Chief Editor: Satya Nugroho. Associate Editors: Adi Santoso, Andi Utama, Wien Kusharyoto, Ines Atmosukarto, Bambang Sunarko. Editorial Assistant: Siti Elly Faisholyah. Cover: Wien Kusharyoto.
Editorial Board: Annales Bogorienses by Research Centre for Biotechnology, Indonesian Institute of Sciences (LlPI) JI.Raya Bogor Km 46, Cibinong 16911, Bogor, Indonesia. P.O. Box 422. Phone +62-21-8751527; Fax +62-21-8754588, Email: annales_bogorienses@yahoo.com.

Penerbit: LIPI Press, Anggota Ikapi, Jl. Gondangdia Lama (RP Suroso) No. 39 Jakarta 10350. Telp.: (021) 314–0228, 314–6942. Fax.: (021) 314–4591. Email: press@mail.lipi.go.id; bmrlipi@centrin.net.id; lipipress@centrin.net.id

Content

Investigation of Functional Difference Between TecIII and TecIV in Mammals COS-1 Cells Using GFP Fusion Proteins
Ines Atmosukarto & Grant W. Booker

Abstract
Signal transduction cascades are critical components of intra- and inter-cellular communication. Key component of such cascades includes tyrosine kinases. One such family of tyrosine kinases is the Tec family of tyrosine kinases. This family of tyrosine kinases is expressed mainly in cells of the hematopoietic lineage, and mutations in at least one of its one member of this family, Btk, has so far been associated with the human immunodeficiency disorder X-Linked Agammaglobulinemia. Two major isoforms of the Tec transcript, referred to as TecIII and TecIV, have been detected in various mouse embryonic and adult tissues, as well as in a number of different hematopoietic cell lines: Tec IV is the full length Tec protein with functional PH, TH, SH3, SH2 and Kinase domains, while TecIII is generated by the splicing out of exon 8 sequences to yield a shorter peptide with a non-functional SH3 domain. Using GFP-TecIII fusion proteins, this shorter isoform of Tec was shown to have biological characteristics that differed from TecIV.

Keywords: tyrosine kinase, Tec, GFP fusion


Identification of Degradation Pathway of Vinylacetate Using Bacterial Isolate V2 and Characterization of The Involved Enzymes
Bambang Sunarko, Nunik Sulistinah, Maria Nieder , Ortwin Meyer

Abstract
Vinyl acetate is a toxic substance, but has a high commercial value. In this study we show that vinyl acetate is subject to microbial degradation at rates of up to 6.38 and 1 mmol/h per g (dry weight) under aerobic and anaerobic conditions, respectively. It was hydrolyzed by bacterium V2 to ethanol, acetaldehyde and acetate. The enzymes involved in the metabolism of vinylacetate were vinyl acetate esterase, aldehyde dehydrogenase, and alcohol dehydrogenase, which localized in the cytoplasmic fraction. The Km values of vinyl acetate esterase and alcohol dehydrogenase were 6.13 mM and 0.24 mM, respectively. Vinyl acetate esterase hydrolyzed the ester to acetate and vinyl alcohol. The latter isomerized spontaneously to acetaldehyde and was then converted to acetate. The acetaldehyde was disproportionated into ethanol and acetate. The acetate was then converted to acetyl coenzyme A and oxidized through the tricarboxylic acid cycle and the glyoxylate bypass.

Key words: Vinylacetate, microbial degradation, vinylacetate-esterase, aldehyde dehydrogenase, alcohol dehydrogenase, bacterial isolate V2


Construction of a Recombinant Virus Between Poliovirus and Coxsackie a Virus 11
Andi Utama & Hiroyuki Shimizu

Abstract
Recent outbreaks of circulating vaccine-derived poliovirus (cVDPV) revealed the possibility of recombination between vaccine strains poliovirus (PV) and cluster C enterovirus. Based on genetic analysis, it is assumed that coxsackie A virus 11 (CAV-11), one of the cluster C enterovirus, may naturally recombine with PV. To elucidate this hypothesis, the chimeric virus between PJ156, a type 1 cVDPV isolate isolated from an acute flaccid paralysis (AFP) case in the Philippines in 2001, and CAV-11 (PJ156/CAV-11) was constructed by using long-PCR method. As the results, PJ156/CAV-11 was viable in HEp-2 cell line. The PJ156/CAV-11 exhibited mostly similar phenotype with parental PJ156 in term of plaque size, viral growth and neurovirulence. These results suggested that recombination between PV and CAV-11 might naturally occur during transmission of vaccine strains in the community. The effect of recombination on the viral phenotype is significantly depending on the counterpart virus.

Key words: Poliovirus, recombination, CAV-11


Subcloning, Expression and Characterisation of a Recombinant Antibody Fab-Fragment Specific Towards 2,4-D
Wien Kusharyoto

Abstract
A generic strategy was established for subcloning the VH and VL gene of antibody variable domains into the plasmid pASK85 for the expression of Fab antibody fragments. pASK85 bears coding sequences for murine constant domains including a His6-tag at the carboxy-terminal end of the constant heavy-chain domain. The VH and VL gene derived from the monoclonal antibody E2/B5 specific towards 2,4-dichlorophenoxyacetic acid (2,4-D) were used in this study. Eschericia coli was used as host cells for the biosynthesis of the Fab-fragment. The Fab-fragment was subsequently purified from the periplasmic extract in a single step by immobilised metal-ion affinity chromatography (IMAC). The production levels obtained were 0.5-0.8 mg purified Fab-fragments per liter E. coli culture. The sensitivity and cross-reactivity of the Fab-fragment determined by direct competitive ELISA were similar to those of the parental monoclonal antibody E2/B5.

Keywords: 2,4-D, antibody, competitive ELISA, Fab-fragment, IMAC


The Effect of Honey on Bacterial Growth, Protein Degradation, Amino Acids Contents and Volatile Compounds of Milks at Storage
Tatik Khusniati & Yantyati Widyastuti

Abstract
Pasteurized milks spoiled at refrigerated storage due to growth of psychrotrophic bacteria. Honey which contain antibacterial and aromatic compounds may be used as supplement to inhibit psychrotrophic bacteria’ activities. To know nutritional and flavor compounds of milks with and without honey, effect of honey on bacterial growth0protein degradation, amino acids and volatile compounds of stored milks were detected. Bacterial growth, protein degradation, amino acids and flavor compounds were detected by total plate counts, formol titration, HPLC and GCMS, respectively. The results show that bacterial growth and protein degradations in honey milks were lower than that without honey. Bacterial growth (5.2x103 - 9.3x106 cfu/mL ) and protein degradations (2.37-2.59% ) in honey whole milks were lower than that (6.2x104 - 6.5x107 cfu/mL)( 2.54-2.88%) in skim milks, respectively (P<0.05). At 10 days after use by date, changing between amino acids’ contents in whole milks with and without honey were more significant than that of skim milks (P<0.05);. and volatile compounds’ percentages in honey whole milks were higher than that without honey, while that in honey skim milks vice versa. Honey caused decreasing bacterial growth and protein degradation, changing amino acids’ contents and producing volatile compounds of stored milks, and honey whole milks were better than honey skim milks.

Keywords: pasteurized milks, skim, honey, protein, amino acids, volatile compounds