Expression of AMPA receptor subunits at synapses in laminae I–III of the rodent spinal dorsal horn

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel α2δ-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S³⁵methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.     

Loss of heterozygosity and K-ras gene mutations in gastric cancer

In order to identify relevant genetic lesions in gastric carcinoma, we searched for tumor suppressor gene inactivation and K-ras gene mutations by analyzing tumor and control DNAs from 34 patients. These were from an epidemiologically defined area of Italy characterized by one of the world's highest incidences of stomach cancer. Allele losses were investigated by the Southern blotting procedure at 16 polymorphic loci on 11 different chromosomes. Our data demonstrate that chromosomal regions 5q, 11p, 17p and 18q are frequently deleted, and that 7q and 13q chromosome arms are also involved, although at a lower frequency. Loss of heterozygosity (LOH) at region 11p was not found during other surveys carried out on patients of different geographic origins. No specific combination of allelic losses could be recognized in the samples analyzed, the only exception being that tumors with 17p allelic loss also showed LOH on the 18q region. When matching frequent LOH events and the stage of progression of the tumors, we observed a trend of association between advanced stages and allelic losses on 17p and 18q chromosome arms. The analysis of K-ras, carried out by the polymerase chain reaction and denaturing gradient gel electrophoresis, demonstrated transforming mutations in only 3 out of 32 cases. Colorectal tumorigenesis proceeds by the accumulation of genetic alterations, including K-ras mutations and inactivation of tumor suppressor genes on the 5q, 17p and 18q regions. Our data indicate that, although gastric and colorectal neoplasias share common genetic alterations, they probably progress through different pathways.     

Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is characterised by pulmonary and systemic inflammation which flare-up during episodes of acute exacerbation (AECOPD). Given the role of Toll-like receptors (TLRs) in the induction of inflammatory responses we investigated the involvement of TLRs in COPD pathogenesis.

Methods

The expression of TLR-2, TLR-4 and CD14 in monocytes was analyzed by flow cytometry. To study the functional responses of these receptors, monocytes were stimulated with peptidoglycan or lipopolysaccharide and the amounts of TNFα and IL-6 secreted were determined by ELISA.

Results

We found that the expression of TLR-2 was up-regulated in peripheral blood monocytes from COPD patients, either clinically stable or during AECOPD, as compared to never smokers or smokers with normal lung function. Upon stimulation with TLR-2 ligand monocytes from COPD patients secreted increased amounts of cytokines than similarly stimulated monocytes from never smokers and smokers. In contrast, the expressions of TLR-4 and CD14 were not significantly different between groups, and the response to lipopolysaccharide (a TLR-4 ligand) stimulation was not significantly different either. At discharge from hospital TLR-2 expression was down-regulated in peripheral blood monocytes from AECOPD patients. This could be due to the treatment with systemic steroids because, in vitro, steroids down-regulated TLR-2 expression in a dose-dependent manner. Finally, we demonstrated that IL-6, whose plasma levels are elevated in patients, up-regulated in vitro TLR-2 expression in monocytes from never smokers.

Conclusion

Our results reveal abnormalities in TLRs expression in COPD patients and highlight its potential relationship with systemic inflammation in these patients.

     

Electrophoretic subtyping of phosphoglucomutase locus 1 (PGM1) polymorphism in the Italian and Czechoslovakian populations

About 3,500 subjects from Italy and Czechoslovakia have been analyzed by acid starch gel electrophoresis for the subtyping of PGM1 polymorphism. The Italian sample included three different subgroups, from Northern, Central and Southern Italy. The allele frequencies found in the three groups do not differ significantly from each other; the observed values in the pooled sample are: PGM1S1 = 0.594, PGM1F1 = 0.118, PGM2S1 = 0.231, PGM2F1 = 0.057. In the Czechoslovakian group, which differs significantly from the Italian population, the following allele frequencies were found: PGM1S1 = 0.639, PGM1F1 = 0.118, PGM2S1 = 0.180, PGM2F1 = 0.063. The analysis of 217 families did not show any exception to Mendelian inheritance of the patterns.     

Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis

The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G > T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.     

An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in β4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 μs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the β4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.     

Trichogynes and Fertilization in Uni- and Bimating Type Colonies of Neurospora tetrasperma

Microscopic examination of living, protoperithecium-bearing colonies of a, A, and a + A that have been challenged by macroconidia from the same three colony types has shown that active trichogynes, i.e., those that grow to and fuse with a conidium, are to be found only in the first two types. Thus, in the a colony the trichogynes respond to conidia from the A and a + A colonies while in the A colony they respond to conidia from a and a + A colonies. In contrast to this ability of conidia from a + A colonies to function as fertilizing elements, the trichogynes of these colonies, if indeed they are formed at all, do not so respond. This nonresponse in a + A colonies may be due to the perithecia that are developing at the time of the challenge. Evidence for this conclusion comes from unimating type colonies in which the two halves of each colony were challenged at different times, 48 h apart. Trichogynes and perithecia developed in the first half; neither developed in the second. This inhibition of trichogyne development and response in the presence of developing perithecia may be only one manifestation of a more general inhibitory action by these structures.     

Cancer incidence in eastern Adriatic isolates, Croatia: examples from the islands of Krk, Cres, Losinj, Rab and Pag

As an extension of previous research this study investigates the incidence of cancer in five genetic isolate island populations of the Eastern Adriatic, Croatia. Thorough anthropological research over the past three decades has established some of those populations as outstanding examples of genetic isolates. A previous study which found higher cancer incidence in 5 Eastern Adriatic islands than in a control population supported a hypothesis that among the founders of these populations there were genetic variants (especially with recessive inheritance) responsible for genetic susceptibility to certain types of cancer. This study sought to investigate cancer incidence in 5 further island populations. All cancer cases in five island populations (Krk, Cres, Losinj, Rab and Pag) over the 20-year period (1971 to 1990) was extracted from the data of the Croatian Cancer Registry. The mainland populations of Istrian and Primorsko-Goranska County, characterized by similar environmental factors but an outbred genetic structure, represented a control population. After standardization by by sex and age, cancer incidence was higher in the island populations than in the control population in both sexes. The cancer sites primarily responsible for the excess incidence were prostate, stomach and pancreatic cancer in males, and ovarian, breast, stomach, bowel, and brain cancer in females. The reasons for the increased cancer incidence are uncertain and may be due to different environmental exposure between the two populations. However, it is possible that genetic isolation and inbreeding are important factors. Further investigations of cancer in these isolate populations are warranted to explore these findings further.     

DNA sequences amplified in cancer cells: an interface between tumor biology and human genome analysis.

There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the 'reverse genetics' methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demonstrated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.