Gene engineering by selectable intraplasmid recombination: construction of novel dihydrofolate reductase minigenes

An intraplasmid recombination system in Escherichia coli has been designed to make possible the engineering of various genes using methods that greatly reduce dependence on appropriately placed restriction enzyme sites. This system has been used to manipulate intervening sequences in dihydrofolate reductase minigenes and to vary the number of 48-bp repeats in the promoter region. In this method, the two fragments to be recombined are cloned into a plasmid separated by a fragment of DNA containing an expressible galactokinase-encoding gene (galK). Selection for loss of the galK gene, but for retention of the plasmid in E. coli, results in a plasmid in which the two fragments have undergone homologous recombination. Several new plasmids are reported here which contain an expressible galK gene flanked by multiple restriction sites. These plasmids should be useful in recombination and as convenient sources of a gene for which both positive and negative selections are available in E. coli.     

Novel thiol- and thioether-containing amino acids: cystathionine and homocysteine families

Natural l-homocysteine and l,l-cystathionine, along with a series of unnatural analogues, have been prepared from l-aspartic and l-glutamic acid. Manipulation of the protected derivatives provided ω-iodoamino acids, which were used in thioalkylation reactions of sulfur nucleophiles, such as the ester of l-cysteine and potassium thioacetate.     

Altered Expression Pattern of Molecular Factors Involved in Colonic Smooth Muscle Functions: An Immunohistochemical Study in Patients with Diverticular Disease

Background

The pathogenesis of diverticular disease (DD) is thought to result from complex interactions among dietary habits, genetic factors and coexistence of other bowel abnormalities. These conditions lead to alterations in colonic pressure and motility, facilitating the formation of diverticula. Although electrophysiological studies on smooth muscle cells (SMCs) have investigated colonic motor dysfunctions, scarce attention has been paid to their molecular abnormalities, and data on SMCs in DD are lacking. Accordingly, the main purpose of this study was to evaluate the expression patterns of molecular factors involved in the contractile functions of SMCs in the tunica muscularis of colonic specimens from patients with DD.

Methods and Findings

By means of immunohistochemistry and image analysis, we examined the expression of Cx26 and Cx43, which are prominent components of gap junctions in human colonic SMCs, as well as pS368-Cx43, PKCps, RhoA and αSMA, all known to regulate the functions of gap junctions and the contractile activity of SMCs.The immunohistochemical analysis revealed significant abnormalities in DD samples, concerning both the expression and distribution patterns of most of the investigated molecular factors.

Conclusion

This study demonstrates, for the first time, that an altered pattern of factors involved in SMC contractility is present at level of the tunica muscularis of DD patients. Moreover, considering that our analysis was conducted on colonic tissues not directly affected by diverticular lesions or inflammatory reactions, it is conceivable that these molecular alterations may precede and predispose to the formation of diverticula, rather than being mere consequences of the disease.     

The different intermolecular interactions of the soluble copper-binding domains of the menkes protein, ATP7A

ATP7A is a P-type ATPase involved in copper(I) homeostasis in humans. It possesses a long N-terminal cytosolic tail containing six domains that are individually folded and capable of binding one copper(I) ion each. We investigated the entire N-terminal tail (MNK1-6) in solution by NMR spectroscopy and addressed its interaction with copper(I) and with copper(I)-HAH1, the physiological partner of ATP7A. At copper(I)-HAH1:MNK1-6 ratios of up to 3:1, thus encompassing the range of protein ratios in vivo, both the first and fourth domain of the tail formed a metal-mediated adduct with HAH1 whereas the sixth domain was simultaneously able to partly remove copper(I) from HAH1. These processes are not dependent on one another. In particular, formation of the adducts is not necessary for copper(I) transfer from HAH1 to the sixth domain. The present data, together with available in vivo studies, suggest that the localization of ATP7A between the trans-Golgi network and the plasma membrane may be regulated by the accumulation of the adducts with HAH1, whereas the main role of domains 5 and 6 is to assist copper(I) translocation.     

ENPP1 affects insulin action and secretion: evidences from in vitro studies

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.     

Vascular-homing peptides for targeted drug delivery and molecular imaging: Meeting the clinical challenges

The vasculature of each organ expresses distinct molecular signatures critically influenced by the pathological status. The heterogeneous profile of the vascular beds has been successfully unveiled by the in vivo phage display, a high-throughput tool for mapping normal, diseased, and tumor vasculature. Specific challenges of this growing field are targeted therapies against cancer and cardiovascular diseases, as well as novel bioimaging diagnostic tools. Tumor vasculature-homing peptides have been extensively evaluated in several preclinical and clinical studies both as targeted-therapy and diagnosis. To date, results from several Phase I and II trials have been reported and many other trials are currently ongoing or recruiting patients. In this review, advances in the identification of novel peptide ligands and their corresponding receptors on tumor endothelium through the in vivo phage display technology are discussed. Emphasis is given to recent findings in the clinical setting of vascular-homing peptides selected by in vivo phage display for the treatment of advanced malignancies and their altered vascular beds.     

Molybdoenzymes in Drosophila. IV. Further characterization of the cinnamon phenotype

Summary Mutations at the cin gene display drastically lowered levels of the molybdoenzymes, xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), and lack pyridoxal oxidase (PO) and sulfite oxidase (SO) activities. Certain mutations at cin also display varying degrees of female sterility, which is maternally affected. Here we characterize five new cin alleles with respect to the molybdoenzyme activities as well as the molybdenum cofactor, commonly required for molybdoenzyme activity. In complementing cin heterozygotes we find that, in addition to the previously reported unusually high levels of XDH and AO activities, there are unusually elevated levels of SO activity, as well as complementation for PO activity. The levels of immunologically crossreacting material in such heterozygotes indicate that the elevated levels of molybdoenzyme activities cannot be due to increases in the number of enzyme molecules. Measurements of the level of molybdenum cofactor activity normally present in XDH, AO, PO, and SO point to the possibility that a larger fraction of the enzyme molecules are active in these heterozygotes. The possible role of SO with respect to cinnamon's female sterility is also discussed.