Extracellular matrix of plant callus tissue visualized by ESEM and SEM

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.     

Estimating Pliocene sea-surface temperatures in the Mediterranean: An approach based on the modern analogs technique

The possibilities of adapting the Modern Analogs Technique (MAT) based on planktonic foraminifers for estimating sea-surface temperatures (SST) in the Mediterranean during the Pliocene are discussed in this article. The calibration database used comprises 684 core-top samples distributed in the North Atlantic and the Mediterranean. MAT estimates show an imperceptible bias (− 0.03 °C; σ = 0.59) and a low mean error of estimates (0.42 °C; σ = 0.42) when applied over the samples of the calibration dataset. The procedure used for assimilating the Pliocene taxonomic categories to those of the modern assemblages results in an increase from 17 to 40 in the number of samples showing an error > 2 °C when applied over the calibration database. However, the precision of MAT does not diminish when these samples are removed from the dataset. This methodology was used for obtaining SST estimates of late-middle Pliocene–earliest Pleistocene samples from ODP-site 975 (Menorca area), which have close modern analogs within the calibration database. In order to compare this technique with an additional proxy, we measured also δ¹⁸O values of G. bulloides tests from these samples. The results obtained show a good agreement on the whole, which corroborates the validity of the technical approach proposed.     

New observations on the Late Miocene–Early Pliocene Lutrinae (Mustelidae: Carnivora,Mammalia) from the Middle Awash,Afar Rift,Ethiopia

New observations on the Late Miocene and Earliest Pliocene mustelids from the Middle Awash of Ethiopia are presented. The Middle Awash study area samples the last six million years of African vertebrate evolutionary history. Its Latest Miocene (Asa Koma Member of the Adu-Asa Formation, 5.54–5.77 Ma) and Earliest Pliocene (Kuseralee and Gawto Members of the Sagantole Formation, 5.2 and 4.85 Ma, respectively) deposits sample a number of large and small carnivore taxa among which mustelids are numerically abundant. Among the known Late Miocene and Early Pliocene mustelid genera, the Middle Awash Late Miocene documents the earliest Mellivora in eastern Africa and its likely first appearance in Africa, a new species of Plesiogulo, and a species of Vishnuonyx. The latter possibly represents the last appearance of this genus in Africa. Torolutra ougandensis is known from both the Late Miocene and Early Pliocene deposits of the Middle Awash. The genus Sivaonyx is represented by at least two species: S. ekecaman and S. aff. S. soriae. Most of the lutrine genera documented in the Middle Awash Late Miocene/Early Pliocene are also documented in contemporaneous sites of eastern Africa. The new observations presented here show that mustelids were more diverse in the Middle Awash Late Miocene and Early Pliocene than previously documented.     

Late Pliocene carnivores from western Macedonia (Greece)

Some carnivores from the late Pliocene (Villafranchian) locality of “Dafnero 1” (DFN) are studied. The site is in the Grevena basin, western Macedonia, Greece. The material is described and compared with other Greek and European samples. Three speciesNyctereutes megamastoides, Vulpes alopecoides andChasmaporthetes lunensis have been determined. The DFN carnivores are similar to those from St.-Vallier (France) and Puebla (Spain). The fauna is dated as Middle Villafranchian (latest Pliocene); more precisely it belongs to the St.-Vallier Faunal Unit, MN 17, with an absolute age of about 2. om. y.     

La microflore de diatomées au passage mio-pliocène en Corse

The “Aleria formation”, described in northeastern Corsica, consists of siliciclastic deposits referred to a deltaic environment and comprises diatomitic lenses exposed at Casabianda. The Aleria formation fills a depression of the Messinian erosional surface and is overlain by Early Pliocene sediments in several places. This suggests a late Messinian-earliest Pliocene age. Diatom microflora is well-preserved and contains 59 species. Pennates indicate a very large diversity with 54 species, while Centrics are represented only by five species. Diatom assemblages show a relative constant composition along the studied interval. The species Aulacoseiragranulata is the dominant taxon with about 90% in all samples. Taxa are of different salinity classes and different modes of life (planktic, tychoplanktic, benthic, epiphytic…), suggesting multiple environmental factor interactions. The environmental model suggested by sedimentological and biological data led as to assume a pond-like coastal receptacle common in deltaic system. This receptacle was slightly influenced by the close marine waters and was supplied by permanent freshwaters from continental run-off and alluvial sheet. This model based on diatom assemblages provides new data for palaeoenvironmental and chronological interpretations of the so-called Messinian Lago-Mare deposits.     

Report of Quambalaria cyanescens in association with the birch (Betula pendula)

Long-term microbiological investigation of the pollen of silver birch (Betula pendula) in the Moscow and Moscow oblast areas revealed that almost one-third of the analyzed samples contained the fungus identified by morphological, cultural, and molecular genetic techniques as Quambalaria cyanescens (de Hoog & G.A. de Vries) Z.W. de Beer, Begerow & R. Bauer. This species was previously known mostly as a symbiont of tropical plants of the genera Eucalyptus and Corymbia and have not been isolated in Russia. We revealed a close association between Quambalaria cyanescens and silver birch. The micromycete was regularly detected in pollen samples, as well as on the inside and outside of the aments, and on the surface of leaves and branches. It was never isolated from other plant species in the investigated area. The data on the morphological and cultural characteristics of the fungus, its cell ultrastructure, and occurrence are presented, as well as the phylogenetic analysis of the isolated strains.     

Les Spatangus du Miocène et du Pliocène de l’Ouest de la France

Numerous fragments of spatangoid echinoids have been discovered in the Pliocene deposits of Challans, in Vendée (western France). In spite of the fragmentary data of the samples, a reconstitution of a complete test could be realized using the different fragments and their symetrization. The general shape of the test, and its architectural and ornemental characters allow establishing the presence of the genus Spatangus in western France during the end of Neogene. It allows to precise the biogeography of the genus Spatangus and of the morphological group S. (S.) purpureus on the Atlantic coast after the Messinian crisis. The Pliocene species is compared to the Miocene Spatangus (Phymapatagus) brittanus, abundant in Anjou, Brittany and Touraine. This older species was refered to the subgenus Phymapatagus according to the presumed lack of primary tubercles on its posterior interambulacrum. The discovery of well-preserved specimens, with primary tubercles on every parts of the test, in the Middle Miocene of Brittany allows to refute this subgeneric distinction and to refer the species brittanus to the subgenus Spatangus (Spatangus). The presence of this subgenus in western France is finally confirmed from Middle Miocene to Pliocene.     

Age-related alterations of mitochondria from muscle tissue

The ultrastructure of mitochondria of cross-striated muscles during aging was studied by electron microscopy. Mitochondrial ultrastructure was analyzed in the flight muscle of D. melanogaster (1- and 36-day-old) and in the cardiomyocytes and skeletal muscle of young and senile Wistar and OXYS rats (3- and 25-month-old). The mitochondria in the flight muscle samples of senile D. melanogaster flies were shown to have several types of peculiar age-related mitochondrial abnormalities corresponding to those described previously. Previously unknown changes were revealed in the ultrastructure of cardiomyocyte mitochondria in senile rats (both Wistar and OXYS). Substantial changes in the ultrastructure of subsarcolemmal mitochondria were found in the fibers of red skeletal muscle of senile OXYS rats. It has been shown that the subsarcolemmal mitochondria of red muscle fibers are a peculiar population of mitochondria with atypical ultrastructure. Initial changes in the ultrastructure of subsarcolemmal mitochondria were revealed even in 3-month-old OXYS rats. At the same time, the skeletal muscle mitochondria of senile Wistar rats maintain their morphological characteristics, and their ultrastructure corresponds to that of skeletal muscle mitochondria in 3-month-old Wistar rats.     

Surface area and porosity of acid hydrolyzed cellulose nanowhiskers and cellulose produced by Gluconacetobacter xylinus

The physical parameters of cellulose such as surface area and porosity are important in the development of cellulose composites which may contain valuable additives which bind to cellulose. In this area, the use of acid hydrolyzed nano-dimensional cellulose nanowhiskers (CNWs) has attracted significant interest, yet the surface area and porosity of these materials have not been explored experimentally. The objective of this work was to characterize the surface area and porosity of CNWs from different origins (plant cotton/bacterium Gluconacetobacter xylinus) and different acid treatments (H₂SO₄/HCl) by N₂ adsorption; as well as to compare surface area and porosity of bacterial cellulose synthesized by static and agitated cultures. Our results showed that CNWs produced from H₂SO₄/HCl exhibited significantly increased surface area and porosity relative to starting material cotton fiber CF11. Micropores were generated in HCl hydrolyzed CNWs but not in H₂SO₄ hydrolyzed CNWs. Bacterial CNWs exhibited larger surface area and porosity compared to plant CNWs. Cellulose synthesized by G. xylinus ATCC 700178 from agitated cultures also exhibited less surface area and porosity than those from static cultures.     

In situ occurrence of a gall midge (Insecta,Diptera, Cecidomyiidae) on fossilized angiosperm leaf cuticle fragments from the Pliocene sediments of eastern India

In situ preservation of fossil insect damage in plant fossils is an excellent tool to study the coevolution of flora and fauna through geological time, but finding both damage and the insect causing that damage in the same specimen is a very rare phenomenon. Galling is a common form of angiosperm leaf damage, which can be regarded as a kind of extended phenotype of the causal insects, essentially the gall midges, but galls usually lack remains of the insects themselves. Here we report the in situ occurrence of a gall midge (Insecta, Diptera, Cecidomyiidae) as well as its pupal exuviae on the abaxial cuticular surface of fossilized leaf cuticle fragments of Fabaceae leaves (cf. Albizia) that also bear galls, recovered from the latest Neogene (Rajdanda Formation, Pliocene) sediments of the Chotonagpur Plateau, Jharkhand, northeastern India. This Pliocene gall midge features well-preserved legs, segmented antenna with distinct and enlarged scape, elongate curved setae, and longer than broad terminal plate of the ovipositor lamellae. The in situ presence of a gall midge on a host fabaceous leaf cuticle indicates the existence of a host-ectoparasite relationship in the ancient warm and humid tropical monsoon-influenced forests of eastern India during the Pliocene. This is the first authentic fossil record of an in situ phytophagous insect of Cecidomyiidae from India, as well as southeast Asia. Although the identification of the recovered phytophagous insect associated with the fossil leaf cuticle is only possible to family level, this find reveals that such plant-insect relationships existed in the Pliocene of eastern India.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : IV. Microfilament Distribution and Cell Shape in Untransformed, Transformed, and Revertant Balb/c 3T3 Cells

A comparison is made of the ultrastructure of the cell periphery in three cloned cell lines: untransformed Balb/c 3T3 cells, SV40-transformed Balb/c 3T3 cells, and revertant cells obtained from the transformed cell line by a selection technique utilizing concanavalin A. Both thin-section and surface replication techniques are used for in situ examination of the cell lines. Microfilaments, 70 Å in diameter (called alpha filaments), are abundant in untransformed and revertant cell lines, particularly in the anterior expansions of the cells, which tend to have many microvilli and small pseudopodia. Alpha filaments are diminished in the anterior expansions of transformed cells, which contain large blunt pseudopodia and relatively few microvilli. Surface replicas confirm the impression gained from thin sections that transformed cells have a greater proportion of their cell surface involved in bulging pseudopodia than either untransformed or revertant cells. Since alpha filaments are shown to bind heavy meromyosin and are similar to F-actin, these filaments are thought to be important in cell motility. These observations suggest that a close relationship exists between decreased alpha filaments, bulging pseudopodia, and loss of contact inhibition of movement in transformed cells.     

Late Pliocene carnivores from Včeláre 2 (Southeastern Slovakia)

Carnivores from the Late Pliocene site of V?eláre 2 are described and their taxonomy is discussed. The carnivore assemblage includes nine specimens of the families Felidae (Homotherium crenatidens), Ursidae (Ursus minimus), and Ailuridae (Parailurus sp.). The scimitar-toothed cat and ursid are common representatives of the Pliocene European fauna, whereas the lesser panda is probably a new species of Parailurus, which differs from both Pliocene species, P. anglicus and P. hungaricus. As a part of a more or less uniform Pliocene carnivore fauna of the Northern Hemisphere, the V?eláre 2 assemblage represents a forest and (or) open grassland environment.     

Nontuberculous mycobacteria: an emerging risk in engineered environmental habitats

Nontuberculous mycobacteria (NTM) are microorganisms commonly living in the environment. Nevertheless, most of them are opportunistic pathogens. To verify concentrations of NTM in some man-made habitats, analyses were performed on water and surface samples, and the data were correlated to the global microbiological quality of water. Most of the drinking water samples (98 %) complied with the microbial requirements established by the European Directive 98/83/EC on drinking water when Escherichia coli was considered. Low counts of heterotrophs were also obtained. NTM were isolated from 72 % of the analyzed samples. Tap water from private buildings, schools and hospitals provided positive results for NTM, with comparable densities ranging from 1 to 6?×10² CFU/L. NTM were also found in swimming pool water samples, with concentrations ranging from 29 to 3?×10⁴ CFU/L, as well as in 70 % of the surface sample. The most frequently isolated species were Mycobacterium mucogenicum, M. intracellulare and M. terrae. These yields confirm that no correlation exists between the monitoring controls carried out by law and the occurrence of these bacteria that may represent a potential risk, especially for immunocompromised people and vulnerable groups.     

On some pliocene echinoids from the area north of mersa alam,red sea coast,Egypt

Twenty three echinoid species are described and illustrated from the Pliocene of the area north of Mersa Alam, Red Sea coast of Egypt. Two new speciesSchizaster (Paraster) ashrafi andBrissus shaimaae, and one new subspeciesMaretia planulata abbassi, are described. Six species are recorded here for the first time from the Pliocene of the Red Sea:Eucidaris metularia (LAMARCK), {ITEchinoneus cyclostomus} LESKE,Clypeaster suffarcinatus DUNCAN & SLADEN, C.fervens KOEHLER, C. {ITtelurus} CLARK, andLaganum depressum delicatum MAZZETTI. A discussion is given of the Miocene - Pliocene boundary, the age of the area studied is referred to the early - late Pliocene.     

New cranial material of Shansirhinus (Rhinocerotidae, Perissodactyla) from the Lower Pliocene of the Linxia Basin in Gansu, China

A well-preserved skull and articulated mandible of Shansirhinus, a horned aceratherine rhinocerotid, is described from an Early Pliocene (Gaozhuangian; approximately 5.3-4.34 Ma) locality of the Linxia Basin in Gansu, China. Comparing the new material of Shansirhinus ringstromi Kretzoi, 1942, we confirm the synonymy proposed previously for the species Chilotherium yunnanensis Tang et al., 1974, Chilotherium cornutum Qiu and Yan, 1982, and Ch. tianzhuensis Zheng, 1982. New knowledge concerning its cranial and mandibular morphology allows insight into its phylogenetic position among aceratherine rhinocerotids. A sister group relationship between Shansirhinus and Chilotherium is proposed on the basis of cranial, mandibular and dental evidence. Shared derived characters that support this relationship include: an expanded mandibular symphysis with a concave ventral surface; retracted premaxillae lacking upper incisors; a robust and right-angled facial crest; a flat or slightly concave dorsal skull profile; a weak or absent parastyle fold; and a constricted protocone on the premolars. Shansirhinus was probably a grazer, which is evidenced by the high crown, strong wear, well-developed secondary folds, and enamel plications on its teeth. The age of S. ringstromi is Late Miocene to the Early Pliocene, corresponding to the Turolian to Ruscinian, MN12-MN15 of Europe. S. ringstromi is likely the ancestor of the more advanced S. brancoi (Schlosser, 1903).     

The pliocene species of Hipparion andtheir biostratigraphical meanings

During the Pliocene, the «Hipparion fauna in the Mediterranean area decreased sharply in comparison with the Upper Miocene. Nevertheless, this species can be used to represent the Hipparion biozones in order to compare them with the mammal units. H. gromovae characterizes the Ventian or MN 13 mammal unit; H. crassum the Ruscinian or the Lower Pliocene or MN 14 unit; H. fissurae the Upper Ruscinian or the Lower Pliocene, MN 15; H. rocinantis, the Middle Pliocene or Lower Villafranchian, MN 16a. This distribution can be correlated in most of the Eurasian region.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

Neogene stratigraphy,foraminifera, diatoms,and depositional history of Maria Madre Island,Mexico: Evidence of early Neogene marine conditions in the southern Gulf of California

Foraminifera and diatoms have been analyzed from an upper Miocene through Pleistocene(?) sequence of marine sediments exposed on Maria Madre Island, largest of the Trés Marias Islands off the Pacific coast of Mexico. The Neogene stratigraphic sequence exposed on Maria Madre Island includes a mid-Miocene(?) non-marine and/or shallow marine sandstone unconformably overlain by a lower upper Miocene to uppermost Miocene upper to middle bathyal laminated and massive diatomite, mudstone, and siltstone unit. This unit is unconformably overlain by lower Pliocene middle to lower bathyal sandstones and siltstones which, in turn, are unconformably overlain by upper Pliocene through Pleistocene(?) upper bathyal to upper middle bathyal foraminiferal limestones and siltstones. These beds are unconformably capped by Pleistocene terrace deposits. Basement rocks on the island include Cretaceous granite and granodiorite, and Tertiary(?) andesites and rhyolites. The upper Miocene diatomaceous unit contains a low diversity foraminiferal fauna dominated by species of Bolivina indicating low oxygen conditions in the proto-Gulf Maria Madre basin. The diatomaceous unit grades into a mudstone that contains a latest Miocene upper to middle bathyal biofacies characterized by Baggina californica and Uvigerina hootsi along with displaced neritic taxa. An angular unconformity separates the upper Miocene middle bathyal sediments from overlying lower Pliocene siltstones and mudstones that contain a middle to lower bathyal biofacies and abundant planktonic species including Neogloboquadrina acostaensis and Pulleniatina primalis indicating an early Pliocene age. Significantly, this Pliocene unit contains common occurrences of benthic species restricted to Miocene sediments in California including Bulimina uvigerinaformis. Pliocene to Pleistocene(?) foraminiferal limestones and siltstones characterize submarine bank accumulations formed during uplift of the Trés Marias Island area, and include abundant planktonic foraminifera such as Pulleniatina obliquiloculata and Neogloboquadrina duterteri. Common benthic foraminifera in this unit are indicative of upper bathyal water depths. The Neogene depositional history recorded on Maria Madre Island involves an early late Miocene subsidence event marking formation of the Trés Marias Basin with relatively undiluted diatomaceous sediment deposited in a low oxygen setting. Subsidence and deepening of the basin continued into the early Pliocene along with rapid deposition of terrigenous clastics. Uplift of the basinal sequence began in late Pliocene time accompanied by deposition of upper Pliocene-Pleistocene foraminiferal limestones on a rising submarine bank. Continued episodic uplift of the Neogene deposits brought the island above sea level by late Pleistocene time.     

The first representative Pliocene assemblages of passerine birds in Asia (Northern Mongolia and Russian Transbaikalia)

Passerine birds from two late Pliocene localities, Shaamar in northern Mongolia and Beregovaya in Transbaikalia (East Russia), represent the first known Neogene fossil assemblages of perching birds from Asia. A total of 11 families and 15 taxa are recognized, including a new genus and species of bunting, Pliocalcarius orkhonensis, apparently related to the Longspurs, and new species within the genera Hirundo and Rhodospiza, which are described. The avifauna is composed of open land and bush dwellers; it contains a few arid species, representing the oldest finds of the corresponding genera or lineages. This may indicate that the living Asian arid avian complex originated in Central Asia in the Neogene. Several genera (Rhodospiza, Paradoxornis) are documented in this paper for the first time in pre-Pleistocene deposits. Report of Calcarius is the first pre-Holocene evidence of the occurrence of this genus in Asia, which together with the remains of Synaptomys lemmings at Shaamar, illustrates faunal interchanges between North America and Asia in the Pliocene.     

Non-selective surface deposit feeding by the Zoophycos producers

The feeding mechanism of Zoophycos producers demonstrates non-selection by particle size or quality. The characteristic morphology and mode of Occurrence of Zoophycos and the presence of pellets with pyroclastic grains imply that the Zoophycos producers were surface deposit-feeders. They dwelt in a burrow within the sediment and ingested the grains with nutrient detritus on the sea floor around the burrow opening. Fecal pellets containing pyroclastic grains were compared with the overlying volcanic ash layers through the analyses of grain size and mineral composition based on samples from two horizons in the Upper Pliocene of Boso Peninsula, central Japan. Judging from the comparative analyses, no significant differences were found between the ash layer and the fecal pellets. This fact strongly suggests that the Zoophycos producers appear to indiscriminately ingest particles on the sediment surface. Environments of low nutrition input such as the deep sea, non-particle size and quality selection in Zoophycos producers may be the feeding strategy for the detritus-feeding burrow-dwellers. □ Zoophycos, surface deposit-feeding, Pliocene Shiramazu Formation, Boso peninsula, Japan.