Antidiabetic properties of zinc-alpha2-glycoprotein in ob/ob mice

Zinc-alpha(2)-glycoprotein (ZAG) is an adipokine associated with fat loss in cancer cachexia. The purpose of this study was to evaluate the ability of recombinant human ZAG to attenuate type 2 diabetes in the ob/ob mouse model. ZAG (50 microg daily, iv) induced a progressive loss of body weight (3.5 g in 5 d), without an effect on food or water intake but with a 0.4 C rise in body temperature, suggesting an increased energy expenditure. Despite an increased plasma glycerol, indicative of increased lipolysis, levels of glucose, triglycerides, and nonesterified fatty acids were decreased by 17, 25, and 62%, respectively, due to an increased use of both glucose and lipids by muscle and brown adipose tissue. The weight of the latter increased 2-fold, and there was increased expression of uncoupling proteins-1 and -3. Plasma insulin levels were reduced by 36%, whereas pancreatic insulin was increased 4-fold, and there was a 53% decrease in the total area under the glucose curve in the glucose tolerance test and reduced insulin requirement. There was an increase in skeletal muscle mass due to an increase in protein synthesis and a decrease in protein degradation. These results suggest that ZAG may potentially be effective in the treatment of type 2 diabetes.

Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing:identification of novel zinc finger proteins.

We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using 'MAX' randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40,000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants.

The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition

Bacteriophage T7 DNA primase recognizes 5'-GTC-3' in single-stranded DNA. The primase contains a single Cys4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys4 motif contacts cytosine of 5'-GTC-3' and may also contribute to thymine recognition. Residues His33 and Asp31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5'-GTC-3', by base pairing. Our deduced mechanism for recognition of ssDNA by Cys4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys2His2 zinc fingers.

A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7

Two colinear bacteriophage T7 gene 4 proteins provide helicase and primase functions in vivo. T7 primase differs from T7 helicase by an additional 63 residues at the amino terminus. This terminal domain contains a zinc-binding motif which mediates an interaction with the basic primase recognition sequence 3'-CTG-5'. We have generated a chimeric primase in which the 81 amino-terminal residues are derived from the primase of phage T3 and the 484 carboxyl-terminal residues are those of phage T7 helicase. The amino-terminal domain of T3 primase is 50% homologous with that of T7 primase. The resulting T3/T7 chimeric protein is a functional primase in vivo. While the primase activity of the purified protein is about one-third that of T7 primase, the recognition sites used and the oligoribonucleotides synthesized from these sites are identical. We conclude that the residues responsible for the interaction with the sequence 3'-CTG-5' are conserved between the chimeric and T7 proteins.

The development of a model system and high throughput assay for the study of interactions between zinc finger proteins and DNA

It has been recognised for some time that a full code of amino acid-based recognition of DNA sequences would be useful. Several approaches, which utilise small DNA binding motifs called zinc fingers, are presently employed. None of the current approaches successfully combine a combinatorial approach to the elucidation of a code with a single stage high throughput screening assay. The work outlined here describes the development of a model system for the study of DNA protein interactions and the development of a high throughput assay for detection of such interactions. A zinc finger protein was designed which will bind with high affinity and specificity to a known DNA sequence. For future work it is possible to mutate the region of the zinc finger responsible for the specificity of binding, in order to observe the effect on the DNA / protein interactions. The zinc finger protein was initially synthesised as a His tagged product. It was not possible however to develop a high throughput assay using the His tagged zinc finger protein. The gene encoding the zinc finger protein was altered and the protein synthesised as a Glutathione S-Transferase (GST) fusion product. A successful assay was developed using the GST protein and Scintillation Proximity Assay technology (Amersham Pharmacia Biotech). The scintillation proximity assay is a dynamic assay that allows the DNA protein interactions to be studied in "real time". This assay not only provides a high throughput method of screening zinc finger proteins for potential ligands but also allows the effect of addition of reagents or competitor ligands to be monitored.

An evaluation of thermal plasmas in pyrometallurgy

The development of reliable, high powered plasma generators has resulted in many plasma processes being proposed as alternatives to existing pyrometallurgical technologies. This work evaluates the advantages and disadvantages of plasma systems by reviewing plasma generators, their integration with reactors and the process economics. Many plasma systems were shown to be technically and economically superior to existing technologies, but some of the plasma system advantages quoted in the literature were found to be impractical because of other system constraints. Process applications were limited by the power inputs available from plasma generators compared to AC electric furnaces. A series of trials were conducted where chromite and steelplant baghouse dusts were smelted in the Tetronics' 2.0 MW transferred arc/open bath reactor to confirm the operating characteristics of the plasma system and its economics. Chromite smelting was technical superior to submerged arc furnace technology, but the economics were unfavourable because of the limited power available from the water-cooled plasma torch and the high electrical energy consumption. A DC graphite electrode plasma furnace using preheated and prereduced chromite concentrates will compete economically with the submerged arc furnace. Ni, Cr and Mo were economically recovered from high alloy content steelplant dusts for recycling. Five Electric Arc Furnace dusts were smelted to produce a non-toxic residue and recover the contained zinc to an enriched zinc oxide product for recycling. It should be possible to condense the zinc vapour directly in a zinc splash condenser to increase the value of the product. Because of the limited power available from plasma generators, plasma processes will be most suitable for treating high and medium value materials such as Au, Pt, Mo, Ni, Ti, V, Cr etc at small production rates, heating metals in tundishes and ladles and remelting superalloy scrap. The treatment of environmentally hazardous waste materials is a particularly interesting application because of the additional financial incentives. Non-transferred arc plasma generators will be used for air and gas preheating in blast furnaces to reduce metallurgical coke consumptions.