Characterisation of pectin subunits released by an optimised combination of enzymes

Pectins from sugar beet, lime and apple were degraded by a rhamnogalacturonan hydrolase associated or not with pectin methylesterases and side chain degrading enzymes (galactanase and arabinanase). The composition of the enzymatic mixture was optimised by following the reaction by viscosimetric means. The reaction products were fractionated by ion exchange chromatography. Treatment with all the enzymes released four fractions: (1). 227-247 mg/g of initial pectins and corresponded to neutral sugars from the side chains; (2,3). represented together 184-220 mg/g of pectins and corresponded to rhamnogalacturonan; (4). 533-588 mg/g of pectins and corresponded to homogalacturonan. Lime pectins have the shortest rhamnogalacturonan regions. The molar masses of homogalacturonans were in the range of 16000-43400 g/mol according to the origin of pectins, corresponding to degrees of polymerisation of 85-250. The mode of action of the enzymes used is also discussed.     

Induction, isolation, and characterization of two laccases from the white rot basidiomycete Coriolopsis rigida

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH(2)). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH(2) extended the reactions catalyzed by these enzymes to the production of H(2)O(2), the oxidation of Mn(2+), the reduction of Fe(3+), and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.     

Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60

SPG13, an autosomal dominant form of pure hereditary spastic paraplegia, was recently mapped to chromosome 2q24-34 in a French family. Here we present genetic data indicating that SPG13 is associated with a mutation, in the gene encoding the human mitochondrial chaperonin Hsp60, that results in the V72I substitution. A complementation assay showed that wild-type HSP60 (also known as "HSPD1"), but not HSP60 (V72I), together with the co-chaperonin HSP10 (also known as "HSPE1"), can support growth of Escherichia coli cells in which the homologous chromosomal groESgroEL chaperonin genes have been deleted. Taken together, our data strongly indicate that the V72I variation is the first disease-causing mutation that has been identified in HSP60.     

Functional role of epsilon-tubulin in the assembly of the centriolar microtubule scaffold

Centrioles and basal bodies fascinate by their spectacular architecture, featuring an arrangement of nine microtubule triplets into an axial symmetry, whose biogenesis relies on yet elusive mechanisms. However, the recent discovery of new tubulins, such as delta-, epsilon-, or eta-tubulin, could constitute a breakthrough for deciphering the assembly steps of this unconventional microtubule scaffold. Here, we report the functional analysis in vivo of epsilon-tubulin, based on gene silencing in Paramecium, which demonstrates that this protein, which localizes at the basal bodies, is essential for the assembly and anchorage of the centriolar microtubules.     

Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer. Reluctant response to antimonial treatment

We investigated the bacterial flora present in skin lesions of patients with chiclero's ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chiclero's ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chiclero's ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chiclero's ulcer.     

Identification of anti-repressor elements that confer high and stable protein production in mammalian cells

The expression of transgenic proteins is often low and unstable over time, a problem that may be due to integration of the transgene in repressed chromatin. We developed a screening technology to identify genetic elements that efficiently counteract chromatin-associated repression. When these elements were used to flank a transgene, we observed a substantial increase in the number of mammalian cell colonies that expressed the transgenic protein. Expression of the shielded transgene was, in a copy number-dependent fashion, substantially higher than the expression of unprotected transgenes. Also, protein production remained stable over an extended time period. The DNA elements are small, not exceeding 2,100 base pairs (bp), and they are highly conserved between human and mouse, at both the functional and sequence levels. Our results demonstrate the existence of a class of genetic elements that can readily be applied to more efficient transgenic protein production in mammalian cells.     

Involvement of an SCF<Superscript>Slmb</Superscript> complex in timely elimination of E2F upon initiation of DNA replication in <Emphasis Type="Italic">Drosophila</Emphasis>

Background  

Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified.