Structure of cytochrome b5 in solution by Fourier-transform infrared spectroscopy

Fourier-transform infrared spectroscopy was used to examine the secondary structure of rabbit liver cytochrome b5 and the polar and nonpolar domains of the protein. The data for both the polar and nonpolar domains agree well with those previously obtained by other physical techniques. In particular it was found that the nonpolar membrane-binding domain was predominantly alpha helix and that the polar domain was also highly helical, but not all alpha helix. The independence of the two domains in the whole molecule was, in general, confirmed by the additivity of the spectra of the two domains. The small differences that were seen indicate that there is a loss of alpha helix when the protein is cut into the two domains. In addition, there appeared to be a slight difference in the exposure to solvent of the amide NH groups in the alpha-helical portion of the nonpolar domain when it was examined in isolation.     

Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.     

Cancer‐associated neochromosomes: a novel mechanism of oncogenesis

Malignant tumours are often characterised by significant rearrangement of the genome. This may be visible in the form of a deranged karyotype with both loss and gain of DNA sequences extending from chromosomal regions to whole chromosomes. In several tumour types, however, gross genomic derangements are minimal, and tumour cells contain one or more additional (supernumerary) chromosomes that may be unrecognisable in terms of a single origin. In this review we term such chromosomes cancer‐associated neochromosomes (CaNCs). In the absence of other identified genomic abnormalities, and because the CaNC is a common feature of the cancer type, it is hypothesised that the genetic alterations required for cell transformation are contained within its structure. In this review, we discuss the potential impact of modern genomic technologies on our understanding of the nature and causes of CaNC formation, which is central to several cancer types, exemplified here by well‐differentiated liposarcoma.     

Plant growth depressions in arbuscular mycorrhizal symbioses: not just caused by carbon drain?

* This study investigated effects of plant density and arbuscular mycorrhizal (AM) colonization on growth and phosphorus (P) nutrition of a cultivar of wheat (Triticum aestivum) that often shows early AM-induced growth depressions. * Two experiments were conducted. Expt 1 had three plant densities and one soil P concentration. Expt 2 had two plant densities and two P concentrations. Plants were grown in calcareous P-fixing soil, inoculated with Glomus intraradices or Gigaspora margarita, or noninoculated (nonmycorrhizal (NM)). Glomus intraradices colonized well and caused a growth depression only in Expt 1. Gigaspora margarita caused large growth depressions in both experiments even though it colonized poorly. * The results showed that growth depressions were mitigated by changes in relative competition for soil P by NM and AM plants, and probably by decreasing carbon costs of the fungi. * The different effects of the two fungi appear to be attributable to differences in the balance between P uptake by the fungal pathway and direct uptake via the roots. These differences may be important in other AM symbioses that result in growth depressions. The results show that mycorrhizal growth responses of plants grown singly may not apply at the population or community level.     

Maternal nicotine exposure increases oxidative stress in the offspring

Fetal and neonatal nicotine exposure causes β-cell apoptosis and loss of β-cell mass, but the underlying mechanisms are unknown. The goal of this study was to determine whether maternally derived nicotine can act via the pancreatic nicotinic acetylcholine receptor (nAChR) during fetal and neonatal development to induce oxidative stress in the pancreas. Female Wistar rats were given saline or nicotine (1 mg/kg/day) via subcutaneous injection for 2 weeks prior to mating until weaning (postnatal day 21). In male offspring, nAChR subunit mRNA expression was characterized in the developing pancreas and various oxidative stress markers were measured at weaning following saline and nicotine exposure. The nAChR subunits 2-4, 6, 7, and β2–β4 were present in the pancreas during development. Fetal and neonatal exposure to nicotine significantly increased pancreatic GPx-1 and MnSOD protein expression, as well as islet ROS production. Furthermore, protein carbonyl formation was higher in nicotine-exposed offspring relative to controls, particularly within the mitochondrial fraction. There was also a nonsignificant trend toward higher serum 8-isoPG levels. These data suggest that β-cell apoptosis in the fetal and neonatal pancreas may be the result of a direct effect of nicotine via its receptor and that this effect may be mediated through increased oxidative stress.     

Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction

BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)(6) and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (His)(6) derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 A resolution.     

Kinetics of Human Serum Butyrylcholinesterase Inhibition by a Novel Experimental Alzheimer Therapeutic,Dihydrobenzodioxepine Cymserine

Cholinergic loss is the single most replicated neurotransmitter deficiency in Alzheimer’s disease (AD) and has led to the use of acetylcholinesterase inhibitors (AChE-Is) and unselective cholinesterase inhibitors (ChE-Is) as the mainstay of treatment. AChE-Is and ChE-Is, however, induce dose-limiting adverse effects. Recent studies indicate that selective butyrylcholinesterase inhibitors (BuChE-Is) elevate acetylcholine (ACh) in brain, augment long-term potentiation, and improve cognitive performance in rodents without the classic adverse actions of AChE-Is and ChE-Is. BuChE-Is thereby represent a new strategy to ameliorate AD, particularly since AChE activity is depleted in AD brain, in line with ACh levels, whereas BuChE activity is elevated. Our studies have focused on the design and development of cymserine analogues to induce selective time-dependent brain BuChE inhibition, and on the application of innovative and quantitative enzyme kinetic analyses to aid selection of drug candidates. The quantitative interaction of the novel inhibitor, dihydrobenzodioxepine cymserine (DHBDC), with human BuChE was characterized. DHBDC demonstrated potent concentration-dependent binding with BuChE. The IC₅₀ and specific new kinetic constants, such as K_T50, P_PC, K_T1/2 and R_I, were determined at dual substrate concentrations of 0.10 and 0.60 mM butyrylthiocholine and reaction times, and are likely attainable in humans. Other classical kinetic parameters such as K_ia, K_ma, V_ma and V_mi were also determined. In synopsis, DHBDC proved to be a highly potent competitive inhibitor of human BuChE in comparison to its structural analogue, cymserine, and represents an interesting drug candidate for AD. Dedicated to Professor Moussa Youdim, Dept. Pharmacology, Technion, Haifa, Israel, in celebrating 45 years in scientific research focused on drug discovery and 30 years at Technion.     

Ribonuclease A homologues of the zebrafish: polymorphism, crystal structures of two representatives and their evolutionary implications

The widespread and functionally varied members of the ribonuclease A (RNase A) superfamily provide an excellent opportunity to study evolutionary forces at work on a conserved protein scaffold. Representatives from the zebrafish are of particular interest as the evolutionary distance from non-ichthyic homologues is large. We conducted an exhaustive survey of available zebrafish DNA sequences and found significant polymorphism among its four known homologues. In an extension of previous nomenclature, the variants have been named RNases ZF-1a-c,-2a-d,-3a-e and-4. We present the first X-ray crystal structures of zebrafish ribonucleases, RNases ZF-1a and-3e at 1.35-and 1.85 Å resolution, respectively. Structure-based clustering with ten other ribonuclease structures indicates greatest similarity to mammalian angiogenins and amphibian ribonucleases, and supports the view that all present-day ribonucleases evolved from a progenitor with three disulphide bonds. In their details, the two structures are intriguing melting-pots of features present in ribonucleases from other vertebrate classes. Whereas in RNase ZF-1a the active site is obstructed by the C-terminal segment (as observed in angiogenin), in RNase ZF-3e the same region is open (as observed in more catalytically efficient homologues). The progenitor of present-day ribonucleases is more likely to have had an obstructive C terminus, and the relatively high similarity (late divergence) of RNases ZF-1 and-3 infers that the active site unblocking event has happened independently in different vertebrate lineages.