Deteksi Brucella abortus dari Sampel Darah-Utuh dengan Uji Polymerase Chain Reaction Tanpa Ekstraksi DNA

Abstract              Brucellosis is a zoonotic disease that cause a significant economic losses for cattle industries worldwide. A rapid, precise and accurate diagnosis technique for diagnosis of brucellosis in all stages of the infection is definitely required.  Blood-samples are widely used for PCR-based DNA analysis because they are easily collected, handled, and processed. Direct PCR analysis without DNA extraction has been attempted to reduce time and  costs for routine analysis. This approach is promising but is still limited by the presence of PCR inhibitors that is naturally found  in the blood samples. The objective of this study was to compare the effectivity of direct PCR technique with or without DNA extraction for detection of Brucella abortus in the blood samples. Three whole-blood samples from brucella infected dairy cattle and five whole-blood samples  from beef cattle that having abortion were used as samples in this study. A pair of  bcsp31 primers and IS711 primers were used for amplification of genus-specific and species-specific of Brucella.  The results showed that amplicon in the position of 223 bp and 498 bp that are specific for B. abortus were detected from all of the samples that were analyzed on 1.5% agarose gels. Based on the result it could be concluded that direct PCR analyses without DNA extraction is a sensitive, specific, simple, rapid  and inexpensive assay for detecting B. abortus in the whole blood samples for either dairy or beef cattle and therefore it could  improve the existing surveillance and control programs for brucellosis. Keywords : brucellosis; direct PCR; PCR inhibitor; whole-blood sample; without DNA extraction                           Abstrak              Brucellosis adalah penyakit zoonosis yang menyebabkan kerugian ekonomi yang signifikan bagi industri ternak di seluruh dunia. Teknik diagnosis yang cepat, tepat dan akurat yang dapat digunakan untuk diagnosis brucellosis pada semua tahap infeksi sangat diperlukan. Sampel darah banyak digunakan untuk analisis PCR berbasis DNA karena mudah untuk dikoleksi, ditangani, dan diproses. Metoda PCR langsung tanpa didahului dengan ekstraksi DNA dikembangkan dengan tujuan penghematan waktu dan beaya untuk analisa secara rutin. Tehnik ini sangat menjanjikan tetapi memiliki keterbatasan karena adanya senyawa penghambat PCR yang secara alami terkandung di dalam sampel darah . Tujuan dari penelitian ini adalah membandingkan efektifitas antara uji PCR secara langsung dengan ekstraksi dan tanpa ekstraksi DNA untuk deteksi Brucella abortus di dalam darah. Tiga ( 3 ) sampel darah-EDTA yang berasal dari  sapi penderita brucellosis dan 5 sampel darah-EDTA dari sapi potong yang mengalami abortus digunakan sebagai sampel dalam penelitian ini. Pasangan primer bcsp31 dan primer IS711 untuk amplifikasi gen dan species specific digunakan dalam penelitian. Hasil menunjukkan bahwa amplikon/pita pada posisi 223 bp dan 498 bp yang spesifik untuk Brucella abortus terdeteksi dari semua sampel yang dianalisa dengan gel agarosa 1,5%. Berdasarkan hasil penelitian dapat disimpulkan bahwa uji PCR secara langsung tanpa didahului dengan ekstraksi DNA merupakan tehnik yang sensitif, spesifik, sederhana, cepat dan murah untuk deteksi B. abortus di dalam sampel darah baik sapi perah maupun sapi potong dan oleh karena itu diharapkan dapat digunakan untuk memperbaiki program kontrol dan survailance yang telah ada untuk brucellosis. Kata kunci : brucellosis; PCR langsung; penghambat PCR; sampel darah-utuh; tanpa ekstraksi DN

Adaptation to ER Stress Is Mediated by Differential Stabilities of Pro-Survival and Pro-Apoptotic mRNAs and Proteins

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling cascade known as the unfolded protein response (UPR). Although activation of the UPR is well described, there is little sense of how the response, which initiates both apoptotic and adaptive pathways, can selectively allow for adaptation. Here we describe the reconstitution of an adaptive ER stress response in a cell culture system. Monitoring the activation and maintenance of representative UPR gene expression pathways that facilitate either adaptation or apoptosis, we demonstrate that mild ER stress activates all UPR sensors. However, survival is favored during mild stress as a consequence of the intrinsic instabilities of mRNAs and proteins that promote apoptosis compared to those that facilitate protein folding and adaptation. As a consequence, the expression of apoptotic proteins is short-lived as cells adapt to stress. We provide evidence that the selective persistence of ER chaperone expression is also applicable to at least one instance of genetic ER stress. This work provides new insight into how a stress response pathway can be structured to allow cells to avert death as they adapt. It underscores the contribution of posttranscriptional and posttranslational mechanisms in influencing this outcome

Perception of the risks and benefits of bt eggplant by Indian farmers

Several researchers—most notably Lennart Sjoberg and his colleagues—have proposed that the moral aspects of risk provide a better explanation of risk perception than the psychometric paradigm or Cultural Theory, neither of which accounts for moral concerns. This study is possibly the first to assess empirically the perception of the risks and benefits of a transgenic food crop—transgenic Bt (Bacillus thuringiensis) eggplant—by farmers in a developing country such as India. It also aims to assess if the moral aspects of risk figure in Indian farmers ’ perception of Bt eggplant and if economic benefits outweigh perceived risks. To answer the research questions, a scenario was used to elicit perceptions of Bt eggplant among 100 eggplant farmers in the state of Maharashtra in India. The findings indicate that economic benefits, safety concerns, and accountability are most salient to Indian farmers’ perception of the risks and benefits of Bt eggplant. Significantly, none of the farmers mentioned moral concerns as an issue. The findings also make clear that economic benefits outweigh perceived risks. This study concludes that economic benefits are more salient than moral concerns to Indian farmers ’ perception Bt eggplant. It also proposes that an alternative theoretical model incorporating economic benefits, safety concerns, and accountability as key variables should be developed and tested for end users in the developing world. KEY WORDS: risk perception, biotechnology, developing countries, farmers 1

Signal recognition particle-dependent targeting of ribosomes to the rough endoplasmic reticulum in the absence and presence of the nascent polypeptide-associated complex

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex

Interaction Between Ribosome-Nascent Chain and sec61 Complexes and Their Role in the Translocation of Proteins Across the Endoplasmic Reticulum Membrane: a Thesis

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) respectively act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the non-selective interaction between the ribosome and the Sec61 complex. The Sec61 complex, serving as both the high affinity ribosome receptor and the translocation channel, is performing two very different functions which presumably requires different activity domains within the Sec61 complex. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the N and C-terminus of Sec61α was used to map the location of proteolysis cleavage sites. We observed a striking correlation between a loss of ribosome binding activity and the digestion of the C-terminal tail or cytoplasmic loop 8 of Sec61α. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether ribosome binding to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind ribosomes with high affinity at physiological ionic strength remain active in SRP-independent translocation indicating that ribosome binding and translocation promotion are separable activities of the Sec61 complex. Translocation promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex

RANCANG BANGUN PENGAWASAN KONDISI HULU SUNGAI UNTUK PENANGGULANGAN DAMPAK BANJIR

Bencana banjir adalah salah satu bencana yang menimbulkan kerugian terbesar di Indonesia baik secara materil maupun non materil. Banjir seringkali disebabkan karena berlebihnya aliran air terhadap daya tampung air pada hulu sungai. Maka dibutuhkan suatu sistem yang dapat melakukan pengawasan terhadap hulu sungai. Adapun parameter yang digunakan dalam penelitian ini mencakup curah hujan, kecepatan air, dan ketinggian air. Berdasarkan hasil  penelitian ini menghasilkan suatu sistem yang dapat mengirimkan data curah hujan, kecepatan air, dan ketinggian air selama 10 hari pada lingkungan hulu sungai menggunakan catu daya power bank berkapasitas 5000 mAh. Kata Kunci : pengawasan hulu sungai, pengiriman data curah hujan, kecepatan air, dan ketinggian air   ABSTRACT Floods in Indonesia is one of the higly loss disaster, usually caused  by water flows that exceed headstream capacity.  Therefore a monitoring system for headstream condition is required. Parameters used in this research are rainfalls, water flow speed, and water level. Based on the result analysis, the system can transmit data for ten days in headstream environment only using 5000 mAh of power bank as power source. Keyword : headstream monitoring, rainfalls, water flow speed, and water level data transmissio

RANCANG BANGUN PENGAWASAN KONDISI HULU SUNGAI UNTUK PENANGGULANGAN DAMPAK BANJIR

Bencana banjir adalah salah satu bencana yang menimbulkan kerugian terbesar di Indonesia baik secara materil maupun non materil. Banjir seringkali disebabkan karena berlebihnya aliran air terhadap daya tampung air pada hulu sungai. Maka dibutuhkan suatu sistem yang dapat melakukan pengawasan terhadap hulu sungai. Adapun parameter yang digunakan dalam penelitian ini mencakup curah hujan, kecepatan air, dan ketinggian air. Berdasarkan hasil  penelitian ini menghasilkan suatu sistem yang dapat mengirimkan data curah hujan, kecepatan air, dan ketinggian air selama 10 hari pada lingkungan hulu sungai menggunakan catu daya power bank berkapasitas 5000 mAh. Kata Kunci : pengawasan hulu sungai, pengiriman data curah hujan, kecepatan air, dan ketinggian air   ABSTRACT Floods in Indonesia is one of the higly loss disaster, usually caused  by water flows that exceed headstream capacity.  Therefore a monitoring system for headstream condition is required. Parameters used in this research are rainfalls, water flow speed, and water level. Based on the result analysis, the system can transmit data for ten days in headstream environment only using 5000 mAh of power bank as power source. Keyword : headstream monitoring, rainfalls, water flow speed, and water level data transmissio

Mitogen-activated protein kinase stimulation by a tyrosine kinase-negative epidermal growth factor receptor

Mutation of the epidermal growth factor receptor (EGF-R) within the ATP binding subdomain results in a receptor that lacks tyrosine kinase activity and is defective in signal transduction. However, this kinase-negative EGF-R is able to activate MAP kinase (Campos-Gonzalez, R., and Glenny, J. R. (1992) J. Biol. Chem. 267, 14535-14538). This observation suggests that signal initiation by the EGF-R can occur by a mechanism that is independent of the receptor tyrosine kinase activity. Here, we report that the kinase-negative EGF-R is phosphorylated on tyrosine in EGF-treated cells. The mechanism of tyrosine phosphorylation can be accounted for by the action of EGF to stimulate a protein kinase activity that is associated with the kinase-negative EGF-R. This protein kinase activity is not intrinsic to the receptor and can be separated from the EGF-R by incubation with 0.5 M NaCl. MAP kinase activation by the kinase-negative EGF-R may therefore occur by a mechanism that requires a receptor-associated tyrosine kinase. Thus, it is unnecessary to propose a novel kinase-independent mechanism of signal initiation to account for MAP kinase activation by the kinase-negative EGF-R

Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases

Two epidermal growth factor-stimulated protein kinases that correspond to ERK1 and ERK2 have been purified from human epidermoid carcinoma cells (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). A consensus primary sequence for substrates of ERK1 has been identified as -Pro-Leu-Ser/Thr-Pro- (Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). However, the structural determinants for substrate recognition are not understood. We performed a systematic analysis of the effect of point mutations in the primary sequence of peptide substrates on the rate of phosphorylation by ERK1 and ERK2. The results of this investigation demonstrate that the substrate specificities of the ERK1 and ERK2 protein kinases are very similar. We propose that the primary sequence of substrates for ERK1 and ERK2 protein kinases can be generalized as -Pro-Xaan-Ser/Thr-Pro- (where Xaa is a neutral or basic amino acid and n = 1 or 2)