Memory services constitute a significant part of old age psychiatry. Part of the memory assessment includes discussion of driving status as legally there is a requirement to disclose a dementia diagnosis to the DVLA (Driving and Vehicle Licensing Agency). At assessment a driving assessment can be undertaken to establish whether a patient is safe to continue driving. To establish if discussions regarding driving are taking place in memory assessment clinics, and if available guidance regarding dementia diagnosis and driving are followed. To then re-audit the department following dissemination of results from the initial audit. Standards set were 100% of patients would have documented discussion of driving. 60 patients were randomly selected from 423 referrals to memory assessment clinic between August 2012 and January 2013. Their electronic records were accessed and audited for evidence regarding documented discussion of driving. The same process was repeated following dissemination of results, with 40 patients records audited between March and May 2014. Data was collected from Bristol Activity of Daily Living Scale (BADLS) and trust pro-forma. Patients given a diagnosis of mild cognitive impairment were excluded. Of others, 55% of patients in the first audit and 56% of patients in the second audit had documented discussions regarding driving status. Standards were not met in either audit for a multitude of reasons, including restructuring of services. Recommendations include improvement of information collection tools and further audit, allowing for a longer length of time for implementation of changes.

Driving is an essential activity of daily living for many patients. A diagnosis of dementia is likely to affect one's ability to continue driving safely due to cognitive decline. It is the role of the memory services team to inform the patient of the above, assess their risk and advise patients about their driving accordingly. Furthermore given that there are currently no gold standard assessment tools available, the method by which patient risk is assessed is often left of the discretion of the assessor. This literature review looks at assessment of driving to see if there is an effective method that could be adopted by memory service assessors. To review literature with regards to assessing competency of driving in patients with dementia (pre and post disclosure). To see if there are effective measures, tools or assessment criteria that can effectively determine if a patient is competent to continue driving. PubMed and OvidSP search using ‘driving assessment’ and ‘dementia’ as search criteria for journal publications in English over the past five years. The search gave 60 papers from pubmed and 14 from OvidSP. 12 papers met the criteria for selection. There are many different tools that can be used to assess cognition, each with varying ability to identify those patients who are at risk when continuing to drive. The problem lies not only in detecting those at risk but there is also the issue over validation for the tests that are available

Introduction Memory services provide assessment to patients with memory difficulties. One important topic discussed is driving. This audit looks into whether discussions surrounding driving are documented in the patient's notes. Objectives The objective was to see if discussions are held about driving with patients at memory clinics. Aims A similar audit was carried out in the south of the county. It was hoped that the findings and recommendations had been implemented in the North of the county, and improvements made. Methods Standards were set that 100% of patients seen by memory services would have a documented discussion about driving at the initial and/or disclosure appointment. A period of 6 months, August 2012 to January 2013 was selected. 423 patients were referred and 60 patient's notes were randomly selected using a random integer set generator to look at. Results We are currently in the process of collecting and analyzing results. The results will be available before the conference. Conclusions By the time people with memory impairment access memory services they are likely to have been driving for most of their lives; it has provided them with independence and access to means. Despite this, requirements are set out by Driver and Vehicle Licensing Agency UK that they must be informed when the licence holder has been diagnosed with dementia. They can suspend a licence based on their assessment, for the safety of the patient and the public. It is therefore of vital importance that these discussions are taking place.

Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia, whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading, and that this association can be regulated by factors in the tumor microenvironment.

In cultured, undifferentiated normal human bronchial epithelial (HBE) cells, transglutaminase activity was localized predominantly in the cytosolic fraction of cell lysates. Upon squamous differentiation, this cytosolic activity declined and was replaced by a 40-fold increase in the activity of particulate (membrane-associated) transglutaminase. Immunoblot analysis demonstrated that the cytosolic transglutaminase was Type II (tissue) transglutaminase and that squamous differentiation shifted gene expression to the Type I (epidermal) transglutaminase. Retinoic acid, an inhibitor of squamous cell differentiation, suppressed the increase in Type I transglutaminase. The decrease in Type II transglutaminase activity was unaffected by retinoic acid. Transforming growth factor-beta-1 (TGF-beta-1) enhanced Type II transglutaminase activity about 10-fold in the undifferentiated cells but did not increase Type I transglutaminase or cholesterol sulfate, two early markers of squamous differentiation. TGF-beta-2 was equivalent to TGF-beta-1 in inducing Type II transglutaminase and in inhibiting the growth of HBE cells. The differentiation-related and TGT-beta-induced changes in transglutaminase activity were reflected in the level of transglutaminase Type I and Type II protein and mRNA. Expression of transglutaminases in lung carcinoma cell lines was variable. No correlation was observed between the expression of Type I transglutaminase and the classification of the cells as squamous cell carcinoma. Several lung carcinoma cell lines exhibited high levels of Type II transglutaminase activity that were increased several-fold by TGF-beta-1 treatment. Retinoic acid was ineffective in altering transglutaminase expression in most cell lines but induced Type II transglutaminase in a time- and dose-dependent manner in NCI-HUT-460 cells. Our results demonstrate that expression of transglutaminases is differentially regulated during squamous differentiation of HBE cells and that TGF-beta and retinoic acid can affect the expression of transglutaminases in normal and neoplastic epithelial cells derived from the human airways.

TRPC and Orai proteins have both been proposed to form Ca 2+ -selective, store-operated calcium entry (SOCE) channels that are activated by store-depletion with Ca 2+ chelators or calcium pump inhibitors. In contrast, only TRPC proteins have been proposed to form nonselective receptor-operated calcium entry (ROCE) cation channels that are activated by Gq/Gi-PLCβ signaling, which is the physiological stimulus for store depletion. We reported previously that a dominant negative Orai1 mutant, R91W, inhibits Ca 2+ entry through both SOCE and ROCE channels, implicating Orai participation in both channel complexes. However, the argument for Orai participating in ROCE independently of store depletion is tenuous because store depletion is an integral component of the ROCE response, which includes formation of IP3, a store-depleting agent. Here we show that the R91W mutant also blocks diacylglycerol (DAG)-activated Ca 2+ entry into cells that stably, or transiently, express DAG-responsive TRPC proteins. This strongly suggests that Orai and TRPC proteins form complexes that participate in Ca 2+ entry with or without activation of store depletion. To integrate these results with recent data linking SOCE with recruitment of Orai and TRPCs to lipid rafts by STIM, we develop the hypothesis that Orai:TRPC complexes recruited to lipid rafts mediate SOCE, whereas the same complexes mediate ROCE when they are outside of lipid rafts. It remains to be determined whether the molecules forming the permeation pathway are the same when Orai:TRPC complexes mediate ROCE or SOCE.

15‐LOX‐1 and its metabolites are involved in colorectal cancer. Recently, we reported that 15‐LOX‐1 overexpression in HCT‐116 human colorectal cancer cells inhibited cell growth by induction of p53 phosphorylation (4). To determine whether the 15‐LOX‐1 protein or its metabolites are responsible for phosphorylation of p53 in HCT‐116 cells, we used HCT‐116 cells that expressed a mutant 15‐LOX‐1. The mutant 15‐LOX‐1 enzyme, with a substitution of Leu at residue His361, was devoid of enzymatic activity. HCT‐116 cells transiently transfected with either native or mutant 15‐LOX‐1 showed an increase in p53 phosphorylation and an increase in the expression of downstream genes. Thus, 15‐LOX‐1 induces p53 phosphorylation independent of enzymatic activity. Treatment of A549 human lung carcinoma cells with IL‐4 increased the expression of 15‐LOX‐1 and also increased the expression of downstream targets of p53. This confirmed that the activation of p53 was also observed in wild‐type cells expressing physiological 15‐LOX‐1. Immunoprecipitation experiments revealed that 15‐LOX‐1 interacts with, and binds to, DNA‐dependent protein kinase (DNA‐PK). The binding of 15‐LOX‐1 to DNA‐PK caused an approximate 3.0‐fold enhancement in kinase activity, resulting in increased p53 phosphorylation at Ser15. Knockdown of DNA‐PK by small interfering RNA (siRNA) significantly reduced p53 phosphorylation. Furthermore, confocal microscopy demonstrated a colocalization of 15‐LOX and DNA‐PK in the cells. We propose that the 15‐LOX‐1 protein binds to DNA‐PK, increasing its kinase activity and results in downstream activation of the tumor suppressor p53, thus revealing a new mechanism by which lipoxygenases (LOX) may influence the phenotype of tumor cells. Published 2008 Wiley‐Liss, Inc.

During hematogenous cancer metastasis, tumor cells separate from a primary mass, enter the bloodstream, disperse throughout the body, migrate across vessel walls, and generate distant colonies. The later steps of metastasis superficially resemble leukocyte extravasation, a process initiated by selectin-mediated cell tethering to the blood vessel wall followed by integrin-mediated arrest and transendothelial migration. Some cancer cells express P-selectin ligands and attach to immobilized P-selectin, suggesting that these cells can arrest in blood vessels using sequential selectin- and integrin-mediated adhesion, as do leukocytes. We hypothesize that selectin binding may regulate subsequent integrin-mediated steps in metastasis. Using a model system of cultured Colo 320 human colon adenocarcinoma cells incubated with soluble P-selectin-IgG chimeric protein, we have found that P-selectin can stimulate activation of the α5β1 integrin resulting in a specific increase of adhesion and spreading of these cells on fibronectin substrates. P-selectin binding also induced activation of p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3-K). PI3-K inhibitors blocked P-selectin-mediated integrin activation, cell attachment, and cell spreading. Inhibition of p38 MAPK activation blocked cell spreading, but not cell attachment. P-selectin binding also resulted in formation of a signaling complex containing PI3-K and p38 MAPK. These results suggest that P-selectin binding to tumor cells can activate α5β1 integrin via PI3-K and p38 MAPK signaling pathways leading to increased cell adhesion. We propose that P-selectin ligands are important tumor cell signaling molecules that modulate integrin-mediated cell adhesion in the metastatic process.

We have investigated the effects of various fatty acids (FAs) on integrinmediated MDA-MB-435 breast carcinoma cell adhesion to type IV collagen (collagen IV) in vitro. Arachidonic acid (AA) and linoleic acid both induced a dose-dependent increase in cell adhesion to collagen IV with no significant increase in nonspecific adhesion to polylysine and BSA. Oleic acid (a monounsaturated FA), AA methyl ester, and linoelaidic acid (a trans-isomer of linoleic acid) failed to stimulate adhesion to collagen IV, suggesting that these effects requiredcis-polyunsaturation and a free carboxylic moiety and that they were not due to membrane perturbations. Calphostin C, a protein kinase C (PKC) inhibitor, blocked cis-polyunsaturated FA (cis-PUFA)-induced cell adhesion in a dose-dependent manner, suggesting a role for a calcium-dependent PKC in this signal transduction pathway. Immunoblotting revealed that cis-PUFAs induced the translocation of PKCe and PKCm, two of the novel PKC isozymes, from the cytosol to the membrane. In contrast, a conventional PKC isozyme, PKCa, as well as the atypical isozymes, PKC z and PKCi, did not translocate aftercis-PUFA treatment. Function-blocking antibodies specific for a1, a2, and b1 integrin subunits inhibited cell adhesion to collagen IV, whereas antibodies toa3 and a5 did not. No increase in the expression of these integrins on the cell surface was detected after the incubation of cells with cis-PUFAs, suggesting that there is an increase in the activity, but not in the amount, of theseb1 integrins. Altogether, these data suggest that cis-PUFAs enhance human breast cancer cell adhesion to collagen IV by selectively activating specific PKC isozymes, which leads to the activation of b1 integrins.

Eukaryotic cells have the ability to degrade proteins and organelles by selective and nonselective modes of micro- and macroautophagy. In addition, there exist both constitutive and regulated forms of autophagy. For example, pexophagy is a selective process for the regulated degradation of peroxisomes by autophagy. Our studies have shown that the differing pathways of autophagy have many molecular events in common. In this article, we have identified a new member in the family of autophagy genes. GSA12 in Pichia pastoris and its Saccharomyces cerevisiae counterpart, CVT18, encode a soluble protein with two WD40 domains. We have shown that these proteins are required for pexophagy and autophagy in P. pastoris and the Cvt pathway, autophagy, and pexophagy in S. cerevisiae. In P. pastoris, Gsa12 appears to be required for an early event in pexophagy. That is, the involution of the vacuole or extension of vacuole arms to engulf the peroxisomes does not occur in the gsa12 mutant. Consistent with its role in vacuole engulfment, we have found that this cytosolic protein is also localized to the vacuole surface. Similarly, Cvt18 displays a subcellular localization that distinguishes it from the characterized proteins required for cytoplasm-to-vacuole delivery pathways.

We have investigated the effects of various fatty acids (FAs) on integrin-mediated MDA-MB-435 breast carcinoma cell adhesion to type IV collagen (collagen IV) in vitro. Arachidonic acid (AA) and linoleic acid both induced a dose-dependent increase in cell adhesion to collagen IV with no significant increase in nonspecific adhesion to polylysine and BSA. Oleic acid (a monounsaturated FA), AA methyl ester, and linoelaidic acid (a trans-isomer of linoleic acid) failed to stimulate adhesion to collagen IV, suggesting that these effects required cis-polyunsaturation and a free carboxylic moiety and that they were not due to membrane perturbations. Calphostin C, a protein kinase C (PKC) inhibitor, blocked cis-polyunsaturated FA (cis-PUFA)-induced cell adhesion in a dose-dependent manner, suggesting a role for a calcium-dependent PKC in this signal transduction pathway. Immunoblotting revealed that cis-PUFAs induced the translocation of PKCepsilon and PKCmu, two of the novel PKC isozymes, from the cytosol to the membrane. In contrast, a conventional PKC isozyme, PKCalpha, as well as the atypical isozymes, PKCzeta and PKCiota, did not translocate after cis-PUFA treatment. Function-blocking antibodies specific for alpha1, alpha2, and beta1, integrin subunits inhibited cell adhesion to collagen IV, whereas antibodies to alpha3 and alpha5 did not. No increase in the expression of these integrins on the cell surface was detected after the incubation of cells with cis-PUFAs, suggesting that there is an increase in the activity, but not in the amount, of these beta1, integrins. Altogether, these data suggest that cis-PUFAs enhance human breast cancer cell adhesion to collagen IV by selectively activating specific PKC isozymes, which leads to the activation of beta1 integrins.

The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dynamic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuole. This process requires the concerted action of various proteins, including Apg5p. Recently, it was shown that another protein required for the import of aminopeptidase I (API) and autophagy, Apg12p, is covalently attached to Apg5p through the action of an E1-like enzyme, Apg7p. We have undertaken an analysis of Apg5p function to gain a better understanding of the role of this novel nonubiquitin conjugation reaction in these import pathways. We have generated the first temperature-sensitive mutant in the Cvt pathway, designated apg5(ts). Biochemical analysis of API import in the apg5(ts) strain confirmed that Apg5p is directly required for the import of API via the Cvt pathway. By analyzing the stage of API import that is blocked in the apg5(ts) mutant, we have determined that Apg5p is involved in the sequestration step and is required for vesicle formation and/or completion.

The haematopoietic system is sensitive to cytotoxic damage and is often the site of dose-limiting toxicity. We previously reported that swainsonine, an inhibitor of protein glycosylation, reduced the bone marrow toxicity resulting from a single dose of anticancer drugs in otherwise healthy mice. However, more important questions are (1) can swainsonine protect tumour-bearing mice without interfering with the anti-tumour effects of the drugs, and (2) can swainsonine stimulate haematopoietic activity of human, as well as murine, bone marrow. We demonstrate here that swainsonine protects C57BL/6 mice bearing melanoma-derived tumours from cyclophosphamide-induced toxicity without interfering with the drug’s ability to inhibit tumour growth. Similar results were obtained in vivo with 3´-azido-3´-deoxythymidine (AZT), a myelosuppressive agent often used in therapy for acquired immune deficiency syndrome. Swainsonine increased both total bone marrow cellularity and the number of circulating white blood cells in mice treated with doses of AZT that typically lead to severe myelosuppression. Swainsonine also increased the number of erythroid and myeloid colony forming cells (CFCs) in short-term cultures of murine bone marrow, restoring the number of progenitor cells to the control level in the presence of AZT doses that reduced CFCs by 80%. With respect to the sensitivity of human haematopoietic cells to swainsonine, we show that swainsonine protected human myeloid progenitor cells from AZT toxicity in vitro. These results suggest that swainsonine may be useful as an adjuvant in several types of human chemotherapy.

Oligosaccharide moieties of cell-surface glycoproteins are thought to be involved in recognition events during cancer metastasis and invasion. Swainsonine, an inhibitor of the Golgi alpha-mannosidase II, has been shown to block pulmonary colonization by tumor cells and stimulate components of the immune system. Swainsonine also abrogates much of the toxicity of chemotherapeutic agents and stimulates bone marrow hematopoietic progenitor cells, suggesting additional therapeutic applications. We are currently characterizing the ability of swainsonine to modify cell growth in human and murine bone marrow progenitor cells. Furthermore, we are examining crucial steps in metastasis that depend upon cell surface molecules that play a role in cell-matrix interactions. Our work shows that tumor cell adhesion to collagen IV in vitro is rapidly stimulated by cis-polyunsaturated fatty acids and is dependent on protein kinase C activity. We are investigating the hypothesis that integrins are critical components of this adhesion and are examining potential signal transduction pathways that lead to the modulation of cell adhesion.