Hui Yee Chee and Sazaly AbuBakar*

Department of Medical Microbiology, Universiti Malaya Medical Center Kuala Lumpur, Malaysia.

(Received 26 February, 1998 / Accepted 20 June 1998)

Abstract.
A recombinant single chain variable fragment (ScFv) of the 3H5-1 monoclonal antibody recognizing dengue 2 virus envelope (E) protein was constructed using a bacteriophage expression system. The variable heavy (V_H) and variable light (V_L) chain genes were amplified and subcloned into the pGEM-T PCR cloning vector. The nucleotide sequences were then determined. Based on the sequencing results, new sets of primers for amplification of the VH and VL genes were designed incorporating the linker peptide, Not 1, Sfi 1, BamH I restriction endonuclease sites and the Flag peptide sequences. A second RT-PCR was performed using the newly designed primers and the resulting V_H and V_L DNA fragments were cloned into a phagemid, pCANTAB 5E. Bacteriophages expressing the ScFv which recognized dengue 2 virus envelope protein were rescued following 'panning'. Two recombinant phage clones which showed significant binding were obtained and soluble forms of the 32 kDa ScFv were expressed following infection of the HB 2151 Escherichia coli.

Keywords: 3H5, dengue, envelope, phage, Scfv

Alexei F. Licea ^1,4, Maria C. Gutierrez ^2,4, Jose Esparza³, Sergio Estrada Parra³, Iris Estrada G3,
Fausto Quesada Pascual3 and Lourival D. Possani4*

¹Facultad de Ciencias Quimicas, Universidad Autonoma de Baja California. Calzada Tecnologico s/n Mesa de Otay.
Tijuana B. C. 22390, Mexico; ²Centro de Biotecnologia, Universidad Autonoma de Morelos, Av. Universidad s/n
Cuernavaca, Mor 62210 Mexico; ³Departamento de Inmunologia, Escuela Nacional de Ciencias Biologicas, Instituto
Politecnico Nacional, Prolongacion de Carpio y Plan de Ayala, Mexico RE 11340 Mexico; ⁴Departamento de
Reconorimiento Molecular y Bioestructura, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico
Apartado Postal 510 3, Cuernavaca, Mor 622 71 Mexico.

(Received 10 March 1998 / Accepted 19 October 1998)

Abstract.
Several proteins from Mycobacterium leprae have been identified as potent antigens. Segments corresponding to the primary stucture of these proteins were synthesized and used for the development of a skin test for leprosy (Gutierrez et al., 1994). Here we describe the synthesis of dimers and a multiple antigenic peptide (MAP) prepared by polymerization of single peptides, and we study the potential enhancement of immunogenicity of such polymerized peptides from various M.Leprae proteins: 65 kDa (P2, P15), 28 kDa (P5) and 18 kDa (P7). For the synthesis of the dimers, an extra cysteine was added either at the amino or carboxyl termini of peptides P2 and P7, whereas for P15 an extra cysteine was added only at the C terminus. Since P5 already had a cysteine at position 2 of the amino terminus no modification was needed. For dimer formation these peptides were air oxidized. Four different dimers were obtained for P2 and P7, and one dimer for each of P5 and P15. A MAP was prepared by combination of Pc2 and P15. From the seven peptides synthesized, only the Pc2 dimer was able to stimulate T cell proliferation in a specific manner. None of the other dimers nor the MAP per se were capable of inducing any type ofproliferation. All peptides synthesized, except the MAP, were also tested in a delayed type hypersensitivity (DTH) assay in M. leprae immunized guinea pigs. Only P7 dimers, Pc7 and P7c, showed an increased reactivity measured by means of DTH, when compared with the parent peptide (P7). Possible interpretations of these data are discussed.

Keywords: leprosy, multiple antigenic peptide (MAP), Mycobacterium leprae, synthetic peptide, T cell activation, mouse

Jorge Olmos ^1*, Alejandro Sanchez¹ and Ramon DeAnda²

¹Molecular Microbiology Laboratory. Centro de Investigacion Cientifica y de Educacion Superior de Ensenada (CICESE),
Ensenada, Mexico.
²Molecular Microbiology Department, Instituto de Bioteenologia, UNAM. Cuernavaca, Mexico.

(Received 21 September 1998 / Accepted 27 October 1998)

Abstract.
The aprE gene of B. subtilis codes for the major extracellular alkaline protease subtilisin. Its expression is controlled by AbrB and other regulators. To understand the regulation of AbrB on subtilisin expression, an aprE::lacZ fusion was examined for the effects induced by combinations of the abrB, hpr and spoOA9V mutations over the aprE expression. We found that during sporulation on the abrB genetic background, the specific activity level of BGal was higher than in the wild type strain. The exception was the abrB/spo0A9Vdouble mutant which had a negative effect on aprE expression.

Keywords: SpoOA9V, hpr, subtilisin, B. subtitis, aprE::lacZ, over production

Salmaan H. Inayat Hussain*, Gerald A Cohen' and Kelvin Cain'

Department of Biomedical Science, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.
'MRC Toxicology Unit, Centre for Mechanisms of Human Toxicity, Hodgkin Building, PO Box 138, Lancaster Rd., Leicester, LE1 9HN, UK

(Received 7 August 19981 Accepted 6 October 1998)

Abstract.
The characteristic DNA cleavage patterns observed in apoptotic nuclei in vivo can be reproduced in isolated nuclei where it has been shown that initially the DNA is degraded into 'large fragments'of >700, 200-300 and 30 -0 kilobase pairs (kbp) by a Mg²⁺ dependent process which is potentiated by Ca²⁺ . These large fragments are subsequently cleaved to mono/ oligonucleosomes by Ca²⁺/Mg²⁺ activated endonuclease(s). In this study, Mn²⁺ alone can substitute for Mg²⁺ and Ca²⁺, thereby activating both 'large fragment' formation and internucleosomal cleavage in isolated rat liver nuclei. The effect of Mn²⁺ is concentration dependent in that low concentrations (<200 µM) of Mn²⁺ stimulate 'large fragment' formation only whereas higher concentrations also activate internucleosomal cleavage. The cleavage of DNA induced by either Mn²⁺, Mg²⁺ or Ca²⁺/Mg²⁺ was equally susceptible to inhibition by Zn²⁺ , N ethylmaleimide and 3,4 dichloroisocoumarin, a serine protease inhibitor. These results suggest that the multi step DNA cleavage is catalysed by a single endonuclease or a class of endonucleases which have multicationic binding sites.

Keywords: apoptosis, DNA cleavage, liver nuclei, Mn²⁺