Open Access Original Research Article

Lectin-Like Oxidized LDL Receptor 1 Mediates the Uptake of the C-Terminal Domain of Hsp70 (A Promising Immune Adjuvant Molecule) and Antigen Peptide Complexes

Miyuki Kumano-Kuramochi, Shoko Kajiwara, Takashi Minowa, Sachiko Machida

International Journal of Biochemistry Research & Review, Page 284-294
DOI: 10.9734/IJBCRR/2014/8699

Aims: Extracellular heat shock protein 70 (Hsp70) is an adjuvant molecule that stimulates the immune system. The C-terminal domain of Hsp70 (C70), without the ATPase domain, is sufficient for antigen cross-presentation.  However, the mechanism by which the receptor mediates the uptake of C70–peptide complex remains unclear.  We therefore aimed to determine the process by which the receptor mediates the uptake of antigenic peptide-bound C70.

Methodology: Hsp70 and C70 individually cloned into pET28a were expressed in Escherichia coli BL21 (DE3) and were purified on Ni-NTA agarose and MonoQ HR5/5.  Hsp70 and C70 were labeled with Alexa 555 and Alexa 633, respectively, to detect cellular binding.  HEK293 cells stably expressing lectin-like oxidized LDL receptor-1 (LOX-1) and KG-1 human dendritic-like cells were incubated with Alexa-labeled Hsp70 and C70 individually or with C70 and antigenic complexes and were observed using fluorescence microscopy.  The affinity of LOX-1 toward Hsp70 and C70 was analyzed by chip assay using surface plasmon resonance, which immobilized LOX-1 ligand recognition domain.

Results: HEK293 cells stably expressing LOX-1 and KG-1 cells accepted the C70–peptide and Hsp70–peptide complexes. Anti-LOX-1-neutralizing antibody inhibited the uptake of the C70–peptide complexes by KG-1 cells. The dissociation constant (KD) of C70 toward the LOX-1 extracellular domain, measured by surface plasmon resonance, was 4.02 × 10−7 M and that of the C70–peptide complex was 6.6 × 10−8 M. C70 increased the LOX-1 affinity by forming a complex with the antigen peptide. 

Conclusion: Our findings suggest that LOX-1 is the primary receptor for the C70–peptide and the Hsp70–peptide complexes. C70 is a promising adjuvant molecule that is internalized via LOX-1. In addition, it is convenient to prepare C70 using an E. coli expression system and C70 is more stable than full-length Hsp70. 

Open Access Original Research Article

Characterization of Serum Complement Activity in Three Species of Crocodilians from Southeast Mexico

Mark Merchant, Rodolfo Falconi, Leah LeFleur, Cassandra Trahan, Shea LeDoux, Armando Escobedo

International Journal of Biochemistry Research & Review, Page 295-305
DOI: 10.9734/IJBCRR/2014/7825

Aims: To characterize the serum complement innate immune system in three species of crocodilians native to southeast Mexico.

Methodology: Plasma collected from three wild crocodilian species native to southeast Mexico were exposed to sheep red blood cells (SRBCs) to measure hemolysis, which is used as an indication of serum complement immune activity. 

Results: Incubation of different volumes of plasma from Crocodylus acutus, Crocodylus moreletii, and Caiman crocodilus resulted in a volume-dependent increase in SRBC hemolysis.  However, while maximum hemolysis for C. acutus and C. moreletii were both approximately five-fold higher than that of Ca. crocodilus.  A kinetic study revealed that the hemolysis was rapid, with near-maximum activity recorded at 30 min for C. acutus and C. moreletii.  However, Ca. crocodilus activity exhibited a significant increase (P<.5) only between one and two hours.  A thermal analysis showed that the SRBC hemolysis was maximal at temperatures to which these species thermoregulate.  The thermal profiles were similar for all three species, although the activity was lower for Ca. crocodilus                 (P<.01).  The SRBC hemolysis was strongly inhibited by mild heat treatment (56°C,30 min) and also by EDTA, indicating that the hemolytic activity was probably due to the presence of crocodilian serum complement activity. The EDTA-inhibited activity was restored by the addition, of a 20mM excess of Ca2+or Mg2+, but not Fe2+orCu2+, thus exhibiting the specific need for Ca2+orMg2+

Conclusions: The serum complement activities of C. acutus and C. moreletii are much higher (P<.01) than Ca. crocodilus, which may be related to the maximum sizes and increased aggressive natures of C. acutus and C. moreletii.

Open Access Original Research Article

Comparative Levels of Total Creatine Kinase and MB-isoenzyme in Cord Blood between Vaginal Delivery and Cesarean Section

Zohre Torabi, Reza Salmani, Afshin Fayaz, Akefeh Ahmadiafshar

International Journal of Biochemistry Research & Review, Page 306-311
DOI: 10.9734/IJBCRR/2014/8394

Aim: Comparing cord blood levels of total Creatine kinase (CK) and Creatine kinase MB  isoenzyme (CK-MB)  in  newborns delivered by vaginal delivery and cesarean section.

Study Design: Descriptive prospective study.

Place and Duration of Study: All neonates delivered from September to October 2012 at the obstetric ward of Mousavi hospital. (Zanjan, Iran).

Methodology: Total cord blood CK (CK) and MB isoenzyme (CK-MB) was measured immediately after delivery. Rout of delivery and APGAR (Appearance, Pulse, Grimace, Activity, and Respiration) Score were also assessed and all data were analyzed by using SPSS 16.0 software.

Results: One hundred and seventy six newborns were recruited (57.4% male and 42.6% female). We found significant association between serum CK-MB level and rout of delivery. There was no significant association between serum levels of total Ck and               CK-MB and APGAR score (p˃0.05).

Discussion and conclusions: This study showed that newborns who were vaginally delivered had elevated CK-MB levels.

Open Access Original Research Article

Ulcero-protective Potentials of Methanolic Extract of Acacia ataxacantha Leaves in Indomethacin and Stress Induced Gastric Ulcer Models

T. C. Akapa, R. O. Arise, O. J. Olajide, I. T. Ikusemoro

International Journal of Biochemistry Research & Review, Page 312-321
DOI: 10.9734/IJBCRR/2014/8747

Aims: This study was carried out to investigate the ulcero–protective activity of methanolic extract of Acacia ataxacantha leaves (MEAAL) against indomethacin and stress induced gastric ulcer in experimental rats.

Study Design: Administration of MEAAL at the dose of 100mg/kg body weight and                  200mg/kg body weight and evaluation of its ulcero-protective activity.

Place and Duration of Study: The experiments were conducted at the Department of Biochemistry, University of Ilorin between September 2012 to May 2013.

Methodology: Acacia ataxacantha leaves were extracted with 95% methanol. MEAAL at the dose of 100 and 200mg/kg body weights were administered to male albino rats 30 minutes before the administration of indomethacin and subjecting to stress. Ranitidine was used as a standard antiulcer drug. Animals were then sacrificed and various gastric parameters assessed were gastric ulcer indices, gastric pH levels, gastric ulcer percentage inhibition, which were done in order to explore the ulcero-protective potential of the plant.

Results: Induction of ulcer by the intraperitoneal administration of indomethacin and forcing rats to undergo stress by swimming resulted in increased ulcer index and decreased pH. Rats pretreated with MEAAL (100 and 200mg/kg body weights) showed significant reduction in ulcer index to indomethacin and stress induced ulcer models in a dose dependent manner when compared to the negative control group. Also, the significant decrease in the gastric pH levels of both ulcer models, were normalized by MEAAL. The various percentages of gastric ulcers inhibition were statistically significant (P<.05) in the groups pretreated with MEAAL. The overall effect of the extract was comparable to that of the standard drug (ranitidine) used.

Conclusion: These findings validated the potentials of Acacia ataxacantha leaves as an ulcero-protective agent and provides a scientific rationale for the use of Acacia ataxacantha in Senegalese folk medicine.

Open Access Original Research Article

Serum Zinc and Copper Levels in Children with Plasmodium falciparum Infection in Nigeria

Caroline A. Okoli, Mariam Solomon, Alphonsus C. Okoli

International Journal of Biochemistry Research & Review, Page 322-332
DOI: 10.9734/IJBCRR/2014/7827

Background: An estimated 2 million children die of malaria yearly, primarily because of Plasmodium falciparum and its complications. Plasmodium falciparum infection could lead to imbalance in micronutrient levels. Micronutrients such as zinc and copper are essential for immune functions.  In this study, we determined the baseline serum zinc and copper levels of children with or without malaria in Jos, Nigeria. This is essential because its result will give us a proper insight whether there is a need for a further study on zinc supplementation in these patients or not.

Study Design: This was an analytical case-control study.

Place and Duration of Study: This study was conducted between August and November 2011 in various hospitals in Jos, North Central Nigeria.

Methodology: The blood samples of 600 children aged zero to18 years from various hospitals in Jos were analyzed for malaria parasite (MP), zinc, copper, albumin and total protein. All statistical analysis were done using SPSS version 17. The results were expressed as means, standard deviation and percentages.

Result: Out of 600 children, 306(51%) had malaria infection. Three hundred and thirteen (52.2%) were females. The overall prevalence of malaria was 51% while that of congenital and neonatal malaria were 2.0% and 1.5% respectively There was no significant difference in the serum zinc and copper levels of children with or without malaria (p=0.404 and p=0.559).  Serum zinc and copper levels were significantly higher in males (p=0.001). There was a significant positive relationship between parasite density and serum zinc and copper levels (p=000).  Serum zinc, copper levels and temperature were not predictors of malaria.

Conclusion: There was no significant difference in the serum zinc and copper levels of children with or without malaria. This implies that there may not be need to supplement children with malaria with zinc or copper, as this may not be necessary in solving the menace of malaria morbidity.

Open Access Original Research Article

Bioaccumulation and Evidence of Hormonal Disruptions in Tilapia Fish (Oreochromis spp.) Exposed to Sub-lethal Concentrations of Pesticides in Sinaloa, Mexico

J. Guillermo Galindo Reyes, Giuseppe R. Galindo Rodríguez, M. Del Carmen Cabrera Osuna, Foday M. Jaward

International Journal of Biochemistry Research & Review, Page 333-343
DOI: 10.9734/IJBCRR/2014/9006

Examination of endocrine disruption in tilapia fish (Oreochromis spp.) after exposure to sub-lethal concentrations of endosulfan, lindane, diazinon and malathion was evaluated by the quantification of 17-β-estradiol, progesterone and testosterone in fish. Female tilapia (96-110 g/fish) were exposed to 2.8, 15, 225 and 315 μg/l of endosulfan, lindane, diazinon and malathion, respectively; while, the male tilapia (same range weight)  were exposed to 2.8 μg/l endosulfan and 315 μg/l of malathion separately   for 21 days.  The obtained results showed that the level of 17-β-estradiol and progesterone concentration in female, were significantly decreased in all treatment.  Similarity, the level of testosterone was decreased significantly in the blood of male fish exposed to endosulfan and malathion pesticides when compared to the control fish group. The bioaccumulation of pesticides in muscular tissue of fishes were 760, 310, 41.3 and 11.9 (μg/g) for endosulfan, lindane, malathion and diazinon, respectively. Results suggest that pesticides assayed, are endocrine disruptors in tilapia, which can affect the reproduction and other biochemical and physiological functions controlled by hormones. On the other hand, pesticides accumulated in fish muscle represent a risk for public health, since tilapia is a popular food in Mexico like in others developing countries.

Open Access Original Research Article

Mode of Oral Ethanol Feeding Affects Liver Oxidative Stress Levels and Methylation Status: Study on NS5A-Transgenic Mice

Natalia Osna, Benita McVicker, Larisa Poluektova, Murali Ganesan, Kusum Kharbanda

International Journal of Biochemistry Research & Review, Page 344-357
DOI: 10.9734/IJBCRR/2014/9689

Background: Alcohol consumption accelerates the progression and worsens the outcomes of hepatitis C viral (HCV) infection in heavy and moderate drinkers.  Our aim was to investigate the effects of two modes of oral ethanol feeding on induction of oxidative stress, impaired methylation status and downstream changes in proteasome activity in livers of NS5A-transgenic (Tg) mice.

Methods: Ethanol was administered either in water (chow fed mice given 20% ethanol in water; designated chow-EtOH) or fed in Lieber De Carli liquid diet (LCD-EtOH). Appropriate controls were used.  The mechanisms of alcohol and NS5A-induced changes in oxidative stress, liver methylation status and 20S proteasome activity were determined after 5 weeks of the feeding regimen.

Results: Ethanol administration using both feeding regimens induced oxidative stress and suppressed cytosolic proteasome activity. However, only LCD-EtOH diet induced fatty changes in the liver which correlated with higher levels of oxidative stress, impaired methylation potential and reduced cytosolic and nuclear proteasome activity. However, LCD diet by itself triggered lipid peroxidation.

Conclusion: We conclude that both modes of oral ethanol feeding (chow and LCD-based) induce oxidative stress in NS5A-Tg mice that suppresses proteasome activity. Nonetheless, impaired methylation potential, higher level of oxidative stress and suppression of nuclear proteasome was observed only in LCD-EtOH mice.  However, the effects of LCD-control liquid diet in inducing lipid peroxidation in NS5A-Tg mice, in certain cases, tended to mask the effects of ethanol.