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"Oksigen merupakan zat penting dalam metabolisme energi tubuh, terutama otak. Neuroglobin (Ngb), salah satu famili hemoprotein berperan sebagai pengikat oksigen di otak. Seperti hemoprotein lainnya, gugus hem pada Ngb rentan mengalami oksidasi Fe2+ menjadi Fe3+ yang dapat menghilangkan fungsi pengikatan oksigennya. Penelitian ini bertujuan untuk menganalisis perubahan spektrum Ngb terhadap oksigen dan karbonmonoksida serta mencari potensi enzim yang dapat mereduksi Ngb teroksidasi (metNgb). Protein Ngb diisolasi dengan teknik fraksinasi menggunakan amonium sulfat kejenuhan 90%, dipurifikasi dengan kromatografi penukar anion (DEAE Selulosa) dan kromatografi imunoafinitas. Hasil isolasi dikonfirmasi dengan SDS-PAGE dan Western blot. Enzim pereduksi metneuroglobin diisolasi dengan RIPA lysis buffer, dipurifikasi dengan kromatografi Affi gel blue, dan dikonfirmasi dengan SDS-PAGE. Ngb hasil purifikasi memiliki berat molekul 17,26 kDa. Analisis spektrum pada rentang panjang gelombang 350-500nm, memperlihatkan puncak soret dari deoksiNgb, oksiNgb, karboksiNgb dan metNgb berturut-turut adalah 415 nm, 405 nm, 405 nm, dan 420 nm. Hasil isolasi enzim pereduksi yang diperoleh terdiri dari 2 bagian, yaitu eluat tidak terikat matriks (eluat-1) dan eluat terikat matriks (eluat-2). Hasil SDS-PAGE dari eluat-1, eluat-2 dan fraksi bebas Ngb (hasil samping purifikasi Ngb) menunjukkan 3 pita yang sama pada berat molekul 72,45; 26,84 dan 16,33 kDa yang diduga berpotensi sebagai enzim pereduksi. Kinetik reduksi diuji dengan mereaksikan fraksi dan metNgb serta mengukur serapan deoksiNgb yang terbentuk tiap satuan waktu. Hasil pengukuran rasio NgbFe3+ menjadi NgbFe2+ dari fraksi bebas Ngb, eluat-1 dan eluat-2, yang memiliki aktivitas reduksi terbaik adalah eluat-1 karena memiliki nilai regresi terbaik.

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Oxygen is an important substance in the body's energy metabolism, especially the brain. Neuroglobin (Ngb), one of the hemoprotein families, acts as an oxygen binder in the brain. Like other hemoproteins, the haem group in Ngb is susceptible to Fe2+ oxidation to Fe3+ which can eliminate its oxygen binding function. This study aims to analyze the changes in the Ngb spectrum towards oxygen and carbon monoxide and to find the potential for enzymes that can reduce oxidized Ngb (metNgb). Ngb protein was isolated by fractionation technique using ammonium sulfate 90% saturation, purified by anion exchange chromatography (DEAE Cellulose) and immunoaffinity chromatography. The isolation results were confirmed by SDS-PAGE and Western blot. The metneuroglobin-reducing enzyme was isolated by RIPA lysis buffer, purified by Affi gel blue chromatography, and confirmed by SDS-PAGE. The purified Ngb has a molecular weight of 17.26 kDa. Spectrum analysis in the wavelength range of 350-500nm, showed the afternoon peaks of deoxyNgb, oxyNgb, carboxyNgb and metNgb were 415 nm, 405 nm, 405 nm, and 420 nm, respectively. The results of the isolation of reducing enzymes obtained consisted of 2 parts, namely the matrix-bound eluate (eluate-1) and the matrix-bound eluate (eluate-2). SDS-PAGE results of eluate-1, eluate-2 and Ngb-free fraction (byproduct of Ngb purification) showed the same 3 bands at a molecular weight of 72.45; 26.84 and 16.33 kDa which are thought to have potential as reducing enzymes. The reduction kinetics was tested by reacting the fraction and metNgb and measuring the deoxyNgb uptake formed per unit time. The results of the measurement of the ratio of NgbFe3+ to NgbFe2+ from the free fractions Ngb, eluate-1 and eluate-2, which has the best reduction activity is eluate-1 because it has the best regression value.Oxygen is an important substance in the body's energy metabolism, especially the brain. Neuroglobin (Ngb), one of the hemoprotein families, acts as an oxygen binder in the brain. Like other hemoproteins, the haem group in Ngb is susceptible to Fe2+ oxidation to Fe3+ which can eliminate its oxygen binding function. This study aims to analyze the changes in the Ngb spectrum towards oxygen and carbon monoxide and to find the potential for enzymes that can reduce oxidized Ngb (metNgb). Ngb protein was isolated by fractionation technique using ammonium sulfate 90% saturation, purified by anion exchange chromatography (DEAE Cellulose) and immunoaffinity chromatography. The isolation results were confirmed by SDS-PAGE and Western blot. The metneuroglobin-reducing enzyme was isolated by RIPA lysis buffer, purified by Affi gel blue chromatography, and confirmed by SDS-PAGE. The purified Ngb has a molecular weight of 17.26 kDa. Spectrum analysis in the wavelength range of 350-500nm, showed the afternoon peaks of deoxyNgb, oxyNgb, carboxyNgb and metNgb were 415 nm, 405 nm, 405 nm, and 420 nm, respectively. The results of the isolation of reducing enzymes obtained consisted of 2 parts, namely the matrix-bound eluate (eluate-1) and the matrix-bound eluate (eluate-2). SDS-PAGE results of eluate-1, eluate-2 and Ngb-free fraction (byproduct of Ngb purification) showed the same 3 bands at a molecular weight of 72.45; 26.84 and 16.33 kDa which are thought to have potential as reducing enzymes. The reduction kinetics was tested by reacting the fraction and metNgb and measuring the deoxyNgb uptake formed per unit time. The results of the measurement of the ratio of NgbFe3+ to NgbFe2+ from the free fractions Ngb, eluate-1 and eluate-2, which has the best reduction activity is eluate-1 because it has the best regression value"

"Latar belakang: Mukopolisakaridosis (MPS) tipe II adalah kelainan genetik yang ditandai dengan gangguan metabolik berupa defisiensi enzim iduronat-2-sulfatase (I2S) karena adanya mutasi pada gen iduronat-2-sulfatase (IDS), sehingga heparan sulfat dan dermatan sulfat tidak terdegradasi dan terakumulasi pada jaringan. Penelitian ini dilakukan untuk menganalisis aktivitas spesifik enzim I2S dan kaitannya dengan varian mutasi gen IDS pada pasien MPS II di Indonesia. Metode: Data sekuen nukleotida gen IDS dari enam pasien MPS II dianalisis untuk melihat jenis mutasi serta dibuat model konformasi proteinnya. Sel peripheral blood mononuclear cell (PBMC) diisolasi menggunakan metode sentrifugasi bertingkat dan dikultur menggunakan medium RPMI. Nilai aktivitas spesifik I2S diperoleh dengan mengukur aktivitas I2S per miligram konsentrasi total protein. Aktivitas enzim I2S diukur menggunakan metode fluorometri, sementara konsentrasi total protein diukur menggunakan bicinchoninic acid (BCA) protein assay. Hasil: Tiga varian mutasi yang ditemukan pada pasien MPS tipe II adalah missense (3/6), delesi (2/6), dan nonsense (1/6). Aktivitas enzim spesifik I2S pada pasien menunjukkan angka yang bervariasi. Mutasi dengan rata-rata aktivitas spesifik I2S paling rendah sampai paling tinggi secara berurutan adalah mutasi delesi (0,026 nmol/min/mg), missense (0,052 nmol/min/mg), dan nonsense (0,052 nmol/min/mg). Kesimpulan: Aktivitas spesifik enzim I2S pada pasien MPS tipe II di Indonesia 0,044 nmol/min/mg, sedangkan pada kontrol adalah 0,172 nmol/min/mg. Nilai rata-rata aktivitas spesifik I2S pada pasien menurun empat kali lipat dibandingkan pada kontrol.

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Background: Mucopolysaccharidosis type II (MPS II) is an inherited metabolic disorder that caused by iduronate-2-sulfatase (I2S) enzyme deficiency due to mutations in the iduronate-2-sulfatase (IDS) gene, so that heparan sulfate and dermatan sulfate do not be degrade and accumulate in tissues. This study was conducted to analyze the specific activity of I2S and its relationship to IDS variant mutation of MPS II patients in Indonesia.

Method: IDS gene nucleotide sequences from six MPS II patients were analyzed to see mutation type and protein conformation model was made. Peripheral blood mononuclear cell (PBMC) were isolated using a stratified centrifugation and cultured using the RPMI medium. I2S specific activity values are obtained by measuring I2S activity per milligram of total protein concentration. I2S enzyme activity was measured using fluorometry method, while total protein concentration was measured using bicinchoninic acid (BCA) protein assay.

Result: There were three variant mutation in MPS II patients, such as missense (3/6), deletion (2/6), and nonsense (1/6). I2S specific enzyme activity shows varying numbers. IDS mutation based on I2S specific activity from lowest to highest mean value are deletion (0.026 nmol/min/mg), missense (0.052 nmol/min/mg), and nonsense (0.052 nmol/min/mg).

Conclusion: I2S specific activity in MPS II patient is 0,044 nmol/min/mg, while the control is 0,172 nmol/min/mg based on the mean value. I2S specific activity in patients decreased four times compared to controls."

"ABSTRAKLatar Belakang. Protein yang berperan penting dalam fungsi sperma berpotensi sebagai target molekul dalam upaya pengembangan bahan kontrasepsi pria. Salah satu protein yang terdapat pada sperma adalah protein kanal Voltage Dependent Anion Channel3 (VDAC3). VDAC3 berfungsi mengatur aliran ion dan metabolit termasuk ATP. Dari penelitian dengan menggunakan teknik knock-out mouse pada gen VDAC3 dilaporkan bahwa mencit jantan mutan VDAC3 homozigot mengalami penurunan yang signifikan dalam motilitas spermanya. Tujuan penelitian ini adalah memproduksi antibodi poliklonal VDAC3 melalui imunisasi protein rekombinan VDAC3 murni dan uji aktivitasnya terhadap motilitas dan viabilitas sperma manusia.Metode. Verifikasi keberhasilan pemotongan His fussion tag beserta 31 asam amino plasmid dari protein rekombinan dilakukan dengan teknik Western blott. ELISA digunakan untuk mengetahui titer IgG anti VDAC3sedangkan uji efektifitas antibodi VDAC3 terhadap fungsi sperma dilakukan dengan menghitung prosentase sperma yang tidak bergerak, waktu yang ditempuh sperma dalam jarak 0,1 mm. Analisa viabilitas sperma dilakukan dengan metode pewarnaan eosin.Hasil. Pada penelitian ini Western blotting dengan menggunakan antibodi Rabbit Anti VDAC Human menghasilkan pita tunggal dengan ukuran ~ 16 kDa, sedangkan penggunaan antibodi terhadap His (C-term) tidak menunjukan adanya pita. Hasil spektofotometri ELISA titer antibodi poliklonal VDAC3 yang berasal dari kelinci menunjukkan adanya peningkatan titer antibodi poliklonal VDAC3 setelah imunisasi dibandingkan dengan titer antibodi sebelum imunisasi (preimun serum). Hasil uji aktivitas antibodi poliklonal VDAC3 menunjukkan terjadi peningkatan jumlah sperma bergerak yang bermakna pada waktu 30 menit (p<0,05) dan 60 menit (p<0,05), juga terjadi peningkatan waktu tempuh sperma yang bermakna pada waktu 0-30 menit (p<0,05) setelah perlakuan. Penambahan antibodi poliklonal VDAC3 juga berpengaruh secara nyata terhadap persentase viabilitas spermatozoa yang hidup (p<0,05).Kesimpulan. VDAC3 poliklonal antibodi berhasil diproduksi melalui imunisasi dari VDAC3 rekombinan murni. Antibodi poliklonal anti-protein rekombinan Voltage Dependent Anion Channel-3 (VDAC3) dapat menurunkan motilitas dan viabilitas sperma manusia invitro secara bermakna.

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ABSTRACT

Background. Sperm-specific proteins that are important for sperm function can potentially be used as a target for developing a male contraceptive. One of the proteins found in the human sperm is Voltage Dependent Anion Channel3 (VDAC3). VDAC3 regulates the flow of ions and metabolites including ATP in the mitochondrial membrane and cell membrane of the eukaryotes. A previous study showed VDAC3 knockout mice had significant reduction in sperm motility. The purpose of this study was to produce polyclonal antibodies through immunization of pure VDAC3 recombinant protein and analyze its effect towards sperm motility and viability.

Methods. Removal of the His fussion tags plus 31 amino acids from the recombinant plasmid was verified using western immunoblotting. The titter of VDAC3 polyclonal antibody was determined by ELISA. The effect of VDAC3 antibodies against sperm qualities namely motility and viability was assessed using standard sperm analyses approved by the WHO.

Results. Western immunoblotting using Rabbit Anti Human VDAC3, produced a single band with size of ~ 16 kDa. No visible band was detected when anti-His (C-term) antibody was used in the analyses. Spectophotometric ELISA showed that the titer of VDAC3 polyclonal antibodies, derived from rabbits, polyclonal antibody increased better than the pre-immune. Analyses of VDAC3 polyclonal antibody against human sperm showed an increase in the number of sperm to move significant at 30 minutes (p < 0.05) and 60 minutes (p < 0.05), as well as an increase in sperm significant travel time at the time of 0-30 minutes (p < 0.05) after treatment. Polyclonal antibodies VDAC3 also significantly affect the percentage of sperm viability (p < 0.05).

Conclusion. Polyclonal antibody anti-VDAC3 was successfully produced via immunization of the pure recombinant VDAC3. Polyclonal antibody anti-recombinant protein Voltage Dependent Anion Channel-3 (VDAC3) may decrease human sperm motility and viability in vitro significantly."

"LATAR BELAKANG: Salah satu tata laksana infertilitas adalah inseminasi intra uterin IIU yang menggunakan spermatozoa hasil pencucian. Ada dua metode pencucian spermatozoa yang umum digunakan yaitu swim-up SU dan density-gradient centrifugation DGC. Tingkat keunggulan metode pencucian spermatozoa terletak pada persentase spermatozoa motil yang dihasilkan. Gangguan motilitas spermatozoa dapat disebabkan oleh ketidakseimbangan transport ion pada spermatozoa. Keseimbangan transpor ion untuk memelihara homeostasis spermatozoa dimediasi oleh enzim ATPase, diantaranya adalah Na ,K -ATPase dan Ca2 -ATPase. Penelitian sebelumnya telah membuktikan bahwa isoform Na ,K -ATPase ?4 dan PMCA4 berperan penting pada motilitas spermatozoa. Oleh karena itu, penelitian ini bertujuan untuk mengevaluasi kembali efisiensi metode SU dan DGC dalam menghasilkan spermatozoa motil berdasarkan aktivitas spesifik Na ,K -ATPase dan Ca2 -ATPase beserta isoformnya. METODE: Pada sampel dilakukan analisis semen, isolasi sel spermatozoa, isolasi protein dan preparasi fraksi membran. Analisis semen dilakukan berdasarkan rujukan dari WHO 2010 , sebelum dan setelah pencucian spermatozoa dengan metode DGC dan SU. Aktivitas enzim diukur berdasarkan kemampuan ATPase melepaskan fosfat organik dari ATP. Deteksi protein Na ,K -ATPase ?4 dan PMCA4 dilakukan dengan metode western blot, sedangkan distribusi proteinnya digunakan metode imunositokimia. HASIL: Terjadi peningkatan rerata konsentrasi, motilitas, morfologi dan kecepatan spermatozoa antara kelompok sebelum dan setelah DGC serta antara sebelum dan setelah SU. Demikian halnya dengan hasil aktivitas spesifik Na ,K -ATPase dan Ca2 -ATPase juga mengalami peningkatan bila dibandingkan antara kelompok sebelum dan setelah pencucian. Terdapat perbedaan bermakna terhadap aktivitas spesifik Na ,K -ATPase pada kelompok sebelum dan setelah DGC serta antara sebelum dan setelah SU. Selain itu, aktivitas spesifik Ca2 -ATPase berbeda tidak bermakna antara sebelum dan setelah DGC dan antara sebelum dan setelah SU. Distribusi protein Na ,K -ATPase ?4 dan PMCA4 tidak mengalami perubahan setelah dilakukan pencucian dengan DGC maupun SU. KESIMPULAN: Aktivitas Na ,K -ATPase dan Ca2 -ATPase yang diperlukan untuk mendukung homeostasis sel spermatozoa meningkat setelah dilakukan pencucian dengan metode DGC dan SU sehingga spermatozoa mempunyai kemampuan motilitas yang lebih baik.

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BACKGROUND: One of the management of infertility is Intra Uterine Insemination IIU by using sperm preparation. There are two methods of sperm preparation that commonly used swim up and density gradient centrifugation. The superiority of sperm preparation method based on the percentage of motile spermatozoa produced. The disorder of sperm motility may caused by the imbalance of ions transport on sperm. The balance of ionic transport to maintain spermatozoa homeostasis is mediated by ATPase, such as Na .K ATPase and Ca2 ATPase enzym. Study has shown that 4 Na ,K ATPase and PMCA4 isoform plays an important role in the sperm motility. Therefore, this study was aimed to evaluate the efficiency of SU and DGC methods in selecting spermatozoa based on the Na ,K ATPase and Ca2 ATPase activity and the isoforms as well.

METHODS: The semen analysis, spermatozoa isolation, protein isolation and membrane fraction preparation were performed. The study analysis was conducted based on WHO 2010, before and after SU and DGC sperm preparation. Enzyme activity was measured by ATPase 39 s ability to release organic phosphate from ATP. The expression of Na ,K ATPase 4 and PMCA4 was done by western blot method, while the protein distribution was used immunocytochemistry method.

RESULT: There was an increase of concentration, motility, morphology and velocity of spermatozoa between normozoospermia group before and after DGC and between before and after SU. Similarly, the specific activity of Na ,K ATPase and Ca2 ATPase also increased when compared to before and after washing. There were significant differences in the specific activity of Na ,K ATPase in the normozoospermia group before and after DGC and between before and after SU. In contrast, the specific activity of Ca2 ATPase not significantly different between before and after DGC and between before and after SU methods. Distribution of Na ,K ATPase 4 and PMCA4 did not change after washing with DGC or SU methods.

CONCLUSIONS: Specific activities of Na ,K ATPase and Ca2 ATPase are needed to support ion homeostasis, so that spermatozoa have better motility abilities after being prepared with DGC and SU methods."

"ABSTRAKJumlah parasitemia pada infeksi malaria yang ada di dalam darah perifer tidak mampu menunjukkan total biomassa parasit malaria. Total biomassa parasit malaria yang diukur dengan pemeriksaan antibodi histidine rich protein HRP dan Plasmodium lactate dehydrogenase pLDH menunjukkan bahwa biomassa parasit malaria lebih tinggi dibandingkan dengan parasitemia di darah perifer. Biomassa parasit malaria berhubungan dengan inflamasi sistemik dan tidak berhubungan dengan aktivasi endotel. Oleh karena itu, biomassa parasit malaria kemungkinan tidak terakumulasi di sel-sel endotel, melainkan di organ non endotel seperti limpa. Penelitian tentang malaria di limpa masih sangat jarang dilakukan, namun ada beberapa yang menunjukkan terdapat perbedaan arsitektur limpa yang terinfeksi oleh P. falciparum dan P. vivax. Perbedaan tersebut diduga karena perbedaan sel inang yang diinfeksi, yaitu eritrosit pada P. falciparum dan retikulosit pada P. vivax. Sebanyak 12 sampel limpa pasien splenektomi digunakan untuk membuktikan apakah limpa manusia merupakan reservoir parasit malaria dan terjadi penghindaran respons imun di limpa. Hasil penelitian menunjukkan bahwa semakin tinggi berat limpa pasien berhubungan dengan tingginya parasitemia, luas pulpa putih dan akumulasi parasit malaria. Akumulasi P. falciparum juga terjadi di limpa dengan tingginya parasitemia di limpa namun stadium hidup yang muda lebih banyak ditemukan di limpa. Hal tersebut berhubungan dengan mekanisme penghindaran respons imun dengan dugaan sekuestrasi di pembuluh darah sehingga menurunkan stadium matang di limpa. Mekanisme penghindaran pada stadium yang lebih muda juga dilakukan dengan cara membentuk rosetting. Akumulasi P. vivax di limpa tidak dapat dideskripsikan di penelitian ini karena jumlah sampel yang sedikit dengan parasitemia rendah. Namun penelitian ini mampu memprediksi kemungkinan akumulasi P. vivax di limpa dengan tingginya retikulosit di limpa.Kata kunci: Limpa, Malaria, P. falciparum, P. vivax, limpa, retikulosit.

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ABSTRACT

The number of parasitemia in malaria infections from peripheral blood was not able to show the total parasite biomass. Total malaria parasite biomass as measured by histidine rich protein HRP and Plasmodium lactate dehydrogenase pLDH antibody test showed higher parasitic biomass than parasitemia in peripheral blood. Total parasite biomass was not correlated with endothelial activation. Therefore parasite biomass was possible to accumulate in non endothelial organ such as the spleen. Research on malaria in the spleen was still very limited, but there were some that showed the differences of splenic architecture in P. falciparum and P. vivax infection. The difference was due to the difference of infected host cell ie red blood cell in P. falciparum and reticulocyte in P. vivax. A total of 12 spleen from splenectomy patients were used to prove whether the human spleen is malaria parasite reservoir and the escaping immune response in the spleen. The results showed that the higher spleen weight was associated with high parasitemia, white pulp area, and accumulation of malaria parasites. P. falciparum accumulation also occurs in the spleen with high parasitemia in the spleen but younger life stages are more common in the spleen. It is related to the mechanism of escaping the immune response with sequestration in the blood vessels thereby decreasing the mature stage in the spleen. The mechanism of escaping immune respons in the spleen at younger stages was also done by forming rosetting. The accumulation of P. vivax in the spleen can not be described in this study because of the limited number of samples with low parasitemia. However, this study was able to predict the possibility of P. vivax accumulation in the spleen with high reticulocytes in the spleen.Keywords Spleen, Malaria, P. falciparum, P. vivax, spleen, reticulocyte."

"Latar belakang: Resistensi progesteron akibat gangguan ekspresi reseptor progesteron pada jaringan endometriosis telah diketahui menjadi faktor yang memperberat kondisi klinis pasien endometriosis. Tujuan penelitian ini adalah untuk menganalisis tingkat metilasi DNA pada promoter gen PR-B pada berbagai jaringan endometriosis seperti eutopik endometrium, lesi ektopik peritoneum, endometrioma dan darah menstruasi serta pengaruhnya terhadap ekspresi mRNAnya dibandingkan dengan kontrol endometrium normal; untuk mengetahui patomekanisme endometriosis pada berbagai lokasi terkait dengan resistensi progesteron.Metode: Penelitian ini menggunakan desain potong lintang yang melibatkan 20 sampel untuk masing-masing kelompok kasus dan kontrol. Tingkat metilasi DNA dari gen PR-B diukur menggunakan metode Methylated Specific PCR MSP lalu intensitas pita di dalam gel agarose dihitung dengan software ImageJ. Presentase intensitas pita pada sampel dibandingkan dengan kontrol positif disebut dengan tingkat metilasi DNA. Pengukuran ekspresi relatif mRNA PR-B menggunakan qRT-PCR dua tahap dan analisis dilakukan dengan metode Livak.Hasil: Dari penelitian ini didapatkan perbedaan bermakna antara tingkat metilasi DNA gen PR-B pada jaringan endometriosis ektopik peritoneum 72,4 termetilasi , endometrioma 85 termetilasi dan eutopik endometrium 72,21 termetilasi dibandingan dengan kontrol p

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Background: Progesterone resistance, due to alteration of progesterone receptor PR expression in endometriosis, was known as a disrupt factor in response to progesterone. The aim of this study is to analyze DNA methylation level on PR B promoter in various tissues include eutopic endometrium, ectopic peritoneal, endometrioma and menstrual blood from endometriosis patient as well as the implication on it's mRNA relatif expression compare with normal endometrium control to know the patomechanisms of endometriosis in various lession in term of progesterone resistance.

Methods: It was a cross sectional study, involved 20 sample for both patient and control. DNA isolate from each sample were converted by bisulfite conversion. DNA methylation level of PR B gene was analysis by Methylated Specific PCR MSP method, then band intensity in gel agarose was measured by ImageJ software. Percentage of band intensity in sample compared with positive control was determined as DNA methylaton level. Quantitative real time PCR was conducted to assess expression of mRNA PR B for each sample and Livak method was used to analysis it's relatif expression compare with control.

Result: There were significant different of methylation level of PR B gene in ectopic peritoneal endometriosis 72,40 methylated , endometrioma 85 methylated and eutopic endometrium 72,21 methylated compared with control p"

"Sel kanker adalah sel yang berproliferasi secara progresif, dan salah satu dasar pengendaliannya hingga saat ini yaitu dengan menghambat kemampuan proliferasinya melalui intervensi sintesis nukleotida purin/pirimidin menggunakan analog purin/pirimidin. Avidin, suatu protein yang ditemukan pada putih telur, diketahui dapat mengikat biotin dengan sangat kuat, yang merupakan koenzim pada reaksi karboksilasi, suatu tahapan penting di biosintesis de novo nukleotida purin. Studi sebelumnya membuktikan bahwa viabilitas dan proliferasi sel mononuklear darah tepi (SMDT) dapat dihambat dengan penambahan avidin yang diakibatkan gangguan ketersediaan biotin. Studi ini bertujuan melihat efek pemberian avidin terhadap sel kanker kolorektal HT-29 dilihat dari viabilitas, proliferasi, ekspresi gen dan protein cyclin D1, serta siklus sel. Penelitian dilakukan dengan mengultur sel kanker kolorektal HT-29 dengan avidin, lalu dianalisis viabilitas, proliferasi, ekspresi gen dan protein cyclin D1, serta siklus selnya pada 24, 48, dan 72 jam. Didapatkan hasil bahwa avidin menghambat viabilitas dan proliferasi sel HT-29, serta menurunkan ekspresi gen dan protein cyclin D1 pada sel HT-29, namun tidak memengaruhi transisi fase G0/G1 ke fase S siklus sel HT-29.

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Cancer cells are progressively proliferating cell, and up to now, one way to control its proliferation is by intervening the formation of purine/pyrimidine nucleotide using its purine/pyrimidine analog. Avidin, a protein from white egg, known to bind biotin strongly, whereas biotin is an important coenzyme in carboxylation reaction, a key step in purine nucleotide de novo pathway. Previous study showed that viability of peripheral blood mononuclear cells (PBMC) was reduced and its proliferation was inhibited caused by lack of biotin due to avidin administration. This study aims to observe the effect of avidin administration to HT-29 cells viability, proliferation, cyclin D1gene and protein expression, also the cell cycle. The experiment done by culturing HT-29 cells, then its viability, proliferation, cyclin D1 gene and protein expression, also the cell cycle analyzed at 24, 48, and 72 hours. The result showed that avidin halted HT-29 cells viability and proliferation, also lower its cyclin D1 gene and protein expression, but did not affect the transition between G0/G1 phase to S phase on HT-29 cell cycle"

"Latar belakang: Proses pematangan spermatozoa membutuhkan interaksi antar protein yang disintesis dan disekresikan oleh epitel epdidimis ke lumen di area tertentu. Gen yang terekspresi secara spesifik di region-region tertentu akan menciptakan lingkungan mikro yang kondusif untuk proses pematangan spermatozoa. Spink2 adalah salah satu gen yang diketahui berperan dalam pematangan spermatozoa yang ekspresinya terdekteksi di epididimis. Studi peran SPINK2 membutuhkan protein yang cukup untuk karakterisasi. Tujuan penelitian ini adalah untuk mengklon, mengekspresikan, dan menguji aktivitas protein rekombinan SPINK2.Metode: Dalam penelitian ini gen mSpink2 dikonstruksikan secara sintetis. Plasmid pQE80L digunakan sebagai vektor ekspresi dan strain sel Escherichia coli yang digunakan adalah Top10 dan BL21. Proses pengklonaan diawali dengan transformasi plasmid pQE80L-mSpink2 ke E. coli Top10 kompeten menggunakan metode heatshock. Proses ekspresi dilakukan dengan penambahan agen induksi Isopropyl-1-Thio-d-Galactopyranoside (IPTG), dipurifikasi dengan kromatografi Ni-NTA. Kemudian dilakukan uji aktivitas protease dengan pembacaan panjang gelombang 660 nm.Hasil: Plasmid pQE80L-mSpink2 terkonfirmasi berhasil diklon dengan analisis enzim restriksi dan sekuensing. Hasil elekstroforesis menunjukan adanya fragmen DNA dengan panjang 4709 pb dan 258 pb. Hasil ekspresi SDS-PAGE dan western blot menunjukkan SPINK2 berhasil terekspresi pada waktu optimum induksi jam ke-4 dan terdapat pita tebal dengan ukuran 14 kDa. Protein rekombinan SPINK2 berhasil dipurifikasi dan ditunjukan dari hasil western blot pada elusi ke-1 hingga ke-4. Hasil uji aktivitas protease menunjukan terdapat penurunan aktivitas enzim tripsin setelah diberi protein rekombinan SPINK2 dengan konsentrasi optimum 0,6 mM.Kesimpulan: Protein rekombinan SPINK2 telah berhasil dikonstruksi secara sintetis, diklon pada vektor plasmid pQE80L, diekspresikan pada E.coli strain BL21 kompeten, dan diketahui dapat menghambat aktivitas enzim protease dengan konsentrasi 0,7 mM.

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Background: The process of spermatozoa maturation requires interaction between proteins synthesized and secreted by the epididymal epithelium to the lumen in a particular area. The interaction between these protein produces a microenvironment for maturation process of spermatozoa. In this condition, it takes genes that are specifically expressed. Spink2 in one of the genes known have a role in the maturation of spermatozoa and expressed in the epididymis. The SPINK2 role study requires sufficient protein for characterization. The aim of this study was to clone, express, and protease assay of the recombinant protein SPINK2.

Methods: In this study the mSpink2 gene was constructed synthetically. The plasmid pQE80L was used as an expression vector and the cell Escherichia coli strains used were Top10 and BL21. The cloning process begins with transformation of the plasmid pQE80L-mSpink2 to a competent E. coli Top10. The expression process was carried out by adding the induction agent Isopropyl-1-Thio-d-Galactopyranoside (IPTG), purified by Ni-NTA chromatography. Then the protease activity assay was carried out with a wavelength 660 nm.

Results: The plasmid pQE80L-mSpink2 was confirmed to be cloned by restriction enzyme and sequencing analysis. The result showed that is formation of DNA with a length of 4709 bp and 258 bp. The SDS-PAGE and western blot result showed that SPINK2 was successfully expressed at the optimum time is 4th hour. There was a thick band with a size of 14 kDa. The SPINK2 recombinant protein was successfully purified and shown form the western blot. The result of the protease activity assay showed that there was a decrease in trypsin enzyme activity after being given SPINK2 recombinant protein with an optimum concentration is 0,6 mM.

Conclusion: Recombinant protein of SPINK2 has been successfully constructed synthetically, cloned, expressed, and tested for its protease inhibitor activity"

"Latar Belakang. Meskipun kondisi pesawat terbang saat ini sudah mengalami modernisasi dengan antematisasi dan disertal dengan kabin bertekanan. Bukan berarti menyingkirkan bahaya hipoksia di dalam dunia penerbangan. Terbukti dengan masih banyaknya angka kecelakaan yang disebahkan oleh karena sebab hipoksia yang terntama disebabkan karena kegagalan sistem kabin bertekanan. Bahaya hipoksin dibidang penerbangan dapat menyebabkan inkspasitas bagi penerbangaya sehingga accident adalah hasil akhimya. Manusia tidak memiliki sistem peringatan dini untuk mengenali adanya hipoksia, sehingga diperlukan pengalaman dalam demonstrasi yang dilakukan di hipobarik chamber. Hasil dari pengalaman itulah ynng diharapkan dalam latihan !LA yang diselenggnrnkan oleh Lakespra Satfanlo TNI AU nntuk dapat segera mengantisipasi ketika terjadi situasi hipoksia baik yang disengaja ataupun tuk disengaja. Metode. 45 orang perwira penerbang dari berbagai usia melaksanakan latihan demostrasi hipoksia di hipobarik chamber di FL 250 lalu melaksanakan tugas hitungan matematika ringan dalam jangka waktu 5 menit. Setiap subjek yang berhenti ditengah selang waktu tsb maka saat itulah waktu sadar efektif (WSE) dicatat. Sebagai parameter fisiologi yang ingin dicari adalah umur, Hb, p.."'tlrentase FVC terhadap ref, persentase FEVJIFVC, dan VO,max sebaga1 variabel independan untuk dicari korelasinya dengan WSE. Setelah diketahui korelasi masing-masing variabel dilakukan ana1isis multivariat untuk menilai faktor dan kekuatan korelasi dan untuk mendapatkan model. Kesimpulan. Dengan diketahuinya model dalam memprediksikan WSE maka akan sangat membantu dalam proses pemilihan dan pembinaan personel dan diharapkan dapat menurunkan angka kejadian akibat hipoksia.

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Background. Even though the condition of the aircrafts have been modernized with automatically equipment and pressured cabin. It doesn't meant we can neglect the danger of hypoxia in aviation as there are a large number of accident bad occurred, caused by hypoxia, particularly due to failure of the system of pressured cabin. The danger of hypoxia in aviation can cause the pilots are incapacity then they can get accident. Human doesn't own early warning system to identifY hypoxia so it requires experience to demonstrate in hypobaric chamber. The result of experience are obtained during hopefully. The training ofil-A which is held in Lakespr:a Saryanto; Indonesian Air Force on the purpose of anticipating the danger of hypoxia whether it occurs consciously or not.

Method. 45 Pilot officers with different ages conduct the training of Hypoxia demonstration in Hypobaric Cbember at FL 250, to complete the test in the form of moderate mathematics in five minute. Every single subject which stops in the mid of time that's the TUC recorded. As parameter of physiology being observed are age<, Hb, percentage of FVC to the reference, percentage of FEVIIFVC, aod V02 as independent variable to find the correlation with rue. After finding the correlation of each variable then there's analysis of multivariate to score the factors, the strength of correlation, and find the model.

Conclusion. After we found the model of predicting TUC, hopefully it can help to selecting and manage the personnel, finally it can reduce the number of accident due to the danger of hypoxia."

"Nefrotoksisitas merupakan efek samping utama yang membatasi penggunaan cisplatin sebagai obat anti-tumor. Kurkumin memeliki beberapa aktivitas farmakologis salah satunya, yaitu sebagai nefroprotektor. Akan tetapi kurkumin kurang larut di dalam air, sehingga digunakan nanokurkumin yang lebih mudah larut/terdispersi dalam air. Tujuan penelitian ini adalah untuk mengetahui efek kurkumin dan nanokurkumin terhadap nefrotoksisitas tikus yang diinduksi cisplatin melalui jalur ERK1/2. Perlakuan hewan coba dilakukan selama 10 hari, menggunakan tikus Sprague Dawley yang dibagi menjadi 5 kelompok, n=6, yaitu kelompok normal, cisplatin CIS, Cisplatin kurkumin 100 mg/kgBB/hari p.o Cis Kurku100, Cisplatin nanokurkumin 50 mg/kgBB/hari p.o Cis Nanokur50, Cisplatin nanokurkumin 100 mg/kgBB/hari p.o Cis Nanokur100 . Pada hari ke-7 dilakukan injeksi cisplatin 7 mg/kgBB, i.p dan 72 jam setelah injeksi cisplatin dilakukan pengambilan darah dan organ ginjal. Cisplatin dosis tunggal pada kelompok CIS menyebabkan peningkatan kadar BUN dan kreatinin dalam plasma, kadar MDA, peningkatan rasio ekspresi BCL-2/Bax, serta peningkatan rasio ekspresi protein p-ERK/ERK secara signifikan, dibandingkan kelompok normal. Pemberian kurkumin 100 mg/kgBB dan nanokurkumin 100 mg/kgBB berperan sebagai antioksidan untuk mencegah progresifitas nefrotoksisitas akibat cisplatin, dilihat melalui terjadinya penurunan kadar BUN dan kreatinin dalam plasma, penurunan kadar MDA, dan peningkatan rasio ekspresi gen BCL-2/Bax secara signifikan dibandingkan kelompok CIS, serta penurunan rasio ekspresi protein p-ERK/ERK secara signifikan dibandingkan kelompok CIS. Cisplatin dosis tunggal 7 mg/kgBB dapat menyebabkan nefrotoksisitas pada tikus yang menyerupai AKI Acute Kidney Injury pada manusia. Kurkumin 100 mg/kgBB cenderung memiliki efek nefroprotektor yang lebih baik dalam mencegah progresifitas nefrotoksisitas akibat cisplatin melalui jalur stress oksidatif dan apoptosis.

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Nephrotoxicity is the major limitation for the clinical use of cisplatin as an antitumor. Curcumin has some pharmacological activity, one of them as nephroprotector. However, curcumin less soluble in water, so it is used nanocurcumin which is readily dispersed in aqueous media. The purpose of this study is to investigate the effects of curcumin and nanocurcumin against ciplatin induced nephrotoxicity in rats through ERK1 2 pathway. This study conducted for 10 days treatment, five groups n 6 of male Sprague Dawley rats were examined normal, cisplatin CIS 7 mg kgBW, Cis curcumin Cis Curcu100 100 mg kg BW day, Cisplatin nanocurcumin 50 mg kg BW day Cis Nanocur50, and Cisplatin nanocurcumin 100mg kg BW day Cis Nanocur100 . After 72 h following injection cisplatin, specimens were collected. This study resulted a single dose of cisplatin in CIS group caused a significant increased in plasma BUN, plasma creatinine, MDA levels, decreased ratio expression of BCL 2 Bax gene, and increased ratio of p ERK ERK as compared to normal group. Pre treatment with curcumin 100 mg kgBW and nanocurcumin 50 and 100 mg kgBW acts as an antioxidant to prevent progression of nephrotoxicity cisplatin, were reduced plasma BUN levels, plasma creatinine levels, MDA levels in kidney, increased GSH level in kidney, increased ratio expression of BCL 2 Bax gene in kidney, and decreased ratio of p ERK ERK protein in kidney compared with cisplatin induced nephrotoxicity rats without treatment. Cisplatin with single dose 7 mg kgBW is able to induced nephrotoxicity in rats that mimicked acute kidney injury in human. Curcumin 100 mg kgBW tend to have a better nephroprotector effect in preventing the progression of cisplatin induced nephrotoxicity through oxidative stress pathways and apoptosis. "