well-for instance, in proboscises of kalyptorhynch platyhelminths, in dorvilleid polychaetes and aplacophoran mollusks-but studies of their ultrastructure show that none of these jaws is homologous with jaws found in Gnathostomulida, Rotifera, and Micrognathozoa. The latter three groups have recently been joined into the monophylum Gnathifera Ahlrichs, 1995, an interpretation supported by the presence of jaw elements with cuticular rods with osmiophilic cores in all three groups. Such tubular structures are found in the fulcrum of all Rotifera and in several cuticular sclerites of both Gnathostomulida and Micrognathozoa. The gross morphology of the pharyngeal apparatus is similar in the three groups. It consists of a ventral pharyngeal bulb and a dorsal pharyngeal lumen. The absence of pharyngeal ciliation cannot be used as an autapomorphy in the ground pattern of the Gnathifera because the Micrognathozoa has the plesiomorphic alternative with a ciliated pharyngeal epithelium. The body of Limnognathia maerski nov. gen. et sp. consists of a head, thorax, and abdomen. The dorsal and lateral epidermis have plates formed by an intracellular matrix, as in Rotifera and Acanthocephala; however, the epidermis is not syncytial. The ventral epidermis lacks internal plates, but has a cuticular oral plate without ciliary structures. Two ventral rows of multiciliated cells form a locomotory organ. These ciliated cells resemble the ciliophores present in some interstitial annelids. An adhesive ciliated pad is located ventrally close to a caudal plate. As in many marine interstitial animals-e.g., gnathostomulids, gastrotrichs, and polychaetes-a special form of tactile bristles or sensoria is found on the body. Two pairs of protonephridia with unicellular terminal cells are found in the trunk; this unicellular condition may be the plesiomorphic condition in Bilateria. Only specimens with the female reproductive system have been found, indicating that all adult animals are parthenogenetic females. We suggest that 1) jaws of Gnathostomulida, Rotifera, and the new taxon, Micrognathozoa, are homologous structures; 2) Rotifera (including Acanthocephala) and the new group might be sister groups, while Gnathostomulida could be the sister-group to this assemblage; and 3) the similarities to certain gastrotrichs and interstitial polychaetes are convergent.

Animals↗

Morphologic characterization of the ice worm Mesenchytraeus solifugus.

Ice worms occupy a unique position in metazoan phylogeny in that they are the only known annelid that completes its life cycle in ice. The mechanism(s) associated with this adaptation are likely to occur at different levels, ranging from modification of their metabolism to changes in morphology. In this study, we examined specimens of Mesenchytraeus solifugus by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in an effort to identify morphologic structures that may aid in its glacial habitation. We report that M. solifugus contains an elongated head pore at the tip of its prostomium, numerous sensory structures, and differentially oriented setae that curve abruptly at their distal end.

Alaska↗

Bioluminescence: from chemical bonds to photons.

The biological transformation of chemical to photic energy involves an enzyme-mediated chemiluminescent reaction, in which one of the products exists in an electronically excited state, emitting a photon as it returns to the ground state. The colour of bioluminescence differs in different organisms, ranging from the deep blue (460 nm) of certain crustacea, through the bluish green (490 nm) of some bacteria, the green (530 nm) of mushrooms to the red (about 600 nm) of the railroad worm. In one case, energy transfer has been demonstrated from the enzyme system to material that emits light with a longer wavelength. The energies involved range from about 165 to 250 kJ/einstein (40 to 60 kcal/einstein). Boyle first showed that air was involved in bioluminescence in 1668 in his experiments with an air pump. Over the past 100 years, it has become clear that most if not all bioluminescent systems require molecular oxygen. The recent isolation and characterization of an oxygen-containing (peroxide) enzyme intermediate from the bacterial system is described and a reaction mechanism is postulated. This scheme is compared with other hypothetical mechanisms, in particular those involving a four-membered ring intermediate, a dioxetane, in which the simultaneous cleavage of two bonds leaves one product in an excited state. I shall discuss the special role of luciferases in bioluminescence, especially in flashing mechanisms involving 'precharged' intermediates.

Acridines↗

A good eye for arthropod evolution.

Insect and crustacean lineages diverged over 500 Myr ago, and there are continuing uncertainties about whether they evolved from a common arthropod ancestor or, alternatively, they evolved independently from annelid worms. Despite the diversity of their limbs and lifestyles, the nervous systems of insects and crustaceans share many common features both in development and in function. Cellular and molecular embryology techniques reveal good evidence for homologies in the developing segmental ganglia. In the visual system, this seemingly common programme of insect and crustacean CNS development culminates in common adult neural function. Comparisons of the cellular anatomy and physiology of animals as diverse as flies and crayfishes indicate that the neural circuits in the lamina of their optic lobe have been inherited largely unchanged from a common ancestor with good compound eyes.

Animals↗

Arenicola marina extracellular hemoglobin: a new promising blood substitute.

The need to develop a blood substitute is now urgent because of the increasing concern over Europe's BSE outbreak and the worldwide HIV/AIDS epidemic, which have cut blood supplies. Extracellular soluble hemoglobin has long been studied for its possible use as a safe and effective alternative to blood transfusion, but this has met with little success. Clinical trials have revealed undesirable side effects-oxidative damage and vasoconstriction-that hamper the application of cell-free hemoglobin as a blood substitute. We have addressed these problems and have found a new promising extracellular blood substitute: the natural giant extracellular polymeric hemoglobin of the polychaete annelid Arenicola marina. Here we show that it is less likely to cause immunogenic response; its functional and structural properties should prevent the side effects often associated with the administration of extracellular hemoglobin. Moreover, its intrinsic properties are of interest for other therapeutic applications often associated with hemorrhagic shock (ischemia reperfusion, treatment of septic shock and for organ preservation prior to transplantation). Moreover, using natural hemoglobin is particularly useful since recombinant DNA techniques could be used to express the protein in large quantities.

Animals↗

Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles of Chaetopterus oocytes and HeLa cells.

The incorporation of tubulin into mitotic spindles in situ was studied by incubating permeabilized mitotic cells in solutions containing [3H]GTP-labeled or dichlorotriazinylamino fluorescein (DTAF)-labeled tubulin. Metaphase HeLa cells or spindle-containing "minicells" from Chaetopterus oocytes were lysed in a microtubule-assembly buffer plus 0.5% Nonidet P-40, 1 mg/ml 120,000g supernatant mammalian brain tubulin, and [3H]GTP. After different periods of incubation, mitotic spindles were isolated in 2 M-glycerol-containing assembly buffer and separated from unbound counts by centrifugation through a 4 M-glycerol cushion; 3H counts per mg protein increase linearly for 8-12 min and then reach a plateau or steady state in both Chaetopterus oocytes and HeLa cells. Addition of 4 mM CaCl2 blocks or reverses incorporation. Little or no [3H]GTP is incorporated if exogenous tubulin or lysed cells are omitted from the assembly mixture. To measure the loss rate of [3H]GTP-tubulin from mitotic spindles, cells were incubated in tubulin plus [3H]GTP for 30 min, and a 20-fold excess of cold GTP (2 mM) was added. Samples were removed after incubation for different periods, and spindles were isolated as described above and counted for 3H content. [3H]GTP is lost from spindles at a rate of about 16%/min until a new steady state is reached in about 8 min. These results are consistent with an incorporation and turnover of [3H]GTP-tubulin in spindle microtubules of these lysed-cell models. The location of this newly incorporated tubulin in the spindle was investigated by incorporating fluorescent DTAF-tubulin into mitotic spindles of these lysed cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Micromanipulation studies of the asymmetric positioning of the maturation spindle in Chaetopterus sp. oocytes: I. Anchorage of the spindle to the cortex and migration of a displaced spindle.

We investigated the nature of the asymmetric positioning and attachment of Chaetopterus oocyte meiotic spindles to the animal pole cortex by micromanipulation. The manipulated spindle's behavior was analyzed in clarified oocyte fragments using video-enhanced polarized light microscopy. As the spindle was drawn towards the cell interior with a microneedle, the cell surface dimpled inwards adjacent to the outer spindle pole. As the spindle was pulled further inwards, the dimple suddenly receded indicating a rupture of a mechanical link between the cell cortex and outer spindle pole. The spindle paused briefly when released from the microneedle; then it spontaneously migrated back to the original attachment site and reassociated with the cell cortex. Positive birefringent astral fibers were seen running between the outer spindle pole and the cortex during the migration. The velocity of the spindle during its migration tended to increase as it came closer to the cortex. Velocities as high as 1.25 micron/sec. were measured. If removed too far from the attachment site cortex (greater than 35 micron), the spindle remained stationary until pushed closer to the original attachment site. Spindles, inverted by micromanipulation, migrated and reattached to the cortical site by their former inner pole; thus either spindle pole can seek out and migrate to the original attachment site. However, spindle poles pushed against other cortical regions did not attach demonstrating that there is only one unique, localized attachment site for spindle attachment.

Animals↗

Movement of Myzostomum spermatozoa: calcium ion regulation of swimming direction.

Spermatozoa of the small myzostomid worm Myzostomum cirriferum usually swim with the flagellum foremost but occasionally stop and then swim with the head foremost. The spermatozoa have axoneme of the 9 + 0 type; thus each lacks the central pair microtubules. The flagellum emerges in the anterior end of the cell body and attaches to it with junctions. To understand the mechanism regulating the swimming direction of the spermatozoa, we recorded the sperm and their flagellar movements using a video camera with a high-speed shutter. The effects of calcium and viscosity on these movements were also examined. The cell body with the flagellum attached to it formed a curved plate during beating, while the free portion of the flagellum beats with small helical bends. Motive force to propel a spermatozoon was mainly due to the bends in the cell body. The spermatozoa reversed the direction of their swimming as a result of a change in the direction of bend propagation. The direction of bend propagation was regulated by calcium; the bends in the cell body propagated from the end of the head toward the free portion of the flagellum at low concentrations of Ca2+, whereas the direction of bend propagation was reversed at high concentrations of this ion. High viscosity of the medium stimulated a change in the direction of bend propagation.

Animals↗

Neuronal development in larval chiton Ischnochiton hakodadensis (Mollusca: Polyplacophora).

Chitons are the most primitive molluscs and, thus, a matter of considerable interest for understanding both basic principles of molluscan neurogenesis and phylogeny. The development of the nervous system in trochophores of the chiton Ischnochiton hakodadensis from hatching to metamorphosis is described in detail by using confocal laser scanning microscopy and antibodies raised against serotonin, FMRFamide, and acetylated alpha tubulin. The earliest nervous elements detected were peripheral neurons located in the frontal hemisphere of posthatching trochophores and projecting into the apical organ. Among them, two pairs of unique large lateral cells appear to pioneer the pathways of developing adult nervous system. Chitons possess an apical organ that contains the largest number of neurons among all molluscan larvae investigated so far. Besides, many pretrochal neurons are situated outside the apical organ. The prototroch is not innervated by larval neurons. The first neurons of the developing adult central nervous system (CNS) appear later in the cerebral ganglion and pedal cords. None of the neurons of the larval nervous system are retained in the adult CNS. They cease to express their transmitter content and disintegrate after settlement. Although the adult CNS of chitons resembles that of polychaetes, their general scenario of neuronal development resembles that of advanced molluscs and differs from annelids. Thus, our data demonstrate the conservative pattern of molluscan neurogenesis and suggest independent origin of molluscan and annelid trochophores.

Animals↗

Regeneration of axons and nerve cell bodies in the CNS of annelids.

The neuron addition hypothesis predicts that species that add CNS neurons during a particular ontogenetic stage should regenerate ablated somata better than species that add few, if any, neurons to the CNS during that same stage. We report that CNS nerve cells do not regenerate in three species of adult (reproductively competent) leeches (Hirudo medicinalis, Haemopus grande, and Macrobdella decora), which do not increase the number of neurons in any portion of the CNS. Nereis virens, a polychaete that adds CNS neurons to newly forming ganglia in the adult stage, also does not regenerate CNS neurons. Conversely, CNS neurons, including a pair of uniquely identifiable somata, do regenerate in Clymenella torquata, a polychaete that has a constant number of neurons in the adult stage. Hence, the results of our study suggest that several versions of the neuron addition hypothesis cannot predict CNS regenerative abilities in adult annelids. Finally, we report that severed stumps of CNS axons do not degenerate rapidly in Nereis or Clymenella, and that both species can regenerate severed CNS axons presumably by morphologic fusion or physiologic activation of surviving stumps.

Animals↗

The lateral giant fibers of the tubificid worm, Branchiura sowerbyi: structural and functional asymmetry in a paired interneuronal system.

Neuroanatomical and ultrastructural studies of the paired lateral giant nerve fibers (LGFs) in posterior segments of the tubificid worm, Branchiura sowerbyi, demonstrate that the fibers are 1) segmental in origin (two cell bodies per segment), 2) joined longitudinally and transversely to form an intersegmental syncytial network, and 3) surrounded by a myelinlike sheath. The LGFs are unique among paired giant fiber systems because of their extreme asymmetry, the diameter of the left fiber being several times greater than that of the right. Electrophysiological studies demonstrate that the small, right fiber has a high input resistance and, during mechanosensory stimulation, functions as the locus for LGF spike initiation. The larger, left fiber contributes by enhancing the speed of LGF spike conduction along the animal. One physiological benefit of this asymmetric arrangement may be optimization of escape reflex sensitivity to mechanosensory inputs.

Action Potentials↗

Sequence analysis of translationally controlled maternal mRNAs from Urechis caupo.

Fertilization of Urechis caupo oocytes stimulates dramatic changes in the pattern of protein synthesis. This shift is brought about entirely through selective translation of the large pool of maternal mRNAs synthesized and stored during oogenesis. My laboratory has identified cDNA clones to more than 20 different Urechis maternal mRNAs. These have been used to determine whether the complementary mRNAs are translated in oocytes or embryos, and to analyze the polyadenylation status of the mRNAs at different stages. For 14 of the mRNAs, multiple, overlapping cDNA clones were isolated, and the complete sequence of the mRNA molecule was determined. Of these 14 mRNAs, half are from the subset that is translated in growing and full-grown oocytes, but not in embryos. These 7 mRNAs have poly(A) tails before fertilization. The other 7 are from the subset that is not translated at any time before fertilization, and has very short poly(A) tails in oocytes. After fertilization these mRNAs are recruited onto polysomes and extensively polyadenylated. The sequence data from the two classes of maternal mRNAs was compared in an attempt to identify consensus sequences that could regulate translation directly, or indirectly, by controlling polyadenylation or secondary structure formation. Two features of the sequences correlate very well with the translation and polyadenylation of the different mRNAs--the identity of the base immediately preceding the AUG start codon, and the presence of the sequences UUUUA and UUUUUA in the 3' untranslated region.

Animals↗