BPTI and N-terminal extended analogues generated by factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli

A recombinant gene for BPTI (bovine pancreatic trypsin inhibitor) is expressed in Escherichia coli using a MBP (maltose-binding protein) fusion vector. BPTI is fused through an FXa (blood coagulation factor Xa protease) target sequence (Ile-Glu-Gly-Arg) to the C-terminus of MBP. The MBP moiety of the hybrid protein enables purification in one step utilizing MBP's affinity to cross-linked amylose, and the FXa target sequence allows specific cleavage of the hybrid protein. Effective FXa cleavage is achieved by spacing the FXa target sequence and Arg-1 of the BPTI sequence with four residues (Met-Glu-Ala-Glu). The resulting N-terminal extended BPTI is readily converted to the wild-type sequence by trimming with cathepsin C exopeptidase, for the activity of which the spacing tetrapeptide is optimized. FXa cleavage is prohibited when the target sequence is placed next to Arg-1. In this construction, off-target cleavage at a somewhat homologous sequence (Val-Pro-Gly-Arg) results in five- or six-residue extended BPTI, indicating new details of the FXa specificity. The yield of highly purified recombinant BPTI is 3-6 mg/liter of culture, making the MBP-BPTI expression system convenient for the production of sufficient amounts of protein for NMR studies. 1H NMR is used to analyze the N-extended BPTI analogues.     

celA from Bacillus lautus PL236 encodes a novel cellulose-binding endo-beta-1,4-glucanase.

celA from the cellulolytic bacterium Bacillus lautus PL236 encodes EG-A, an endo-beta-1,4-glucanase. An open reading frame of 2,100 bp preceded by a ribosome-binding site encodes a protein with a molecular mass of 76,863 Da with a typical signal sequence. The NH2-terminal active domain of EG-A is not homologous to any reported cellulase or xylanase and may represent a new family of such enzymes. A 150-amino-acid COOH-terminal peptide is homologous to noncatalytic domains in several other cellulases (A. Meinke, N.R. Gilkes, D.G. Kilburn, R.C. Miller, Jr., and R.A.J. Warren, J. Bacteriol. 173:7126-7135, 1991). Upstream of celA, a partial open reading frame encodes a 145-amino-acid peptide which also belongs to the family mentioned. Zymogram analysis of extracts from Escherichia coli and supernatants of Bacillus subtilis and B. megaterium, including protease-deficient mutants thereof, which express celA, revealed two active proteins, EG-A-L and EG-A-S, with Mrs of 74,000 and 57,000, respectively. The proportion of EG-A-L to EG-A-S depends on the extracellular proteolytic activity of the host organism, indicating that EG-A-S arises from posttranslational proteolytic modification of EG-A-L. Since EG-A-S has an NH2 terminus corresponding to the predicted NH2-terminal sequence of EG-A, processing appears to take place between the catalytic and noncatalytic domains described. EG-A-L and EG-A-S were purified to homogeneity and shown to have almost identical characteristics with respect to activity against soluble substrates and pH and temperature dependency. EG-A-L binds strongly to cellulose, in contrast to EG-A-S, and has higher activity against insoluble substrates than the latter. We conclude that the COOH-terminal 17,000-Mr peptide of EG-A-L constitutes a cellulose-binding domain.     

Metal thiolate coordination in the E7 proteins of human papilloma virus 16 and cottontail rabbit papilloma virus as expressed in Escherichia coli.

The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma virus (CRPV) have been purified from an expression system in Escherichia coli. The proteins as purified from E. coli contain one tightly bound Zn(II) ion per molecule. The metal site shows facile exchange with either Cd(II) or Cu(I). The HPV 16 E7 maximally bound one Cd(II) or two Cu(I) ions, while the CRPV E7 bound two Cd(II) or three Cu(I) ions. The Cd(II) and Cu(I) E7 molecules exhibited optical transitions in the ultraviolet suggestive of metal:thiolate coordination. E7 proteins from HPV 16 and CRPV contain 7 and 8 cysteines/molecule, respectively. Reaction of the E7 proteins with the sulfhydryl reagent, dithiodipyridine, revealed that all the cysteinyl sulfurs are present in the reduced thiol state. Cu(I)-E7 molecules are luminescent with maximal emission at 570 nm. The observed emission at room temperature is indicative of metal coordination within a compact protein environment shielded from solvent interactions. The emission maxima occurs at the same wavelength (570 nm) as Cu(I)-cysteinyl sulfur clusters in Cu(I)-metallothioneins. The single Zn(II) atom in each protein can be removed from E7 in the presence of EDTA. The resulting apoE7 molecules remain soluble and can be partially reconstituted with Cd(II) to regain the ultraviolet charge transfer transitions.     

Pharmacokinetics of stealth versus conventional liposomes: effect of dose

Liposomes which substantially avoid uptake into the mononuclear phagocyte system (MPS), termed Stealth liposomes, have recently been formulated (Allen, T.M. and Chonn, A., (1987) FEBS Lett. 223, 42-46). The pharmacokinetics of stealth liposomes as a function of liposome dose and a comparison to conventional liposome pharmacokinetics, was the subject of the present study. We have examined the tissue distribution of two different formulations of stealth liposomes, i.e., sphingomyelin:egg phosphatidylcholine:cholesterol:monosialoganglioside GM1 (SM:PC:CHOL:GM1) 1:1:1:0.2 and SM:PC:CHOL:polyethylene glycol distearoylphosphatidylethanolamine (PEG(1990)-DSPE) 1:1:1:0.2, and compared them with the tissue distributions seen for a liposomal formulation which is avidly removed from circulation by the cells of the MP system (PC:CHOL, 2:1). Tissue distribution in mice was examined over a 100-fold concentration range (0.1 to 10 mumol phospholipid/mouse) and at several time points over a 48 h time period. Liposome size ranged from 92-123 nm in diameter for all compositions. Clearance from blood of PC:CHOL liposomes following intravenous administration showed a marked dose dependence (i.e., saturation-type or Michaelis-Menten kinetics), with MPS uptake decreasing and % of injected dose in blood increasing as dose increased, over the entire dosage range. Injection of stealth liposomes, on the other hand, resulted in % of injected doses of liposomes in MPS, blood and carcass which were dose-independent and log-linear (first order kinetics) over the entire dosage range. The doses of stealth liposomes containing PEG(1900)-DSPE required for MPS saturation was higher than 10 mumol phospholipid/mouse or 400 mumol/kg. The dosage-independence of the pharmacokinetics of stealth liposomes and their lack of MPS saturation within the therapeutic dose range are two more assets, in addition to the prolonged circulation half-lives, leading towards their eventual use as drug delivery systems in the clinic.     

The amino acid sequence of a major protein component in the light harvesting complex of the green photosynthetic bacterium Chlorobium limicola f. thiosulfatophilum

A 7.5-kDa protein has been isolated from chlorosomes of Chlorobium limicola f. thiosulfatophilum and the complete primary structure determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The 74-residue protein shows great homology to a similar protein of unknown function which has been isolated from Pelodictyon luteolum but otherwise no significant homology to other proteins can be found. The possible role of the protein in the structure and function of the chlorosome is discussed.     

A controlled study of eight months of physical training and reduction of blood pressure in children: the Odense schoolchild study.

OBJECTIVE--To examine the effect of physical training on physical fitness and blood pressure in children aged 9-11 years. DESIGN--Prospective randomised controlled intervention study of a sample of children drawn from a population survey of coronary risk factors in children. SETTING--Odense, Denmark. SUBJECTS--69 children with mean blood pressure greater than or equal to 95th centile (hypertensive group) and 68 with mean blood pressure less than 95th centile (normotensive group), randomly selected from a population of 1369 children. INTERVENTION--67 children were randomised to receive three extra lessons a week of an ordinary school physical education programme for eight months. MAIN OUTCOME MEASURES--Physical fitness assessed by calculation of maximum oxygen uptake and blood pressure recorded by one unblinded observer. RESULTS--After three months neither blood pressure nor physical fitness had changed significantly. After adjustment for values in weight, height, heart rate, and the variable in question before training physical fitness rose significantly at the end of eight months'' training, by 3.7 mlO2/kg/min (95% confidence interval 2.2 to 5.3) in the normotensive training subgroup and by 2.1 mlO2/kg/min (0.1 to 4.2) in the hypertensive training subgroup compared with that in the controls. Systolic and diastolic blood pressures in the training subgroups fell significantly by 6.5 mm Hg (3.2 to 9.9) and 4.1 mm Hg (1.7 to 6.6) respectively in the normotensive group and by 4.9 mm Hg (0.7 to 9.2) and 3.8 mm Hg (0.9 to 6.6) respectively in the hypertensive group. CONCLUSIONS--Physical training lowers blood pressure and improves physical fitness in children and might have implications for an important non-pharmacological approach to primary prevention of essential hypertension.     

Incidence of chromosome abnormalities in newborn children. Comparison between incidences in 1969–1974 and 1980–1982 in the same area

Summary As part of an ongoing study of the influence of environmental factors on pregnancy, childbirth, and fetuses, comparisons have been made between incidences in 1969–1974 and in 1980–1982 of chromosome aberrations in liverborn children in the same area of Denmark. The incidence of chromosome aberrations in the first period was 2.6 per 1000, compared with 4.1 per 1000 during the latter period. However, the difference was mainly due to an increase in inversions, and this in turn was due to a difference in chromosome staining methods between the two periods.It is concluded that the Danish study and similar studies in the United States, Canada, and Scotland indicate that early detection of chromosome aberrations by chromosome examination at birth is indicated in order to be able to inform and counsel parents of children with chromosome aberrations. Chromosome examination at birth is also of importance in the diagnosis of structural inheritable chromosome aberrations and consequent family investigation and genetic counseling.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species.

Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography Their structures are suggested to be (4R)- and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, and rat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4-hydroxyochratoxin A.     

Rats chronically injected with urine from Huntington's chorea patients do not striatal damage

Rats were injected subcutaneously for 147 consecutive days with large volumes of urine from control subjects and from patients with Huntington's chorea (HC) in an effort to test for presence of a possible neurotoxic substance in HC. No evidence of illness was observed in animals treated with HC urine, and their behavior did not differ from animals treated with control urine. After rats were sacrificed, striatum was examined for the biochemical and neuropathological changes seen in human striatum in HC. No deficiency of γ-aminobutyric acid content, nor reduction in activities of glutamic acid decarboxylase and choline acetyltransferase, was found in striatum of rats chronically treated with HC urine. Also, no significant differences were found between striatum of control and experimental rats by light or electron microscopy. These results neither support for exclude the possibility of a neurotoxic mechanism for the neuronal loss characteristic of HC.