Mutational analysis of human papillomavirus type 11 E5a oncoprotein

In this study, we investigated the structural basis of human papillomavirus type 11 (HPV-11) E5a transforming activity at the amino acid level. The effects of insertion, deletion , and substitution mutations on teh E5a transforming activity were determined by the assay of anchorage-independent growth. In the conserved Cys-X-Cys structure, substitution of Ser for Cys-73 resulted in indistinguishable transforming activity, whereas substitution of Ser for Cys-75 or Ser for both Cys-73 and Cys-75 retained 50 and 42% transformation, respectively. This suggests that Cys at position 75 may be important for transformation. Charge and structural changes at teh COOH termini of several mutants impaired transformation significantly, but those at the middle region did so only mildly. In addition, the 16,000-molecular-weight pore-forming protein (16K protein) is known to associate with BPV-1, HPV-6, and HPV-16 E5 proteins. In this study, we investigated the correlation between E5a-16K binding affinity and the transforming activity of E5a by the use of 11 E5a mutants. Results show that E5a and these 11 E5a mutants could bind to the 16K protein when these proteins were coexpressed in COS cells, suggesting that simple binding of the 16K protein by E5a may not be sufficient for cell transformation.     

The direct action of 17 beta-estradiol in isolated omental artery from nonpregnant and pregnant women is related to calcium antagonism

OBJECTIVE: Our purpose was to study the mechanism by which 17 beta-estradiol modulates contractile activity in isolated rings of omental artery from nonpregnant and pregnant patients. STUDY DESIGN: Rings of omental artery with intact endothelium from nonpregnant and pregnant women were mounted in organ chambers for isometric tension recording. The concentration-relaxation relationship to 17 beta-estradiol (10(-7) mol/L to 3 x 10(-5) mol/L) was studied in rings contracted with 60 mmol/L potassium chloride (in both the absence and the presence of tamoxifen, 10(-6) mol/L). The effect of 17 beta-estradiol (10(-5) mol/L) on the contraction induced by 60 mmol/L potassium chloride and on the concentration-contraction relationships to both norepinephrine (10(-9) mol/L to 10(-5) mol/L) and calcium ion (0.05 mmol/L to 2.5 mmol/L in calcium-free depolarizing solution) were studied in the presence and absence of tamoxifen (10(-6) mol/L). The maximal contraction, negative logarithm of the concentration producing 50% relaxation or 50% contraction to the reference 60 mmol/L potassium chloride contraction, and the area under the curve were calculated. Data analysis was by one-way analysis of variance, Newman-Keuls test, and two-sample tests as appropriate. Probability values less than 0.05 in a two-tailed test were considered statistically significant. RESULTS: 17 beta-Estradiol relaxed omental arteries contracted with 60 mmol/L potassium chloride, and this effect was potentiated by tamoxifen in both groups. Incubation of the omental arteries with 17 beta-estradiol inhibited contractions induced by 60 mmol/L potassium chloride in rings from both groups of patients, and tamoxifen did not antagonize this effect in either group. Rings of omental artery from the nonpregnant patients (expressed as percentage of the reference potassium chloride contraction) showed greater contraction than rings from the pregnant women when exposed to norepinephrine, a statistically significant difference. 17 beta-Estradiol decreased the norepinephrine-induced contraction in omental arteries from nonpregnant but not pregnant women in a statistically significant way. Tamoxifen did not influence the effect of norepinephrine for either group. 17 beta-Estradiol inhibited calcium ion-induced contraction similarly in rings of omental artery from both nonpregnant and pregnant patients. Tamoxifen potentiated estradiol-induced inhibition in arteries from pregnant patients. CONCLUSIONS: 17 beta-Estradiol inhibits norepinephrine-induced contractions in omental arteries from nonpregnant but no pregnant patients. The inhibition of the ter sion developed after exposure to potassium chloride, norepinephrine, and calcium ion is caused by a calcium channel blocking action.     

Effects of injectio reduqing on plasma IL-8 and nitric oxide levels in rabbits with endotoxin induced disseminated intravascular coagulation

Experiments were performed for investigating the effects of Injectio Reduqing (RDQ) on plasma interleukin-8 (IL-8), NO2-/NO3-, complement 5a(C5a) and polymorphonuclear neutrophilic leukocyte (PMN) Chemotaxis Index (CI) in rabbits with endotoxin-induced disseminated intravascular coagulation (DIC). The results showed that plasma IL-8, NO2-/NO3-, C5a and CI levels of PMN increased markedly in model group, which were confirmed pathologically with obvious damage of tissues or organs. While in RDQ group the abov-mentioned parameters and damage of tissues or organs were reduced significantly (P < 0.01). The results suggested that the IL-8 and NO might be involved in pathogenesis of endotoxin-induced DIC, and RDQ could be used in preventing or treating DIC through mechanism of regulation of cytokines network.     

The expression, origin and function of tenascin during peripheral nerve formation in the chick

During development of the peripheral nervous system, the extracellular matrix molecule tenascin has been found to be closely associated with growing axons. However, its origin and function in peripheral nerve formation are far from clear. In this study, we examined the expression of tenascin during outgrowth of sensory, motor and sympathetic preganglionic axons, and assessed its origin and function in peripheral nerve formation. During outgrowth of sensory and motor axons, a high concentration of tenascin and its mRNA was found to surround sensory and motor axons in the newly formed spinal nerves. The source of this tenascin was examined through a series of surgical manipulations. Neural crest removals did not alter the distribution of tenascin protein or its mRNA surrounding the spinal nerves. Transplantation of quail somites into chick embryos showed that, similar to the distribution of tenascin, there is a high concentration of somitic cells surrounding the spinal nerves. Moreover, somite removals resulted in a reduction of the tenascin and tenascin mRNA surrounding the spinal nerves. Taken together, these results suggest that the majority of the tenascin surrounding the spinal nerves is of somitic origin. Possible functions of tenascin associated with peripheral nerve formation were examined through injections of tenascin or its antiserum into individual somites prior to or during axon outgrowth. Injections of tenascin or its antiserum did not alter the trajectory of peripheral axons in the anterior half of the somite, nor produce gross abnormalities in the morphology of peripheral nerves, suggesting that tenascin does not play a crucial role in the early formation of peripheral nerves.