Mechanism of Action of Antivirals against Nasopharyngeal Carcinoma at the Molecular Level
Chu WL, Shar Mariam, Mak JW, Ling SN, Phang SM, Rakesh Naidu, Pauline Balraj, Khoo Soo Beng, Patricia Lim Kim Chooi, Kok Yih Tih, Lai Pey Jiun

Algae are lower plants which are important sources of pharmaceuticals and nutraceuticals. We are screening microalgae for products with bioactivity against nasopharyngeal carcinoma (NPC) and Epstein Barr Virus (EBV) under this project funded by the Ministry of Science, Technology and Innovation Malaysia (MOSTI). This project is in collaboration with Universiti Malaya and Institute for Medical Research. Nasopharyngeal carcinoma is a type of cancer arising in the epithelial cells of the nasopharynx, and one of the main aetiological factors is the EBV. The major objectives of this project are to screen indigenous microalgae of Malaysia for bioactivity against NPC cell lines and EBV, and to unravel the mechanisms of action of the bioactive compounds.

The solvent extracts from 19 microalgae were screened for their cytotoxic effect against NPC cell lines. Results from the screening showed that methanol extracts from three microalgae, Synechococcus elongates (Fig. 1a), Spirulina platensis (Fig. 1b), and Ankistrodesmus convolutes (Fig. 1c), showed cytotoxic activity against NPC cell lines (Figs. 2a & b). Fractionation of the crude extracts using chromatographic techniques and further testing with the fractions are being carried out. The antiviral activity of the algae extracts was assessed based on their effect on the expression of latent proteins by EBV and the assay of the viral DNA in the lytic cycle. The methanol extracts caused significant reduction of the EBV DNA load in the lytic cycle and the expression of latent membrane proteins of EBV in lymphoblastoid cells. The effect of the algae extracts on the gene expression of NPC cell lines is being investigated.

Fig. 1. Microalgae which showed bioactivity against nasopharyngeal carcinoma (NPC) cell lines and Epstein Barr Virus (EBV); a) Synechococcus elongatus ; b) Spirulina platensis; c) Ankistrodesmus convolutus

Fig. 2. (a) Nasopharyngeal carcinoma cell line (control); (b) Nasopharyngeal carcinoma cell line treated with methanol extract from Synechococcus elongatus. The cells round up and there is loss of contact between the cells

 

Bioactive Compounds in Bacillus thuringiensis
Vishna Devi Nadarajah, Shar Mariam Mohammed, Chan Kok Keong, Kanakeswary Karisnan, Rebecca Wong, Barathy Rani Ramasamy

Our research group at IMU previously reported the identification of a Malaysian Bacillus thuringiensis strain designated Bt 18 to be specifically cytotoxic against CEM-SS cell lines, (leukaemic T lymphocytes). Bt 18 produces parasporal inclusions, which after trypsin activation is non-haemolytic to human or rat erythrocytes, and is non-cytotoxic to uterus, breast and colorectal cancer cell lines. Importantly, Bt 18 is non toxic to purified T- 1a 1b 1c 2a 2blymphocytes isolated from healthy individuals, indicating its potential as a therapeutic agent for leukaemia cells. N-terminal sequencing of the trypsin activated Bt18 inclusion, showed that this protein has a low sequence similarity to a mosquitocidal Bt protein, found in Bt sub species jegathesaniensis, also isolated from the Malaysian soil.

We have successfully raised polyclonal antibodies against Bt 18 toxins, and have used them to perform cross-reactivity and immunohistochemical studies. Quantitative cross-reactivity studies using ELISA were performed with 15 Bt isolates, 2 Bacillus sphaericus (Bs) isolates, 1 Bacillus substilis (Bsub) isolate and 1 Bacillus cereus (Bc) isolate. It was observed that the Bt isolates share similar antigenic properties with the 54 kDa protein from Bt 18. Very low cross-reactivity was observed with the Bs, Bsub and Bc isolates (<3.9 %, 5.8% and 16.7% respectively). Qualitative cross reactivity studies using Western Blot analysis showed that the proteins from Bt strains cross react with the anti-Bt18 antibody at molecular weights around or higher than 54kDa. Immunohistochemical studies show that the Bt18 toxins are localized around the CEM-SS cells, indicating binding on the membrane plasma of the cells (Fig. 3). Future work will be focused on the binding mechanism and gene expression of Bt18.

Fig. 3. Immunostaining of CEM-SS cells incubated with Bt 18 parasporal inclusion protein at 100 μg/ml for 1 hour. Cells harvested by centrifugation and fixed in acetic acid: alcohol fixative for 10 minutes. The fixed cells smeared on poly-L-lysine coated slides and air dried. Smear blocked for non-specific binding with BSA for 20 minutes. Primary antibody (anti-Bt 18 antibody) dilution 1:10, Secondary antibody (anti-rabbit HRP labelled) dilution 1: 500 and developed using liquid DAB + substrate chromogen system, DAKO. Counter stained with haematoxylin for 10 seconds. Arrows indicate brownish ring formation around the cell indicating localization of Bt18.

As the long term usage of insecticidal Bt strains can lead to the development of insect resistance, it is important to search for strains that display novel vegetative insecticidal polypeptides (Selvapandiyan, 2001). Unlike the insecticidal Bt-endotoxins, the vegetative insecticidal protein, (Vip), a 88-kDa protein, is secreted during the vegetative stage by Bt and displays high toxicity against a range of insects. Our group in IMU have isolated bioactive compounds, expressed during the vegetative growth phase of the Malaysian Bt strains, and several of these compounds show potential mosquitocidal and anti-cancer activity. Our research group in IMU is leading this initiative on Bt bioactive compounds, and our main collaborators are senior researchers from the Entomology Department of IMR and the Medical Faculty of UPM.



Development of Immunoassays for the Detection of Candida Antigens in Systemic Candidiasis
Wong SF, Mak JW, Peter Pook CK, Ng KP, Seow HF and Ngah Zasmy

The increased incidence of candidiasis in the elderly, critically ill or immunocompromised has been attributed to the use of broad-spectrum antibiotics, immunosuppressive and antineoplastic agents, prosthetic devices and graft, organ and bone marrow transplantation, HIV infection, burns and other debilitating diseases. As there are no pathognomonic signs or symptoms of systemic candidiasis, the early diagnosis of invasive candidiasis remains a laboratory and clinical challenge. Thus, this collaborative project between researchers of IMU, Universiti Putra Malaysia and Universiti Malaya, seeks to develop diagnostic assays for the detection of systemic candidiasis, identify Candida virulence factors and associated pathogenesis through immunohistochemistry.

Monoclonal antibodies against C. albicans and C. parapsilosis were produced, purified, and characterized. The monoclonal antibodies were able to bind to the surface of the yeast cells and germ tubes. The monoclonal antibodies were used for the development of immunoassays using ELISA, dot blot, immunoperoxidase (Fig. 4a) and immunofluorescence (Fig. 4b) techniques for the detection of antibody, antigen or immune complexes in tissues.

Fig. 4a. Immunoperoxidase staining of Candida using monoclonal antibody; 1000x mag.	Fig. 4b. Immunofluorescence staining of Candida using monoclonal antibody; 1000x mag.

In rodent models induced with systemic candidiasis through intravenous injection of cultured C. albicans and C. parapsilosis, cystic lesions with large clumps of fungal hyphae and some yeast were found in the kidneys (Figs. 5a & b). Clusters of yeast cells were also detected in lungs, brains and spleen. The correlation between the histopathological changes, immune response and cytokine expression are being investigated.

Fig. 5a. Kidney, showing Candida fungal hyphae and yeast in cystic lesion; Gomori stain; 400x mag.	Fig. 5b. Kidney, indirect fluorescent antibody staining of Candida using monoclonal antibody; 400x mag.

Molecular and Immunological Studies on Acanthamoeba Isolated in Malaysia
Chan Li Li, Mak JW, Shar Mariam Mohamed, Init Ithoi

Acanthamoeba are free-living opportunistic protozoan parasites that are ubiquitous in the environment. Based on cyst morphology, at least 24 species have been named within this genus. However, as morphology of the organism is inconsistent, Acanthamoeba is reclassified into 15 different genotypes on the basis of the 18S rDNA sequences. Under appropriate conditions and host susceptibility, the pathogenic Acanthamoeba can cause a sight-threatening keratitis, which is usually associated with contact lens use, or fatal granulomatous encephalitis, which affects mostly immunocompromised patients.

A total of 86 dust samples were collected from the air chillers or air condition units located in two large educational institutions (building A & B) in Kuala Lumpur. The dust samples were inoculated onto the surface of non-nutrient agar (NNA) plates seeded with Escherichia coli and the plates were sealed with parafilm and incubated at room temperature (28-30ºC). Sixty three out of 86 samples (59 out of 82 samples from building A, and all 4 samples from the building B respectively) were found positive for amoeba growth under direct microscopic observation.

These isolates were further studied through PCR analysis of nucleic acid sequences, utilising a pair of Acanthamoeba genus specific primers developed by Schroeder et al. (2001). Acanthamoeba was present in 24 out of the 63 amoeba positive growth samples (20 samples from A, and all 4 samples from B respectively).

Since most of the Acanthamoeba cultures were present as mixed isolates, 3-4 single cysts were isolated from each sampling site and were established in xenic cultures. These cell lines were then subjected to axenisation to eliminate bacteria contaminants in the Acanthamoeba cultures. Axenisation was carried out on 40 amoeba cell lines derived from single cyst isolates from 16 sampling sites. The ability of the amoeba to adapt from xenic to axenic cultures varied greatly among strains; only 16 amoeba cell lines were found able to adapt and expand/grow under axenic culture conditions, that is in peptone-yeast-glucose medium (Figs. 6a & b).

Fig. 6a. Established axenic Acanthamoeba culture.	Fig. 6b. Non-adapted axenic Acanthamoeba culture.

18S rDNA partial fragments from 9 of these axenic Acanthamoeba cultures were sequenced by direct PCR sequencing. All the isolates showed high levels of similary (>98%) to various Acanthamoeba strains sequenced to date. Isolated strains identified through sequence data are: castellanii (isolate-11, G11/4/4), culbertsoni (isolate-1, 6, SL), griffini (L3-4/4/1), hatchetti (L3-21/1), polyphaga (isolate-G5/1), and quina (L1-25/1).

The pathogenic potential of selected axenic Acanthamoeba cultures was investigated in vitro studies. Human glial cells were seeded with different concentrations of trophozoites and incubated at 37°C. The degree of cytopathic effects of Acanthamoeba on glial cells was measured at 24, 48 and 96 hours, using the Roche Cytotoxicity Detection Kit (LDH). It was found that several Acanthamoeba spp. isolated showed the ability to engulf and kill glial cells in vitro. Transmission electron microscopic studies of 5 selected isolates indicate that phagocytosis was a possible mechanism of glial cell injury (Fig. 7).

Three stable clones producing IgM monoclonal antibodies against an environmental isolate of Acanthamoeba have been developed using the hybridoma technique. The three clones are currently used for ascetic fluid production in Balb/c mice for further studies.

Fig. 7. Transmission microscopy image showing the engulfment of Acanthamoeba trophozoite (T) of a glial cell (G). The trophozoite is characterised by the presence of acanthopodia (A).

Genetic Polymorphism of Cytochromes P450 and Their Involvement in Drug-herb Interactions
Ong Chin Eng, Pan Yan, Tiong Kai Hung, Peter Pook, Mak Joon Wah, Yiap Beow Chin, Tan Eng Lai, Badrul Amini Abd-Rashid, Zakiah Ismail, Rusli Ismail

The cytochromes P450 (CYP) constitute a superfamily of haem-thiolate enzymes present in most species and are associated with Phase I metabolism of a large number and variety of drugs and other xenobiotics. In humans, a relatively small number of hepatic microsomal CYPs catalyse the oxidation of most foreign compounds and these enzymes constitute CYPs from the families CYP1, CYP2 and CYP3. Numerous population studies revealed a pronounced interindividual variability in CYP levels and activities, with some individuals completely lacking some isoforms of this enzyme superfamily. This variability can be attributed to genetic polymorphism in the genes of these enzymes.

Since early 2002, our research has been focusing on developing in vitro methods in studying the functional consequences of genetic polymorphism of some CYP isoforms. CYP2C8 cDNA, provided as a gift from Professors Donald Birkett and John Miners from Australia, was used as a template to generate a number of mutants using site-directed mutagenesis. These mutants, carrying nucleotide changes as reported in known alleles in human populations, were expressed in E. coli system, and the expressed recombinant proteins were characterised using various biochemical assays. Mutants that have been characterised include CYP2C8*2, CYP2C8*3 and CYP2C8*4. Spectral scanning and proteinase K assays revealed that most of these mutants were inherently unstable exhibiting altered structural folds in their tertiary structures as revealed by higher degree of degradation from the protease digestion and the abnormal absorbance spectra. Our recent studies have also focused on cloning of CYP2A6 cDNA from liver RNA using the RT-PCR (reverse transcription-polymerase chain reaction) technique. The cDNA was successfully cloned and modified for expression in E. coli. The recombinant CYP2A6 was expressed adequately as revealed by immunoblotting and spectral analyses. Site-directed mutagenesis is currently carried out to generate common allelic variants as detected in human population studies and their biochemical characterization will commence soon.

Parallel to these studies on cDNA cloning and protein expression, we have also established a series of HPLC (high performance liquid chromatography)-based incubation assays to investigate CYP activities. Assays that have been developed include tolbutamide methylhydroxylase, paclitaxel 6a-hydroxylase, dextromethorphan O-demethylation, testosterone 6b-hydroxylation and coumarin 7-hydroxylase assays. These assays serve as activity markers allowing comparison of catalytic activities of mutant proteins generated through site-directed mutagenesis as mentioned above. Studies on CYP2C8 mutants using tolbutamide methylhydroxylase and paclitaxel 6a-hydroxylase assays have indicated that mutations in the protein sequence resulted in drastic reduction in enzymatic activities. This implies that these amino acids play important roles in substrate catalysis and protein stability. Another focus of our work is to use the developed incubation assays as a screening tool to investigate drug-herb interactions. This was achieved by co-incubation of herbal extracts and active constituents with the probe substrates in the assays followed by characterization of the kinetic behaviours of the enzymes involved in the assays using various pharmacokinetic parameters such as Km, Vmax, IC50 and Ki. A study investigating the modulatory effects of flavonoids on CYP2C8 activity was completed in 2004. The results indicated that the flavonoids inhibited CYP2C8 with different potency. A number of structural factors were found to be important for CYP2C8 inhibition; these include the molecular shape (volume to surface ratio), the number of hydroxyl groups as well as glycosylation of the hydroxyl group in the flavonoid structure. Data from this kind of structure-function relationship analysis has enhanced our understanding of structural principles of flavonoid-CYP2C8 interaction. More recently, we have expanded the work on screening of interaction involving two commonly used local herbs, pegaga and hempedu bumi. Various extracts of the plants (provided by the Institute for Medical Research), as well as active constituents, are being screened using tolbutamide methylhydroxylase, dextromethorphan O-demethylation, testosterone 6b-hydroxylation assays. It is envisaged that this screening work will provide insights on the potential of these two herbs to cause pharmacokinetic interactions with other drug substrates, and allow us to elucidate the mechanisms in the interactions.


Formulation and Characterisation of Lipogels
Khalid Ahmad Sheikh, Kang Yew Beng and Peter Pook

There are two main types of gels recognised to date i.e. aqueous (hydrogels or chemical gels) and non-aqueous (organogels or physical gels). Most of the pharmaceutical and cosmetic gels available belong to hydrogels and little emphasis is given to organogels. We have developed an interest in the lipogels, a class of organogels, containing hydrophobic metallic stearates (gelling agents), which are known to gel many non-polar solvents. It is fascinating to note that how such a small amount of gelling agent (5-15%) can gel an enormous amount (85-95%) of the liquid. There is little literature explaining the gelling mechanism of lipogels and most of it provides information only about aluminium stearate because of its extensive use in the lubricating greases. We are trying to understand the gelling mechanism of lipogels of magnesium stearate, which is commonly used in the tablet manufacture as a lubricant. Magnesium stearate exists in various polymorphic forms, which are sensitive to moisture and shear. For these reasons, lipogels of magnesium stearate show varying appearances and stability. These lipogels are characterised by microscopy, DSC, TGA, rheology, FTIR, XRD and texture analyses. The microstructure of the lipogels shows the presence of star-shaped clusters of tubules (Fig. 8a) as well as Maltese crosses (Fig. 8b), which in turn support the existence of liquid crystalline gel network structure. DSC, TGA, FTIR and XRD results have shown that the presence of bound moisture in magnesium stearate is vital for the formulation of stable and elastic lipogels because the dried magnesium stearate resulted in crumbly lipogels with syneresis. Homogenisation (shear) provided elastic lipogels unlike unhomogenised ones, which were crumbly and experienced syneresis. These results show that magnesium stearate exists in different polymorphic forms in homogenized and unhomogenised lipogels. Dihydrate form of magnesium stearate provides stable gels, whereas amorphous (dried) form provides crumbly lipogels. We are now trying to determine the forces which are holding the crystalline gel network together, that can lead us to understand the gelling mechanism and to predict the stability of these systems.

The lipogels can be used as non-aqueous media for material synthesis, for purification and separation purposes, as temperature and moisture sensors and as transdermal delivery vehicles and carriers for liquid crystals. The lipogels are excellent replacement for ointment bases due to their silky and non-greasy nature.

Fig. 8a. Star-shaped clusters of tubules in lipogels	Fig. 8b. Maltese crosses in lipogels

Pregnancy Induced Hypertension (PIH) Study
John Paul Judson, Vishna Devi V Nadarajah, Sivalingam Nalliah, Richard Lee, Elena How, Kamalan Jeevaratnam, Tee Kok Chuan

Pregnancy-induced hypertension (PIH) complicates between 5-10% of all pregnancies, and is said to be the leading cause of death and disability in mothers and infants. Although its exact pathogenesis is still not known, recent reports have indicated that the imbalance of circulating angiogenic factors is important in the onset of PIH and preeclampsia. At the International Medical University, research has been ongoing to investigate the role of serum markers in the pathophysiology of PIH. One recent study, examines the levels of the anti-angiogenic factor soluble Fms-like tyrosine kinase-1 (sFlt-1), pro-angiogenic factor placental growth factor (PlGF), novel hormones like leptin and adiponectin in pregnant mothers recruited from Hospital Tuanku Jaafar, Seremban.

Among normotensive mothers, both sFlt-1 and PlGF levels were found to be increased throughout the 24-28 weeks and 28-32 weeks of pregnancy. When normotensive and PIH mothers were compared, the PIH mothers were found to have lower PlGF with higher sFlt-1 levels compared to normotensives. In approximately 40% of normotensives who were traced till delivery and developed PIH, this inversed pattern of marker levels was also noted. This study established sFlt-1/PlGF ratios in normotensive and PIH mothers may also be used to evaluate PIH (see Table 1). This ratio method, together with the inversed pattern of marker levels, was found to improve the overall detection of normotensive mothers who were at risk of developing PIH later in pregnancy.

Leptin levels were significantly increased in PIH mothers compared to normotensive mothers, but this difference became not significant when mothers were matched by BMI. Mothers who developed both PIH and GDM had elevated leptin levels compared to mothers who only developed PIH or GDM alone. Adiponectin levels indicated no significant difference in PIH mothers compared to normotensive mothers. We suggest that the high levels of leptin and adiponectin in PIH mothers may be the consequence of the high BMI and hence contribute to the pathophysiology of the signs and symptoms seen in PIH mothers. Leptin and adiponectin may not make good markers for PIH as they are influenced by BMI.

There are several other ongoing projects associated to PIH, including the role of auto immunity in PIH, morphometric analysis of PIH placenta and the role of emerging markers in PIH. More importantly, these projects are made possible, because of the good collaboration with the O&G Departments of both Hospital Tuanku Jaafar, Seremban, and Hospital Tunku Ampuan Rahimah, Klang. We thank both Dr Ravindran Jegasothy and Dr Farouk Abdullah for their continuous support.

	Gestational Weeks
	24-28 Weeks		28-32 Weeks
	N ormotensives	PIH	Normotensives	PIH
Mean sFlt-1 (pg/mL)	1,838.28	1,127.09	1,251.00	1,819.63
Mean P1GF (pg/mL)	1,183.00	653.73	1,449.94	522.53
Average Ratio	1.55	1.72	0.86	3.48

Table 1. General ratios of sFlt-1/PlGF for normotensive and PIH mothers*.

*PlGF and sFlt-1 values were determined by ELISA. Normotensive and PIH mothers were matched based on maternal age. For each sample, its sFlt-1 concentration was divided by the PlGF concentration to obtain a ratio of sFlt-1/PlGF.