Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein

PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.     

Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone

The heavy-chain and kappa light-chain variable region genes of an antizearalenone hybridoma cell line (2G3-6E3-2E2) were isolated by PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a single-chain Fv (scFv) DNA fragment. The scFv DNA fragment was cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 in Escherichia coli TG1. In the presence of helper phage M13K07, the scFv fusion protein was displayed on the surfaces of recombinant phages. High-affinity scFv phages were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjugate. The selected recombinant phages were used to infect E. coli HB2151 for the production of soluble scFv antibodies. One selected clone (pQY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5) with a high zearalenone-binding affinity (concentration required for 50% inhibition of binding, 14 ng/ml), similar to that of parent monoclonal antibody in a competitive indirect enzyme-linked immunosorbent assay. However, scFv QY1.5 exhibited higher cross-reactivity with zearalenone analogs and had greater sensitivity to methanol destabilization than the parent monoclonal antibody did. Nucleotide sequence analyses revealed that the light-chain portion of scFv QY1.5 had a nucleotide sequence identity of 97% to a mouse germ line gene VK23.32 in mouse kappa light-chain variable region subgroup V, whereas the heavy-chain nucleotide sequence was classified as mouse heavy-chain subgroup III (D) but without any closely related members having highly homologous complementarity-determining region sequences. The potential of soluble scFv QY1.5 for routine screening of zearalenone and its analogs was demonstrated with zearalenone-spiked corn extracts.     

A Numerical Treatment of the rf SQUID: I. General Properties and Noise Energy

We investigate the characteristics and noise performance of rf Superconducting Quantum Interference Devices (SQUIDs) by solving the corresponding Langevin equations numerically and optimizing the model parameters with respect to noise energy. After introducing the basic concepts of the numerical simulations, we give a detailed discussion of the performance of the SQUID as a function of all relevant parameters. The best performance is obtained in the crossover region between the dispersive and dissipative regimes, characterized by an inductance parameter β′ _L ≡2π LI ₀/Φ ₀≈1; L is the loop inductance, I ₀ the critical current of the Josephson junction, and Φ ₀ the flux quantum. In this regime, the lowest (intrinsic) values of noise energy are a factor of about 2 above previous estimates based on analytical approaches. However, several other analytical predictions, such as the inverse proportionality of the noise energy on the tank circuit quality factor and the square of the coupling coefficient between the tank circuit and the SQUID loop, could not be well reproduced. The optimized intrinsic noise energy of the rf SQUID is superior to that of the dc SQUID at all temperatures. Although for technologically achievable parameters this advantage shrinks, particularly at low thermal fluctuation levels, we give examples for realistic parameters that lead to a noise energy comparable to that of the dc SQUID even in this regime.      

STABILITY OF RESINS IN PELLETISED HOPS

The α-acids content and Lead Conductance Value of hop pellets, manufactured from seedless Pride of Ringwood hops grown in Australia and stored at 5°C, 20°C or 30°C, were monitored over a twelve month storage period. No decrease in α-acids content occurred during storage at 5°C, whereas at 20°C the decrease (ca. 7%) was approximately one-third that experienced for baled hops of the same variety. At both 5°C and 20°C the Lead Conductance Value decreased at approximately one-third the rate of that in baled hops. During twelve months storage at 30°C the α-acids content of the hop pellets decreased by approximately 40% and the Lead Conductance Value by 30%. The hop pellets stored at 20°C and 30°C developed rancid odours during the twelve months storage period.     

Filtration in coal liquefaction: preparation of filter aid from filter cake

In this study the preparation of filter aid from filter cake is considered. Two methods of preparation, carbonization at 550–600 °C and combustion at 800–1000 °C, are used and the optimum conditions determined. The performance of the products is assessed as both a precoat and body feed by comparison with a commercial grade of diatomite. It is shown that efficient filter aids, with performances comparable to that of diatomite, may be produced from filter cake.     

Estimation of the concentration of donatable hydrogen in a coal solvent by n.m.r.

¹H and ¹³C nuclear magnetic resonance (n.m.r.) spectroscopies have been used to study the changes in composition of a hydrogenated anthracene oil solvent that occur during extraction of a UK bituminous coal. It has been shown that the principal hydrogen donor groups in the solvent are hydroaromatic and naphthenic rings in hydroaromatic species. Good agreement was achieved between the concentration of donatable hydrogen estimated from n.m.r., and the amount of hydrogen transferred from the solvent during exhaustive extraction.     

Filtration in coal liquefaction. Influence of digestion conditions in the filtration of non-hydrogenated coal digests

A series of experiments has been carried out to study the way in which preparation conditions of coal digests influence filtration rate. It was shown that a relation exists between cake resistivity and digestion temperature and time, and therefore good control over digestion conditions is important for rapid filtration. Particular attention must be paid to the design of the reactor to ensure that all the material is given the same heat-treatment. The optimum residence time occurs when repolymerization of the dissolved coal commences.     

Low temperature age hardening in U-13 at.% Nb: An assessment of chemical redistribution mechanisms

Low temperature aging (<350 °C) of U-13 at.% Nb martensite results in increased strength levels accompanied by significant ductility loss. To determine the decomposition mechanism(s) responsible for these mechanical property changes, atom probe tomography was used to examine the niobium and impurity distributions after aging at 200 or 300 °C for times ranging from 2 h to 70 days. No patterns of niobium or impurity atoms were observed that would indicate segregation to the martensitic twin interfaces, making this hardening mechanism unlikely. Phase separation into roughly equiaxed regions of high and low niobium concentration was clearly observed after aging at 300 °C for 70 days. However, only subtle niobium concentration changes were observed after aging at 200 °C relative to the as-quenched condition, indicating that conventional phase separation is an unlikely explanation for the dramatic mechanical property changes at 200 °C. Therefore, consideration of aging mechanisms other than segregation and phase separation may be warranted.