Nucleotide sequence of an actin-encoding gene from Hydra attenuata: structural characteristics and evolutionary implications

We have determined the complete nucleotide sequence of an actin-encoding gene from Hydra attenuata as well as partial sequences of cDNA clones from two additional actin-encoding genes. The gene from the genomic clone contains a single intron, and has promoter and polyadenylation signals similar to those found in other species. The hydra genome has a very A + T-rich base composition (71%). This is reflected in the codon usage of the actin-encoding genes, which is strongly biased towards codons having A or T in the third position. The hydra actin-encoding gene family consists of three or more transcribed genes, two of which are very closely related to each other and probably arose by a recent gene duplication. Hydra actin, like other invertebrate actins, is more similar to the non-muscle isotypes of vertebrates than to the vertebrate muscle actins. Hydra actin is more similar to animal actins than to those of plants or fungi, which is consistent with the view that all metazoans arose from a single protist ancestor.     

Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues.

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.     

Phytoplankton biomass and production in shelf waters off NW Spain: spatial and seasonal variability in relation to upwelling

Summary Chlorophyll-a and primary production on the euphotic zone of the N-NW Spanish shelf were studied at 125 stations between 1984 and 1992. Three geographic areas (Cantabrian Sea, Rías Altas and Was Baixas), three bathymetric ranges (20 to 60 m, 60 to 150 m and stations deeper than 200 m), and four oceanographic stages (spring and autumn blooms, summer upwelling, summer stratification and winter mixing) were considered. One of the major sources of variability of chlorophyll and production data was season. Bloom and summer upwelling stages have equivalent mean and maximum values. Average chlorophyll-a concentrations approximately doubled in every step of the increasing productivity sequence: winter mixing — summer stratification — high productivity (upwelling and bloom) stages. Average primary production rates increased only 60% in the described sequence. Mean (± sd) values of chlorophyll-a and primary production rates during the high productivity stages were 59.7 ± 39.5 mg Chl-a m^–2 and 86.9 ± 44.0 mg C m^–2 h^–1, respectively. Significant differences in both chlorophyll and primary production resulted between geographic areas in most stages. Only 27 stations showed the effects of the summer upwelling that affected coastal areas in the Cantabrian Sea and Rías Baixas shelf, but also shelf-break stations in the Rías Altas area. The Rías Baixas area had lower chlorophyll than both the Rías Altas and the Cantabrian Sea areas during spring and autumn blooms, but higher during summer upwelling events. On the contrary, primary production rates were higher in the Rías Baixas area during blooms in spring and autumn. Mid-shelf areas showed the highest chlorophyll concentrations during high productivity stages, probably due to the existence of frontal zones in all geographic areas considered. The estimated phytoplankton growth rates were comparable to those of other coastal upwelling systems, with average values lower than the maximum potential growth rates. Doubling rates for upwelling and stratification stages in the northern and Rías Altas shelf areas were equivalent, despite larger biomass accumulations during upwelling events. Low turnover rates of the existing biomass in the Rías Baixas shelf in upwelling stages suggests that the accumulation of phytoplankton was due mainly to the export from the highly productive rías, while the contribution of in situ production to these accumulations was relatively lower.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Plankton distribution across a slope current-induced front in the southern Bay of Biscay

Relatively warm (12.50–12.75°C) and high-salinity[<35.640 practical salinity units (PSU)] water flowing eastwardwas detected at the shelf-break during a cruise carried Outin the southern Bay of Biscay in Spring 1987. The slope currentinduced the formation of a convergent front separating well-mixedoceanic waters from haline-stratified coastal waters. Very highconcentrations of dissolved oxygen (295 µmol kg^–1)and chlorophyll a(>4.5 mg m^–3) were found at the outeredge of the frontal boundary. Small autotrophic flagellatesdominated the phytoplankton community. Primary production peakedat the boundary region. Estimated phytoplankton growth ratesindicated that active growth was taking place, with lower turnovertimes integrated over the water column at the frontal station(2.5–5 days) than at coastal (1.5–2.8 days) or oceanic(1.5–3.5 days) stations. The lowest doubling times (1–2days) were calculated for surface frontal populations. Accumulationof zooplankton was also observed associated with the convergentphysical structure, although this relationship was less markedthan for phytoplankton. Copepods, mainly Paracalanus parvus,Acartia clausi and Oithona helgolandica, formed the bulk ofthe mesozooplankton biomass. Compatibility between the sizeof phytoplankton cells and copepod size spectra indicate highfood availability for these animals, particularly in the vicinityof the front. The distribution of fish eggs and fish larvaewas also coupled with the slope current-induced front. Sardinelarvae were more abundant at the coastal side of the front,whereas larval stages of blue whiting reached the highest densitiesat off-shelf stations. Larvae of lamellibranch molluscs andbryozoa were restricted to nearshore waters, as the frontalboundary prevented larval dispersion to the open ocean. Theresults presented in this paper suggest that the Iberian slopecurrent and its associated shelf-break frontal structure werecrucial in controlling phytoplankton primary production, activityof grazers, distribution of larvae of fishes and benthic invertebrates,and ultimately in determining the structure of the pelagic foodweb in the southern Bay of Biscay during the seasonal periodof vertical mixing.     

Fed-batch cultivation of recombinant escherichia coli JM103 and production of the fusion protein SPA::EcoRI in a 60-L working volume airlift tower loop reactor

SPA::EcoRI fusion protein was produced by Escherichia coli JM103 carrying the multicopy expression plasmid pMTC48, the multicopy repressor plasmid pRK248, and the multicopy protection plasmid pEcoR4 in a 60-L working volume airlift tower loop reactor on M9 minimal medium with glucose. Cell mass concentration, total cell count, number of colony-forming units, specific growth rate, yield coefficient, and metabolite (acetate, pyruvate, succinate, lactate, ethanol) concentrations were monitored during the growth phase and gene expression. Gene expression was induced by temperature shift or chemically by isopropyl-thiogalactosidase in the airlift tower loop reactor (ALTR) at constant cultivation time and in a small stirred tank reactor at different cultivation times. During induction, the cultivation medium was supplemented with concentrated Luria-Bertani (LB) medium. The intracellular enzyme activity was evaluated as a function of the time after the start of the induction. It was found that the reduction of the glucose concentration and increase of the dissolved oxygen concentration reduced the acetate produced and increased the intracellular enzyme activity. (c) 1993 John Wiley & Sons, Inc.     

The role of active oxygen in iron tolerance of rice (Oryza sauva L.)

Summary Iron tolerance of rice (Oryza sativa L.) was investigated using an oxygen depleted hydroculture system. Treatment with high concentrations of Fe²⁺ induced yellowing and bronzing symptoms as well as iron coatings at the root surface. Root and shoot growth were inhibited by increasing iron concentration in the medium. All symptoms were more pronounced in an iron sensitive cultivar (IR 64) compared to an iron tolerant one (IR 9764-45-2). Superoxide dismutase and peroxidase activity of root extracts of IR 97 were about twice that of IR 64 in untreated control plants. No significant increase of peroxidase activity was detected with increasing iron concentration in the medium. Catalase activity of IR 64 was slightly higher than that of IR 97, independent of iron concentration.Abbreviations SOD Superoxide dismutase (EC 1.15.1.1) - POD peroxidase (EC 1.11.1.7) - EDTA ethylenediamintetraacetic acid - fwt fresh weight - Hepes (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) - BSA bovine serum albumin - IR 97 IR 9764-45-2 an iron tolerant rice cultivar - IR 64 iron sensitive rice cultivar - PM plasma membrane     

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.     

Formation of pattern in regenerating tissue pieces of Hydra attenuata. III. The shaping of the body column

Excised pieces of hydra body tissue of varying size and shape regenerate into cylinders with a head and foot at opposite ends. The numbers of cells along the axial and circumferential dimensions were determined before, during, and after regeneration. The main process in shaping the excised tissue into a body column was found to be a rearrangement of the cells. When regenerates of different size were measured, the proportions of the body columns were found to vary, such that the smaller the animal the squatter the body column was. The presence of the head in regenerates was necessary for the formation or maintenance of the cylindrical shape, while the size of the head determined the proportions of the cylinder. The formation of a gradient of adhesivity induced by the developing head is suggested as the basis for the rearrangement of the cells into the cylindrical form.     

Head regeneration and polarity reversal inHydra attenuata can occur in the absence of DNA synthesis

Summary Regeneration in hydra is considered to be morphallactic because it can occur in the absence of cell division. Whether DNA synthesis is required for regeneration or other repatterning events is not known. The question was investigated by blocking DNA synthesis with hydroxyurea and examining several developmental processes. Head regeneration, reversal of regeneration polarity and battery cell differentiation all took place in the absence of DNA synthesis. Hence, morphallactic regulation in hydra is independent of both DNA synthesis and mitosis.