The contractility of elongated microvilli in early sea urchin embryos

Summary Elongated microvilli attach the early sea urchin embryo to the fertilization envelope and support it in a concentric position within the perivitelline space. The contractility of the elongated microvilli was demonstrated in several ways. (1) During normal cleavage, these microvilli change their length to adapt to the change in shape and numbers of blastomeres. (2) When treated with calcium-free sea water, embryos become eccentrically located and the microvilli extend further than normal on one side; when returned to normal sea water, the embryos become centered again. (3) Several agents cause the fertilization envelope to become higher and thinner than normal and the elongated microvilli to extend correspondingly if treated within ten min after fertilization. In some cases, both elongated microvilli and fertilization envelope return to normal size when returned to normal sea water. (4) Fertilization in a papain solution causes the elongated microvilli and the fertilization envelope to contract to the surface of the embryo. (5) Refertilization after the papain-induced contraction can bring about the elongation of these microvilli and the elevation of the fertilization envelope a second time. It was also shown that elongated microvilli are extended immediately upon fertilization, at the same time as the short microvilli. The firm adherence of the tips of elongated microvilli to the fertilization envelope by means of extracellular matrix fibers is shown in a high voltage electron microscope stereoimage. This allows us to understand why it is that when the elongated microvilli extend or contract, the fertilization envelope also extends and contracts accordingly.     

The extracellular matrix of Xenopus laevis eggs: a quick-freeze, deep- etch analysis of its modification at fertilization

Eggs of the amphibian, Xenopus laevis, were quick-frozen, deep-etched, and rotary-shadowed. The structure of the extracellular matrix surrounding these eggs, including the perivitelline space and the vitelline envelope (VE), was visualized in platinum replicas by electron microscopy. The perivitelline space contains an elaborate filamentous glycocalyx which connects microvillar tips to the plasma membrane, to adjacent microvilli, and to the overlying VE. The VE is comprised of two layers, the innermost of which is a thin network of horizontal fibrils lying on the tips of the microvilli. The outermost is a thicker layer of large, cable-like fibers which twist and turn throughout the envelope. Upon fertilization, three dramatic modifications of the matrix occur. A thin sheet of smooth material, termed the smooth layer, is deposited on the tips of the microvilli and separates the egg from the overlying envelopes. The VE above is transformed from a thick band of cable-like fibers to concentric fibrous sheets, the altered VE. Finally, an ornate band of particles, corresponding to the fertilization layer in previous studies, is deposited at the altered VE/jelly interface. The altered VE and the fertilization layer comprise the fertilization envelope, which effects the structural block to polyspermy.     

Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

All animal embryos begin development by modifying the egg extracellular matrix. This protein-rich matrix protects against polyspermy, microbes and mechanical stress via enzyme-dependent transformations that alter the organization of its constituents. Using the sea urchin fertilization envelope, a well-defined extracellular structure formed within minutes of fertilization, we examine the mechanisms whereby limited permeability is established within this matrix. We find that the fertilization envelope acquires a barrier filtration of 40,000 daltons within minutes of insemination via a peroxidase-dependent mechanism, with dynamics that parallel requisite production of hydrogen peroxide by the zygote. To identify the molecular targets of this free-radical modification, we developed an in vivo technique to label and isolate the modified matrix components for mass spectrometry. This method revealed that four of the six major extracellular matrix components are selectively crosslinked, discriminating even sibling proteins from the same gene. Thus, specific free-radical chemistry is essential for establishing the embryonic microenvironment of early development.     

Cellular transglutaminase has affinity for extracellular matrix

Summary Cellular transglutaminase (TGase) was demonstrated as an intracellular enzyme by immunofluorescence in WI-38 cells. Following cell membrane perturbation by Triton X-100 treatment, TGase was bound to the extracellular matrix and was found to coexist with fibronectin as visualized by immunofluorescence microscopy. The binding of TGase to the cell matrix was blocked by anti-fibronectin antibody. Exogenous sources of soluble TGase were transferred to the extracellular matrix of an untreated or methanol fixed cell. The experimental data indicated that “particulate bound” TGase is a consequence of soluble TGase binding to the extracellular matrix following cell rupture. Editor's statement This report suggest that “particulate bound” transglutaminase may be a consequence of affinity of soluble enzyme for specific molecules in extracellular matrix and opens up a means to characterize transglutaminase binding sites in the matrix.     

Ramp edges,Mach bands,and the functional significance of the simple cell assembly

The responses of “complex” simple cells to sharp and blurred ramp edges were studied. These responses are quite similar to those in the case of lines, which implies that phase information cannot be used to discriminate between ramp edges and lines. Furthermore, if the maximum of the modulus is used as a position estimate, a systematic bias toward the ramp side results, and this bias increases with edge blur. In contrast, a local extremum in the real part of the cell responses provides a precise position estimate, even for strongly blurred edges. Possible multiscale detection strategies are discussed in the context of a syntactical visual reconstruction. This is illustrated by an explanation of Mach bands as perceived at trapezoidal edges, including Ratliff’s Mach-band cancellation stimulus, and criteria for local probability summation in the prediction of Mach-band detection thresholds are presented. Received: 10 December 1992/Accepted in revised form: 6 August 1993     

Temporo-spatial distribution of matrix and microfilament components during odontoblast and ameloblast differentiation

Summary Several extracellular matrix components (procollagen type III, fibronectin, collagen type IV, laminin and nidogen) and microfilament constituents (actin, α-actinin and vinculin) were localized by indirect immunofluorescence microscopy in frozen sections of embryonic mouse molars. Nidogen was present at the epithelio-mesenchymal junction during polarization and initial steps of functional differentiation of odontoblasts. Nidogen disappeared at a stage where direct contacts between preameloblasts and predentin were required to allow the initiation of ameloblast polarization. Our observations concerning the distribution of procollagen type III and fibronectin during odontoblast differentiation add to current knowledge. Procollagen type III and fibronectin surrounding preodontoblasts accumulated at the apical part of polarizing and functional odontoblasts secreting “initial” predentin. Procollagen type III, but not fibronectin, disappeared in front of functional odontoblasts synthesizing “late” predentin and dentin. Fibronectin, present in “initial” predentin, was no longer detected in “late” predentin and dentin but was found between odontoblasts secreting “late” predentin and dentin. Actin, α-actinin and vinculin were concentrated in the peripheral cytoplasm of preameloblasts and accumulated at the apical and basal poles of functional ameloblasts. During differentiation of odontoblasts, the three proteins accumulated at the apical pole of these cells. Time and space correlations between matrix and microfilament modifications during odontoblast and ameloblast differentiation are documented. The possibility is discussed that there is transmembranous control of the cytoskeletal activities of odontoblasts and ameloblasts by the extracellular matrix.     

Organisms can essentially be classified according to two codon patterns

We recently classified 23 bacteria into two types based on their complete genomes; “S-type” as represented by Staphylococcus aureus and “E-type” as represented by Escherichia coli. Classification was characterized by concentrations of Arg, Ala or Lys in the amino acid composition calculated from the complete genome. Based on these previous classifications, not only prokaryotic but also eukaryotic genome structures were investigated by amino acid compositions and nucleotide contents. Organisms consisting of 112 bacteria, 15 archaea and 18 eukaryotes were classified into two major groups by cluster analysis using GC contents at the three codon positions calculated from complete genomes. The 145 organisms were classified into “AT-type” and “GC-type” represented by high A or T (low G or C) and high G or C (low A or T) contents, respectively, at every third codon position. Reciprocal changes between G or C and A or T contents at the third codon position occurred almost synchronously in every codon among the organisms. Correlations between amino acid concentrations (Ala, Ile and Lys) and the nucleotide contents at the codon position were obtained in both “AT-type” and “GC-type” organisms, but with different regression coefficients. In certain correlations of amino acid concentrations with GC contents, eukaryotes, archaea and bacteria showed different behaviors; thus these kingdoms evolved differently. All organisms are basically classifiable into two groups having characteristic codon patterns; organisms with low GC and high AT contents at the third codon position and their derivatives, and organisms with an inverse relationship.     

Holocene sea level changes and reef development in the southwestern Indian Ocean

 The sedimentological and chronological study of Holocene reef sequences recovered in drill cores through modern reefs of Mauritius, Réunion Island and Mayotte allows the reconstruction of sea level changes and reef growth patterns during the Holocene. The branching-coral facies systematically predominates over coral head facies throughout the Holocene reef sequences, and Acropora is the main frame builder among the branching forms. The reconstructed sea level curves, based both on identification of coral assemblages and on radiometric U/Th ages, are characterized by a rapid rise between 10 and 7.5 ky BP, followed by a clear inflection between 7.5 and 7 ky BP. The stabilization of sea level at its present level occurred between 2000 and 3000 years ago, probably without a higher sea level stand. Rates of vertical reef accretion range between 0.9 and 7 mm. y^-1. In Mauritius, and also probably in Réunion Island, the reef first tracked, then caught-up to sea level to reach an equilibrium position (“catch-up” growth), while the barrier reef margin off Mayotte has been able to keep pace with rising sea level (“keep-up” growth). Accepted: 1 March 1997     

Reflex responses of the spinal cord under conditions of the spinal “superreflexia” evoked by pharmacological agents increasing its excitability

We studied monosynaptic reflex discharges (MRD) recorded from the ventral roots (VR) of rats subjected to systemic administration with thyroliberin, thyroxin, or 4-aminopyridine (4-AP). Under such conditions, in some of the experiments the MRD amplitude reached values sufficient to excite non-active VR fibers. In these cases, immediately after the MRD peak had been reached, abnormally increased responses (AIR) developed, whose amplitude was 2–2.5 times higher than the amplitude of highly facilitated MRD. Proofs are presented that AIR reflect excitation of “neighboring” VR fibers, which were not involved in the reflex response, and MRD plays the role of a “stimulus” exciting these fibers. Therefore, we demonstrate the possibility of transmission of excitation from “active” to “silent” fibers within a nerve trunk under conditions of the development of “superreflexia”. This state can be considered a model of excitation spreading via a non-synaptic pathway under conditions of hyperexcitability of the CNS structures, in seizure states in particular. neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 120–127, March–April, 2000.     

Pulses of movement across the sea ice: population connectivity and temporal genetic structure in the arctic fox

Lemmings are involved in several important functions in the Arctic ecosystem. The Arctic fox (Vulpes lagopus) can be divided into two discrete ecotypes: “lemming foxes” and “coastal foxes”. Crashes in lemming abundance can result in pulses of “lemming fox” movement across the Arctic sea ice and immigration into coastal habitats in search for food. These pulses can influence the genetic structure of the receiving population. We have tested the impact of immigration on the genetic structure of the “coastal fox” population in Svalbard by recording microsatellite variation in seven loci for 162 Arctic foxes sampled during the summer and winter over a 5-year period. Genetic heterogeneity and temporal genetic shifts, as inferred by STRUCTURE simulations and deviations from Hardy–Weinberg proportions, respectively, were recorded. Maximum likelihood estimates of movement as well as STRUCTURE simulations suggested that both immigration and genetic mixture are higher in Svalbard than in the neighbouring “lemming fox” populations. The STRUCTURE simulations and AMOVA revealed there are differences in genetic composition of the population between summer and winter seasons, indicating that immigrants are not present in the reproductive portion of the Svalbard population. Based on these results, we conclude that Arctic fox population structure varies with time and is influenced by immigration from neighbouring populations. The lemming cycle is likely an important factor shaping Arctic fox movement across sea ice and the subsequent population genetic structure, but is also likely to influence local adaptation to the coastal habitat and the prevalence of diseases.     

Stereotyped sequence of mating behavior in the Far Eastern catfish, Silurus asotus, from Lake Biwa

 Mating behavior of the Far Eastern catfish, Silurus asotus (Siluriformes: Siluridae), was observed in a ricefield system facing the shore of Lake Biwa in mid-May to early June in 1990–1997. A set behavioral sequence similar to those of two other silurid fishes, S. biwaensis and S. lithophilus, both endemic to the Lake Biwa system, was observed: “chasing,”“clinging,”“enfolding” while “squeezing” by the male, and “circling” by the spawning pair. This form of mating behavior is quite different from that of S. asotus reported from the Ooi River system in Kyoto Prefecture, which mainly spawns in running water in ditches. Received: April 10, 2001 / Revised: November 5, 2001 / Accetped: November 20, 2001     

An innovative LC/MS approach applied to the determination of zeralenone in maize: Alternate Isotope-coded Derivatization Assay (AIDA)

Zearalenone is a mycotoxin mainly produced by severalFusarium species, which are known to colonize grains in temperate climates. The purpose of the study is to provide a reliable isotope dilution method for the quantification of this mycotoxin. A derivative of the analyte to be used as standard is obtained by reaction with acetic anhydride, which is available in two pure isotopic forms, a protonated (“light”) and a hexadeuterated (“heavy”). The derivatized standards are added to the matrix split intwo parts. Then, the derivatization procedure is repeated on both matrices derivatizing the part containing the “heavy” labelled standard with the “light” acetic anhydride and the part containing the “light” labelled standard with the “heavy” acetic anhydride. Both extracted mixtures are analyzed by LC/MS, monitoring the “light” and the “heavy” labelled analytes and using the former as standard for the latter in one case and viceversa in the other case. The method allowed to obtain very good results, without the need of IAC purification. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005. Financial support: The Italian Ministry of Health     

Morphology of coral desmocytes, cells that anchor the calicoblastic epithelium to the skeleton

 We used light, scanning, and electron microscopy to investigate the ultrastructure of desmocytes in the scleractinian Stylophora pistillata from the Red Sea. Desmocytes are abundant on the calicoblastic epithelium, numbering up to 150 per mm² in the coenosarc. The surface of the skeleton bears shallow pits which may represent desmocyte attachment scars. Previously described as cell remnants or extracellular products, coral desmocytes appear to be bona fide cells as they manifest plasma membranes, organelles, and nuclei. Desmocytes attach to the mesoglea in mortise and tenon fashion. A field of 40 or more tenons protrude fingerlike from the proximal surface of the desmocyte and interdigitate with the mesoglea. Each tenon is coated extracellularly with short fibers which are joined to fibers of the mesoglea. The arrangement resembles previously described “fascial” hemidesmosomes. The short fibers pass through the plasma membrane and connect with relatively long intracellular fibers which occupy the center of each tenon. The long fibers extend distally and attach to structures resembling vertebrate hemidesmosomes. These, in turn, attach to the skeleton. The fiber arrangement and orientation seems designed to resist tensile forces. The dynamic adhesion potentially provided by the distal hemidesmosomes may enable desmocytes to detach and reattach to the skeleton during episodes of mineral accretion. Accepted: 15 April 1997     

Dynamic role of “illite-like” clay minerals in temperate soils: facts and hypotheses

Analysis of new data and reinterpretation of published information for clay minerals found in temperate climate soil profiles indicates that there is often a gradient of “illite-like” clay minerals with depth. We used the term “illite-like” because these observations are based on X-Ray Diffractogram patterns and not on layer charge measurements which allow to define properly illite. It appears that “illite-like” layers are concentrated in the upper, organic - rich portion of the soil profile both under grassland and forest vegetation. “Illite-like” layer quantity seems directly related to soil potassium status. Indeed, intensive agriculture practises without potassium fertilization reduce “illite-like” content in surface soils, whereas several years of potassic fertilization without plant growth can increase “illite-like” content. The potassic soil clay mineral, illite, is particularly important in that it can be the major source of readily available potassium for plants. Spatial and temporal dynamics of clay minerals should be related to the potassium cycle. We propose that the frequently observed general trend of increasing exchangeable potassium in the top soil can be correlated with an increase in “illite-like” in the clays and that the decrease of potassium caused by intensive agricultural practices leads to “illite-like” layer destabilization. This vision of “illite-like” layer as a potassium reservoir refueled by plants and emptied by intensive cropping renews the concept of potassium availability and indicates a need to be discussed as well in natural ecosystems as in cultivated ecosystems.     

Renovation of the egg extracellular matrix at fertilization

Extracellular matrices are essential for cell survival and function. This is especially relevant for eggs, which establish a physical barrier at fertilization to protect a new embryo from additional sperm and pathogens. Formation of an extracellular matrix is most dramatic in sea urchins, in which fertilization was first observed in animals with the "sudden appearance of a perfectly transparent envelope" (A. Derbs, 1847). The process of assembling this extracellular "envelope" has been a topic of intense study ever since. Here we integrate the cellular and molecular events necessary to form this fertilization envelope within the first few minutes of a new embryo's life.     

Changes in terrestrial biota before the Permian-Triassic ecological crisis

The period around the Permian-Triassic boundary was marked by one of the most important and interesting events in the evolution of life. The diversity of both marine and continental biotas decreased. The changes were global and led to the establishment of the new Mesozoic World. Transformations of the organic world constituted a single process with changes in the inorganic components of the biosphere. The preceding glacial period had ended and the “cool,” zonal, and markedly seasonal climate was replaced by a “warm,” “equable,” virtually non-seasonal and azonal climate. The new climatic organization remained on Earth for more than two hundred million years. The biotic crisis was global: it involved the sea, the land, and inland waters. The changes on land began earlier and more superficial. The principal events were in the Kazanian and Vyatkian, before the end of the Permian. The crisis was caused to a greater extent by biospheric processes than by momentary external influences, the latter at most triggering the crisis.     

Ultrastructural and immunochemical features of the cell wall sac formed in mulberry (Morus alba) idioblasts

A peculiar inward growth, named a “cell wall sac”, formed in mulberry (Morus alba) idioblasts, is a subcellular site for production of calcium carbonate crystals. On the basis of ultrastructural observations, a fully expanded cell wall sac could be divided into two parts—an amorphous complex consisting of multi-layered compartments with multiple fibers originating from the innermost cell wall layer, and a peripheral plain matrix with fiber aggregates. Immunofluorescent localization showed that low and highly esterified pectin epitopes were detected at the early stages of development of the cell wall sac, followed by complete disappearance from the both parts of fully enlarged mature sac. In contrast, the xyloglucan epitope remained in the compartment complex; this was supported by the observation that the xyloglucan epitope labeled with immuno-gold particles is found on fibers in the complex part.     

Longitudinal changes of dominance rank among the females of the Koshima group of Japanese monkeys

The changes of dominance rank among female Japanese monkeys of the Koshima group over a period of 29 years from 1957 were studied. The dominance rank order was relatively stable in the early population growing phase, while large scale-changes of dominance rank order occurred successively in the phase of population decrease brought about by the severe control of artificial feeding after 1972. Nevertheless, the rank order of several females of the highest status was stable. Furthermore, the reproductive success of these highest status females was high (Mori, 1979a;Watanabe et al., in prep.). Divergence of the dominance rank order fromKawamura's rules (Kawamura, 1958) was observed in the following respects: (1) Some females significantly elevated their rank depending on the leader males. (2) If mothers died when their daughters were still juveniles or nulliparous, the dominance rank of some of these offspring females was significantly lower than the mother's one. However 55% of daughters which lost their mothers at a young age inherited the mother's rank. (3) Dominance among sisters whose mother had died when at least one of the daughters was under 6 years old followed the rule of youngest ascendancy in 60% (Kawamura, 1958), and in 80% when both of the daughters were nulliparous at the mother's death. The mean rate of aggressive interactions for each female with subordinates to her was calculated by dividing the total aggressive interactions between the female in question and her subordinates by the number of subordinate females to the female in question. A female which showed a high rate of aggressive interactions with her subordinates was categorized as an “Attacker”, and a female showing a lower rate was categorized as a “Non-attacker”. Similarly, categories of “Attacked”, and “Non-attacked” were distinguished by using the rate of aggressive interactions with dominant females. Several females which were once categorized in one category in a year were repeatedly categorized in the same category over different years. The “Attacked” tended to be females of higher rank, and “Non-attackers” tended to be females of lower rank. “The second-higher-status females”, were “Attacked”, and their rank was unstable. In particular, females of lower rank within the lineage of the highest rank suffered this kind of severe status. Most of the daughters of these females showed a sharp drop of rank, and died when they were still at a young age, i.e. “the second-higher-status females” displayed low fitness. “Non-attackers” were significantly “Non-attacked”; i.e. they were females which showed a non-social attitude. Females which underwent a drop of rank tended to be “Non-attackers”. The most important factor which determined the females' rank was the memory of their dominance relations under the influence of their mother [dependent rank (Kawai, 1958)] in their early life during development. This finding corresponds well with the results in baboons obtained byWalter (1980); the target females of aggressive interactions by adolescent females were determined by the rank of the mothers when these adolescent females were born.     

Mating system and stigmatic behaviour during flowering of Alpinia kwangsiensis (Zingiberaceae)

 Little is known about the breeding systems of tropical perennial ginger species. In this paper, we provide information about the breeding system of Alpinia kwangsiensis in Yunnan, Southwest China, specifically self-compatibility, mechanisms promoting outcrossing, and the visitation characteristics of pollinators. Populations of A. kwangsiensis have two specific phenotypes that differ in flowering behaviour: 1) “cataflexistyle” individuals in which the stigma is held erect above the dehiscent anther when anthesis begins in the morning and becomes decurved under the anther at noon and 2) “anaflexistyle” individuals in which the receptive stigma is decurved under the indehiscent anther first and moves into a reflexed superior position above the anther as it begins to shed pollen at mid-day. The stigmatic movements in the two floral phenotypes, which occur in a ratio of 1:1 in natural populations, are synchronous and correlate with the foraging behaviour of floral visitors; pollination is effected only between floral forms. Field experiments indicate that A. kwangsiensis is self-compatible and dependent upon insects for fertilization. This newly reported floral mechanism, which we have named “flexistyly,” adds to the repertoire of devices that have evolved in flowering plants to insure outcrossing. Received November 29, 2001 Accepted January 8, 2002