Gene expression in Xenopus oocytes

1. Gene expression in Xenopus oocytes is now an integral part of many molecular cloning strategies. 2. For some genes, such as those encoding the ion channels, this system has emerged as the only available means to authenticate and examine the biological activities of the cloned DNA. 3. This review discusses some of the current applications of Xenopus oocytes in modern molecular biology.     

Reduction of the prenatal hypoxic-ischemic brain edema with noscapine

Cytotoxic free radicals and release of several neurotransmitters such as bradykinin contribute to the pathogenesis of hypoxic-ischemic brain damage. We have studied the efficacy of noscapine, an opium alkaloid and a bradykinin antagonist, in reducing post-hypoxic-ischemic damage in developing brain of 7-d-old rat pups. Hypoxic-ischemic injury to the right cerebral hemisphere was produced by legation of the right common carotid artery followed by 3 h of hypoxia with 8% oxygen. Thirty to 45 min before hypoxia the rat pups received noscapine (dose = 0.5-2 mg/kg) or saline. Pups were scarified at 24 h post recovery for the assessment of cerebral damage by histological methods. Our results showed that noscapine was an effective agent in reducing the extent of brain injury after hypoxic-ischemic insult to neonatal rats. Therefore, it is concluded that noscapine may be a useful drug in the managements of patients after stroke.     

Process of simultaneous hydrogen sulfide removal from biogas and nitrogen removal from swine wastewater

The feasibility of a new flowchart describing simultaneous hydrogen sulfide removal from biogas and nitrogen removal from wastewater was investigated. It took 30 days for the reactor inoculated with aerobic sludge to attain a removal rate of 60% for H₂S and NO_x–N simultaneously. It took 34 and 48 days to attain the same removal rate for the reactor without inoculated sludge and the reactor inoculated with anaerobic sludge respectively. The reactor without inoculated sludge still operated successfully, despite requiring a slightly longer startup time. The packing material was capable of enhancing the removal efficiency of reactors. Based on the concentration of NO_x–N and H₂S in the effluent, the loading rate and the ability of the system to resist shock loading, the performance of the reactor filled with hollow plastic balls was greater than that of the reactor filled with elastic packing and the reactor filled with Pall rings.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

Developmental Regulation of β-Thymosins in the Rat Central Nervous System

HPLC analysis of guanidinium hydrochloride extracts of neonatal and adult rat brain revealed a polypeptide that is present in high concentration in the immature nervous system, but whose levels decline dramatically in the adult. This polypeptide has been isolated and its complete amino acid sequence determined by gas-phase Edman degradation following specific chemical and enzymatic cleavages. The molecule is identified as thymosin beta 10, a member of a multigene family that encodes a structurally conserved series of small acidic polypeptides of uncertain function. Thymosin beta 10 is present in the developing nervous system as early as embryonic day 9. Levels subsequently increase to peak values between embryonic day 15 and postpartum day 3, before falling to adult values (about a 20-fold reduction) by postpartum day 14. The elevated levels of thymosin beta 10 in fetal and neonatal brain correlate with high levels of thymosin beta 10 mRNA, whereas the low values of the polypeptide in the adult and juvenile are mirrored by an approximate 15-fold reduction in specific mRNA. In comparison, the levels of thymosin beta 4 polypeptide, a homologue of thymosin beta 10, only decline by about 20% during the same developmental period. However, the mRNA encoding thymosin beta 4 is elevated in fetal brain, and its levels decrease approximately four-fold to a stable value around the time of birth. The reason for this discrepancy between thymosin beta 4 protein and mRNA levels is unknown. Thymosin beta 10 can also be detected by HPLC in fetal liver, where levels are approximately 5% of those in brain. In liver, thymosin beta 10 also declines following birth. It is concluded that beta-thymosin expression (as measured by steady-state mRNA and polypeptide levels) is both up- and down-regulated during different phases of maturation of the mammalian nervous system.     

Correlation between ACE activity and mean blood pressure in Iranian normotensive subjects after oral administration of a single dose of enalapril

Relationship between serum ACE activity and mean blood pressure (MAP) after administration of a single oral dose of the ACE inhibitor enalapril 10 and 20 mg tablets was investigated in 19 Iranian normotensive male subjects. Enalapril at doses, which maximally inhibit ACE activity, reduced MAP dose dependently. The t(max) of ACE inhibition decreased significantly by increasing the enalapril doses, but t(max) of MAP reduction did not change by increasing the dose. The AUC (area under the curve) of ACE inhibition versus time was significantly larger in 20 mg enalapril group compare to 10mg enalapril group (p<0.001). A significant correlation was found between log of residual ACE activity and MAP (r=0.4927; p<0.001). It is concluded that in Iranian normal subjects, after administration of a single oral dose of enalapril, MAP related to residual ACE activity.     

Umbelliprenin shows antitumor,antiangiogenesis, antimetastatic,anti‐inflammatory,and immunostimulatory activities in 4T1 tumor‐bearing Balb/c mice

Umbelliprenin (UMB) has shown various pharmacological properties in vitro. We investigated the antineoplastic and immunostimulatory effects of UMB in 4T1 mammary‐tumor‐bearing mice. Two‐hundred microliter of UMB (12.5 mg/ml) was intraperitoneally administrated to healthy and tumor‐bearing female Balb/c mice for a period of 18 days. Data was analyzed using GraphPad Prism 5 software for Windows (version 5, La Jolla, CA). UMB caused a significant decrease in tumor size (P < 0.01). Serum interferon gamma (IFNγ) was augmented in both healthy and tumor‐bearing animals (P < 0.01), and IL‐4 declined in healthy animals (P < 0.01) treated with UMB. Expressions of Ki‐67, VEGF, CD31, MMP2, MMP9, VCAM1, and NF‐κB were significantly decreased in tumors from UMB‐treated animals (P < 0.001), whereas E‐Cadherin and TNFR1 expressions were markedly increased (P < 0.001). The rates of liver and lung metastases in UMB‐administrated animals were smaller compared to the control. UMB can potently inhibit tumor growth, angiogenesis, metastasis, and inflammation and potentiate an antitumor immune response in vivo. However, further investigations are required to evaluate the UMB mechanisms of action in cancerous cells.     

Purification by ammonium sulfate precipitation of bacteriophage lambda gt11 DNA for restriction analysis of cloned cDNA inserts

A rapid and efficient method to purify lambda gt11 DNA is described. This technique involves precipitation of intact bacteriophage particles with ammonium sulfate, followed by phage lysis with sodium dodecyl sulfate, proteinase K, and alkaline treatment. The quality of DNA for subsequent restriction analysis, infectivity, subcloning, and radiolabeling is comparable to that isolated by cesium chloride banding or ion exchange chromatography. The yield of the phage DNA is, however, two to eight times higher than that obtained by other conventional methods of lambda gt11 purification. Furthermore the time required to process the bacteriophage lysate is approximately 2 h and therefore more rapid than other currently used methods.