Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.     

Alizarin-beta-D-galactoside: a new substrate for the detection of bacterial beta-galactosidase

We describe the synthesis of a new substrate for the detection of bacterial beta-galactosidase. This substrate, alizarin-beta-D-galactoside, is readily hydrolysed to release alizarin which complexes with various metal ions to form brightly coloured chelates. A total of 367 strains of Gram-negative bacteria were examined for their ability to hydrolyse three chromogenic substrates: alizarin-beta-D-galactoside (Aliz-gal), cyclohexenoesculetin-beta-D-galactoside (CHE-gal) and 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). A total of 182 strains (49.6%) were found to hydrolyse at least one of the three substrates. All of these 182 strains (100%) hydrolysed Aliz-gal whereas only 170 (93.4%) and 173 (95.1%) hydrolysed CHE-gal and X-gal, respectively. We conclude that alizarin-beta-D-galactoside is a highly sensitive substrate for the demonstration of beta-galactosidase.     

Discovery and structure–activity relationships of 6-(benzoylamino)benzoxaboroles as orally active anti-inflammatory agents

Structure–activity relationships of 6-(benzoylamino)benzoxaborole analogs were investigated for the inhibition of TNF-α, IL-1β, and IL-6 from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compound 1q showed potent activity against all three cytokines with IC₅₀ values between 0.19 and 0.50 μM, inhibited LPS-induced TNF-α and IL-6 elevation in mice and improved collagen-induced arthritis in mice. Compound 1q (AN4161) is considered to be a promising lead for novel anti-inflammatory agent with an excellent pharmacokinetic profile.     

TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.     

Isolation and identification of products from alkylation of nucleic acids: ethyl- and isopropyl-purines.

Ethylation and isopropylation of guanine in alkaline solution, or of adenine in formic acid, by alkyl methanesulphonates gave the following products: 1-, N2-, 3-, O6-, 7- and 9-alkylguanines; 1-, 3-, 7- and 9-alkyladenines. The products were identified from their characteristic u.v-absorption spectra, by comparison with either known ethyladenines or with the corresponding known methyladenines, and were also characterized by mass spectrometry. Their chromatographic properties on paper, t.l.c. and various columns were determined. DNA was alkylated in neutral solution with 14C-labelled alkyl methanesulphonates and the ratios of the alkylpurines formed were obtained, and compared for alkylation by methyl, ethyl and isopropyl methanesulphonates and by N-methyl-N-nitrosourea. The extents of alkylation at O-6 of guanine relative to those at N-7 of guanine varied with the reactivity of the methylating agents according to the predictions of Swain & Scott (1953) relating nucleophilicity of the groups alkylated with the substrate constants of the alkylating agents. The relative extents of alkylation at N-3 of adenine did not follow this correlation.     

Purification and characterization of fatty acid beta-oxidation enzymes from Caulobacter crescentus.

Acetoacetyl coenzyme A (acetoacetyl-CoA) thiolase, an enzyme required for short-chain fatty acid degradation, has been purified to near homogeneity from Caulobacter crescentus. The relative heat stability of this enzyme allowed it to be separated from beta-ketoacyl-CoA thiolase. The purification scheme minus the heating step also permitted the copurification of crotonase and 3-hydroxyacyl-CoA dehydrogenase. These activities are in a multienzyme complex in Escherichia coli, but a similar complex was not observed in C. crescentus. Instead, separate proteins differing in enzymatic activity were detected, analogous to the beta-oxidation enzymes that have been isolated from Clostridium acetobutylicum and from mitochondria of higher eucaryotes. In these cells, as appears to be the case with C. crescentus, the individual enzymes form multimers of identical subunits.     

Effect of deamidation on folding of ribonuclease A

The folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65-72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn --> isoAsp where the local structure of the loop 65-72 has been modified keeping intact the C65-C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild-type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65-72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.     

ACAT-1, Cav-1 and PrP expression in scrapie susceptible and resistant sheep

Scrapie is a prion disease for which no means of ante-mortem diagnosis is available. We recently found a relationship between cell susceptibility to scrapie and altered cholesterol homeostasis. In brains and in skin fibroblasts and peripheral blood mononuclear cells from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype, the levels of cholesterol esters were consistently higher than in tissues and cultures derived from animals with a scrapie-resistant genotype. Here we show that intracellular accumulation of cholesterol esters (CE) in fibroblasts derived from scrapie-susceptible sheep was accompanied by parallel alterations in the expression level of acyl-coenzymeA: cholesterol-acyltransferase (ACAT1) and caveolin-1 (Cav-1) that are involved in the pathways leading to intracellular cholesterol esterification and trafficking. Comparative analysis of cellular prion protein (PrPc) mRNA, showed an higher expression level in cells from animals carrying a susceptible genotype, with or without Scrapie. These data suggest that CE accumulation in peripheral cells, together with the altered expression of some proteins implicated in intracellular cholesterol homeostasis, might serve to identify a distinctive lipid metabolic profile associated with increased susceptibility to develop prion disease following infection.     

Expression of ATP7B in normal human liver

ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.     

INFLORESCENCE DEVELOPMENT IN BRASSICA CAMPESTRIS L.

Vegetative seedlings of the Ceres strain Brassica campestris L., a quantitative, long-day plant, were induced to flower by exposure to a 16-hr, long-day cycle. Cytohistological and cytohistochemical changes associated with inflorescence development were examined. Developing shoot apices were classified in vegetative, transitional, and reproductive stages. The vegetative apex possessed a biseriate tunica, central zone, peripheral zone and pith-rib meristem. The transitional stage at 48 hr was marked by an increase in size and by a stratification of the upper cell layers of the shoot apex with a concurrent decrease of apical cytohistochemical zonation. The reproductive stage was initiated at 58 hr by periclinal cell divisions in the 3rd and 4th cell layers of the flank region. Cytohistochemical zonation in the vegetative apical meristem was restored in the floral apex. An “intermediate developmental” phase was not observed between the vegetative and reproductive stage.