Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented byHLA-B7 and is encoded by a new gene that we have namedRAGE1. No expression ofRAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.     

Yeast proteins can activate expression through regulatory sequences of the amdS gene of Aspergillus nidulans

The upstream regulatory region of the amdS gene of Aspergillus nidulans contains a CCAAT sequence known to be important in setting both basal and derepressed levels of expression. We have investigated whether the CCAAT-binding HAP2/3/4 complex of the yeast Saccharomyces cerevisiae can recognise this sequence in an amdS context. Sequences from the 5′ region of amdS were cloned in front of the CYCI-lacZ fusion gene bearing a minimal promoter and transformed into wild-type and hap2 strains of yeast. This study has indicated that amdS sequences are capable of promoting regulated expression of the fusion gene in response to carbon limitation. The yeast HAP2/3/4 complex can recognise the amdS CCAAT sequence and activate expression from this sequence. In addition, the results indicate that other yeast proteins can also regulate expression from the A. nidulans amdS 5′ sequences under carbon-limiting conditions.     

Free l-amino acids and d-aspartate content in the nervous system of Cephalopoda. A comparative study

We have determined the content of free l-amino acids and d-aspartate in the nervous tissue of three representative cephalopods: Sepia officinalis, Octopus vulgaris, and Loligo vulgaris, and the optic lobes of adult and embryo Sepia officinalis. Taurine is the most abundant amino acid in the cephalopod nervous tissue. Its content amounts to more than 50% of the total free amino acids. The other most concentrated amino acids are Glu, Ala, Asp, and GABA. High concentrations of d-aspartate were found in the nervous tissue of all cephalopods examined (7–12 μmol/g wet tissue) which represents 50–80% of the total aspartate (d + l), depending on the animal. Among the various regions of the brain of Octopus vulgaris, d-aspartate was found to be evenly distributed in the various regions of the brain. In nerve tissue of Sepia officinalis, there is no significant difference in the pattern of free l-amino acids, in particular of the d-aspartate concentration, between adults and embryos, except for GABA, Gly, His and Thr. This suggests that d-aspartate in nerve tissue of the Cephalopoda is of endogenous origin and not a product of accumulation from exogenous sources. From a comparative study of the content of d-aspartate in the nervous tissue of different animals, we found that protostomia contain a significantly higher amount than deuterostomia. Thus, d-aspartate could be a criterion to distinguish the protostomia phyla from the deuterostomia phyla.     

GAPs for rho-related GTPases

Ras-related GTP-binding proteins (GTPases) of the rbo subfamily play important roles in regulating the organization of the actin cytoskeleton. A large number of multifunctional proteins that can stimulate their intrinsic GTPase activity have been identified. Here, we discuss the nature of such GTPase-activating proteins (GAPs) and their potential importance for cell signalling.     

Cysteine proteinase forms in sprouting potato tuber

Transformation of plants with exogenous proteinase inhibitor genes represents an attractive strategy for the biological control of insect pests. However, such a strategy necessitates a thorough characterization of endogenous proteinases. which represent potential target enzymes for the exogenous inhibitors produced. In the present study. changes in general endoproteolytic activity were monitored during sprouting of potato ( Solanum tuberosum L. cv. Kennebec) tuber. Quantitative data obtained using standard procedures showed that an increase in cysteine proteinase (EC 3.4.22) activity occurs during sprouting. This increased activity results from the gradual appearance of new cysteine proteinase forms, as demonstrated by the use of class-specific proteinase activity gels. While only one cysteine proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, oryzacystatins I and II. two cysteine proleinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase delected. Similar results were obtained with leaf, stem and stolon proteinases. This apparent absence of direct interference supports the potential of oryzacystatin genes for production of insect-tolerant transgenie potato plants.     

Gene transfer to inflorescence and flower meristems using ballistic micro-targeting

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.     

Binding of malate dehydrogenase and NADH channelling to complex I

As previously reported, mitochondrial malate dehydrogenase (MDH) binds to purified complex I of the electron transport system. With conditions used in previous reports, MDH binds even more extensively, but probably predominantly non-specifically, to the matrix side of the inner mitochondrial membrane of submitochodrial particles (SMP). Herein we report experimental conditions for highly specific binding of malate dehydrogenase to complex I within SMP. These conditions permit us to demonstrate NADH channelling from malate dehydrogenase to complex I using the completing reaction test. This test, though not ideal for all situations, has several advantages over the enzyme buffering test previously used. These advantages should facilitate further studies elucidating NADH channeling to complex I from MDH and other dehydrogenases. Independent evidence of NADH channelling to the electron transport chain and the potential advantages of substrate channelling in general are also discussed. Substrate channelling from MDH in particular may be especially beneficial because of the unfavourable equilibrium and kinetics of this enzyme reaction.