Beyond the blot: cutting edge tools for genomics, proteomics and metabolomics analyses and previous successes.

The skin has an amazing array of complex interacting biological processes. Recent advances in investigational techniques now allow evaluation of these processes at the level of the gene, protein and metabolite. Sometimes collectively known as the omics, these fields of inquiry, known as genomics, proteomics and metabolomics, respectively, are yielding new and important insights into skin structure and processes, its responses to injury and age, and the mechanisms by which new interventions and compounds may work to improve the health and integrity of this crucial organ.

Dissociating the effects of distraction and proactive interference on object memory through tests of novelty preference.

Encoding information into memory is sensitive to distraction while retrieving that memory may be compromised by proactive interference from pre-existing memories. These two debilitating effects are common in neuropsychiatric conditions, but modelling them preclinically to date is slow as it requires prolonged operant training. A step change would be the validation of functionally equivalent but fast, simple, high-throughput tasks based on spontaneous behaviour. Here, we show that spontaneous object preference testing meets these requirements in the subchronic phencyclidine rat model for cognitive impairments associated with schizophrenia. Subchronic phencyclidine rats show clear memory sensitivity to distraction in the standard novel object recognition task. However, due to this, standard novel object recognition task cannot assess proactive interference. Therefore, we compared subchronic phencyclidine performance in standard novel object recognition task to that using the continuous novel object recognition task, which offers minimal distraction, allowing disease-relevant memory deficits to be assessed directly. We first determined that subchronic phencyclidine treatment did not affect whisker movements during object exploration. Subchronic phencyclidine rats exhibited the expected distraction standard novel object recognition task effect but had intact performance on the first continuous novel object recognition task trial, effectively dissociating distraction using two novel object recognition task variants. In remaining continuous novel object recognition task trials, the cumulative discrimination index for subchronic phencyclidine rats was above chance throughout, but, importantly, their detection of object novelty was increasingly impaired relative to controls. We attribute this effect to the accumulation of proactive interference. This is the first demonstration that increased sensitivity to distraction and proactive interference, both key cognitive impairments in schizophrenia, can be dissociated in the subchronic phencyclidine rat using two variants of the same fast, simple, spontaneous object memory paradigm.

Sex, but not maternal protein or folic acid intake, determines the fatty acid composition of hepatic phospholipids, but not of triacylglycerol, in adult rats.

The aim of the study was to investigate whether the protein and folic acid content of the maternal diet and the sex of the offspring alter the polyunsaturated fatty acid content of hepatic phospholipids and triacylglycerol (TAG). Pregnant rats were fed diets containing 18% or 9% protein with either 1 or 5mg/kg folic acid. Maternal diet did not alter hepatic lipid composition in the adult offspring. Data from each maternal dietary group were combined and reanalysed. The proportion of 18:0, 20:4n-6 and 22:6n-3 in liver phospholipids was higher in females than in males, while hepatic TAG composition did not differ between sexes. Delta5 Desaturase expression was higher in females than in males. Neither Delta5 nor Delta6 desaturase expression was related to polyunsaturated fatty acid concentrations. These results suggest that sex differences in liver phospholipid fatty acid composition may reflect primary differences in the specificity of phospholipid biosynthesis.

Structures of poliovirus complexes with anti-viral drugs: implications for viral stability and drug design.

BACKGROUND: Picornaviruses, such as the structurally related polioviruses and rhinoviruses, are important human pathogens which have been the target of major drug development efforts. Receptor-mediated uncoating and thermal inactivation of poliovirus and rhinovirus are inhibited by agents that bind to each virus by inserting into a pocket in the beta barrel of the viral capsid protein, VP1. This pocket, which is normally empty in human rhinovirus-14 (HRV14), is occupied by an unknown natural ligand in poliovirus. Structural studies of HRV14-drug complexes have shown that drug binding causes large, localized changes in the conformation of VP1. RESULTS: We report the crystal structures of six complexes between poliovirus and capsid-binding, antiviral drugs, including complexes of four different drugs with the Sabin vaccine strain of type 3 poliovirus, and complexes of one of these drugs with two other poliovirus strains that contain sequence differences in the drug-binding site. In each complex, the changes in capsid structure associated with drug binding are limited to minor adjustments in the conformations of a few side chains lining the binding site. CONCLUSIONS: The minor structural changes caused by drug binding suggest a model of drug action in which it is the conformational changes prevented by the bound drug, rather than obvious conformational changes induced by drug binding, which exert the biological effect. Our results, along with additional structures of rhinovirus-drug complexes, suggest possible improvements in drug design, and provide important clues about the nature of the conformational changes that are involved in the uncoating process.

Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.

Beyond the "recognition code": structures of two Cys2His2 zinc finger/TATA box complexes.

BACKGROUND: Several methods have been developed for creating Cys2His2 zinc finger proteins that recognize novel DNA sequences, and these proteins may have important applications in biological research and gene therapy. In spite of this progress with design/selection methodology, fundamental questions remain about the principles that govern DNA recognition. One hypothesis suggests that recognition can be described by a simple set of rules--essentially a "recognition code"--but careful assessment of this proposal has been difficult because there have been few structural studies of selected zinc finger proteins. RESULTS: We report the high-resolution cocrystal structures of two zinc finger proteins that had been selected (as variants of Zif268) to recognize a eukaryotic TATA box sequence. The overall docking arrangement of the fingers within the major groove of the DNA is similar to that observed in the Zif268 complex. Nevertheless, comparison of Zif268 and the selected variants reveal significant differences in the pattern of side chain-base interactions. The new structures also reveal side chain-side chain interactions (both within and between fingers) that are important in stabilizing the protein-DNA interface and appear to play substantial roles in recognition. CONCLUSIONS: These new structures highlight the surprising complexity of zinc finger-DNA interactions. The diversity of interactions observed at the protein-DNA interface, which is especially striking for proteins that were all derived from Zif268, challenges fundamental concepts about zinc finger-DNA recognition and underscores the difficulty in developing any meaningful recognition code.

Analysis of symmetry and three-dimensional reconstruction of thin gp32*I crystals.

Thin, multilayered crystals of gp32*I were analyzed by negative stain electron microscopy and image processing. Images of untilted crystals exhibited different projection symmetries and structural motifs. Systematic analysis of these images categorized the projections into four types. Areas producing the type 1 projection were reconstructed in three-dimensions from four tilt series containing 111 images. The three-dimensional data has excellent p121 plane group symmetry and reveals that the gp32*I molecule contains two large domains linked together by a small domain. Computer simulations utilizing projection data suggested that the type 2 and 3 projections arise from superposition of type 1 projections related by a 21 screw axis along the projection axis. The three-dimensional reconstruction was utilized in a final simulation that explained the occurrence of the fourth type of projection. This work provides a firm foundation for future high-resolution analysis of the crystal by electron cryomicroscopy.

Inhibition by FK506 of formyl peptide-induced neutrophil activation and associated protein synthesis.

The macrolide FK506 inhibited, by up to 50%, neutrophil migration and the production of the superoxide radical in response to the formyl peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). The production of the superoxide radical in response to phorbol 12-myristate 13-acetate (PMA) was unaffected by FK506. The inhibition of neutrophil functions was accompanied by a partial reversal of FMLP-induced synthesis of cellular proteins, despite a rise in intracellular Ca2+. Neutrophils treated with FK506 demonstrated a small (average 23%) though significant decrease in formyl-peptide receptor numbers but receptor binding affinity was unaffected. The effects of FK506 on neutrophil activation appear to be analogous to those in T-lymphocytes. The incomplete inhibition, by FK506, of neutrophil responses suggests further that activation by FMLP is mediated via distinct multiple signalling pathways, including protein kinase activation and protein synthesis. The inability of FK506 to reduce FMLP-induced rises in cellular Ca2+ or PMA-induced activation of neutrophils suggests that its action is distal to Ca2+ mobilization and distinct from pathways relying on PKC activation. Thus the immunosuppressive effects of FK506 in vivo might be mediated through the inhibition of inflammatory cells other than lymphocytes and the drug therefore has therapeutic potential in a variety of inflammatory conditions. The drug also has potential in vitro for the characterization of signalling pathways from the plasma membrane to the nucleus.

Dermal collagen implants.

The feasibility of using preparations of cell-free, fibrous dermal collagen, prepared by trypsin-treatment of skin, for the repair of soft body tissues has been examined both as subcutaneous implants and as a replacement for dermis in skin wounds in rats. Increased collagen stability, and suppression or reduction of tissue antigenicity in collagen heterografts, was achieved by crosslinking with weak solutions of aldehydes while still allowing implant recellularization and revascularization. Tritium-labelled collagen turnover studies have shown that maintenance of collagen mass in implants crosslinked with glutaraldehyde occurs primarily by inhibition of loss of original implant collagen. Some of the in vitro growth characteristics of human fibroblasts on animal collagen preparations are also described.

Specimen preparative methods for electron crystallography of soluble proteins.

Technical factors which influence the choice of specimen preparation method for electron crystallographic study of thin crystals of soluble proteins are discussed. Ice embedding appears to be the most desirable choice of preparation method. However, in terms of the yield of major structural information, we conclude that negative stain remains a useful method for low resolution and glucose embedding for high resolution.

Potentiometric determination of plutonium by argentic oxidation, ferrous reduction and dichromate titration.

A simple and rapid method is described for the routine determination of plutonium with a coefficient of variation of better than 0.2%. It is directly applicable to nitrate solutions containing a large amount of uranium; moderate amounts of iron, molybdenum, fluoride and phosphate do not interfere. Chromium, cerium and manganese interfere quantitatively, and the procedure may also prove convenient for the determination of these elements. The plutonium is oxidised to the sexivalent state with argentic oxide in nitric acid solution, and the excess of oxidant is destroyed by reaction with sulphamic acid. A weighed small excess of iron(II) solution is then added, and the excess is titrated potentiometrically with standard potassium dichromate solution using polarised gold indicator electrodes. The whole determination is performed in one vessel at room temperature, and takes about 20 min.

The effect of deamination and/or blocking arginine residues on the molecular assembly of acid-extracted rat tail tendon collagen.

We describe the effect of deamination of lysine and blocking of arginine residues on the assembly of collagen into native fibrils and SLS aggregates. Treatment of collagen solutions with one or both of these procedures does not prevent the formation of fibrils or SLS aggregates but reduces their ability to form assemblies with accurate longitudinal registration. These observations provide direct confirmation that hydrophobic interactions are important in collagen assembly. Unbanded fibrils were formed within the first 24 h at 4 degrees C from both acidic and neutralized deaminated and from neutralized control collagen solutions, transversely banded fibrils appearing later. This is compatible with the suggestion that initially, collagen fibrils are assembled by lyotropic liquid crystallization and with other observations which suggest that collagen molecules are initially free to move laterally within the fibril before being locked into place. Fibrils assembled from deaminated collagen solution show two variant longitudinal registration patterns which grade into one another. This suggests that, with a reduction in positively charged side chains, the thermodynamic energy minima responsible for longitudinal registration are less sharp compared with control collagen solutions. Reduction of positive charge by chemical modification helps to explain why the chemical modifications reduce swelling of collagen fibres. It also helps to explain why fibrils form spontaneously at 4 degrees C in both arginine-blocked and deaminated collagen solutions. Thus chemical modifications of rat tail tendon provides new insight into the mechanisms in collagen assembly.