Ross K. McCulloch

Fatty acid and amino acid composition in haruan as a potential role in wound healing

1. Two species of snakehead fish are available in Sabah, i.e. Channa striatus and Channa melanosoma, and are commonly known as haruan. Haruan is consumed by many Malaysians to induce healing after a clinical operations. However, there is no scientific evidence as yet to substantiate the claim, and so it was decided to analyse the biochemical composition in haruan to determine which compounds may have a possible role or potential in wound healing. 2. Samples (midline fillet) of both species were extracted separately in hexane for the qualitative analysis of fatty acids by a gas chromatography, Hewlett-Packard 5890A, using a 10 meter superox 11 column (Alltech) at temperature between 190 and 245 degrees C. Peak areas were calculated automatically using Hewlett-Packard 3393A computing integrator. Subsequently, the amino acid composition was analysed using a precolumn derivatization reverse phase HPLC waters PICO-TAG system. 3. Haruan is found to contain unusually high arachidonic acid (AA) but almost no eicosapentaenoic acid (EPA). AA which is a precursor of prostaglandin may initiate blood clotting and be responsible for growth. Haruan also contains all the essential amino acids for wound healing, particularly glycine which is the most important component of human skin collagen. Therefore, haruan contained all the basic biochemical requirements for wound healing.

Differential Effect of Aspirin on Platelet Aggregation in IDDM

Diabetes mellitus is associated with a high incidence of cardiovascular diseases not directly attributable to hyperlipidemia, smoking, or hypertension, but which in part may be explained by an enhanced tendency to thrombosis due to increased platelet activity. The aim of this study was to evaluate platelet function and compare the effectiveness of the antiplatelet drug aspirin on platelet aggregation in diabetic and nondiabetic subjects. Platelet aggregation and composition were examined in 20 male Insulin-dependent diabetes mellitus (IDDM) patients and 20 nondiabetic control subjects matched for age and body mass Index. All were normotensive with serum total cholesterol <6.5 mM. Although within the clinically acceptable normal range, blood pressure was significantly higher in diabetic patients (130/75 mmHg) than in control subjects (123/70 mmHg) (P < 0.05). Serum thromboxane B2 and ex vivo aggregation of platelets In response to two doses of the agonists collagen and platelet-activating factor (PAF) were similar to nondiabetic subjects. However, after taking 100 mg/day aspirin for 5 days, platelet aggregation to collagen was reduced by 76% in control subjects compared to 56% in IDDM patients (P < 0.001). Aspirin treatment also reduced the slope of the aggregation curve and increased the lag time (the period between the addition of collagen and the start of irreversible aggregation) significantly more in control than In diabetic platelets. This difference in platelet aggregation could not be attributed to differences in platelet serotonin or thromboxane A2 secretion, the latter being almost completely suppressed by aspirin in each group. Platelet aggregation to PAF was similar in both groups and was not affected by aspirin. IDDM patients had a significantly higher percentage of linoleic acid but lower percentage of ω-3 fatty acids in plasma and platelet phospholipids. The phospholipid-cholesterol ratio and membrane fluidity were not altered in diabetic platelets. These results suggest that platelets from IDDM patients are resistant to the effect of aspirin by mechanisms independent of both the cyclooxygenase and PAF pathways of aggregation.

MADM, a Novel Adaptor Protein That Mediates Phosphorylation of the 14-3-3 Binding Site of Myeloid Leukemia Factor 1

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3ζ and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3ζ via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3ζ, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

HLS5, a Novel RBCC (Ring Finger, B Box, Coiled-coil) Family Member Isolated from a Hemopoietic Lineage Switch, Is a Candidate Tumor Suppressor

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1α, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.

The incidence of microsatellite instability and loss of heterozygosity in fibroadenoma of the breast

A majority of studies have shown an increase in the risk of breast cancer among women previously diagnosed with fibroadenoma (FA). At present there is conflicting evidence whether some of the chromosome abnormalities frequently found in breast carcinoma, such as loss of heterozygosity (LOH), are already present in FAs and other types of benign breast disease and, if present, whether such abnormalities are associated with the observed increase in risk. Microsatellite instability (MSI) is also recognised as a marker of genetic damage and is thought to occur when there has been damage to the cells mismatch repair (MMR) system. We have analysed 39 cases of FA obtained from paraffin-embedded tissue for the presence of MSI and LOH at 11 loci to determine if these types of genetic alterations occur in FA. The incidence of MSI and LOH found were 4 of 395 (1.0%) and 5 of 271 (1.8%) informative loci tested respectively. Approximately 8% of cases were positive for MSI and 10% were positive for LOH, with one specimen having multiple occurences of both MSI and LOH. We conclude that these forms of genetic alteration do occur in FAs but that the incidence is low.

Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule

Myeloid leukemia factor 1 (MLF1) is an oncoprotein associated with hemopoietic lineage commitment and acute myeloid leukemia. Here we show that Mlf1 associated with a novel binding partner, Mlf1-associated nuclear protein (Manp), a new heterogenous nuclear ribonucleoprotein (hnRNP) family member, related to hnRNP-U. Manp localized exclusively in the nucleus and could redirect Mlf1 from the cytoplasm into the nucleus. The nuclear content of Mlf1 was also regulated by 14-3-3 binding to a canonical 14-3-3 binding motif within the N terminus of Mlf1. Significantly Mlf1 contains a functional nuclear export signal and localized primarily to the nuclei of hemopoietic cells. Mlf1 was capable of binding DNA, and microarray analysis revealed that it affected the expression of several genes, including transcription factors. In summary, this study reveals that Mlf1 translocates between nucleus and cytoplasm, associates with a novel hnRNP, and influences gene expression.

Enhancement of platelet 12-HETE production in the presence of polymorphonuclear leukocytes during calcium ionophore stimulation

This study investigates the effect of platelet/neutrophil interactions on eicosanoid production. Human platelets and polymorphonuclear leukocytes (PMNs) were stimulated alone and in combination, with calcium ionophore A23187 and the resulting eicosanoids 12S-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), 12S-heptadecatrienoic acid (HHT), 5S,12R-dihydroxy-(6Z,8E,10E,14Z)-eicosatetraenoi c acid (LTB4) and 5S-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) were measured by HPLC. The addition of PMNs to platelet suspensions caused a 104% increase in 12-HETE, a product of 12-lipoxygenase activity, but had only a modest effect on the cyclooxygenase product HHT (increase of 18%). By using PMNs labelled with [14C]arachidonic acid it was shown that the increases in these platelet eicosanoids could be accounted for by translocation of released arachidonic acid from PMNs to platelets and its subsequent metabolism. The observation that 12-lipoxygenase was about five times more efficient than cyclooxygenase at utilising exogenous arachidonic acid during the platelet/PMN interactions was confirmed in experiments in which platelets were stimulated with A23187 in the presence of [14C]arachidonic acid. Stimulations of platelets with thrombin in the presence of PMNs resulted in a decrease in 12-HETE and HHT levels of 40% and 26%, respectively. The presence of platelets caused a small increase in neutrophil LTB4 output but resulted in a decrease in 5-HETE production of 43% during stimulation with A23187. This study demonstrates complex biochemical interactions between platelets and PMNs during eicosanoid production and provides evidence of a mechanism to explain the large enhancement in 12-HETE production.

Reduced expression and normal nucleotide sequence of androgen receptor gene coding and promoter regions in a family with partial androgen insensitivity syndrome

BACKGROUND AND OBJECTIVES Androgen insensitivity syndrome (AIS) is an X‐linked disorder of XY males characterized by varying degrees of impaired masculinization. In many AIS cases, mutations have been identified in the coding sequence of the human androgen receptor (AR) gene which impair receptor function. Cases have also been reported in which reduced AR mRNA expression may contribute to AIS in association with AR gene mutations. The purpose of this study was to define the molecular basis of AIS in members of a family with clinical and laboratory features of partial androgen insensitivity (PAIS).