Biotecnologia Aplicada, vol. 16, no. 3, 1999

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BIOTECNOLOGIA APLICADA
CC Vol. 16 No. 3 (July-September, 1999)
CC ISSN 0864-4551 (printed)
CC ISSN 1027-2852 (electronic)
CC Copyright 1999, Elfos Scientiae.

CC Founded in 1983 and published as Interferon y
CC Biotecnologia until 1990, Biotecnologia Aplicada is a
CC peer-reviewed scientific journal. It appears four times
CC per year, sponsored by the Ibero-Latin-American Society
CC of Biotechnology Applied to Health.

CC BIOTECNOLOGIA APLICADA is a vehicle of Iberian-Latin-
CC American scientists working in different fields of modern
CC biotechnology for the diffusion of their experiences and
CC results. Some of the main topics that are considered for
CC publication are the following:
CC a) Molecular genetics and recombinant DNA technology;
CC b) Therapeutic and prophylactic uses of molecules
CC obtained by biotechnological methods;
CC c) Immunotechnology and development of diagnostic
CC methods;
CC d) Chemistry, biochemistry and design of molecules with
CC biological activity;
CC e) Application of recombinant DNA techniques and its
CC products in agriculture and animal science;
CC f) Transgenesis;
CC g) Fermentation, recovery and purification of biotech-
CC nological products;
CC h) Scaling-up, plant design and economic evaluation;
CC i) Control and automation of processes.

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CC Correspondence and subscriptions
CC Ave. 31 entre 158 y 190,
CC Cubanacan, Playa
CC Ciudad de La Habana,
CC Apdo. 6072, Habana 6, Cuba.
CC Telephones: (53-7) 33 1917 / 21 8466 / 21 8164
CC Fax: (53-7) 33 1917 / 21 8070 / 33 6008
CC E-mail: el...@cigb.edu.cu

CC Full papers are available online through BIOLINE System
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AU Peter Lindblad
IN Department of Physiological Botany, Uppsala University,
Villavägen 6, S-752 36 Uppsala, Sweden.
Phone/Fax: +46-18 471 28 26;
E-mail: Peter.L...@Fysbot.uu.se
TI Cyanobacterial H2 Metabolism: Knowledge and
Potential/Strategies for a Photobiotechnological
Production of H2
DE biotechnology
DE cyanobacteria
DE H2 evolution/uptake
DE hydrogenase
DE Nostoc
SO Biotecnologia Aplicada 1999;16:141-144
AB Molecular hydrogen is an environmentally clean source of
energy that may be a valuable alternative to the limited
fossil fuel resources of today. For photobiological H2
production, cyanobacteria are among the ideal candidates
since they have the simplest nutritional requirements:
they can grow in air (N2 and CO2), water (electrons and
reducing agents), and simple mineral salts with light as
the only source of energy. In N2-fixing cyanobacteria, H2
is mainly produced by nitrogenases, but its partial
consumption is quickly catalyzed by a unidirectional
uptake hydrogenase. In addition, a bidirectional
(reversible) enzyme may also oxidize some of the molecular
hydrogen. Filamentous cyanobacteria have been used in
bioreactors for the photobiological conversion of H2O to
H2. However, the conversion efficiencies achieved are low
because the net H2 production is the result of H2 evolution
via a nitrogenase and H2 consumption mainly via an uptake
hydrogenase. Consequently, the improvements of the
conversion efficiency are achieved through the
optimization of the conditions for H2 evolution by the
nitrogenase and through the production of mutants
deficient in H2 uptake activity. Symbiotic cells are of
fundamental interest since they in situ "function as a
bioreactor", possess a high metabolism and there is
transfer of metabolite(s) from symbiont to host, but
almost no growth. This communication presents the general
knowledge about hydrogen metabolism/hydrogenases in
filamentous cyanobacteria, outline strategies for
improving the capacity of H2 production by filamentous
strains, and stresses the importance of international
cooperations and networks.
CC Review article
CC Language: English

AU Juan M Rivera, Maria del Carmen Dominguez, Maira Ponce,
Ramon E Narciandi
IN Division de Inmunotecnologia y Diagnostico. Centro de
Ingenieria Genetica y Biotecnologia. AP 6162, CP 10600,
Ciudad de La Habana, Cuba. Telf: (53-7) 21 3313, 21 8164;
Fax: (53-7) 33 6008, 21 8070; E-mail: vlad...@cigb.edu.cu
TI Production of Recombinant Treponema pallidum Membrane
Protein A in Escherichia coli Using Fed-batch Fermentation
DE Escherichia coli
DE fed-batch fermentation
DE recombinant protein
DE TmpA
SO Biotecnologia Aplicada 1999;16:145-148
AB There is special interest on the 42-kDa Treponema pallidum
membrane protein A (TmpA) due to its potential use for
syphilis serological assay. Different genetic
constructions and cloning in bacteria of recombinant DNA
containing the synthetic TmpA gene have been reported, but
in all cases the expression levels and fermentation
productivity have been affected by different factors and,
on the other hand, its purification has involved a series
of laborious steps. A fed-batch culture of Escherichia
coli W3110 capable of achieving high expression levels
(36%) and high production yields (1.74 g/L) of recombinant
TmpA was developed using the expression vector pR2M6.
Different host strains and induction conditions were
studied in order to maximize protein expression. In this
study, a rapid and simple procedure for the purification
of recombinant TmpA to over 85% of purity and about 60% of
recovery is reported using immobilized metal ion affinity
chromatography.
CC Research article
CC Language: English

AU Diana S Leal-Klevezas, Alberto Barbabosa-Pliego, Marcial
Flores-Trujillo, Ahide Lopez-Merino, Juan P Martinez-
Soriano
IN Centro de Investigacion Biomedica del Noreste. Instituto
Mexicano de Seguro Social. San Luis Potosi y 2 de abril,
Colonia Independencia, 64720 Monterrey, NL, Mexico.
Telf: (52-8) 343 1251; Fax: (52-8) 344 4116;
E-mail: dsl...@academ01.mty.itesm.mx
Universidad Autonoma del Estado de Mexico.
Escuela Nacional de Ciencias Biologicas del Instituto
Politecnico Nacional. 4Centro de Investigaciones y de
Estudios Avanzados.
TI Epidemiologia molecular de un foco primario de brucelosis
en el Estado de Mexico
DE brucellosis
DE epidemiology
DE molecular diagnosis
DE PCR
DE polymerase chain reaction
SO Biotecnologia Aplicada 1999;16:149-153
AB A polymerase chain reaction (PCR) assay was used to
diagnose and analyze a brucelosis outbreak. In this study
Brucella spp. were detected in humans by serological
means (rose Bengal, buffered acidified antigen, rivanol
and complement fixation) and were confirmed by direct and
semi-nested PCR. These procedures were also used on farm
animals, demonstrating the presence of this pathogen in
bovines, ovines, swines, and canines. The molecular
diagnosis was also confirmed by the isolation and
typification of Brucella abortus as the pathogen
associated with the outbreak. Herein it is discussed that
the initial infection likely started in bovines and spread
horizontally to the other species. PCR could be considered
as a concluding test for the "molecular isolation of
Brucella", since the pathogen can be efficiently detected
by such procedures in early or latent infections of
symptomless animals or humans.
CC Research Article
CC Language: Spanish

AU Hector Santana, Eduardo Martinez, Julio C Sanchez, Galina
Moya, Raudel Sosa, Eugenio Hardy, Alejandro Beldarrain,
Vivian Huerta, Luis J Gonzalez, Lazaro Betancourt,
Vladimir Besada, Rudka Brito, Tania Curras, Joel Ferrero,
Vivian Pujols, Miriela Gil, Luis Herrera
IN Divisions of Production, Quality Control, Physical-
Chemistry. Center for Genetic Engineering and
Biotechnology, PO Box 6162, Havana 10600, Cuba.
Fax: (53-7) 21 8070, 33 6008; E-mail: Santana@.cigb.edu.cu
TI Molecular Characterization of Recombinant Human
Interferon Alpha-2b Produced in Cuba
DE IFN
DE molecular characterization
DE recombinant human interferon alfa 2b
SO Biotecnologia Aplicada 1999;16:154-159
AB The recombinant human interferon a 2b (IFN-a2b) produced by
Cuban technology is obtained from Escherichia coli. This
is the active principle of the product registered in Cuba
as Heberon alfa R® trademark (IFN-a2b, CIGB, Havana), which
has been successfully used worldwide for the therapy of
several viral diseases and neoplasms. Here we describe the
purity and identity tests used for its molecular
characterization. The data show a product with a well-
established identity, a high purity and a specific
activity higher than 1.4 x 108 IU/mg of proteins. We also
compared the final preparation with other IFN-a2 products
available in the international market. It behaved very
similar to Intron A® and Roferon A®, and showed a higher
homogeneity when compared with Bioferon® and Interimmun®.
CC Research Article
CC Language: English

AU Angela E Sosa Espinosa, Jorge Sotolongo Peña,
Vladimir Martinez Santana, Rossana Garcia Fernandez,
Juan Morales Grillo
IN Subdireccion de Regulaciones, Division de Formulacion y
Envase, Division de Desarrollo, Division de Bioindusrtia
Division de Vacunas. Centro de Ingenieria Genetica y
Biotecnologia. AP 6162, Ciudad de La Habana, Cuba.
E-mail: ang...@cigb.edu.cu
TI Comparacion de la quimiosina bovina secretada al medio de
cultivo por cepas recombinantes de las levaduras Pichia
pastoris y Kluyveromyces lactis
DE Kluyveromyces lactis
DE Pichia pastoris
DE recombinant chymosin
SO Biotecnologia Aplicada 1999;16:160-164
AB Bovine chymosin, an aspartic protease that is used for
the industrial production of cheese, is extracted from
the stomachs of calves. To be obtaines from another
source, chymosin has been cloned in different
microorganisms. However, the post-translational
modificatons of the recombinant enzyme that frequently
occur in the host cells may change its biochemical
properties. In this work, the properties of a raw material
wich contains the bovine chymosin produced in recombinant
strains of the yeasts Pichia pastoris and Kluyveromyces
lactis are studied. Recombinant chymosin glycosilation was
analyzed by the periodic acid-Schiff reaction on
polyacrylamide slab gels. Besides, the effect of several
parameters (the molar concentration of Ca2+ ions, pH,
temperature) on the chymosin coagulant activity and the
proteolytic activity and the termal stability of the
enzyme in solution, were studied. In comparison with the
yeast K. lactis, it was observed that P. pastoris can be a
better host for the expression of chymosin, since the
properties of the P. pastoris-derived enzyme seemed to be
closer to those of the natural enzyme.
CC Research Article
CC Language: Spanish

AU Jorge Aguiar, Maria Vazquez, Julio Fernandez, Yssel
Mendoza, George Auburger
IN Division of Pharmaceutics. Center for Genetic Engineering
and Biotechnology. PO Box 6162, Cubanacan, Havana 10600,
Cuba. Phone: (53-7) 21 8008; Fax: (53-7) 21 8070;
E-mail: far...@cigb.edu.cu
Division of Neurology. University Hospital, D-40225
Duesseldorf, Germany.
TI Cloning and Sequencing of the 5' Region from Human
Spinocerebellar Ataxia 2 Gene
DE CpG island
DE disease
DE spinocerebellar ataxia 2
DE transgenic mouse
SO Biotecnologia Aplicada 1999;16:165-168
AB A restriction fragment containing the 5' region of the
spinocerebellar ataxia type 2 (SCA2) gene was subcloned
from PAC65i22 into pBluescript and totally sequenced using
an ABI 373A automated sequencer, in combination with the
direct sequencing of the mentioned PAC genomic clone
through the DOP-vector PCR method. A typical CpG island
with several potential sites for Sp1 transcription factor
has been found, where the promoter activity of the SCA2
gene can be situated, maybe like it occurs with the gene
for Huntington disease.
CC Research Article
CC Language: English

AU Javier E Vazquez, Freya Freyre, Marta Ayala, Leonardo
Canaan-Haden, Hanssel Bell, Jorge V Gavilondo
IN Division of Immunotechnology and Diagnostics, Division of
Physical Chemistry. Center for Genetic Engineering and
Biotechnology. PO Box 6162, Havana 10600, Cuba.
E-mail: Jorge.G...@cigb.edu.cu
TI Single-chain Fv Antibody Fragment as a Cytoplasmic Protein
in Escherichia coli
DE antibody fragments
DE carcinoembryonic antigen
DE CEA
DE E. coli
DE scFv
SO Biotecnologia Aplicada 1999;16:169-172
AB In this paper, a strategy to increase expression in
Escherichia coli and simplify the purification of a
single-chain Fv (scFv) antibody fragment specific for the
carcinoembryonic antigen (CEA), is reported. The anti-CEA
scFv fragment was expressed as an insoluble fusion protein
in the cytoplasm of E. coli, with an N-terminal domain
related in part with the first amino acids of human
interleukin 2, and a C-terminal hexahistidine tag. The
scFv fragment was extracted by solubilization, purified by
immobilized metal ion affinity chromatography and refolded
in a slightly alkaline buffer. With this system, the yield
of active scFv increased four-fold when compared to a
periplasmic variant of the same scFv, previously produced
by the authors.
CC Paper on Techniques
CC Language: English

AU Rolando Ochoa Azze, Mayte Nerey Olivares, Juan C Martinez
Rodriguez
IN DACTA. Instituto Finlay. Ave 27 No. 19805. AP 16017,
CP 11600. La Lisa, Ciudad de La Habana, Cuba.
Fax: (53-7) 33 6754; E-mail: oc...@finlay.edu.cu
TI Use of Poly-L-lysine Precoating in an ELISA for the
Detection of Antibodies against Serogroup C Neisseria
meningitidis Capsular Polysaccharide
DE ELISA for anti-polysaccharide C antibodies
DE poly-L-lysine
DE poly-L-lysine precoating
DE serogroup C Neisseria meningitidis
SO Biotecnologia Aplicada 1999;16:173-175
AB Precoating with poly-L-lysine was analyzed in an ELISA
used for the quantitative determination of antibodies
against serogroup C Neisseria meningitidis capsular
polysaccharide. Different coatings were analyzed: purified
capsular polysaccharide of serogroup C N. meningitidis did
not attach well to polystyrene, but precoating with poly-
L-lysine and the use of methylated human serum albumin
attached to polysaccharide had a good performance. Cross
reactivity to poly-L-lysine was not detected in 48 adults
immunized with a divalent (serogroups B and C)
meningococcal vaccine (VA-MENGOC-BC®). Seroconversion and
seroresponse were detected in 89.58 and 97.92% of the
vaccinated individuals, respectively.
CC Article on Techniques
CC Language: Spanish

AU Alejandro Moro, Andrea Calixto, Eduardo Suarez, Manuel de
J Araña, Silvio E Perea
IN Centro de Ingenieria Genetica y Biotecnologia. AP 6162,
La Habana 10600, Cuba. Fax: (53-7) 33 6008;
E-mail: far...@cigb.edu.cu Centro de
Inmunologia Molecular, La Habana, Cuba.
TI La oncoproteina E7 del virus del papiloma humano tipo 16
(VPH 16) es capaz de inducir resistencia al efecto
citostatico del IFN-a2b
SO Biotecnologia Aplicada 1999;16:177-178
CC Report
CC Language: Spanish

AU Jose A Cremata, Omar Quintero, Raquel Montesino,
Rossana Garcia
IN Laboratorio de Glicoproteinas. Division de Bioindustrias.
Centro de Ingenieria Genetica y Biotecnologia, AP 6162,
Ciudad de La Habana, Cuba. Fax: (53-7) 21 8070;
E-mail: glyc...@cigb.edu.cu
TI Desarrollo de una nueva metodologia para la
caracterizacion estructural de la N-glicosilacion de
proteinas naturales o recombinantes
SO Biotecnologia Aplicada 1999;16:178-180
CC Report
CC Language: Spanish

AU Yamila Martinez Zubiaur, Pedro L Ramos Gonzalez,
Rudy Peral, Yenne Marrero, Madelaine Quiñones,
Ramon G Guevara, Carmen de Blas Beorlegui, Orlene Guerra
Peraza, Esther L Peralta, Pedro Oramas Frenes,
Rafael Rivera Bustamante
IN Centro Nacional de Sanidad Agropecuaria. AP 10, San Jose
de las Lajas, La Habana, Cuba. Fax: 64 63897;
E-mail: je...@censa.cu
Centro de Ingenieria Genetica y Biotecnologia. AP 6162,
Playa, Ciudad de La Habana, Cuba. Fax: 21 7080, 33 6008;
E-mail: virp...@cigb.edu.cu
Centro de Investigacion y Estudios Avanzados del IPN.
Irapuato, Mexico.
Instituto Nacional de Investigaciones Agrarias. Madrid,
España.
TI Identificacion y caracterizacion molecular de geminivirus
bipartitos en Cuba
SO Biotecnologia Aplicada 1999;16:180-181
CC Report
CC Language: Spanish

AU Lilian M Mederos Cuervo, Pedro L Valero-Guillen,
Jose A Valdivia Alvarez
IN Instituto de Medicina Tropical "Pedro Kouri". AP 601,
Marianao 13, Ciudad de La Habana, Cuba.
Fax: (53-7) 24 6051; E-mail: med...@ipk.sld.cu
Universidad de Murcia. Facultad de Medicina. Murcia,
España.
TI Aportes quimiotaxonomicos al estudio del Mycobacterium
habana, especie cubana candidato vacunal
SO Biotecnologia Aplicada 1 999;16:181-182
CC Report
CC Language: Spanish

AU Rafael Perez Cristia, Pedro Fleites Mestre, Tomas Verdura
Barrios, Jacques Barnouin, Michelle Chassagne, Josiane Arnaud
IN Centro Nacional de Toxicologia. 2Instuto Finlay, Ciudad de
La Habana, Cuba. Unidad de Ecopatologia, INRA de Theix,
Francia. Laboratorio de Biologia del Estres Oxidativo,
Universidad "J. Fourier", Grenoble, Francia.
TI Factores toxico-nutricionales y neuropatia epidemica.
Programa de investigaciones "SECUBA"
SO Biotecnologia Aplicada 1999;16:182-184
CC Report
CC Language: Spanish

AU Gil A Enriquez-Obregon, Roberto I Vazquez-Padron,
Ariel D Arencibia, Dmitri Prieto-Sansonov, Elva R Carmona,
Pilar Tellez-Rodriguez, Gustavo A de la Riva,
Luis E Trujillo, Guillermo Selman-Houssein, Pedro Oramas,
Fidel Hernandez, Dongo Blanco, Marlen Perez, Ariel Cruz
IN Centro de Ingenieria Genetica y Biotecnologia. AP 6162,
La Habana, Cuba. Fax: (53-7) 21 4764;
E-mail: gustav...@cigb.edu.cu.
EPICA de Jovellanos, Matanzas, Cuba.
TI Obtencion de plantas transgenicas de caña de azucar
(Saccharum officinarum L) mediada por Agrobacterium
tumefaciens: una nueva metodologia para la transformacion
genetica de esta graminea
SO Biotecnologia Aplicada 1999;16:184-186
CC Report
CC Language: Spanish

AU Maria T Cornide, Orlando Coto, Eduardo Canales,
Antonio Sigarroa, Jesus E Sanchez, Miguel Ramos Leal,
Hector Leonard Olivares, Florencio de Prada Esquivel,
Ariel Arencibia, Dania Calvo, Gelasio Perez Oramas,
Jose M Mesa, Ernesto Fernandez, Roberto Peña Urrutia
IN Dpto. de Biotecnologia de las Plantas. Centro Nacional de
Investigaciones Cientificas. Ave 25 y 158, Cubanacan,
AP 6990. Ciudad de La Habana. Cuba. Fax: (53-7) 33 0497;
E-mail: bla...@biocnic.cneuro.edu.cu
TI Desarrollo de las aplicaciones de los marcadores
moleculares en el mejoramiento de la caña de azucar en
Cuba
SO Biotecnologia Aplicada 1999;16:186-190
CC Report
CC Language: Spanish

AU Maria T Gonzalez-Arnao, Caridad Urra, Marcos Martinez,
Maria de los Angeles Torres, Manuel Martinez,
Tomas Moreira, Rodobaldo Ortiz
IN Dpto. Biologia Vegetal. Facultad de Biologia. Universidad
de La Habana. Calle 25 No. 455, entre I y J, Vedado,
Ciudad de La Habana, Cuba. Fax: (53-7) 32 1321;
E-mail: ar...@fbio.oc.uh.cu
Centro Nacional de Investigaciones Cientificas. Ciudad de
La Habana, Cuba.
Centro de Bioplantas. Universidad de Ciego de Avila.
Ciego de Avila, Cuba.
Instituto Nacional de Investigaciones Fundamentales de la
Agricultura Tropical. La Habana, Cuba.
Instituto Nacional de Ciencias Agricolas. La Habana, Cuba.
TI Desarrollo de la crioconservacion de germoplasma
vegetal en Cuba
SO Biotecnologia Aplicada 1999;16:190-191
CC Report
CC Language: Spanish

AU Cristina M Mateo de Acosta del Rio, Josefa Lombardero
Valladares, Ernesto Moreno, Rolando Perez Rodriguez
IN Centro de Inmunologia Molecular. Calle 206 No. 1926,
Cubanacan, Playa, Ciudad de La Habana.
Fax: (53-7) 33 5049; E-mail: cris...@ict.cim.sld.cu
TI Humanizacion de epitopos T en las regiones variables
murinas de un anticuerpo que reconoce al receptor del
factor de crecimiento epidermico: obtencion de
inmunoglobulinas de menor inmunogenicidad
SO Biotecnologia Aplicada 1999;16:191-193
CC Report
CC Language: Spanish

AU Eli E Sercarz
IN Division of Immune Regulation, La Jolla Institute of
Allergy and Immunology. La Jolla, California 92121.
Phone: (619) 558-3505; Fax: (619) 678-4595;
E-mail: e...@liai.org
TI Why Autoimmunity?
SO Biotecnologia Aplicada 1999;16:195-196
CC Focus
CC Language: English