Development of a scalable recombinant system for cyclic beta-1,2-glucans production.

BACKGROUND: Cyclic ß-1,2-glucans (CßG) are bacterial cyclic homopolysaccharides with interesting biotechnological applications. These ring-shaped molecules have a hydrophilic surface that confers high solubility and a hydrophobic cavity able to include poorly soluble molecules. Several studies demonstrate that CßG and many derivatives can be applied in drug solubilization and stabilization, enantiomer separation, catalysis, synthesis of nanomaterials and even as immunomodulators, suggesting these molecules have great potential for their industrial and commercial exploitation. Nowadays, there is no method to produce CßG by chemical synthesis and bacteria that synthesize them are slow-growing or even pathogenic, which makes the scaling up of the process difficult and expensive. Therefore, scalable production and purification methods are needed to afford the demand and expand the repertoire of applications of CßG. RESULTS: We present the production of CßG in specially designed E. coli strains by means of the deletion of intrinsic polysaccharide biosynthetic genes and the heterologous expression of enzymes involved in CßG synthesis, transport and succinilation. These strains produce different types of CßG: unsubstituted CßG, anionic CßG and CßG of high size. Unsubstituted CßG with a degree of polymerization of 17 to 24 glucoses were produced and secreted to the culture medium by one of the strains. Through high cell density culture (HCDC) of that strain we were able to produce 4,5 g of pure unsubstituted CßG /L in culture medium within 48 h culture. CONCLUSIONS: We have developed a new recombinant bacterial system for the synthesis of cyclic ß-1,2-glucans, expanding the use of bacteria as a platform for the production of new polysaccharides with biotechnological applications. This new approach allowed us to produce CßG in E. coli with high yields and the highest volumetric productivity reported to date. We expect this new highly scalable system facilitates CßG availability for further research and the widespread use of these promising molecules across many application fields.

Porphyrin photosensitised processes in the prevention and treatment of water- and vector-borne diseases.

Water- and vector-borne diseases are a global burden which is estimated to cause several million deaths and innumerable cases of sickness every year. These infectious illnesses are emerging or resurging as a result of several factors, such as changes in climate, in public health and demography policy, as well as the spread of resistance to insecticide and drug, and genetic changes in pathogens. Integrated prevention strategies must be developed and implemented in endemic disease areas to reverse the trend of emergent/resurgent water- and vector-borne diseases. With this perspective porphyrins and their analogues, that have been shown to act as very efficient photosensitising agents against a broad number of microbial pathogens (bacteria, fungi, protozoa) and parasitic animals, could represent an important tool for the prevention and control of these pathologies. The application of photosensitised processes can be exploited to address environmental problems of high significance, including the decontamination of waste waters, the disinfection of fish-farming tanks and the control of populations of noxious insects. Such diversified applications take advantage of the availability of a truly large number of porphyrin derivatives with chemical structures which can be tailored to comply with the physical and chemical properties, as well as the biological features of several milieus. In addition, the property typical of porphyrins to absorb essentially all the wavelengths in the sun emission spectrum allows the promotion of processes largely based on natural resources with significant energy saving and low impact on the ecosystems.

Mechanisms involved in the photosensitized inactivation of Acanthamoeba palestinensis trophozoites.

AIMS: To advance our understanding of the mechanisms involved in the RLP068 phthalocyanine-photosensitized inactivation of Acanthamoeba palestinensis trophozoites through a precise identification of the targets of the photoprocess in both the cytosolic and mitochondrial compartments. METHODS AND RESULTS: We followed the activities of selected marker enzymes as well as we performed fluorescence and transmission electron microscopy investigations of the alterations induced by the photoprocess in the fine structure of subcellular compartments. RLP068 is preferentially located in the contractile vacuole: the fluorescence in that site is particularly evident in the unirradiated cells and becomes more diffused after irradiation. Electron microscopic analysis of photosensitized A. palestinensis cells clearly shows that the swelling of trophozoites and the appearance of vacuoles spread throughout the cytoplasm after phototreatment. The activity of a typical cytoplasmic enzyme, such as lactate dehydrogenase, underwent a 35% decrease as a consequence of the photoprocess, reflecting the photodamage induced by migrating phthalocyanine molecules in their micro-environment. CONCLUSIONS: The presence of multiple targets for the phthalocyanine-photosensitized process is of utmost importance because this pattern of cell damage makes it unlikely that photoresistant A. palestinensis strains are gradually selected or mutagenic phenomena are developed as a consequence of the photoinduced damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Photosensitization via phthalocyanines appears to represent an efficient and safe approach for achieving a close control of the population of a potentially pathogenic protozoan such as A. palestinensis, opening new perspectives for the disinfection of microbiologically polluted waters.

Functional mapping of Brucella abortus cyclic beta-1,2-glucan synthase: identification of the protein domain required for cyclization.

Cyclic beta-1,2-glucans (CbetaG) are periplasmic homopolysaccharides that have been shown to play an important role in several symbiotic and pathogenic relationships. Cyclic beta-1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of CbetaG, is an integral membrane polyfunctional protein that catalyzes the four enzymatic activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of CbetaG. Recently, we have identified the glycosyltransferase and the beta-1,2-glucooligosaccharide phosphorylase domains of Brucella abortus Cgs. In this study, we performed large-scale linker-scanning mutagenesis to gain further insight into the functional domains of Cgs. This analysis allowed us to construct a functional map of the enzyme and led to the identification of the minimal region required for the catalysis of initiation and elongation reactions. In addition, we identified the Cgs region (residues 991 to 1544) as being the protein domain required for cyclization and demonstrated that upon cyclization and releasing of the CbetaG, one or more glucose residues remain attached to the protein intermediate that serves as a primer for the next round of CbetaG synthesis. Finally, our results indicate that the overall control of the degree of polymerization of CbetaG is the result of a balance between elongation, phosphorolysis, and cyclization reactions.

A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides.

Cyclic beta-1,2-glucans (CbetaG) are osmolyte homopolysaccharides with a cyclic beta-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the CbetaG synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked beta-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a beta-1,2-glucan phosphorylase present at the CbetaG synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the beta-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this "length-controlling" phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides.

Malformations in offspring of women with epilepsy: a prospective study.

PURPOSE: The incidence of malformations among infants of mothers with epilepsy treated with antiepileptic drugs (AEDs) during pregnancy is higher than that found in the general population. The aim of this study was to contribute to providing a definition of the rate of congenital anomalies in the offspring of mothers with epilepsy and to detect possible risk factors. METHODS: Since 1977, 517 pregnancies were followed up at the San Paolo Hospital in Milan by a team of epileptologists and obstetricians. The patients received monthly obstetric and neurologic examinations, and the blood levels of AEDs were tested monthly. During pregnancy the patients underwent ultrasound investigations to evaluate fetal morphology and development. At the time of delivery, the infants were submitted to a standardized examination by a pediatrician, and a more detailed clinical examination was performed on day 5. Malformations were classified as (a) genetic and chromosomic, (b) severe and mild malformations, and (c) deformities. RESULTS: The overall rate of malformations was 9.7%: of these, 5.3% were structurally severe, 2.2% were mild, 0.4% were chromosomic-genetic, and 1.8% were deformities. No malformation was detected in the 25 untreated patients. CONCLUSIONS: The risks of teratogenicity have been regarded as multifactorial, involving such factors as genetic predisposition, although most prospective studies show that AED-related factors are the primary risk factors for an increased incidence of congenital malformations.

Intrauterine growth in the offspring of epileptic women: a prospective multicenter study.

The aim of the present study was to evaluate the risk of intrauterine growth delay in the offspring of epileptic mothers and to quantify the risks of intrauterine exposure to antiepileptic drugs (AEDs). Data concerning 870 newborns, prospectively collected in Canada, Japan and Italy, using the same study design, were pooled and analyzed. The overall proportion of newborns whose body weight (7.8%) or head circumference (11.1%) at birth were below the 10th percentile was not increased. However, logistic regression analysis showed that the risk of small head circumference was significantly higher in Italian than in Japanese (RR 4.2; 95% CI: 2.2-8.0) or Canadian children (RR 2.6; 95% CI: 1.1-6.5), and in children exposed to polytherapy (RR 2.7; 95% CI: 1.2-6.3), phenobarbital (PB) (RR 3.6; 95% CI: 1.4-9.4) and primidone (PRM) (RR 4.5; 95% CI: 1.5-13.8). Country was also the only factor affecting low body weight, with Italian children having a higher risk than Japanese (RR 5.2; 95% CI: 2.6-10.4) or Canadian (RR 8.8; 95% CI: 2.0-38.1) children. Due to the small categories, the influence of AED doses and plasma concentrations was studied for each individual AED, without adjustment for the other potential confounding factors. A clear dose-dependent effect was found for PB and PRM in terms of both small head circumference and low body weight, and a concentration-dependent effect for PB in terms of small head circumferences. The size of the difference between the Italian and the other two populations, which is only partially explained by differences in therapeutic regimens, suggests that genetic, environmental and ethnic factors also need to be taken into account when considering possible explanations.

Purification and characterization of a humoral opsonin, with specificity for D-galactose, in the colonial ascidian Botryllus schlosseri.

A humoral agglutinin from the hemolysate of the colonial ascidian Botryllus schlosseri was purified by affinity chromatography. This agglutinin does not require metal cations for its activity and is specific for derivatives of D-galactose. On SDS-PAGE analysis, it was resolved in two bands, of 17 and 19 kDa in reducing conditions and 15 and 16 kDa in non-reducing conditions. This behavior is due to the establishment of disulfide bridge between the thiols of cysteine, well represented in the molecule as revealed by amino acid analysis. The latter also indicated high percentages of hydrophilic residues, probably involved in sugar recognition. The lectin is an opsonin, as it increases both the phagocytic index and the number of phagocytized yeast cells. The hypothesis that this Botryllus agglutinin belongs to the galectin family of lectins is discussed.

Felbamate in refractory partial epilepsy.

This open-label study was performed to evaluate efficacy and safety of Felbamate (FBM) add-on therapy in drug-refractory partial epilepsy. We evaluated 36 patients (12 males) aged 11-68 years (mean 29.8) in which FBM was titrated gradually from 300 mg/day to a mean total maintenance daily dose of 1936 mg. Patients were monitored according to clinical practice and performed regularly laboratory tests. Mean follow-up of FBM therapy was 10 months (range 2-27). In this study, 5% of patients resulted to be seizure-free, 11% showed a seizure reduction more than 75%, 23% decreased their seizure frequency between 50% and 75% (P = 0.0001). The adverse events which were reported more frequently were: nausea, vomiting, anorexia and weight loss. Even if the patients sample is small FBM proves its efficacy in partial epilepsy, showing a relatively well tolerated profile.

Chromosome 20 ring: a chromosomal disorder associated with a particular electroclinical pattern.

PURPOSE: The chromosome 20 ring [r(20)] is a rare chromosomal disorder without clear phenotypical markers. We describe the electroclinical pattern in a group of patients with r(20). METHODS: We observed 3 patients (a boy, patient 1; his mother, patient 2; and an unrelated man, patient 3), performing prolonged video-EEG and cytogenetic studies and fluorescent in situ hybridization (FISH) with chromosome-specific telomeric probes. RESULTS: All 3 patients had a very similar abnormal electroclinical pattern characterized by long bursts or trains of rhythmic theta waves, which were sharply contoured or had a notched appearance (with no detectable clinical correlate), and generalized spike waves (SW) associated with seizures of probable frontotemporal origin (SFT). In all 3 patients, the cytogenetic analysis of T lymphocytes showed mosaicism with a normal cell line and a second cell line with a chromosome 20, although the latter was little represented in patients 2 and 3. A few cells with a single chromosome 20 were also found. The same cytogenetic findings were confirmed in the lymphoblastoid cell line of patient 1 and in the fibroblasts of patient 3. FISH with chromosome-specific telomeric probes and TTAGGG sequences demonstrated the integrity of the ring chromosomes. CONCLUSIONS: The clinical picture of these patients appears to be related to the instability of the r(20)-generating cells monosomic for chromosome 20 and is thus haploinsufficient for a gene. In these patients, the electroclinical pattern of theta waves (probably unrelated to epilepsy) and the SW and SFT, even with mild mental retardation (MR) or no MR and without dysmorphic features, suggest that the r(20) syndrome may be present.

Worsening in seizure frequency and severity in relation to folic acid administration.

We report a case of increase in seizure frequency and severity in a 26-year-old woman receiving folic acid at a dosage of 0.8 mg/day. She had symptomatic partial epilepsy with simple and complex seizures treated with carbamazepine. She was planning pregnancy and we prescribed folic acid for prevention of neural-tube defects. In the next few days she had a generalized tonic-clonic seizure for the first time and a significant increase in seizure frequency. Because of the temporal relation between the seizure worsening and the administration of folic acid, we hypothesize a role of folic acid in provoking seizures, as has been reported in the literature.Copyright Lippincott-Raven Publishers

Cadmium-thionein in Tetrahymena thermophila and Tetrahymena pyriformis.

The treatment of Tetrahymena thermophila with cadmium causes a reduction in growth rate according to dose; almost all the metal is accumulated in the cytosol where the Zn content is also increased threefold. Bio-Gel and Water 160 (HPLC) column chromatography show that Cd and Zn are bound to a protein with an ultraviolet (UV) spectrum that appears to be similar to that of Cd-metallothioneins isolated by higher organisms, but its molecular weight is greater: about 28 000 D, comparable to that of metallothionein isolated from Tetrahymena pyriformis. Further purification of these proteins by ion exchange chromatography revealed the presence of two peaks, considered as two isoforms of the metallothioneins present in both T. thermophila and T. pyriformis (MT 1 and MT 2). Their amino acid analyses confirmed that they are different isometallothioneins, MT 1 and MT 2, with about 30% cysteine, and aspartic acid, glycine and lysine as major amino acids. From our analyses we may conclude that Tetrahymena pyriformis MTs are similar to those present in invertebrates and vertebrates, while Tetrahymena thermophila MTs are peculiar in that they have cyclic amino acid histidine in both MT 1 and MT 2; furthermore, aromatic amino acid phenylalanine is also present in MT 2.

Biochemical and ultrastructural data on Tetrahymena pyriformis treated with copper and cadmium.

The addition of copper (10 micrograms ml-1) or cadmium (5 micrograms ml-1) to the medium is well tolerated by Tetrahymena pyriformis GL. Both metals are accumulated by cells, cadmium to a greater extent than copper. The growth rate is not affected and from the micrographs it is evident that the ultrastructure is not altered by the treatments. After 3 days of culture the macronucleus contains dense masses of chromatin and numerous nucleolar fusion bodies. Granules, cytolysomes and many food vacuoles are present in both control and treated cells. Cadmium induces the formation of a chelating protein; the amino acid analyses and the ultraviolet spectrum indicate that it is similar to the metallothionein isolated by higher organisms. The molecular weight of native protein is about 27,000. After treatment by sulphitolysis or oxidation we obtained a peak of molecular weight at about 6,000. The treatment with copper does not appear to induce metallothioneins or other chelatins. The high tolerance of Tetrahymena towards cadmium is believed to be due to the formation of a Cd-Zn metallothionein. The different chelating proteins induced by copper and cadmium in other groups of Protozoa and the different detoxification mechanisms present in these organisms are discussed.

Chelatins in Euglena gracilis and Ochromonas danica.

Amino acid analysis was performed on low molecular weight copper binding proteins purified from two species of Protozoa after exposure to a high level of this metal. The compound from Ochromonas is similar to Cu-chelatins. The two peptides from Euglena have a different molecular weight and a very dissimilar amino acid composition. Peptide No. 1 has a peculiar composition with a high content of aspartic acid and arginine. Some speculations are made about its detoxification role in comparison with other compounds found in blue-green algae.

Globin evolution in some species of the genus Bufo.

The globins of 10 species of toads of the genus Bufo have been analysed. From the values of the reciprocal rates between their amino acid residues a 'dissimilarity matrix' was made. In this have been included the values related to Discoglossus pictus and Xenopus laevis. The numerical data, derived from a computer program, gave a dendrogram representing the evolution of the analysed globins. This appears correlated to the geographical distribution and to the metabolic adaptation of the corresponding species of Bufo more than their chronological appearance.