Breast cancer incidence trends in European women aged 20-39 years at diagnosis.

An increase in the incidence of breast cancer in women aged<40 years has been reported in recent years. Increased incidence could be partly explained by subtle detection biases, but the role of other risk factors cannot be ruled out. The purpose of the present study was to investigate the changes in temporal trends in breast cancer incidence in European women aged 20-39 years at diagnosis. Age specific breast cancer incidence rates for 17 European Cancer Registries were retrieved for the calendar period 1995-2006. Cancer registries data were pooled to reduce annual fluctuations present in single registries and increase incidence rates stability. Regression models were fitted to the data assuming that the number of cancer cases followed the Poisson distribution. Mean annual changes in the incidence rate (AIC) across the considered time window were calculated. The AIC estimated from all European registries was 1.032 (95% CI=1.019-1.045) and 1.014 (95% CI=1.010-1.018) in women aged 20-29 and 30-39 years old at diagnosis, respectively. The major change was detected among women aged 25-29 years at diagnosis: AIC=1.033 (95% CI=1.020-1.046). The upward trend was not affected when registries with high or low AIC were removed from the analysis (sensitivity analysis). Our findings support the presence of an increase in the incidence of breast cancer in European women in their 20s and 30s during the decade 1995-2006. The interpretation of the observed increase is not straightforward since a number of factors may have affected our results. The estimated annual increase in breast cancer incidence may result in a burden of the disease that is important in terms of public health and deserves further investigation of possible risk factors.

Cancer incidence in pet dogs: findings of the Animal Tumor Registry of Genoa, Italy.

BACKGROUND: The occurrence of spontaneous tumors in pet animals has been estimated in a few European and North American veterinary cancer registries with dissimilar methodologies and variable reference populations. OBJECTIVES: The Animal Tumor Registry (ATR) of Genoa, Italy, was established in 1985 with the aim of estimating the occurrence of spontaneous tumors in dogs. METHODS: Six thousand seven hundred and forty-three tumor biopsy specimens were received from local veterinarians in the Municipality of Genoa between 1985 and 2002. Three thousand and three hundred and three (48.9%) biopsy specimen samples were diagnosed as cancer and were coded according to the International Statistical Classification of Diseases (ICD-9). RESULTS: Mammary cancer was the most frequently diagnosed cancer in female dogs, accounting for 70% of all cancer cases. Incidence of all cancers was 99.3 per 100,000 dog-years (95% CI: 93.6-105.1) in male dogs and 272.1 (95% CI: 260.7-283.6) in female dogs. The highest incidence rates were detected for mammary cancer (IR = 191.8, 95% CI: 182.2-201.4) and for non-Hodgkin's lymphoma (IR = 22.9, 95% CI: 19.7-26.5) in bitches and for non-Hodgkin's lymphoma (IR = 19.9, 95% CI: 17.4-22.7) and skin cancer (IR = 19.1, 95% CI: 16.6-21.8) in male dogs. All cancer IR increased with age ranging between 23.7 (95% CI: 18.4-30.1) and 763.2 (95% CI: 700.4-830.1) in bitches and between 16.5 (95% CI: 12.8-21.1) and 237.6 (95% CI: 209.1-269.0) in male dogs aged < or =3 years and >9-11 years. CONCLUSION: This study summarizes the work done by the ATR of Genoa, Italy, between 1985 and 2002. All cancer incidence was 3 times higher in female than in male dogs, a difference explained by the high rate of mammary cancer observed in bitches. Because a biopsy specimen was required to make a cancer diagnosis, cancer rates for internal organs cancers, such as respiratory and digestive tract cancers may have been underestimated in the study population.

Salts induce structural changes in elongation factor 1alpha from the hyperthermophilic archaeon Sulfolobus solfataricus: a Fourier transform infrared spectroscopic study.

Elongation factor 1alpha from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1alpha) carries the aminoacyl tRNA to the ribosome; it binds GDP or GTP, and it is also endowed with an intrinsic GTPase activity that is triggered in vitro by NaCl at molar concentrations [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376-20379]. The structural properties of SsEF-1alpha were investigated by Fourier transform infrared spectroscopy. The estimation of the secondary structure of the SsEF-1alpha*GDP complex, made by curve fitting of the amide I' band or by factor analysis of the amide I band, indicated a content of 34-36% alpha-helix, 35-40% beta-sheet, 14-19% turn, and 7% unordered structure. The substitution of the GDP bound with the slowly hydrolyzable GTP analogue Gpp(NH)p induced a slight increase in the alpha-helix and beta-sheet content. On the other hand, the alpha-helix content of the SsEF-1alpha*GDP complex increased upon addition of salts, and the highest effect was produced by 5 M NaCl. The thermal stability of the SsEF-1alpha*GDP complex was significantly reduced when the GDP was replaced with Gpp(NH)p or in the presence of NaBr or NH4Cl, whereas a lower destabilizing effect was provoked by NaCl and KCl. Therefore, the extent of the destabilizing effect of salts depended on the nature of both the cation and the anion. The data suggested that the sodium ion was responsible for the induction of the GTPase activity, whereas the anion modulated the enzymatic activity through destabilization of particular regions of SsEF-1alpha. Finally, the infrared data suggested that, in particular region(s) of the polypeptide chain, the SsEF-1alpha*Gpp(NH)p complex possesses structural conformations which are different from those present in the SsEF-1alpha*GDP complex.

The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with GDP reveals novel features in nucleotide binding and exchange.

The crystal structure of elongation factor 1alpha from the archaeon Sulfolobus solfataricus in complex with GDP (SsEF-1alpha.GDP) at 1.8 A resolution is reported. As already known for the eubacterial elongation factor Tu, the SsEF-1alpha.GDP structure consists of three different structural domains. Surprisingly, the analysis of the GDP-binding site reveals that the nucleotide- protein interactions are not mediated by Mg(2+). Furthermore, the residues that usually co-ordinate Mg(2+) through water molecules in the GTP-binding proteins, though conserved in SsEF-1alpha, are located quite far from the binding site. [(3)H]GDP binding experiments confirm that Mg(2+) has only a marginal effect on the nucleotide exchange reaction of SsEF-1alpha, although essential to GTPase activity elicited by SsEF-1alpha. Finally, structural comparisons of SsEF- 1alpha.GDP with yeast EF-1alpha in complex with the nucleotide exchange factor EF-1beta shows that a dramatic rearrangement of the overall structure of EF-1alpha occurs during the nucleotide exchange.

Phenylmethanesulfonyl fluoride inactivates an archaeal superoxide dismutase by chemical modification of a specific tyrosine residue. Cloning, sequencing and expression of the gene coding for Sulfolobus solfataricus superoxide dismutase.

The gene encoding the superoxide dismutase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expression in Escherichia coli obtained. The chemicophysical properties of the recombinant SsSOD were identical with those of the native enzyme. The recombinant SsSOD possessed a covalent modification of Tyr41, already observed in native SsSOD [Ursby, T., Adinolfi, B.S., Al-Karadaghi, S., De Vendittis, E. & Bocchini, V. (1999) J. Mol. Biol. 286, 189--205]. HPLC analysis of SsSOD samples prepared from cells treated or not with phenylmethanesulfonyl fluoride (PhCH(2)SO(2)F), a protease inhibitor routinely added during the preparation of cell-free extracts, showed that the modification was caused by PhCH(2)SO(2)F. Refinement of the crystal model of SsSOD confirmed that a phenylmethanesulfonyl moiety was attached to the hydroxy group of Tyr41. PhCH(2)SO(2)F behaved as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modification increased. The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD. These results show that Tyr41 plays an important role in the enzyme activity and the maintenance of the structural architecture of SsSOD.

Effects of microenvironment on morphology and function of the microglial cell line BV-2.

Effects of microenvironmental changes were examined in the microglial cell line BV-2. In serum supplemented medium cells were ameboid shaped and exhibited thin cytoplasmatic processes at lower concentration or in absence of serum. High levels of acetylated low-density lipoprotein (LDL) receptor and of phagocytic and proliferative activity were detected. Lipopolysaccharide (LPS) and the neuropeptide substance P (SP) induced secretion of interleukin-6. Low interleukin-3 secretion was detected only occasionally and was not influenced by LPS and SP. In defined medium, "process-bearing" cells were evident. Compared to cultures in serum supplemented medium, the cells expressed lower acetylated LDL-binding and phagocytic activity while actively proliferated, the response to LPS was reduced and to SP absent. Granulocyte/macrophage colony-stimulating factor increased the number of process-bearing cells, of acetylated LDL-binding and of IL-6 secretion induced by LPS. Cell morphology was not influenced by neurotrophins like nerve growth factor and brain-derived neurotrophic factor. The described phenotypical and functional plasticity makes the BV-2 cell line a useful model to investigate mechanisms of microglial activation.

Psychrophilic elongation factor Tu from the antarctic Moraxella sp. Tac II 25: biochemical characterization and cloning of the encoding gene.

The elongation factor Tu was isolated from a psychrophilic eubacterial Antarctic Moraxella strain (MoEF-Tu) and its molecular and functional properties were determined. It catalyzed the synthesis of poly(Phe) and bound specifically guanine nucleotides with an affinity for GDP about 12-fold higher than that for GTP. The affinity toward guanine nucleotides was lower than that of other eubacterial EF-Tu. The intrinsic GTPase activity of MoEF-Tu was hardly detectable but was accelerated by 2 orders of magnitude in the presence of the antibiotic kirromycin (GTPase(k)). Such a property resembled Escherichia coli EF-Tu (EcEF-Tu) even though the affinity of MoEF-Tu for the antibiotic was lower. MoEF-Tu showed a thermophilicity higher than that of EcEF-Tu; its temperature for half-denaturation was 44 degrees C. The MoEF-Tu encoding gene corresponding to E. coli tufA was cloned and sequenced. The translated protein had a calculated molecular weight of 43 288 and contained the GTP-binding sequence motifs. Concerning its primary structure, MoEF-Tu showed sequence identity with E. coli and Thermus thermophilus EF-Tu equal to 84% and 74%, respectively, while the identity with EF-1 alpha from the archaeon Sulfolobus solfataricus was equal to 32%.

Interleukin-1beta induces apoptosis in GL15 glioblastoma-derived human cell line.

Interleukin 1-beta (IL-1beta) induces apoptosis in a glioblastoma-derived human cell line, exhibiting a poorly differentiated astrocytic phenotype. The apoptotic effect was demonstrated by analyzing nuclear morphology, in situ DNA fragmentation, and by ELISA detection of cytoplasmatic nucleosomes. We correlated the degree of differentiation of GL15 cells with the apoptotic response: 1) 4',6-diamidino-2-phenylindole staining, combined with glial fibrillary acidic protein (GFAP) immunofluorescence, showed that the cells with apoptotic nuclei express low levels of GFAP; and 2) at 13 days of subculture, in a more differentiated state, GL15 cells did not respond with apoptosis to IL-1beta. In this cell line, nonrandom chromosome changes and the expression of SV40 early region have been previously shown. The involvement of p42/p44 mitogen-activated protein kinase (MAPK) pathway in the induction of apoptosis by IL-1beta was hypothesized. Previous studies have shown that SV40 small T antigen partially inhibits phosphatase 2A, leading to an enhancement of the steady-state activity of p42/p44 MAPK pathway. PD-098059, specific inhibitor of p42/p44 MAPK pathway, counteracts the apoptotic effect of IL-1beta, whereas SB-203580, specific inhibitor of p38 stress-activated protein kinase (SAPK) pathway, is ineffective. The imbalance between MAPK and SAPK pathways has been proposed as a key factor in determination of cell fate. Our results demonstrate that a further stimulation of p42/p44 MAPK pathway can constitute a death signal in tumor cells in which genomic damage and MAPK pathway control alterations occur.

The archaeal elongation factor 1alpha bound to GTP forms a ternary complex with eubacterial and eukaryal aminoacyl-tRNA.

The archaeal Sulfolobus solfataricus elongation factor 1alpha (SsEF-1alpha) bound to GTP or to its analogue guanyl-5'-yl imido diphosphate [Gpp(NH)p] formed a ternary complex with either Escherichia coli Val-tRNAVal or Saccharomyces cerevisiae Phe-tRNAPhe as demonstrated by gel-shift and gel-filtration experiments. Evidence of such an interaction also came from the observation that SsEF-1alphaz.rad;Gpp(NH)p was able to display a protective effect against either the spontaneous deacylation or the digestion of aminoacyl-tRNA by RNase A. Protection against the deacylation of aminoacyl-tRNA allowed evaluatation of the affinity of SsEF-1alphaz. rad;Gpp(NH)p for both aminoacyl-tRNAs used. The K'd values of the ternary complex containing S. cerevisiae Phe-tRNAPhe or E. coli Val-tRNAVal were 0.3 microM and 4.4 microM, respectively. In both cases, the affinity of SsEF-1alphaz.rad;Gpp(NH)p for aminoacyl-tRNA was three orders of magnitude lower than that of the homologous eubacterial ternary complexes, but comparable with the affinity shown by the ternary complex involving eukaryal EF-1alpha [Negrutskii, B.S. & El'skaya, A.V. (1998) Prog. Nucleic Acids Res. 60, 47-77]. As already observed with eukaryal EF-1alpha, SsEF-1alpha in its GDP-bound form was also able to protect the ester bond of aminoacyl-tRNA, even though with a 10-fold lower efficiency compared with SsEF-1alphaz.rad;Gpp(NH)p. The overall results indicated that the archaeal elongation factor 1alpha shares several properties with eukaryal EF-1alpha but not with eubacterial EF-Tu.

A NAD(P)H oxidase isolated from the archaeon Sulfolobus solfataricus is not homologous with another NADH oxidase present in the same microorganism. Biochemical characterization of the enzyme and cloning of the encoding gene.

A NAD(P)H oxidase has been isolated from the archaeon Sulfolobus solfataricus. The enzyme is a homodimer with M(r) 38,000 per subunit (SsNOX38) containing 1 FAD molecule/subunit. It oxidizes NADH and, less efficiently, NADPH with the formation of hydrogen peroxide. The enzyme was resistant against chemical and physical denaturating agents. The temperature for its half-denaturation was 93 and 75 degrees C in the absence or presence, respectively, of 8 M urea. The enzyme did not show any reductase activity. The SsNOX38 encoding gene was cloned and sequenced. It accounted for a product of 36.5 kDa. The translated amino acid sequence was made of 332 residues containing two putative betaalphabeta-fold regions, typical of NAD- and FAD-binding proteins. The primary structure of SsNOX38 did not show any homology with the N-terminal amino acid sequence of a NADH oxidase previously isolated from S. solfataricus (SsNOX35) (Masullo, M., Raimo, G., Dello Russo, A., Bocchini, V. and Bannister, J. V. (1996) Biotechnol. Appl. Biochem. 23, 47-54). Conversely, it showed 40% sequence identity with a putative thioredoxin reductase from Bacillus subtilis, but it did not contain cysteines, which are essential for the activity of the reductase.

A chimeric elongation factor containing the putative guanine nucleotide binding domain of archaeal EF-1 alpha and the M and C domains of eubacterial EF-Tu.

A recombinant chimeric elongation factor containing the region of EF-1 alpha from Sulfolobus solfataricus harboring the site for GDP and GTP binding and GTP hydrolysis (SsG) and domains M and C of Escherichia coli EF-Tu (EcMC) was studied. SsG-EcMC did not sustain poly(Phe) synthesis in either S. solfataricus or E. coli assay system. This was probably due to the inability of the chimera to interact with aa-tRNA. The three-dimensional modeling of SsG-EcMC indicated only small structural differences compared to the Thermus aquaticus EF-Tu in the ternary complex with aa-tRNA and GppNHp, which did not account for the observed inability to interact with aa-tRNA. The addition of the nucleotide exchange factor SsEF-1 beta was not required for poly(Phe) synthesis since the chimera was already able to exchange [(3)H]GDP for GTP at very high rate even at 0 degrees C. Compared to that of SsEF-1 alpha, the affinity of the chimera for guanine nucleotides was increased and the k(cat) of the intrinsic GTPase was 2-fold higher. The heat stability of SsG-EcMC was 3 and 13 degrees C lower than that displayed by SsG and SsEF-1alpha, respectively, but 30 degrees C higher than that of EcEF-Tu. This pattern remained almost the same if the melting curves of the proteins being investigated were considered instead. The chimeric elongation factor was more thermophilic than SsG and SsEF-1 alpha up to 70 degrees C; at higher temperatures, inactivation occurred.

The interaction between the archaeal elongation factor 1alpha and its nucleotide exchange factor 1beta.

In Sulfolobus solfataricus the binding of the exchange factor 1beta (SsEF-1beta) to SsEF-1alpha-GDP displaces the nucleotide and the SsEF-1alpha-SsEF-1beta complex is formed. The complex itself is stable, but it dissociates upon the addition of GDP or Gpp(NH)p but not ATP. Since the rate of the formation of the SsEF-1alpha-SsEF-1beta complex is significatively slower than the rate of the nucleotide exchange catalyzed by SsEF-1beta it can be inferred that in vivo the GDP/GTP exchange reaction proceeds via an SsEF-1alpha-SsEF-1beta interaction without involving the formation of a stable binary complex as an intermediate.

The A26G replacement in the consensus sequence A-X-X-X-X-G-K-[T,S] of the guanine nucleotide binding site activates the intrinsic GTPase of the elongation factor 2 from the archaeon Sulfolobus solfataricus.

A recombinant form of the elongation factor 2 from the archaeon Sulfolobus solfataricus (SsEF-2), carrying the A26G substitution, has been produced and characterized. The amino acid replacement converted the guanine nucleotide binding consensus sequences A-X-X-X-X-G-K-[T,S] of the elongation factors EF-G or EF-2 into the corresponding G-X-X-X-X-G-K-[T,S] motif which is present in all the other GTP-binding proteins. The rate of poly(U)-directed poly(Phe) synthesis and the ribosome-dependent GTPase activity of A26GSsEF-2 were decreased compared to SsEF-2, thus indicating that the A26G replacement partially affected the function of SsEF-2 during translocation. In contrast, the A26G substitution enhanced the catalytic efficiency of the intrinsic SsEF-2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scarano, G., & Bocchini, V. (1997) Biochimie 78, 832-837]. Surprisingly, A26GSsEF-2 was able to hydrolyse GTP even in the absence of ethylene glycol; furthermore, the alcohol increased the affinity for GTP without modifying the catalytic constant of A26GSsEF-2 GTPase. Compared to SsEF-2, the affinity of A26GSsEF-2 for [3H]GDP was significantly reduced. These findings suggest that A26 is a regulator of the biochemical functions of SsEF-2. The involvement of this alanine residue in the guanine nucleotide-binding pocket of EF-2 or EF-G is discussed.

Iron superoxide dismutase from the archaeon Sulfolobus solfataricus: analysis of structure and thermostability.

The crystal structure of superoxide dismutase (SOD) from the hyper thermophile Sulfolobus solfataricus has been determined at 2.3 A resolution by molecular replacement and refined to a crystallographic R-factor of 16.8 % (Rfree 19.8 %). The crystals belong to the space group C2 (a=76.3 A, b=124.3 A, c=60.3 A, beta=128.8 degrees) with two identical monomers in the asymmetric unit. The monomer has a molecular weight of 24 kDa and consists of 210 amino acid residues of which 205 are visible in the electron density map. The overall fold of the monomer of S. solfataricus SOD is similar to that of the other known Fe or Mn-SODs. S. solfataricus SOD forms a very compact tetramer of a type similar to that of SOD from the hyperthermophile Aquifex pyrophilus. Both structures show an elevated number of inter-subunit ion-pairs compared with the mesophilic SOD from Mycobacterium tuberculosis and the thermophilic SOD from Thermus thermophilus. However, in contrast to the A. pyrophilus SOD structure, the number of intra-subunit ion-pairs as well as inter- subunit hydrogen bonds is not higher than in the compared mesophilic and thermophilic SOD structures. The electron density also revealed an unexpected and unusual covalent modification of a conserved tyrosine in the active site. Its involvement in the specific activity of the enzyme is discussed.

The effect of ribosome-inactivating proteins on the ribosome from the hyperthermophilic archaeon Sulfolobus solfataricus.

Protein synthesis in the thermoacidophilic archaeon Sulfolobus solfataricus (Ss) was inhibited by polynucleotide:adenosine glycosylase activity of some type 1 ribosome-inactivating proteins (RIP). The target of RIP was S. solfataricus rRNA that was depurinated thus producing inactive ribosomes. The amount of RIP required to half-inactivated Ss-ribosomes was comparable to that needed for eubacterial ribosomes, but two orders of magnitude higher than that required for mammalian ribosomes. In addition, RIP treated Ss-ribosomes were also less efficient in stimulating the ribosome dependent GTPase activity of the S. solfataricus elongation factor 2 (SsEF-2) thus suggesting that the inhibition of protein synthesis was probably due to the lack of the interaction between depurinated Ss-ribosomes and SsEF-2. Since SsEF-2 protects Ss-ribosomes against RIP activity it can be hypothesised that also on Ss-ribosomes the sites of interaction for the translocation factor 2 and the RIP are topographically close.

Expression in Escherichia coli of the elongation factor 1beta gene and its nucleotide T160C mutant from the archaeon Sulfolobus solfataricus.

The guanine nucleotide exchange factor EF-1beta gene from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1beta) was amplified by PCR and cloned into the pT7-7 expression vector. One of four selected clones harbored the T160C nucleotide substitution leading to the Y54H amino acid change in a hydrophobic region of SsEF-1beta, caused by a nucleotide misincorporation of the Taq DNA polymerase during PCR. The resulting plasmids were used to transform the Escherichia coli BL21(DE3)pLysE strain. Upon induction with isopropyl beta-d-thiogalactopyranoside about 1.4 mg of the recombinant SsEF-1beta (recSsEF-1beta) and Y54HSsEF-1beta were obtained from 1 liter of cell culture. recSsEF-1beta and Y54HSsEF-1beta were both able to catalyze the GDP/GTP exchange on SsEF-1alpha as observed with the wild-type SsEF-1beta. In addition, the heat inactivation profiles of recSsEF-1beta and Y54HSsEF-1beta were identical, being both half inactivated after 30 min treatment at 105 degrees C. These results suggest that Tyr 54 is not essential for the nucleotide exchange activity and is not involved in the thermostability of SsEF-1beta.

Protein engineering on enzymes of the peptide elongation cycle in Sulfolobus solfataricus.

The present article is a review of the work done on the elongation factors EF-1 alpha, EF-2 and EF-1 beta isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. The molecular, physical and biochemical properties of the intact, truncated, mutant or chimeric forms are described and compared.

A time-dependent increase in glial fibrillary acidic protein expression and glutamine synthetase activity in long-term subculture of the GL15 glioma cell line.

1. Astrocytes are the most numerous cellular elements in the central nervous tissue, where they play a critical role in physiological and pathological events. The biological signals regulating astrocyte growth and differentiation are relevant for both physiology and pathology, but they are still little understood. 2. Using a poorly differentiated glioma cell line, GL15, we investigated whether, in long-term subculture, this could upregulate the expression of glial fibrillary acidic protein (GFAP), as described in some rodent astrocyte cell lines. Under the same culture conditions, we investigated glutamine synthetase (GS) activity, growth-associated protein (GAP)-43 expression, and expression of several neutrotrophic factors. 3. A dramatic increase in GFAP expression was evidenced by Western blotting during progressive in vitro growth of GL15 cells. GS specific activity was also upregulated in long-term culture. The time spent in vitro by GL15 cells did not affect GAP-43 and neutrophic factor BDNF and NT3 expression as revealed by RT-PCR analysis. 4. Our results suggest that, in GL15, GFAP and GS genes may have common or integrated regulatory mechanisms elicited at the cell confluency which could be relevant for both astrocyte physiology and astrocyte pathology. These mechanisms are not involved in GAP-43 and neutrophic factor BDNF and NT3 expression.

Constitutive expression of beta-N-acetylhexosaminidase in a microglial cell line: transcriptional modulation by lipopolysaccharide and serum factors.

We investigated the expression of the alpha- and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase in the BV-2 microglial cell line under different culture conditions. Beta-N-acetylhexosaminidase from BV-2 microglia cells was separated into its constituent isoenzymes on diethylaminoethyl (DEAE) cellulose, and its activity was monitored with 4-methylumbelliferyl-beta-N-acetylglucosamine and 4-methylumbelliferyl-beta-N-acetylglucosamine-6-sulphate substrates. Forms corresponding to the mouse isoenzymes A and B were present in the cells incubated in serum-supplemented medium as well as in serum-free medium. Lipopolysaccharide, a well-known activator of microglia in vitro, added to the BV-2 cells in serum-supplemented medium induced a decrease in the specific enzymatic activity determined with the 4-methylumbelliferyl-beta-N-acetylglucosamine substrate. Lipopolysaccharide had no effect on hexosaminidase isoenzyme pattern of BV-2 cells in serum-supplemented medium. The level of alpha-subunit mRNA was increased and the level of beta-subunit mRNA was decreased in BV-2 cells incubated in serum-supplemented medium plus lipopolysaccharide. In the cells incubated in a serum-free medium no significant changes in the hexosaminidase-specific activities towards the above substrates were observed. Interestingly, increased expression of alpha- and beta-subunit mRNA was evident in comparison with cultures in serum-supplemented medium. The present results suggest that the BV-2 cell line may be a useful tool to study the possible role of microglia in the metabolism of brain glycolipids.

Heterologous expression in Escherichia coli of the gene encoding an archaeal thermoacidophilic elongation factor 2. Properties of the recombinant protein.

The gene encoding the elongation factor 2 from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-2) was expressed in Escherichia coli using the pT7-7 expression vector. The synthesis of the heterologous product did not increase upon addition of isopropyl-beta-thiogalactopyranoside. The amount of purified intact recombinant SsEF-2 (SsEF-2rec) was about 3 mg from 60 g of transformed wet cells. Recombinant and naturally occurring SsEF-2 showed identical electrophoretic mobility, immunological properties and the N-terminal amino acid sequence; both were lacking the initial methionine. Differently from SsEF-2, SsEF-2rec did not undergo post-translational modification of His603 into diphthamide, as indicated by its inability to be ADP-ribosylated. SsEF-2rec appeared indistinguishable from SsEF-2 in the fulfillment of its biological functions; in fact, it was fully capable to support poly(Phe) synthesis, to bind GDP and to display either the intrinsic or the ribosome-dependent GTPase. Finally, SsEF-2rec was endowed with the same heat stability as SsEF-2. Altogether these findings proved that SsEF-2rec was functionally active as SsEF-2. The used expression system could allow to produce mutated forms of SsEF-2 obtained by mutagenesis of the corresponding gene.