The purification and characterization of a fatty acid binding protein specific to pig (Sus domesticus) adipose tissue.

Western-blot analysis using antiserum to 3T3-L1-cell fatty acid binding protein (FABP) revealed that pig adipose tissue contains a 15 kDa protein immunologically similar to the murine protein. This 15 kDa protein was purified from pig adipose tissue by sequential application of Sephadex G-50 gel filtration, cation exchange and covalent chromatography on Thiol-Sepharose-4B. The purity of the pig protein was established by two-dimensional polyacrylamide-gel electrophoresis. Isoelectric focusing indicated that the pig adipose FABP (a-FABP) exists with two charge isoforms (pI 5.1 and 5.2), both of which persist after delipidation. The N-terminus of the purified pig a-FABP was blocked; however, cleavage with CNBr allowed recovery of a 12-amino-acid peptide which was identical with the murine a-FABP sequence (residues 36-48) at 10 of 12 positions. The pig a-FABP bound 12-(9-anthroyloxy)oleic acid saturably and stoichiometrically, with an apparent dissociation constant of 1.0 microM. Northern-blot analysis using the cDNA for the murine 3T3-L1 FABP revealed that the pig a-FABP was expressed exclusively in adipose tissue.     

The heterogeneous nature of microbial products as shown by solid-state13C CP/MAS NMR spectroscopy

Homoionic Na-, Ca-, and Al-clays were prepared from the <2 m fractions of Georgia kaolinite and Wyoming bentonite and mixed with sand to give artificial soils with 5, and 25% clay. The artificial soils were inoculated with microbes from a natural soil before incubation. Unlabelled and uniformly¹³C-labelled (99.9% atom) glucose were incorporated into the artificial soils to study the effects of clay types, exchangeable cations and clay contents on the mineralization of glucose-carbon and glucose-derived organic materials. Chemical transformation of glucose-carbon upon incorporation into microbial products and metabolites, was followed using solid-state¹³C CP/MAS NMR spectroscopy.There was a significant influence of exchangeable cations on the mineralization of glucose-carbon over a period of 33 days. At 25% clay content, mineralization of glucose-carbon was highest in Ca-soils and lowest in Al-soils. The influence of exchangeable cations on mineralization of glucose-carbon was more pronounced in soils with bentonite clay than those with kaolinite clay. Statistical analysis of data showed no overall effect of clay type on mineralization of glucose-carbon. However, the interactions of clay type with clay content and clay type with clay content and exchangeable cations were highly significant. At 25% clay content, the mineralization of glucose-carbon was significantly lower in Na- and Al-soils with Wyoming bentonite compared with Na- and Al-soils with Georgia kaolinite. For Ca-soils this difference was not significant. Due to the increased osmotic tension induced by the added glucose, mineralization of glucose-carbon was slower in soils with 5% clay than soils with 25% clay.Despite the differences in the chemical and physical characteristics of soils with Ca-, Na- and Al-clays, the chemical composition of organic materials synthesised in these soils were similar in nature. Assuming CP/MAS is quantitative, incorporation of uniformly¹³C-labelled glucose (99.9% atom) in these soils resulted in distribution of carbon in alkyl (24–25%), O-alkyl (56–63%), carbonyl (11–15%) and small amounts of aromatic and olefinic carbon (2–4%). However, as decomposition proceeded, the chemistry of synthesised material showed some changes with time. In the Ca- and Na-soils, the proportions of alkyl and carbonyl carbon decreased and that of O-alkyl carbon increased with time of incubation. However, the opposite trend was found for the Al-soil.Proton-spin relaxation editing (PSRE) subspectra clearly showed heterogeneity within the microbial products. Subspectra of the slowly-relaxing (long T₁(H)) domains were dominated by alkyl carbon in long- and short-chain structures. The signals due to N-alkyl (55 ppm) and carbonyl carbon were also strong in these subspectra. These subspectra were very similar to those obtained for microbial and fungal materials and were probably microbial tissues attached to clay surfaces by polysaccharide extracellular mucilage. Subspectra of fast-relaxing (short T₁(H)) domains comprised mostly O-alkyl and carbonyl carbon and were probably microbial metabolites released as neutral and acidic sugars into the extracellular environment, and strongly sorbed by clay surfaces.     

The Bcl-2 family of proteins: regulators of cell death and survival

The Bcl-2 protein inhibits apoptosis induced by a variety of signals, in a range of cell types and in diverse organisms, and it is implicated in both normal development and oncogenesis. Despite this central role, the mechanism of action of Bcl-2 is not yet clear. Recent studies have uncovered a number of Bcl-2-related gene products that regulate apoptosis either negatively or positively, and Bcl-2 forms heterodimers with at least one of these proteins, Bax. This article discusses the role of the Bcl-2 family of proteins in the light of these findings.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

Malignant cerebral glioma--I: Survival,disability, and morbidity after radiotherapy.

OBJECTIVE: To describe survival, disability, and morbidity after radiotherapy for malignant glioma. DESIGN: Two year prospective study with home interviews with patients and relatives. SETTING: Seven neurosurgical and radiotherapy centres in London. SUBJECTS: 105 patients aged 21 to 75: 59 had biopsy; 46 had partial macroscopic resection; 92 received radiotherapy; and 13 received steroids alone. MAIN OUTCOME MEASURES: Survival, time free from disability, and changes in disability after treatment. RESULTS: Six and 12 month survival for radiotherapy patients was 70% and 39%, respectively. Age, World Health Organisation clinical performance status, extent of surgery, and history of seizures before diagnosis each influenced survival. The Medical Research Council prognostic index was also significantly related to survival. Multivariate analysis showed that initial clinical performance status was the most important component of the index. Most (80%; 49/61) patients with a clinical performance status of 0, 1, or 2 lived at least six months before becoming permanently disabled. Most patients who had initially had a good clinical performance status (0-2) and who were alive six months after radiotherapy (68%; 36/52), however, had experienced either clinical deterioration or severe tiredness after treatment. In 17% (9/52) of these some permanent loss of function remained. These adverse effects were associated with increasing radiotherapy dose. Severely disabled patients (clinical performance status 3 or 4) gained little benefit. CONCLUSION: Severely disabled patients gain little physical benefit from radiotherapy, whereas those not so disabled may experience considerable adverse effects.     

Alignment of clonedamiE gene ofPseudomonas aeruginosa with theN-terminal sequence of amidase

A restriction enzyme map was constructed for 5.1-kb fragment of Pseudomonas aeruginosa DNA inserted into plasmid pBR322. Restriction enzyme sites were matched to the N-terminal amino acid sequence of amidase to obtain alignment of the amiE gene within the cloned fragment.