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Biomedical subjects

F Marks

Publications and source records attributed to F Marks.

At least 19 recordsLinked to original sources
PubMed2005

Pre-treatment with chloroquine and parasite chloroquine resistance in Ghanaian children with severe malaria.

BACKGROUND: Self-medication with anti-malarial drugs is widespread, and chloroquine (CQ) resistance is increasing. The effect of these factors on the incidence and presentation of severe malaria is uncertain. AIM: To investigate subtype of severe malaria, duration of illness, previous CQ treatment and prevalence of Plasmodium falciparum CQ-resistance markers among children presenting with severe malaria to a teaching hospital in Ghana. DESIGN: Prospective clinical study. METHODS: Consecutive patients (n = 189) presenting with severe malaria were examined clinically, and blood was taken for routine haematology and malaria films. Plasma and blood cells were stored and subsequently analysed by ELISA for CQ levels (n = 168) and by PCR and restriction digest for P. falciparum chloroquine resistance transporter gene (pfcrt) mutations (n = 139). RESULTS: Of 47 presenting with cerebral malaria, 21 had severe anaemia and 13 respiratory distress (RDS). Twenty-nine had prostration or RDS alone, 41 severe anaemia with prostration or RDS, and 72 severe anaemia not associated with coma, prostration or RDS. Of the patients studied, 77% had CQ in their plasma, and 88% were carrying P. falciparum with a CQ-resistance genotype. Significant associations were found (i) between the CQ-resistance genotype of parasites and plasma CQ levels, (ii) between the presence of CQ in plasma and the reported duration of illness, and (iii) between the reported duration of illness and the occurrence of severe but otherwise uncomplicated anaemia. DISCUSSION: There was extensive prior CQ use in our patients presenting with severe malaria, and a high prevalence of parasites with the CQ-resistance genotype. CQ resistance in P. falciparum may contribute to the development of severe but otherwise uncomplicated anaemia in this setting.

Animals↗
PubMed2001

Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin.

In prostanoid biosynthesis, the first two steps are catalyzed by cyclooxygenases (COX). In mice and humans, deregulated expression of COX-2, but not of COX-1, is characteristic of epithelial tumors, including squamous cell carcinomas of skin. To explore the function of COX-2 in epidermis, a keratin 5 promoter was used to direct COX-2 expression to the basal cells of interfollicular epidermis and the pilosebaceous appendage of transgenic mouse skin. COX-2 overexpression in the expected locations, resulting in increased prostaglandin levels in epidermis and plasma, correlated with a pronounced skin phenotype. Heterozygous transgenic mice exhibited a reduced hair follicle density. Moreover, postnatally hair follicle morphogenesis and thinning of interfollicular dorsal epidermis were delayed. Adult transgenics showed a body-site-dependent sparse coat of greasy hair, the latter caused by sebaceous gland hyperplasia and increased epicutaneous sebum levels. In tail skin, hyperplasia of scale epidermis reflecting an increased number of viable and cornified cell layers was observed. Hyperplasia was a result of a disturbed program of epidermal differentiation rather than an increased proliferation rate, as reflected by the strong suppression of keratin 10, involucrin, and loricrin expression in suprabasal cells. Further pathological signs were loss of cell polarity, mainly of basal keratinocytes, epidermal invaginations into the dermis, and formation of horn perls. Invaginating hyperplastic lobes were surrounded by CD31-positive vessels. These results demonstrate a causal relationship between transgenic COX-2 expression in basal keratinocytes and epidermal hyperplasia as well as dysplastic features at discrete body sites.

Aging↗
PubMed2001

A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression.

Epidermis-type lipoxygenases, a distinct subclass within the multigene family of mammalian lipoxygenases (LOX), comprise recently discovered novel isoenzymes isolated from human and mouse skin including human 15-LOX-2, human and mouse 12R-LOX, mouse 8S-LOX, and mouse e-LOX-3. We have isolated the human homologue of mouse e-LOX-3. The cDNA of 3362 bp encodes a 711-amino-acid protein displaying 89% sequence identity with the mouse protein and exhibiting the same unusual structural feature, i.e., an extra segment of 41 amino acids, which can be located beyond the N-terminal beta-barrel domain at the surface of the C-terminal catalytic domain. The gene encoding e-LOX-3, ALOXE3, was found to be part of a gene cluster of approximately 100 kb on human chromosome 17p13.1 containing in addition the 12R-LOX gene, ALOX12B, the 15-LOX-2 gene, ALOX15B, and a novel 15-LOX pseudogene, ALOX15P. ALOXE3 and ALOX12B are arranged in a head-to-tail fashion separated by 8.5 kb. The genes are split into 15 exons and 14 introns spanning 22 and 15 kb, respectively. ALOX15P was found on the opposite DNA strand directly adjacent to the 3'-untranslated region of ALOX12B. ALOX15B is located in the same orientation 25 kb downstream of ALOX12B, and is composed of 14 exons and 13 introns spanning a total of 9.7 kb of genomic sequence. RT-PCR analysis demonstrated a predominant expression of ALOXE3, ALOX12B, and ALOX15B in skin.

Amino Acid Sequence↗
PubMed2001

Enzymic characterization of epidermis-derived 12-lipoxygenase isoenzymes.

Substrate selectivity and other enzymic characteristics of two epidermis-derived lipoxygenases (LOXs), the epidermis-type (e) (12S)-LOX and (12R)-LOX, were compared with those of the platelet-type (p) (12S)-LOX. In contrast with p(12S)-LOX, e(12S)-LOX and (12R)-LOX exhibited no or very low reactivity towards the customary substrates linoleic acid and arachidonic acid but metabolized the corresponding fatty acid methyl esters, which, in contrast, were not accepted as substrates by p(12S)-LOX. Other esters of arachidonic acid and linoleic acid, including propan-2-yl and cholesterol esters, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphoethanolamine, and ceramide 1 carrying an omega-linoleic acid ester, were not metabolized by these three LOX isoenzymes. Among various polyunsaturated fatty acids the isomeric eicosatrienoic acids were found to be oxygenated by e(12S)-LOX but not by (12R)-LOX. 4,7,10,13,16,19-Docosahexaenoic acid as a substrate was restricted to p(12S)-LOX. Variations in the pH and the Ca(2+) content of the incubation medium affected the catalytic potential only slightly. Whereas (12R)-LOX activity increased in the presence of Ca(2+) and with an acidic pH, Ca(2+) had no effect on p(12S)-LOX and e(12S)-LOX; an acidic pH decreased the catalytic activity of the latter two. However, the catalytic activity of the epidermis-type isoenzymes, but not of p(12S)-LOX, was found to be markedly increased in the presence of DMSO. Under these conditions, e(12S)-LOX and (12R)-LOX oxygenated 4,7,10,13,16,19-docosahexaenoic acid to 14-hydroxy-4,7,10,12,16,19-docosahexaenoic acid and 13-hydroxy-4,7,10,14,16,19-docosahexaenoic acid respectively. In addition, (9R)-hydroxyoctadeca-10,12-dienoic acid methyl ester was generated from linoleic acid methyl ester by (12R)-LOX. Independently of the substrate, the catalytic activity of e(12S)-LOX and (12R)-LOX was always at most 2% of that of p(12S)-LOX with arachidonic acid as substrate.

Animals↗
PubMed2001

Aberrant cyclooxygenase isozyme expression in human intrahepatic cholangiocarcinoma.

METHODS: Cellular localisation of the cyclooxygenase (COX) isozymes COX-1 and COX-2 was analysed in 24 cholangiocarcinomas, including 17 matched tissues originating from non-tumorous liver tissue adjacent to tumours and seven biopsies of normal human liver, by immunohistochemistry using isozyme selective antibodies. RESULTS: In normal liver, constitutive expression of COX-2 protein was a characteristic feature of hepatocytes whereas no COX-2 immunosignal was detectable in normal bile duct epithelium, Kupffer, and endothelial cells. In cholangiocarcinoma cells, COX-2 protein was strongly expressed at high frequency. The intensity, percentage of positive cells, and pattern of COX-2 expression were found to be independent of the stage of tumour differentiation. In hepatocytes of matched non-tumorous tissue, COX-2 expression was unaltered. In contrast, strong COX-1 expression was frequently localised to Kupffer cells, endothelial cells, and occasionally to hepatocytes, but not to bile duct epithelial cells. In approximately half of moderately and poorly differentiated but not well differentiated cholangiocarcinomas, weak to moderate COX-1 staining was found in tumour cells while COX-1 expression in Kupffer cells was much more pronounced. CONCLUSION: Aberrant COX-2 expression occurs during the early stage while COX-1 over expression seems to be related to later stages of cholangiocarcinogenesis.

Adult↗
PubMed2001

Successful use of cyclooxygenase-2 inhibitor in a patient with aspirin-induced asthma.

We describe the case of an aspirin-sensitive asthma patient with a history of anaphylactic reactions to nonsteroidal anti-inflammatory drugs. The patient was subsequently diagnosed with rheumatoid arthritis and treated with a cyclooxygenase (COX)-2 inhibitor without an adverse response. Current prescribing information warns to avoid using COX-2 inhibitors in aspirin-sensitive asthma patients. New evidence suggests that aspirin sensitivity may be linked to the COX-1 pathway, and COX-2 inhibitors, as a result of their selectivity, may be beneficial in patients with aspirin-induced asthma.

Aged↗
PubMed2000

DIK, a novel protein kinase that interacts with protein kinase Cdelta. Cloning, characterization, and gene analysis.

A novel serine/threonine kinase, termed DIK, was cloned using the yeast two-hybrid system to screen a cDNA library from the human keratinocyte cell line HaCaT with the catalytic domain of rat protein kinase Cdelta (PKCdelta(cat)) cDNA as bait. The predicted 784-amino acid polypeptide with a calculated molecular mass of 86 kDa contains a catalytic kinase domain and a putative regulatory domain with ankyrin-like repeats and a nuclear localization signal. Expression of DIK at the mRNA and protein level could be demonstrated in several cell lines. The dik gene is located on chromosome 21q22.3 and possesses 8 exons and 7 introns. DIK was synthesized in an in vitro transcription/translation system and expressed as recombinant protein in bacteria, HEK, COS-7, and baculovirus-infected insect cells. In the in vitro system and in cells, but not in bacteria, various post-translationally modified forms of DIK were produced. DIK was shown to exhibit protein kinase activity toward autophosphorylation and substrate phosphorylation. The interaction of PKCdelta(cat) and PKCdelta with DIK was confirmed by coimmunoprecipitation of the proteins from HEK cells transiently transfected with PKCdelta(cat) or PKCdelta and DIK expression constructs.

Amino Acid Sequence↗
PubMed2000

A causal relationship between unscheduled eicosanoid signaling and tumor development: cancer chemoprevention by inhibitors of arachidonic acid metabolism.

Cancer results from disturbances of cellular signal transduction and data processing at the genetic and epigenetic level. In the early phase of the disease these disturbances are mainly caused by environmental toxic agents, i.e. genotoxic and non-genotoxic carcinogens, whereas endogenous agents derived from dys-regulated metabolic reactions may take over this role at later stages, thereby leading to a state of 'genetic instability' and 'growth autonomy'. Among these metabolic reactions becoming dys-regulated in the course of tumorigenesis, eicosanoid biosynthesis from arachidonic acid seems to play a particular role. A steadily increasing body of evidence indicates a causal relationship between cancer development and an abnormal overexpression of eicosanoid-forming enzymes, i.e. cyclooxygenases and lipoxygenases, in a wide variety of human and animal tumors. This overexpression seems to result from disturbances of cellular signaling cascades such as the Ras-Raf-MAPkinase cascade due to oncogenic gene mutations. Presently, research is focussed on the proinflammatory enzyme cyclooxygenase-2 (COX-2) the pathological overexpression of which has been found to be related to key events of tumor promotion such as cellular hyperproliferation, inhibition of programmed cell death, and tumor angiogenesis. In the mouse skin model of multistage carcinogenesis COX-2-derived prostaglandin F(2alpha) has been indentified as an endogenous tumor promoter. Moreover, genotoxic byproducts of both cylooxygenase and lipoxygenase-catalyzed arachidonic acid metabolism (such as active oxygen species, free radicals etc.) are suspected to contribute to 'genetic instability' and thus to malignant progression of tumor cells. The enzymes of eicosanoid biosynthesis rank therefore among the most attractive targets for cancer chernoprevention. In fact, both nonsteroidal antiinflammatory drugs, i.e. non-specific COX inhibitors, and isozyme-specific COX-2 inhibitors have been shown to inhibit experimental and human cancer development, in the latter case in particular in the large bowel. Beside their role as indicators of neoplastic development eicosanoids may be also used as reporters of skin irritation. Based to this concept an in vitro test system for skin toxicity has been developed in which the release of arachidonic acid and interleukin-1alpha, i.e. two key mediators of acute inflammation, from a human keratinocyte cell line is measured. The excellent correlation found between this mediator release and the effects of various chemical irritants on human skin in vivo indicates that, in the near future, this and related methods may help to do without animal experiments in toxicological testing.

Animals↗
PubMed2000

Biphasic effect of protein kinase C activators on spontaneous and glucocorticoid-induced apoptosis in primary mouse thymocytes.

Spontaneous and glucocorticoid (fluocinolone acetonide, FA)-induced apoptosis of primary mouse thymocytes was inhibited by protein kinase C (PKC) activators such as bryostatin-1 and phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate (TPA) within the first 2-4 h of incubation but was enhanced upon prolonged treatment. Only the anti-apoptotic but not the pro-apoptotic effect of TPA was completely suppressed by the PKC inhibitor Goe 6983 and moderately inhibited by Goe 6976. Immunoblot analysis revealed distinct PKC alpha, beta, delta, eta, theta, mu and zeta signals, a very faint PKCepsilon and no PKCgamma signal. Upon prolonged TPA treatment all PKC isoenzymes became downregulated, albeit at different rates (PKCdelta>alpha>mu>beta,theta>>eta,zeta). No significant generation of caspase-derived catalytic PKC fragments, as found to be produced upon induction of apoptosis and to be pro-apoptotic in other systems, was observed in FA- or TPA-treated thymocytes. It is concluded that the early anti-apoptotic effect of TPA depends on the activation of n-type PKC isoenzymes, whereas stimulation of spontaneous and FA-induced apoptosis by TPA ensues, at least partially, from a downregulation (or inactivation) of anti-apoptotic PKC species, i.e. in primary thymocytes PKC activation is primarily involved in a negative regulation of apoptosis.

Animals↗
PubMed2000

Positional- and stereo-selectivity of fatty acid oxygenation catalysed by mouse (12S)-lipoxygenase isoenzymes.

A quantitative stereochemical analysis of the products generated by recombinant mouse (12S)-lipoxygenase isoenzymes was performed with arachidonic acid and linoleic acid as substrates. The leucocyte-type (12S)-lipoxygenase generated, in addition to 12-hydroxyeicosatetraenoic acid (12-HETE) as the main product, 15- and 8-HETE from arachidonic acid and 13- and 9-hydroxyoctadecadienoic acid (13- and 9-HODE) from linoleic acid. The platelet-type enzyme oxygenated arachidonic acid to 12- and 8-HETE and linoleic acid to 13- and 9-HODE, whereas the epidermis-type (12S)-lipoxygenase reaction was essentially mono-specific with arachidonic acid but oxygenated linoleic acid to both 13- and 9-HODE. 12-HETE and 13-HODE were almost exclusively the S enantiomers. 8-HETE was the R enantiomer as a side-product of the platelet-type (12S)-lipoxygenase reaction but the S enantiomer as a side-product of the leucocyte-type reaction. 9-HODE was generated as the R enantiomer by the platelet-type and the epidermis-type isoenzymes and as the S enantiomer by the leucocyte-type (12S)-lipoxygenase. On the basis of published models of lipoxygenase-substrate interaction, the stereochemistry of the products generated by the platelet- and epidermis-type (12S)-lipoxygenases is in agreement with a fixed 'tail-to-head' orientation of the substrate fatty acid in the binding pocket of these enzymes, whereas that of the reaction products of the leucocyte-type (12S)-lipoxygenase can be explained only when the inverse orientation of the substrate or a rotational isomerism along the longitudinal axis of the substrate is allowed. Both the product spectra generated and the sensitivity towards the 12-lipoxygenase selective inhibitors N-benzyl-N-hydroxy-4-phenylpentanamide and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate indicated the platelet-type and the epidermis-type isoenzymes to be biochemically more related to each other than to the leucocyte-type (12S)-lipoxygenase.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2000

Evidence against a role of general protein kinase C downregulation in skin tumor promotion.

Using isoenzyme-specific antibodies, we have performed an immunoblot analysis of the PKC isoenzyme pattern during the course of TPA-induced tumor promotion in the epidermis of NMRI mice. The TPA-sensitive PKC isoforms alpha, delta, straightepsilon, eta, nu (and TPA-insensitive PKCzeta), but not PKCbeta and gamma, were found to be expressed in both normal and neoplastic epidermis. The immune signals of all TPA-sensitive PKC isoforms were moderately and reversibly attenuated upon a single TPA treatment. Using different antibodies against PKCeta and PKCmu, this apparent downregulation could mainly be attributed to epitope changes of these enzymes, whereas for the other PKC species no such conclusion could be drawn. Except for PKCstraightepsilon, no substantial long-term attenuation of the immune signals of the other PKC isoforms occurred upon chronic phorbol ester treatment (i.e., 14 applications of 5 nmol TPA each over 7 weeks), which led to tumor development in initiated mouse skin. Specific PKC activity (related to tissue weight) was 40-50% lower in TPA-treated as compared with control epidermis whereby no clearcut difference was found between single and chronic TPA treatment. Benign and malignant skin tumors generated according to the initiation-promotion protocol did not exhibit consistent alterations in the immune pattern of the PKC isoenzymes with the exception of a decrease of PKCstraightepsilon and an increase of PKCmicro signal in carcinomas. Our data indicate that, in contrast with earlier assumptions, no general long-lasting PKC downregulation plays a critical role in skin tumor promotion.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2000

Diversity of mouse lipoxygenases: identification of a subfamily of epidermal isozymes exhibiting a differentiation-dependent mRNA expression pattern.

By using reverse transcription-polymerase chain reaction technology (RT-PCR) and Northern blot analysis, the tissue-specific mRNA expression patterns of seven mouse lipoxygenases (LOX)--including 5S-, 8S-, three isoforms of 12S-, 12R-LOX, and a LOX of an as-of-yet unknown specificity, epidermis-type LOX-3 (e-LOX-3)--were investigated in NMRI mice. Among the various tissues tested epidermis and forestomach were found to express the broadest spectrum of LOX. With the exception of 5S- and platelet-type 12S-LOX (p12S-LOX) the remaining LOX showed a preference to exclusive expression in stratifying epithelia of the mouse, in particular the integumental epidermis. The expression of the individual LOX in mouse epidermis was found to depend on the state of terminal differentiation of the keratinocytes. mRNA of epidermis-type 12S-LOX (e12S-LOX) was detected in all layers of neonatal and adult NMRI mouse skin, whereas expression of p12S-LOX, 12R-LOX, and e-LOX-3 was restricted to suprabasal epidermal layers of neonatal and adult mice. 8S-LOX mRNA showed a body-site-dependent expression in that it was detected in stratifying epithelia of footsole and forestomach but not in back skin epidermis. In the latter, 8S-LOX mRNA was strongly induced upon treatment with phorbol esters. With the exception of e12S-LOX and p12S-LOX, the isozymes that are preferentially expressed in stratifying epithelia are structurally related and may be grouped together into a distinct subgroup of epidermis-type LOX.

Aging↗
PubMed2000

Cancer chemoprevention through interruption of multistage carcinogenesis. The lessons learnt by comparing mouse skin carcinogenesis and human large bowel cancer.

Whilst in the early stages, neoplastic development is predominantly triggered by environmental genotoxic and non-genotoxic carcinogens, tumour progression becomes more and more autonomous at later stages. In this context a dysregulation of arachidonic acid metabolism seems to play a disastrous role. Conversely, non-steroidal anti-inflammatory drugs (NSAIDs) rank among the most potent and most promising agents for cancer chemoprevention probably because of their ability to inhibit prostaglandin biosynthesis, in particular, at the level of the 'pro-inflammatory' enzyme cyclooxygenase-2 (COX-2). A pathological overexpression of COX-2 resulting in excessive prostaglandin production has been found already in early stages of carcinogenesis and seems to be a consistent feature of neoplastic development in a wide variety of tissues. COX-2 overexpression is thought to occur along signalling pathways of inflammation and tissue repair which become activated in the course of tumour promotion and, due to autocrine and auto-stimulatory mechanisms, finally lead to some autonomy of tumour development (self-promotion). Prostaglandins formed along a dysregulated COX pathway have been shown to mediate tumour promotion in animal experiments and may play a role, in addition, in other processes involved in tumour growth such as angiogenesis, metastasis and immunosuppression. Moreover, genotoxic byproducts such as organic free radicals, reactive oxygen species and malondialdehyde produced in the course of prostanoid biosynthesis may contribute to genetic instability (mutator phenotype) of neoplastic cells thereby promoting malignant progression. Such mixtures of physiologically highly active mediators and genotoxic byproducts are, in addition, formed along the various lipoxygenase-catalysed pathways of arachidonic acid metabolism some of which also become dysregulated during tumour development and, therefore, provide novel targets of future chemopreventive approaches.

Animals↗