Iranian Biomedical Journal
Volume:6 Issue: 4, Oct 2002

The characteristics of cellular and molecular mechanisms associated with cell proliferation and differentiation is important to understand malignancy. In this report we characterise a leukemic model, D5A1, to study the action of differentiation agent, cellular events and gene expression of the selected transcription factors. Cells induced with 4 mM hexamethylene bisacetamide (HMBA) caused signs of erythroid differentiation (changes in morphology and size, haemoglobinisation) and cessation of proliferation including accumulation of cells in G0/G1. Treatment with HMBA caused a time-related decrease of tumorigenicity detectable by 48 hour. Northern-blotting showed induction of -amino levulinic acid synthase-erythroid (ALAS-E) mRNA at 48 hours and appeared in a strong level subsequently. C-myc (myelocytomatosis) and c-myb (myeloblastoma) mRNA levels decreased transiently in early hours returning to control values by 24 hour and decreased again. Stem cell leukaemia (SCL) and GATA-1 mRNA were markedly down regulated in early hours and then returned back. A later time point, upregulation of GATA-1 and SCL was relevant to maturation phenotype. These data provide a useful model to study the cellular and molecular events in leukomogenesis and action of differentiation therapy in leukaemia.

Despite the availability of various alkaline phosphatase (ALP) isoenzymes, the calf enzyme is being used in current enzyme assays as the detector enzyme. The glycosylation pattern of this enzyme makes it a suitable ligand for binding to wheat germ agglutinin lectin (WGA). As a result of this property, the enzyme can not be used as a conjugate with this lectin, and the calf enzyme conjugates can not be used when lectin is on the solid phase in lectin based ELISA systems. The purpose of this study was to evaluate the activity and lectin binding property of an ALP from the hepatopancreas of shrimp Pandalus borealis as an alternative for the calf enzyme. While the problem of non-specific binding to WGA lectin was circumvented with the shrimp enzyme, the activity of the new studied enzyme was seen to be higher than the calf enzyme. Studies of the effect of magnesium ion concentration on both enzymes demonstrated a major effect of the cation on shrimp enzyme and a relatively minor effect on the calf ALP. Conjugation of the shrimp ALP with streptavidin can be used in enzyme amplification technique in lectin based ELISA using WGA.

The affinity of low density lipoprotein (LDL) to its receptor is very important, because most of LDL-uptake pathway is done by the LDL receptor and the change in size of LDL particle and the modification in its components may affects the LDL affinity for its receptor. In this study, the effects of lipophilic agents such as vitamin E and seven volatile oils: anethol, eugenol, geraniol, limonene, linalool, pulegone and thymol have been investigated on the affinity of LDL to its receptor. LDL receptor was purified of bovine adrenal tissue. LDL was isolated by sequential density ultracentrifugation from normolipidemic human plasma. Then, LDL was labeled with fluoresein isothiocyanate (FITC) at 4°C for 24 h. Native LDL was incubated with various concentrations of each of the volatile oils and vitamin E for 2 h. Finally, the native LDL treated with volatile oils and vitamin E was incubatd with the LDL receptor in the presence of labeled-LDL at 37°C for 30 min. After incubation, the medium was centrifuged at 4000 ×g for 20 min and the fluorescence intensity (FI) of supernatant from each sample was determined at excitation 495 nm and emission 515 nm. The elevation of FI in each fraction demonstrates increasing the affinity of non-labeled-LDL to its receptor. We showed that vitamin E and volatile oils increased the affinity of LDL to its receptor, and among these compounds, vitamin E and thymol are the best agents that increase the affinity of native LDL to its receptor. The effects of these compounds are as follows: vitamin E > thymol > eugenol > anethol > geraniol > linalool > limonene > pulegone. These findings raise the possibility that vitamin E and some of the volatile oils may decrease the effect of LDL in formation of atherosclerotic lesions.

Apolipoprotein E (apo E) is a structural constituent of several serum lipoprotein classes. It plays an important role in lipid metabolism by acting as a ligand for low-density lipoprotein (LDL) and chylomicron remnant receptors. Three common alleles called e2, e3 and e4 have been described, which code for three protein isoforms (E2, E3 and E4). The polymorphism is clinically significant, and it has therefore become very important in epidemiological studies attempting to obtain complete information about apo E allele frequencies in populations around the world. In the present study, two point mutations coding amino acid residues 112 and 158 were amplified using the polymerase chain reaction (PCR) from DNA extracted from 100 Iranian subjects. Apo E genotypes were determined by restriction fragment length polymorphism (RFLP) and allelic frequencies were estimated by gene counting (e2 = 0.01, e3 = 0.88, e4 = 0.11). The Hardy-Weinberg expectation of genotype distribution was calculated from the estimated allele frequencies and a c2-test was used to test for equilibrium (c2 = 0.487, P>0.05). Student’s t-test indicated that subjects with the two most common apo E genotypes (E4/E4, E3/E3) were different from each other in means serum total cholesterol (t = 3.1, P<0.01). Allele frequencies of the present study performed on Iranian subjects were similar to those of Japanese, Korean (Asian groups) Mexican and African American populations.

The immune responses of mice immunized with ovalbumin (OVA) together with killed L. major (KLM) promastigotes as adjuvant were studied. Three doses (5 × 107, 1 × 108 and 2 × 108) of KLM combined with OVA (100 mg) were injected into the groups of C57BL/6 mice. BCG and complete Freund’s adjuvant (CFA) were used as control adjuvants. Lymphocyte proliferation and antibody titers were determined, and IFN-g and IL-4 were measured in the supernatants of lymph node cell cultures. Results showed that immunization using OVA mixed with KLM enhanced the in vitro proliferative response of T-cells to the antigen and resulted in the production of increased levels of IFN-g (2800-3700 pg/ml) relative to the mice injected with OVA alone (1750 pg/ml). In the mice receiving OVA + 5 × 107 KLM, the production of IL-4 remained lower (18, 20 pg/ml) than OVA alone (105, 109 pg/ml) and almost was similar to that of observed in mice inoculated with OVA + BCG, leading to high IFN-g/IL-4 ratios. Using higher doses of KLM (1 × 108), the IL-4 responses were of the same magnitude as or higher than the responses of mice inoculated with OVA + CFA. Antibody titers to OVA were also strongly boosted at the highest KLM dose. These findings indicate that KLM may function as an adjuvant, and its dose plays a role in the eventual outcome of the response. Inoculation of the mice with a low dose of KLM (5 × 107) tends to promote a Th1-type response.

Random peptide libraries (RPL) displayed on the surface of filamentous bacteriophages have been extensively used as a tool to map epitopes or to identify antigenic mimics (mimotpoes) of disease-specific monoclonal antibodies or polyclonal sera. These RPL are engineered by the insertion of degenerate oligonucleotides, encoding a specific number of random amino acids, in frame with a bacteriophage gene specifying a virion surface protein. The RPL are constructed by inserting 21 to 36 random nucleotides into the gene for an appropriate coat protein in a phage that is propagated in E. coli. Particularly, clear mimotopes have been obtained with monoclonal antibodies and with a few polyclonal antibodies against viruses. The choice and processing of sera for the selection of the disease related mimotopes and sera to remove mimotopes reacting with ubiquitous antibodies are important in studies of RPL. We have used RPL to select mimotopes of antigens to be used for immunization against dermatophilosis. IgG from sheep protected from dermatophilosis with an enzyme preparation from Dermatophilus congolensis, was used to select peptides displayed on phage in the Ph.D.-7 random peptide library which contains 109 peptides. Phage displaying peptides that were unrelated to the mimotopes associated with protection to dermatophilosis was removed with IgG from sheep that were vaccinated with the enzyme preparation but were not protected. The IgG from the protected sheep, before vaccination, was also used for the negative selection. The selected mimotopes, those with clearly repeating motifs, were chosen and used to immunize sheep. A mixture of four phage mimotopes induced antibody to a recombinant protease from D. congolensis. The immunized sheep recovered more rapidly from the lesions caused by the strains when challenged with two strains of D. congolensis.

Sodium salt of N-acetyl-DL-tryptophan (AT) and octanoic acid (caprylic acid CA) individually and in combination were studied as thermal stabilizer with different concentrations in heat treatment of human albumin solution. HPLC protein profiles before pasteurization of albumin showed less than 1% polymer. In free stabilizer albumin solution during pasteurization visible clot was formed. But by using variation of the above mentioned stabilizers, the amount of polymer was increased to about 4% during pasteurization (60° C, 10 h). By increasing the heat to the visible clot formation point, the most effective stabilizer against visible clot formation was found to be CA. However, in drug formulation it is important to choose the variety and concentration of stabilizers in accordance with regulations.

Cuminum cyminum Linn. (Umbelliferae) is a plant, which has been used as a toothache remedy in folk medicine of Iran. In this study, the potential anti-nociceptive and anti-inflammatory activities of the fruit essential oil of C. cyminum has been evaluated in chemical (formalin test) and thermal (tail-flick test) models of nociception and formalin model of acute inflammation in rats and mice. The essential oil at the doses ranging between 0.0125 and 0.20 ml/kg exhibited a significant and dose-dependent analgesic effect in the model of chronic and inflammatory pain. However, the essential oil was devoid of anti-inflammatory activity. Moreover, the essential oil had no analgesic effect in tail flick test as a model of acute pain. The LD50 value of 0.59 ml/kg was obtained for the essential oil. This low toxicity of the essential oil makes it worthy for further studies.