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Abstract Background Dengue virus remains a major public health problem with one of the hallmark pathologies is the vascular leakage caused by endothelial dysfunction which can lead to Dengue Hemorrhagic Fever (DHF) manifestation. In the status quo, no specific therapy has been discovered but rather heavily relies on judicious and frequent monitoring of intravenous fluids administration. The current guideline has discussed the roles of fluid therapy during the Dengue Shock Syndrome (DSS) stage, however, administration of early fluid intervention for DHF grade I and II remains uncharted territory. In addition, the choice and timing of colloid administration remains underexplored. As one of the widely available colloids, 5% albumin has known physiological properties that potentially minimize plasma leakage. Therefore, this study aimed to evaluate the benefit of early intervention of 5% albumin in adults with DHF in the hope of preventing the lethal progression to DSS and further, shorten the length of stay (LOS) for patients. Methods We conducted a multicenter, open-labeled, randomized controlled trial in Jakarta and Banten to compare the effect of early intervention with 5% albumin in adult patients with DHF compared to Ringer’s Lactate (RL). Statistical analyses were conducted using unpaired t-test and Mann-Whitney for normally and abnormally distributed data respectively. Results Adult patients with a diagnosis of DHF grade I and II that being hospitalized to receive the early intervention of 5% albumin had significantly lower levels of hemoconcentration 4, 12, and 24 h (p = 0.002, 0.001, 0.003, respectively), higher platelet counts 4 h (p = 0.036), higher serum albumin levels 48 h (p = 0.036), lower proteinuria 24 and 48 h post-albumin administration (p

Tuberculosis is caused by Mycobacterium tuberculosis infection. During M. tuberculosis infection, there is a decrease in the partial pressure of oxygen in the granuloma microenvironment, which causes the hypoxia-inducible factor (HIF) to become stable. HIF functions as a transcription factor that regulates the expression of genes crucial for metabolic adaptation in hypoxic conditions. Recent research suggests that HIF plays a vital role in infectious and inflammatory conditions. Several studies have demonstrated that HIF signaling can enhance macrophages antimicrobial activity and bactericidal effect against M. tuberculosis, such as increasing macrophage autophagy, enhancing the effects of rifampicin, inhibiting p38 MAPK signaling, enhancing the regulation of effector antimicrobial pathways mediated by human β defensin 2 (hBD2) and vitamin D receptor (VDR), redirecting energy metabolism to glycolysis, and producing various cytokines. All these responses ultimately result in the inhibition of intracellular M. tuberculosis growth. HIF has therapeutic implications, potentially being a new candidate for host-directed therapy as a complement to existing antituberculosis drugs. Understanding the role of HIF in macrophages during M. tuberculosis infection and comprehending the host-pathogen relationship with M. tuberculosis is advantageous for developing future therapies. Keywords: Mycobacterium tuberculosis, macrophages, hypoxia-inducible factor

Background: Initially, contact lenses were recommended only for daily wear because of infection and discomfort concerns. With rising demand, particularly for overnight use, extended wear lenses have gained significance. Despite concerns, intensive development of materials like Lotrafilcon B aims to meet this demand, focusing on comfort and safety. Aim: This study aimed to evaluate the mucin quantity and quality changes between daily and extended wear (Lotrafilcon B), impacting eye health and comfort. Setting: The study was conducted at Kirana Eye Center, Cipto Mangunkusumo Hospital, Jakarta, Indonesia. Methods: This study was a single-blinded randomised clinical trial with two parallel groups. Forty (40) eligible subjects willingly participated. Mucin 5AC (MUC5AC) and Ferning-type tests were conducted. The 40 subjects with moderate myopia were then divided into a daily wear and an extended wear group. The MUC5AC test was performed at pre-fitting and at 4 weeks, whereas the Ferning-type tests were taken at pre-fitting and 1st and 4th weeks. Results: For both groups, there was a significant increase in MUC5AC levels from pre-fitting to week four, but with no significant difference between them in final MUC5AC levels. Additionally, the comparison of eyes with normal and abnormal Ferning-types between the two groups showed no significant differences at pre-fitting, 1st and 4th weeks. Conclusion: There is no significant difference in MUC5AC levels with the use of daily or extended wear (with Lotrafilcon B). Contribution: This study compares the impact of daily and extended wear Lotrafilcon B contact lenses on eye health and comfort in moderate myopia patients.

Hypobaric hypoxia is situation that might occur to helicopter pilots in Indonesia who must fly at an altitude of more than 3,048 m such as in Papua. It can be dangerous because hypoxic condition can affect person's performance. So far, the heart is known as an aerobic organ and very sensitive to hypoxic conditions. Hitherto, the effects of hypobaric hypoxia exposure on biomolecular aspects of the heart are still unclear. Therefore, this study assessed cardiac response in rats exposed to intermittent hypobaric hypoxia (IHH) (equivalent to 3,048 meters/10,000 feet). Sprague-Dawley rats were divided into six groups: control; acute hypobaric hypoxia (AHH); and IHH, for 7; 14; 21; and 28 days. We measured super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, reduced glutathione (GSH), malondialdehyde (MDA), cytoglobin, myoglobin, HIF-1α, and Nrf2 level as our parameters. Activities of SOD, CAT, GPx and GSH increased while the levels of MDA, cytoglobin, myoglobin, HIF-1α, and Nrf2 decreased in all IHH groups compared with the AHH group. A biphasic pattern was observed as IHH sessions increased from 14 to 21 or 28. Where the IHH treatment for more than 14 sessions caused a decrease in endogenous antioxidants, but the response to hypoxia and oxidative stress increased. Our findings presented the molecular alterations of cardiac rats exposed to intermittent hypobaric hypoxia.

BACKGROUND: Cancer stem cells (CSCs) is defined as tumor initiating cells within tumor that maintain stemness properties and tumorigenicity. Extracellular pH of CSCs in in vitro condition is important for supporting cell proliferation which may also regulate the expression of stemness markers such as OCT4. This work aimed to examine the effect of cell culture media on the proliferation and stemness of human breast cancer stem cells (BCSCs). METHODS: Human CD24-/CD44+ BCSCs were grown in Dulbecco's Modified Eagle Medium/F-12 (DMEM/F-12) with 15mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), without HEPES and adjusted to pH 7.4, or without HEPES but pH was not adjusted. BCSCs were grown under standard conditions for various days. Viable cell number was measured using trypan blue exclusion, whereas proliferation rate using MTS assay. OCT4 mRNA and protein were analyzed using quantitative real time PCR (qRT-PCR) and Western Blot assay, respectively. In vitro tumorigenic activity was determined using mammosphere formation unit (MFU) assay. RESULTS: Our results showed a higher viable cell number and proliferation of BCSCs in DMEM/F-12 HEPES (-) compared to HEPES (+) medium until 4 day incubation. OCT4 mRNA and protein level, as well as MFU of BCSCs were significantly higher in HEPES (-) compared to HEPES (+) medium on day 2. CONCLUSION: DMEM/F-12 medium without HEPES facilitates CD24-/CD44+ BCSCs to have higher proliferation and stemness on day 2 incubation compared to those with HEPES. KEYWORDS: breast cancer, cancer stem cell, OCT4, stemness, proliferation

Chitosan-based particles are desirable materials for drug delivery because of their muco-adhesiveness in tissues, biocompatibility, low toxicity and effectiveness in antigen transport. It is still a challenge to prepare chitosan-based particles with high stability and by an effective method. Here we developed chitosan-based nanoparticles for the delivery of toll-like receptor 9 ligands, cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs), which can induce the change from Th-2 type to Th-1 type immune response. Because of the low tolerance to DNase and less uptake into the cells of the free CpG ODNs, the development of positively charged carriers is necessary for the effective delivery of CpG ODNs into immune cells. Chitosan nanoparticles were prepared by ionic gelation, and the nanoparticles size and surface charge were measured by dynamic light spectroscopy and zeta potential analyzer, respectively. The synthesized chitosan nanoparticles showed two dispersion peaks, at 196 ± 29 nm and 1.33 ± 0.36 μm, with a zeta potential of +3.3 ± 0.4 mV. The toxicity of the chitosan nanoparticles to murine RAW264 macrophages was measured by formazan dye assay utilizing the water-soluble tetrazolium salt WST-8. Our chitosan nanoparticles exhibited no cytotoxicity to RAW264 cells. Finally, we evaluated the immunostimulatory activity of CpG ODNs loaded on chitosan nanoparticles using human peripheral blood mononuclear cells. CpG ODNs significantly enhanced the secretion level of interleukin-6 and interferon-γ by cells compared to the free CpG ODNs. These results indicated that chitosan nanoparticles could be a good candidate for the delivery of CpG ODNs.

This study examined the hypothesis that there is an impairment of macrophageal function in spinal TB. We examined macrophageal functions in spinal TB patients. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of five spinal TB patients and five healthy persons as control. The isolated monocytes were cultured with stimulation of macrophage colony-stimulating factor (M-CSF) for seven days for maturation. The phagocytic ability of the macrophages derived from monocytes was measured. Also, nitric oxide (NO), myeloperoxidase (MPO), beta-glucuronide, and acid phosphatase activity was investigated. We found that the monocytes collected from patient PBMCs were significantly fewer than those of the control group (2992.103 vs. 6474.103 (cells/mL)). There were also fewer macrophages that had adhered to sheep red blood cells (SRBC) (598.103 vs. 264.103 (cells/mL)). However, NO production (2346 vs. 325.17 (µmol/gram of protein)), and the MPO (570.7 vs. 17.4 (unit/mg), beta-glucuronide (0.149 vs. 0.123 (μmol/hour/100 mg of protein)), and acid phosphatase activities (1776.9 vs. 287.9 (μmol/hour/100 mg of protein)) of the macrophages in the spinal TB group were markedly higher than in the healthy group. Despite the low adhesion to foreign bodies, the intracellular processing of TB macrophages, including oxidative activity and lysosome function, was significantly high. These results suggested the impairment of macrophageal function in spinal TB. Possibly, there is a dominance of innate non-specific immunity in spinal TB infection.

Background: The kidneys in physiological conditions are always in a state of relative hypoxia. Cytoglobin (Cygb) is the newest globin protein found of the globin family. One of the functions of Cygb is in oxygen supply. Cygb expression is found to increase in hypoxic conditions, which are thought to be an adaptation response to hypoxia. Objective: This study aimed to analyze the expression of Cygb in rat kidneys which were exposed to chronic systemic hypoxia. Methods: Twenty five male Sprague-Dawley rats, weighing 150-200 g were used in this experiment. Rats were divided into 5 groups: The control group was exposed to normoxia; the hypoxia groups (10% oxygen / 90% nitrogen) for 1 day; 3 days; 7 days and 14 days. After treatment, rats were sacrificed and their kidneys were taken. Cygb mRNA expression was measured by qRT-PCR, while Cygb protein expression was measured by the ELISA method. Results: The expressions of Cygb mRNA and protein were found to be highest on day 3 of hypoxia and was correlated very strongly and significantly (r2 = 0.96; p

Background: Lipoic acid is a substance contained in intra and extracellular that act as a coenzyme of Pyruvate Dehydrogenase, also as an antidote, chelating agent and antioxidant. Measurement of lipoic acid is needed to determine the amount of lipoic acid that performs its functions either as a coenzyme or an antioxidant. Besides, this measurement requires a special tool such as High Performance Liquid Chromatography (HPLC) and a process that is available in rural or simple laboratories. Objective: A common and easy tool such as a spectrophotometer was conducted and could expected to be a tool of lipoic acid determination in body fluid such as plasma. Methods: Measurement of lipoic acid using spectrophotometry with UV methanol and visible PdCl2 has been tested and compared to HPLC measurement that was valid and reliable in drug measurement or pharmaceutical preparations. Results: Determination of lipoic acid in plasma and leukocytes using PdCl2 produced replicable, reliability and valid result, with high accuracy, precision and was not different from lipoic acid measurement using HPLC, p=0.99. While UV methanol was different compare to HPLC p =0.0001 or was not valid. Conclusion: The measurement of lipoic acid using PdCl2 visible method can be applied to determine the levels of lipoic acid (LA) and DHLA in plasma and equal to HPLC result.

The aim of this study was to investigate the influence of intermittent hypobaric hypoxia on the expression hypoxia adaptation proteins, namely hypoxia inducibla factor-1a (HIF-1a) and myoglobin (Mb). Twenty five male Sprague-Dawley rats were exposed to intermittent hypobaric hypoxia in a hypobaric chamber in Indonesian Air Force Institute of Aviation Medicine, for 49.5 minutes at various low pressure, 1 week interval for 4 times (day 1, 8, 15 and 22). HIF-1α and Mb protein were measured with ELISA. mRNA expression of Mb was measured with one step real time RT-PCR. HIF-1α protein levels increased after induction of hypobaric hypoxia and continues to decrease after induction of intermittent hypobaric hypoxia 3 times (ANOVA, p = 0.0437). mRNA expression and protein of Mb increased after induction of hypobaric hypoxia and continues to decrease after induction of intermittent hypobaric hypoxia 3 times (ANOVA, p = 0.0283; 0.0170), and both are strongly correlated (Pearson, r = 0.6307). The heart of rats adapted to intermittent hypoxia conditions by upregulation the expression of HIF-1a and myoglobin and then both return to normal level.

Background: intestinal glycocalyx plays a role in bacterial translocation as the pathogenesis sepsis derived from intra-abdominal infections that vulnerable in certain blood types. However, the link between intestinal glycocalyx in specific types of blood groups and abdominal infections remains unknown. This study aims to find out the condition of intestinal glycocalyx in certain blood types with intraabdominal sepsis. Methods: descriptive study involved subjects with intraabdominal infections who underwent laparotomy. Samples are in the form of intestinal specimens. The measurement of intestinal glycocalyx proceeded by the ELISA method using blood group antigens (A and B). Expression data on the secretors were analyzed using the Kolmogorov – Smirnov test followed by parametric comparisons using ANOVA and t-tests. Results: there were 32 subjects with intra-abdominal infections studied in this study. All of them are secretors and express A and B antigens strongly. We found no difference between intraabdominal infections in those with complications or without complications. Blood type O is a predominant blood type found (43.8%). Escherichia coli is the most commonly found microbe in the culture (61.3%). Conclusion: this study shows there is no disrupted intestinal glycocalyx of sepsis patients caused by intraabdominal infection.

Background: High altitude can cause hypobaric hypoxia (HH), resulted from the lower barometric pressure and hence partial pressure of oxygen. Hypoxia can lead to a lot of deleterious molecular and cellular changes, such as generation of free radicals or reactive oxygen species (ROS). Increasing of ROS can cause oxidative stress if the antioxidant enzyme does not increase simultaneously. Oxidative damage in brain has toxic effect on cognitive functions. Objective: In this study, we investigate effect of acute intermittent HH on oxidative stress and antioxidant enzyme activity in rat brain. Method: Wistar rats divided into 5 groups, consisting control group and four experimental groups which treated to HH. Rats were exposed to simulated HH equivalent to 35.000 feet in hypobaric chamber for 1 minute, repeated once a week. Results: Level of malondialdehyde and carbonyl in rat brain under acute HH increased at HH exposure (group I) compare to control group. These levels decreased afterward at intermittent HH exposure (group II-IV). Specific activity of superoxide dismutase (SOD) shows increasing level at intermittent HH exposure, especially group IV was increasing of SOD level significantly. The increasing pattern of specific activity of catalase was inversely from SOD pattern, but it still has higher activity in intermittent HH compare to control group. Conclusion: Brain tissue seems to be able to perform an adequate adaptive response to hypobaric hypoxia after the training, shown by its significantly decreased MDA and carbonyl level and also increased specific activity of SOD and catalase.

Background: Folate is an important substance used for purine and pyrimidine nucleotide synthesis. One measurement of folate that already establishes is using ELISA (Enzyme-linked immunosorbent assay) method. Folate binding protein is a protein that can bind folate, therefore it considered can be used as a tool that can replace antibody dependent ELISA method. Objectives: The aim of this research was to create a method for folate measurement in serum called Enzyme-labeled protein ligand binding assay (ELPLBA) by replacing antibody as used in ELISA method with folate binding protein (FBP) that purified from the whey of milk. Methods: The method is tested using 20 serum samples and compared to ELISA. Folate binding protein was purified from bovine’s milk using ammonium sulfate up to 90% saturated, DEAE-cellulose anion exchange chromatography and affinity chromatography. SDS-PAGE and western blot were used to establish the protein band of FBP that has molecular weight of ~25-35 kDa. ELPLBA was arranged with stationary phase using aminohexyl-agarose, and folic acid linked on it using carbodiimide. Results: The result show there was no significant difference of folate concentration between ELPLBA (14.804 ± 2.795) and ELISA method (13.859 ± 3.638), p = 0.363. Conclusion: ELPLBA method show similarity for determination of folate in serum which was the same as standard folate measurement (ELISA).

This study aimed to investigate whether an ethanolic extract of Garcinia mangostana pericarp is able to modulate the glutathione peroxidase and glutathione system in the liver of CCl4-induced hepatic injury. Thirty one male Sprague Dawley rats were divided into five groups, including control (untreated) group; CCl4 group (at dose 0.55 mg/kg body weight dissolved in coconut oil, at day eight); EEGM group (at doses 900; 1080; and 1296 mg/kg body weight/day for eight days then received CCl4 at dose 0.55 mg/kg body weight dissolved in coconut oil). Glutathione peroxidase and GSH/GSSH ratio were assayed by colorimetric method. The level of GPx was insignificantly lower in the CCl4 group compared to the untreated control group (P > 0.05). Out of the 900 mg/kg BW, 1080 mg/kg BW, and 1296 mg/kg BW doses of Garcinia mangostana extract, only the two highest doses significantly decreased in GPx level compared to the CCl4 group or untreated control group (P < 0.05). The level of the GSH/GSSG ratio in the liver homogenate were not significantly different between groups (P > 0.05). Mangosteen pericarp extract decrease the activity of glutathione peroxidase liver on CCl4-induced hepatic injury of rats. [Med-Science 2016; 5(3.000): 809-12]

Asam folat merupakan kelompok vitamin B. Asam folat terikat dengan suatu protein yang disebut protein ikat folat (PIF). PIF merupakan suatu glikoprotein dengan 222 residu asam amino dan berat molekul sekitar 31000 Dalton. Struktur tiga dimensi PIF dipertahankan oleh enam sampai delapan jembatan disulfida. PIF memiliki kemampuan mengikat asam folat dengan perbandingan ikatan 1:1 mol. Situs aktif PIF mengandung gugus imidazol atau gugus sulfhidril. Kofaktor ion kalsium tidak dibutuhkan PIF dalam mengikat asam folat. Di dalam tubuh, PIF berperan menghimpun, mengangkut, dan membantu penyimpanan, penggunaan serta pendistrubusian asam folat ke jaringan. PIF juga berperan mengurangi ekskresi asam folat di ginjal dan meningkatkan penyerapan asam folat di intestinum. Dalam bidang kedokteran PIF dapat dimanfaatkan untuk mengukur kadar asam folat serum dengan metode analog ELISA.

Background: Carbonic anhydrases (CAs) are metalloenzymes which catalyze the reversible hydration/dehydration reaction of CO2, in order to maintain the cell homeostasis. These enzymes are found in various tissues and involve in a number of different physiological processes, including ion transport, acid-base balance, bone formation, and gluconeogenesis. Objective: To examine the specific activity of CA and to observe the liver tissue respond to oxidative stress by measured the malondialdehyde (MDA) concentration, in rat liver tissue induced by chronic systemic hypoxia for 1, 3, 5, 7, and 14 days of hypoxia. Results: The study showed that the activity of CA induced by chronic systemic hypoxia significantly increasing at early exposure to the hypoxic condition, at day 1 and days 3 of hypoxia (0.281 and 0.262 nmol/mg protein/minute compared to control 0.155 nmol/mg protein/minute) (p

Background: There is now increasing evidence that HIF-1 is also responsive to a variety of non-hypoxic stimuli. However, the mechanisms by which these non-hypoxic stimuli induce HIF-1α are not completely known, yet, although some evidence points to a role of ROS as messengers regulating HIF activity. Objective: To determine the expression of HIF-1α in liver rat tissue induced by carbon tetrachloride under normoxic conditions, with or without N-acetylcysteine protection. Methods: Twenty-five male Sprague-Dawley rats were divided into 5 groups: normal control rats, normal rats orally administered with coconut oil (1 mL/200 g body weight) for 1 day, rats orally administered with CCl4 (0.55 mg/g body weight) for 1 day, rats injected i.v. with NAC (0.15 mg/g body weight) for 8 days and then orally administered with CCl4 (0.55 mg/g body weight) for 1 day, rats orally administered with CCl4 (0.55 mg/g body weight) for 1 day and then injected i.v. with NAC (0.15 mg/g body weight) for 2 days. The expression of HIF-1α mRNA was measured by real-time RT-PCR using the Livak method. The expression of HIF-1α protein was measured by ELISA assay. Results: The highest HIF-1α mRNA and protein expression found in the group treated by CCl4 and then was gradually lowered in the pre-NAC group, post-NAC group, control group, and last, in the oil group. Conclusion: Our study shows the effect of CCl4-treated rats under normoxic conditions increased the mRNA and protein HIF-1α. NAC post-treatment provide a better protective effect compared with NAC pre-treatment

The aim of this study was to investigate the influence of intermittent hypobaric hypoxia on the expression hypoxia adaptation proteins, namely hypoxia inducibla factor-1a (HIF-1a) and myoglobin (Mb). Twenty five male Sprague-Dawley rats were exposed to intermittent hypobaric hypoxia in a hypobaric chamber in Indonesian Air Force Institute of Aviation Medicine, for 49.5 minutes at various low pressure, 1 week interval for 4 times (day 1, 8, 15 and 22). HIF-1α and Mb protein were measured with ELISA. mRNA expression of Mb was measured with one step real time RT-PCR. HIF-1α protein levels increased after induction of hypobaric hypoxia and continues to decrease after induction of intermittent hypobaric hypoxia 3 times (ANOVA, p = 0.0437). mRNA expression and protein of Mb increased after induction of hypobaric hypoxia and continues to decrease after induction of intermittent hypobaric hypoxia 3 times (ANOVA, p = 0.0283; 0.0170), and both are strongly correlated (Pearson, r = 0.6307). The heart of rats adapted to intermittent hypoxia conditions by upregulation the expression of HIF-1a and myoglobin and then both return to normal level.

The purpose of this study was to get optimum medium composition and agitation to trypsin-like protease production by Lactobacillus plantarum FNCC 0270. The medium composition and agitation for enzyme production was optimized using Central Composite Design and Response Surface Method with Design Expert software version 7.1.5 Fermentation was carried out in erlenmeyer flasks at initial pH 8, 37 °C, using an incubator shaker at 87.5 rpm. The best results showed an enzyme activity of 1.0 mU/mL, a protein level of 0.557 mg/mL, and desirability value of 0.740. Numerical optimization was performed to approach the ideal state of the fermentation or the desirability value of 1. The medium composition containing of 3.64% baker's yeast, 1.21% glucose, and 0.13% skim milk was used for the fermentation. The enzyme activity of 1.51 mU/mL and protein level of 0.205 mg/mL can be achieved. After numerical optimization, the fermentation process was verified in erlenmeyer flasks with incubator shaking at 77 rpm, initial pH 8, 37 °C, and 15 h fermentation. The verification results showed that the enzyme activity of 1.273 ± 0.227 mU/mL and protein level of 0.248 ± 0.012 mg/mL.

Trypsin is an enzyme that has a unique mechanism of cutting peptide bonds specifically at the carboxyl side of lysine or arginine amino acids, with another amino acid. This study aims to analyze a trypsin-like protease (TLP) found in Lactobacillus plantarum FNCC 0270, by performing partial proteomic tests, i.e. MALDI-TOF/TOF, and standard bioinformatics tools. SDS-PAGE analysis showed 4 protein bands. Two bands of the (P1 and P2) showed molecular weights equivalent to 47.35 and 38.42 kD, each generating 8 and 11 peptide fragments respectively. According to information in www.ncbi.nlm.nih.gov/genbank/structures, the structure of serine protease HtrA (subs. plantarum L. plantarum ST-DT) consists of three domains. Using Clone Manager® software by aligning two sequences we obtained eleven. The Lactobacillus produces of the trypsin-like serine protease has 40-90% similarity. Using the Clustal W2 software we passed the 11 sequences through multiple alignments, and found that the isolate L. plantarum is closely related to L. buchneri, L. brevis, and L. malefermentans on the phylogenetic tree. Alignment analysis results showed that all 8 peptide fragments of band 1 and 11 peptide fragments of band 2, of the SDS-PAGE, were located in the active domain region of the fourth trypsin-like serine protease producing Lactobacilli.

The aim of this research was to obtain the different characteristics of haemoglobin molecules in subjects under hypoxic condition, namely eel, catfish, suckermouth fish, green sea turtle using an electrophoresis technique. We used human umbilical cord blood and thalassemia patient blood, as well as a normal adult-human blood as controls. The proteins obtained after electrophoresis process were stained with two different colouring techniques, each based on different principles. Both staining techniques gave practically identical results. Subject that live in hypoxic condition has a different haemoglobin in comparison to the one found in adult human live in normal oxygen condition (normoxia). These hypoxia-adapted or -needed hemoglobin migrate slower than adult human hemoglobin from normoxia. This observation suggests that hemoglobin which is needed to live in hypoxic condition or environment is a different molecule. Whether this hemoglobin from hypoxic condition has a higher affinity to oxygen is not yet known. Further study is needed to clarify this issue.

Thiamine has fundamental role in energy metabolism. The organs mostly sensitive to the lack of thiamine levels in the body are the nervous system and the heart. Thiamine deficiency causes symptoms of polyneuritis and cardiovascular diseases. Because of its importance in the metabolism of carbohydrates, we need to measure the levels of thiamine in the body fluids by using an easy and inexpensive way without compromising the sensitivity and selectivity. An option to it is thiamine measurement based on the principle of which is analogous to ELISA, in which a thiamine binding protein (TBP) act by replacing antibodies. The presence of TBP in several seeds have been reported by previous researchers, but the presence of TBP in mung beans has not been studied. This study was aimed to isolate and purify TBP from mung bean. The protein was isolated from mung bean through salting out by ammonium sulphate of 40, 70, and 90% (w/v). TBP has a negative charge as shown by cellulose acetate electrophoresis. The result obtained after salting out by ammonium sulphate was further purified bymeans of DEAE-cellulose chromatography and affinity chromatography. In precipitation of 90% of salting out method, one peak protein was obtained by using affinity chromatography. The protein was analyzed by SDS PAGE electrophoresis. The result of SDS PAGE electrophoresis showed that TBP has a molecular weight of 72.63 kDa.

Thiamine has fundamental role in energy metabolism. The organs mostly sensitive to the lack of thiamine levels in the body are the nervous system and the heart. Thiamine deficiency causes symptoms of polyneuritis and cardiovascular diseases. Because of its importance in the metabolism of carbohydrates, we need to measure the levels of thiamine in the body fluids by using an easy and inexpensive way without compromising the sensitivity and selectivity. An option to it is thiamine measurement based on the principle of which is analogous to ELISA, in which a thiamine binding protein (TBP) act by replacing antibodies. The presence of TBP in several seeds have been reported by previous researchers, but the presence of TBP in mung beans has not been studied. This study was aimed to isolate and purify TBP from mung bean. The protein was isolated from mung bean through salting out by ammonium sulphate of 40, 70, and 90% (w/v). TBP has a negative charge as shown by cellulose acetate electrophoresis. The result obtained after salting out by ammonium sulphate was further purified bymeans of DEAE-cellulose chromatography and affinity chromatography. In precipitation of 90% of salting out method, one peak protein was obtained by using affinity chromatography. The protein was analyzed by SDS PAGE electrophoresis. The result of SDS PAGE electrophoresis showed that TBP has a molecular weight of 72.63 kDa.

Folic acid played an important role in the metabolism of the body. To measure the serum folic acid levels could use the folate binding protein (FBP) from cow’s milk with a technique analogous to ELISA. The aims of this study were to identify characteristics of FBP from cow’s milk and binding capacity of FBP to folic acid and to purify FBP from other whey protein passed through DEAE-cellulose chromatography column. Each of DEAE-cellulose peaks was passed in affinity chromatography column. FBP was released from affinity column with sodium acetate buffer pH 3.5. The purity of obtained FBP was demonstrated by a single spot in SDS-PAGE analysis and the estimated molecular weight of FBP was around 31 kDa. Our study indicated that 1 mol FBP bound 1 mol folic acid. Alkylation with iodoacetic acid decreased the binding capacity of FBP which suggested the presence of a–SH or imidazol group in its active site. The importance of disulfide bridge was proven by decreasing of folate binding capacity of FBP after -mercaptoethanol treatment. In contrary, the folate binding didn need Ca2+ ion, as indicated by EDTA test which gave the same result as control.

Folic acid played an important role in the metabolism of the body. To measure the serum folic acid levels could use the folate binding protein (FBP) from cow's milk with a technique analogous to ELISA. The aims of this study were to identify characteristics of FBP from cow's milk and binding capacity of FBP to folic acid and to purify FBP from other whey protein passed through DEAE-cellulose chromatography column. Each of DEAE-cellulose peaks was passed in affinity chromatography column. FBP was released from affinity column with sodium acetate buffer pH 3.5. The purity of obtained FBP was demonstrated by a single spot in SDS-PAGE analysis and the estimated molecular weight of FBP was around 31 kDa. Our study indicated that 1 mol FBP bound 1 mol folic acid. Alkylation with iodoacetic acid decreased the binding capacity of FBP which suggested the presence of a–SH or imidazol group in its active site. The importance of disulfide bridge was proven by decreasing of folate binding capacity of FBP after β-mercaptoethanol treatment. In contrary, the folate binding didn need Ca2+ ion, as indicated by EDTA test which gave the same result as control.

Green sea turtle – Chelonia mydas is a lung respiration animal that is able to dive and stay uder sea water for hours without needing to surface for oxygen. As oxygen supply in muscles is assured by myoglobin, we propose to study some characteristic of this muscle protein in green sea turtles. To achieve this objective, pure green sea turtle myoglobin has to be made available. Therefore, our first task is to purify this muscle protein from green sea turtles. Skeletal muscles from 3 green sea turtle hatchlings were studied icroscopically and biochemically. Microscopy observation showed a general structure of striated muscle. Biochemical studies revealed that green sea turtle myoglobin could be more purely isolated to a certain degree by ephadex G-75 gel filtration and purified by immunoaffinity gel chromatography rather than direct purification by DEAE-Sepharose ion exchange chromatography technique. SDS-PAGE analysis showed that green sea turtle myoglobin ran together with horse myoglobin as 17 kDa molecular weight proteins.

The aim of this research was to gain the profile of blood gasses of green turtle (Chelonia mydas) hatch. Blood gas of the green turtle was analysed after exposuring them at 28 °C and 50% of humidity for 24 hours in a pvc tube and at 34 °C under sunlight exposured with 47% of humidity for 30 minutes. The result showed the different values of blood gas contents. This result showed indication of metabolism activities and poikilothermic adaptation of green turtle hatch. This information can be used to support for turtle hatchery in Indonesia.

This study was to analyze the distribution of liposomal-methylprednisolone palmitate (L-MPLP) as a new drug formulation, in several organs of mice after intra-peritoneal injection. In a previous study, in vitro, the stability and the incorporation of methylprednisolone palmitate into liposome membranes were increased, from 70% to approximately 95% using tetra-ether lipid as a stabilizer of the liposome membrane. Based on this result, the stability of L-MPLP should also be proved, in vivo, that the drug, methylprednisolone palmitate, could be distributed into several organs more effective than in a control group (methylprednisolone palmitate and methylprednisolone as a standard of drug and liposome). Forty-two mice of C3H were divided into 5 study groups. Each group of animals was divided into 6 sub-groups of time from 10 minutes to 48 hours. Each drug was injected intra-peritoneal, blood was drawn from the vein of the tail and the organs i.e. liver, kidneys, spleen, thymus, and bone marrow were extirpated after sacrificing the mice using ether. The distribution of the drug or their metabolites was higher at the minute of 180 and tended to decrease at the time of 48 hours after injection. The higher distribution was shown in the liver and rather high in the spleen, thymus, kidney, and bone-marrow respectively.

An understanding of the underpinning physiology and biochemistry of animals is essential to properly understand the impact of anthropogenic changes and natural catastrophes upon the conservation of endangered species. An observation on the tissue location of the key respiratory protein, myoglobin, now opens up new opportunities for understanding how hypoxia tolerance impacts on diving lifestyle in turtles. The respiratory protein, myoglobin has functions other than oxygen binding which are involved in hypoxia tolerance, including metabolism of reactive oxygen species and of the vascular function by metabolism of nitric oxide. Our work aims to determine whether myoglobin expression in the green turtle exists in multiple non muscle tissues and to confirm the hypothesis that reptiles also have a distributed myoglobin expression which is linked to the hypoxia-tolerant trait. This initial work in turtle hatch Chelonia mydas confirms the presence of myoglobin transcriptin brain, heart and liver tissues. Furthermore, it will serve as a tool for completing the sequence and generating an in situ hybridization probe for verifying of cell location in expressing tissues.

Simian retrovirus type-D (SRV) is a causative agent of simian acquired immunodeficiency syndrome in Asian macaques, and can serve as a viral model in understanding of retrovirus infection because of some similarities to human AIDS pathogenesis. Study of infection and pathogenesis of SRV in macaques could be a strategy of vaccine and antiviral development for preventive and therapeutic purposes. We expressed the SRV-2 envelope gene using baculovirus expression vector system and transfected it to Spodoptera frugiferda insect cell line for SRV-2 recombinant protein production. Analysis using PCR and sequencing technique of recombinant in the passage-3 viral stock indicated the occurrence of recombination between SRV-2 envelope and baculovirus genome. Purification using immobilized metal ion affinity chromatography Ni2+-NTA to recombinant protein could minimize the presence non-specific proteins. The SDS-PAGE analysis showed a specific protein for SRV-2 gp70 envelope. Western blot analysis of this purified protein indicated a specific reaction with anti-SRV-2 antibody positive of Macaca fascicularis serum shown as SRV-2 gp70 envelope band.

Background : Intermittent hypobaric hypoxia is suggested to possess a protective effect toward the hypoxic condition. The aim of this study is to analyze the expression of cytoglobin (Cygb), neuroglobin (Ngb) and the specific activity of acetylcholinesterase (AChE) in brain tissue as adaptive responses to intermittent hypobaric hypoxia. Methods: Twenty-five adult Sprague-Dawley male rats were divided into 5 groups: 1) The control group (normoxia); 2) group exposed to acute hypobaric hypoxia (AHH); 3) group exposed to hypobaric hypoxia (HH) on day-1 and re-exposed on day-8 (intermittent hypobaric hypoxia once or IHH1x); 4) group that is exposed to HH on day-1, re-exposed to HH on day-8 and day-15 (intermittent hypobaric hypoxia two times or IHH2x); 5) group exposed to HH on day-1, re-exposed to HH on day-8, day-15 and day-22 (intermittent hypobaric hypoxia 3x or IHH3x). Homogenized brain tissue was then measured and analyzed for Cygb and Ngb protein expression, and also AChE specific activity. Results: Cytoglobin and Ngb were decreased in the acute induction and increased significantly along with the increasing frequency of the IHH induction. There were significant differences in Cygb expression between IHH2x and IHH3x groups compared to normoxia group, and between IHH1x, IHH2x and IHH3x compared to AHH group. There were significant differences in Ngb expression between IHH2x and IHH3x groups compared to normoxia group and between IHH2x and IHH3x groups compared to AHH group. The specific activity of AChE was increased significantly since the first induction of AHH, but then decreased in IHH3x. There were significant differences in the specific activity of AChE between IHH2x and IHH3x groups compared to normoxia and between IHH2x and IHH3x groups compared to IHH1x groups. Conclusions: We conclude that IHH, especially IHH3x, seems to induce the protective adaptive response in the rat brain tissue through the changes of these three parameters.