Phytoplasmas are small (0.2–0.8 μm), wall-less, pleiomorphic prokaryotes responsible of numerous economically important plant diseases. They are characterized by a very small genome and are obligate parasites of phloem tissues and some insects that act as vectors of infection. To investigate molecular mechanisms involved in pathogenesis, the differential display technique was here applied to identify plant genes whose transcription was significantly altered in leaves of Madagascar periwinkle (Catharanthus roseus (L.) G.Don) infected by ‘Candidatus Phytoplasma pyri’. We detected, reamplified, cloned, and sequenced 16 putative differentially expressed cDNA fragments. Northern blot analysis revealed that seven of the 16 genes identified were up-regulated following phytoplasma infection, while three genes were down-regulated. The remaining six genes did not show significant changes in the level of expression. Identified genes are mainly involved in plant defence/stress responses, protein metabolism and transport, transcriptional regulation, vesicle trafficking, and carbohydrate metabolism. The possible role played by these genes in the phytoplasma infection is discussed.

To study the host plant-viroid interaction, green bark samples of Troyer citrange, sour orange and Alemow seedlings inoculated with a Citrus viroid-IIIb (CVd-IIIb) source were processed by a rapid and sensitive RT-SYBR Green I-based real time assay to assess molecular events taking place at an early stage of infection.The messenger RNA (mRNA) differential display technique was applied for the identification and isolation of genes whose transcription was altered significantly in Etrog citron leaves infected by CVd-IIIb.Seven of them, known to be related to plant stress defense, were selected to evaluate the variation of the expression level in Troyer citrange, sour orange, and alemow seedlings by Northern Blot analysis and reverse-transcriptase quantitative real time PCR (RT-qPCR).Both methods were valuable, but the latter was more sensitive.The seven genes were detected, in the three rootststocks, as up-or down-regulated.The inoculation of CVd-IIIb in Troyer citrange seedlings modified the expression of metallothionein (MT), alcohol dehydrogenase (ADH), ethylene-responsive binding protein (EREBP), regulator of gene silencing (RGS), peroxidase (PRX) and CONSTANS-like at a lower level than in Etrog citron but with a similar pattern.Alemow seedlings showed a slight over-expression of MT and ADH; in contrast, PRX was severely down regulated.Sour orange seedlings did not reveal any signal with the exception of PRX and CONSTANS, which were up regulated as in Etrog citron.RT-qPCR allowed the detection of the genes, which escaped the Northern Blot analysis, showing in sour orange the same pattern (over-expression) as Etrog citron for ADH, EREBP and AP; whereas MT and RGS were down regulated.In alemow seedlings, EREBP, RGS, AP and CLP showed a reduction of the expression level as well as AP in Troyer citrange.

A bacterium which can grow on chicken feathers and which exhibits keratinolytic activity was isolated from solfataric muds. It was classified as belonging to the genus Clostridium and closely related to C. sporogenes. Based on its unique capability to degrade chicken feathers, it was designated as Clostridium sporogenes bv. pennavorans bv. nov. The keratinase purified from the culture supernatant is a monomer of 28.7kDa molecular mass. The enzyme is relatively thermostable and is active over a broad range of temperature and pH. Specific enzymatic assays demonstrate that keratinase can act on a large variety of soluble and insoluble protein substrates.

Bryophytes are valuable bioaccumulators, because they have high absorbing and ion exchange capacities. Cadmium (Cd) is a heavy metal naturally present in soil; it is non-essential and highly toxic to most organisms, having toxicity 2 to 20 times higher than many other heavy metals. The presence of elevated levels of Cd ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To investigate the ability of Cd to affect gene transcription, the messenger RNA (mRNA) differential display technique was applied to the identification and isolation of genes whose transcription was altered in cultured Lunularia cruciata plants that were grown in the presence of cadmium salts. Four genes whose mRNA levels significantly changed in response to cadmium exposure were isolated and identified. The first gene identified in our analysis is up-regulated by Cd: it encodes the enzyme cystathionine gamma-synthase. The other genes are down-regulated by cadmium. These genes encode a methyltransferase, a tyrosine phosphatase and the EST 408 of the diatom Fragilariopsis cylindrus, whose function is unknown. Our findings demonstrate the usefulness of mRNA differential display technique for the detection of plant metabolic pathways affected by cadmium stress.

The cleavage specificity of Pernisine, a subtilisin-like protease from the hyperthermophilic archaeon Aeropyrum pernix, was established by mass spectrometry, analysing the peptides generated by digestion of oxidised bovine insulin B chain. The specificity was explored by changing several factors such as substrate/enzyme ratio, temperature and reaction media. Using a S/E ratio of 1000 (w/w) and a temperature of 60°C, five primary cleavage sites in the insulin B chain were detected suggesting a broad specificity of Pernisine, which is different from that found for other bacterial subtilisin-like proteases. When the S/E ratio and/or temperature were increased, a higher selectivity of Pernisine was observed with a unique cleavage site occurring between Leu15 and Tyr16. In addition, the influence on the enzymatic hydrolysis of different organic solvent concentrations was investigated. The results demonstrated that Pernisine could specifically digest the peptide substrate even in the presence of 80% acetonitrile solution or 30% dimethyl sulfoxyde. Thereby the cleavage specificity of Pernisine can be opportunely modulated by controlling the in vitro digestion conditions, suggesting that this enzyme could be an attractive candidate to use in a variety of biotechnological applications.

The messenger RNA (mRNA) differential display technique was applied to the identification and isolation of genes whose transcription was altered in leaves of Prunus armeniaca infected by European stone fruit yellows (ESFY) phytoplasma belonging to ribosomal subgroup 16SrX-B. Four genes whose steady-state levels of expression significantly changed in response to phytoplasma infection were isolated and identified. The results obtained show that two group of genes are affected by phytoplasma infection in apricot leaves. The first group comprises genes that are up-regulated by phytoplasma presence: in particular, a gene encoding the heat-shock protein HSP-70, a gene encoding a metallothionein (MT) and another homologous to the EST 673 cDNA clone of P. armeniaca, whose function was unknown. The other gene identified in our analysis is down-regulated by phytoplasma presence. It encodes a protein having homology to an amino acid transporter of Arabidopsis thaliana. Our findings demonstrate the usefulness of mRNA differential display approach for the detection of plant metabolic pathways affected by phytoplasma infection.

A novel extracellular serine protease designated Pernisine was purified to homogeneity and characterized from the archaeon Aeropyrum pernix K1. The molecular mass, estimated by SDS-PAGE analysis and by gel filtration chromatography, was about 34 kDa suggesting that the enzyme is monomeric. Pernisine was active in a broad range of pH (5.0-12.0) and temperature (60-120 degrees C) with maximal activity at 90 degrees C and between pH 8.0 and 9.0. In the presence of 1 mM CaCl(2) the activity, as a function of the temperature, reached a maximum at 90 degrees C but at 120 degrees C the enzyme retained almost 80% of its maximal activity. Activity inhibition studies suggest that the enzyme is a serine metalloprotease and biochemical data indicate that Pernisine is a subtilisin-like enzyme. The protease gene, identified from the sequenced genome of A. pernix, was amplified from total genomic DNA by PCR technique to construct the expression plasmid pGEX-Pernisine. The Pernisine, lacking the leader sequence, was expressed in Escherichia coli BL21 strain as a fusion protein with glutathione- S-transferase. The biochemical properties of the recombinant enzyme were found to be similar to those of the native enzyme.

A new enzyme showing a dehydrogenase activity towards aromatic aldehydes was isolated, purified and characterized from a halophilic strain isolated from saline environment. The enzyme is a monomer of 54 kDa; it is rather thermostable (optimal temperature: 50degreesC) showing a broad spectrum of activity in a large pH range with the maximum at pH 9.5. The substrate specificity and the effect of ions were evaluated and compared with analogous described proteins.

Caco-2, a human cell line, displays several biochemical and morphological characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concentrations of the metal. High pressure liquid chromatography fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins), were present in the apical and basal media of controls as well as in cells grown in the presence of high concentrations of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling experiments with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concentrations of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells.

Cathepsin D was purified to homogeneity from the liver of Antarctic icefish by anion-exchange chromatography followed by affinity chromatography on concanavalin-A Sepharose. The purified enzyme showed a molecular mass of 40 kDa and displayed optimal activity at pH 3.0 with a synthetic chromogenic substrate. The N-terminal sequence of this proteinase was determined by automated Edman degradation and was used to design a primer for use in reverse-transcriptase polymerase chain reaction. The open reading frame of the cloned cDNA encoded an aspartic proteinase, which contained the experimentally determined N-terminal sequence. The predicted sequence (396 residues) had a high similarity with those of cathepsin D from various vertebrate sources, but was considerably different from that of nothepsin, a distinct aspartic proteinase described previously from Antarctic fish [1]. Determination of kinetic parameters for substrate hydrolysis showed that, at temperatures between 8 and 50°C, the icefish cathepsin D had a higher specificity constant (kcat/Km) than human cathepsin D. The stability of both enzymes was measured at 50°C and half-lives of 55 and 3 min were derived for icefish and human cathepsin D, respectively.

Icefish (Notothenioidei, Channichthyidae) are a group of Antarctic fish displaying unique phenotype characteristics. Besides the reported lack of hemoglobin, icefish have a very low metallothionein (MT) content compared to Antarctic red-blooded fish. Despite the low amount of MT protein in icefish, cDNA encoding two MT isoforms could be produced from total hepatic RNA by RT-PCR. The mRNA 5' end fragments were amplified by 5'-RACE. Steady-state mRNA levels were assessed in both icefish and red-blooded fish by high stringency hybridization of the MT probe with total RNA. The results showed the presence of large amounts of MT mRNA in icefish liver. A further increase in MT mRNA was observed in Cd-treated icefish which was accompanied by the appearance of large amounts of MT protein. A comparison of the sequences of the 5' ends of the mRNAs showed the lack of a short motif in the 5'-UTR of only one of the two MT isoforms. On the basis of these results, we suggest that translation of the mRNA encoding an MT isoform is regulated in icefish by specific motifs in the 5' end.

Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.

Metallothionein levels were determined in the eggs of two sea urchin species, the Mediterranean Sphaerechinus granularis and the Antarctic Sterechinus neumayeri. While appreciable levels of metallothionein were found in S. granularis eggs, a negligible amount was detected in S. neumayeri. Two metallothionein isoforms were purified from S. granularis. and metallothionein cDNAs were obtained by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Two distinct cDNA species were cloned and sequenced. The translated amino acid sequences of these two forms consisted of 67 residues and differed in two amino acid substitutions. Despite the lack of metallothionein in S. neumayeri eggs, a metallothionein cDNA was obtained by RT-PCR amplification and a single amino acid sequence coding for a 63 residues MT was deduced. A comparative analysis of the primary structure of S. granularis and S. neumayeri metallothioneins with those of the other sea urchin metallothioneins has been performed. Sea urchin metallothioneins appear to be less similar to each other than metallothioneins of closely related vertebrates.

Icefish (family Channichthyidae, suborder Nothothenioidei) are a group of Antarctic fish that have evolved unique phenotypes in order to adapt to the environment in which they live. Besides the lack of haemoglobin and the drastic reduction in the number of erythrocyte-like cells, another striking feature of the icefish is that their liver is devoid of metallothionein. These cysteine-rich heavy-metal-binding proteins are usually present in large amounts in a large variety of organisms, from bacteria to mammals. Despite the failure to detect appreciable levels of metallothionein in icefish liver, a cDNA encoding metallothionein was produced from total RNA by reverse transcriptase PCR. The icefish metallothionein showed high percentage identity with metallothionein from Trematomus bernachii, a red-blooded Antarctic fish in which a normal content of hepatic metallothionein was found. Steady-state mRNA levels were assessed in fish liver by high-stringency hybridization of the metallothionein probe with total RNA. The results showed that icefish livers retain large amounts of untranslated metallothionein mRNA. The stability of the icefish transcript might be correlated with the lack of specific motifs in the untranslated 3' ends of mRNA.

Two low-molecular-weight zinc-binding proteins were purified from eggs of the sea urchin Strongylocentrotus intermedius (Echinodermata: Echinoidea) by gel-permeation and anion-exchange chromatography followed by high performance liquid chromatography. The characteristics of these proteins are distinct from those of metallothionein. Amino acid compositions show a low or intermediate content of cysteine and high amounts of acidic amino acids, glycine and histidine; they also contain methionine and aromatic residues. On the basis of these characteristics, the S. intermedius zinc-binding proteins appear similar to other metalloproteins isolated from both vertebrates and invertebrates. No typical metallothionein was detected in the extracts from S. intermedius eggs.

Bacillus acidocaldarius grown in the presence of Cu++ was capable of accumulating the metal in the form of a protease-sensitive high molecular weight (HMW) moiety whose formation was inhibited by actinomycin D. Only cells preadapted in Cu++ were able to grow in a Cd(++)-containing medium. A cell-free extract from cadmium-stressed cells was fractionated by gel-permeation chromatography. The majority of cadmium was found associated with a HMW protein fraction which was further purified by anion exchange chromatography and high-performance liquid chromatography. The molecular weight of the purified protein was estimated to be 23,000 by SDS-PAGE. Amino acid analysis showed a low cysteine content and an abundance of aspartate and glutamate. It is likely that the cadmium-binding protein is an essential component of the mechanism mediating recovery from heavy metal toxicity.

Recent reports have assigned to activation of phospholipase A2 (PLA2), with subsequent production of arachidonic acid (AA) and its derivatives, and to stimulation of protein kinase C (PKC), a key role in the control of body pattern, tentacle regeneration, and bud formation in two Hydra species. Experiments conducted in vivo suggested also the occurrence of a bidirectional interaction between the two enzymes during these processes. Here we describe for the first time the simultaneous partial characterization of PLA2 and PKC activities in a hydrozoan species, the freshwater Hydra vulgaris. PLA2 activity was found to be associated with membrane fractions, dependent on pH and on millimolar Ca2+ concentrations and counteracted by a specific inhibitor of mammalian membrane PLA2, oleyloxyethyl-phosphoryl-choline (OOPC). A PKC-like enzyme with a molecular weight of about 70 kDa was partially purified from cytosolic extracts. Its activity was also found to depend on Ca2+ as well as phosphatidylserine, but was not influenced by AA. Conversely, the PKC activator tetradecanoyl-phorbol-II-acetate (TPA) induced PLA2 activation and AA liberation in H. vulgaris polyps in vivo. While PKC-PLA2 interactions have been extensively investigated in mammals, the present study represents the first example of PKC-induced activation of an invertebrate PLA2.

A low-molecular-mass zinc-binding protein was purified from the eggs of the sea urchin Paracentrotus lividus using procedures that included gel-permeation and anion-exchange chromatography followed by HPLC. The primary structure of this protein was derived from the sequences of peptide fragments obtained by digestion with trypsin and thermolysin. The reconstructed sequence showed the presence of 20 cysteinyl residues, thus resembling that of a metallothionein. The Paracentrotus protein was most similar to the metallothionein of Strongylocentrotus purpuratus, another member of the order of Echinoida, living along the coast of the Pacific Ocean. However, the presence of non-conservative amino acid substitution, together with a deletion of two residues in the Strongylocentrotus metallothionein, make the similarity scores of the two sea urchin proteins lower than that of metallothioneins from vertebrates of the same order. In addition, the present data show that sea urchin metallothioneins display no homology with metallothioneins of any other species.

[3H]‐dopamine ([3H]‐DA) uptake was measured in the presence or absence of the catecholamine uptake inhibitor nomifensine in both unfertilized and fertilized eggs. Specific [3H]‐DA uptake depended on time and [3H]‐DA concentration; it was high in unfertilized eggs, declined 20–30 min after fertilization, and rose again during cleavage. Irreversible inactivation of dopamine receptors by N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) resulted in a complete loss of sensitivity of egg adenylate cyclase to dopamine stimulation. In fertilized eggs treated with EEDQ for 1 hr, restoration of adenylate cyclase activity sensitive to dopamine stimulation could be observed 4 hr after the end of treatment, thus suggesting the appearance of new dopamine receptors in cleaving eggs. Short‐term EEDQ treatment on unfertilized eggs, although not impairing fertilization, resulted in cleavage inhibition; the same treatment carried out soon after fertilization, on the other hand, elicited no effect on development. On the contrary, in embryos subjected to continuous treatment with EEDQ, development was impaired independent of the stage at which the treatment was started. © 1995 Wiley‐Liss, Inc.

Metallothionein presence and amount were determined in the unfertilized eggs of six sea urchin species by silver saturation assay and gel-chromatography of cell extracts. The results showed high levels of metallothionein in the egg cytoplasm of the two Mediterranean species Paracentrotus lividus and Sphaerechinus granularis. No metallothionein was found either in the eggs of Arbacia lixula, or in those of the three Eastern species Strongylocentrotus intermedius, Temnopleurus hardwickii and Clypeaster japonicus. However, the extracts of the latter three species revealed the presence of zinc bound in a macromolecular form, thus suggesting the existence of metal-binding proteins distinct from metallothioneins.