Membrane activator of the 72 kDa type IV collagenase in malignant breast carcinoma patients: Expression of membrane-type 1-matrix metalloproteinase (MT1-MMP)

In this study, we determined proMMP-2 activating capacity of membrane extract prepared from the tissue of invasive ductal carcinoma of breast by zymogram gel analysis. We compared the effect of membrane extract on the activation of the latent type IV collagenases with that of the organic mercurial compound leg, APMA)-induced self cleavage of the latent type IV collagenases. We also compared the expression levels of MT1-MMP between invasive carcinoma and normal tissue by Western blot, Northern blot and semi-quantitative RT-PCR analysis. Our result demonstrated that the specificity of processing by breast carcinoma membrane activator corresponds to the specificity of MT1-MMP, which clearly showed the conversion of 72-kDa proMMP-2 to the activated form while APMA processed both 72- and 92-kDa proMMPs to their activated forms. MT1-MMP protein and mRNA were expressed both in invasive carcinoma and normal tissues, and the expression levels in both tissues were comparable. Quantitative analysis of the mRNA level by RT-PCR revealed that the difference of MT1-MMP mRNA between carcinomas and normal tissues was not statistically significant on Wilcoxon signed-ranks test (P>0.05). The results from the study on the expression of MT1-MMP gene suggest that the cellular activation of MMP-2 in breast tissue, requires additional effects in addition to up-regulation of MT1-MMP.

Effect of Carbon Monoxide-Induced Hypoxia on Synaptosomal Uptake and Release of Dopamine in Rat Striatum

We studied the effect of carbon monoxide (CO)-induced hypoxia on synaptosomal uptake and release of dopamine (DA) in rat striatum. When the rats were intoxicated at a blood level of carboxyhemoglobin (HbCO), 60-70% for 3-4hrs, [3H] DA uptake was inhibited as much as 80% of control activity. This suppressed activity remained as long as 12 hrs after termination of the intoxication. After a week recovery period, the suppressed uptake activity was restored completely. When the rats were intoxicated maintaining a blood level of HbCO at 30-40% for 6-7hrs, the uptake was inhibited to 57% of the control actvity and this suppressed activity was restored within 12hrs. For the rats maintaining a blood level of HbCO at 15-25% for 6-7hrs, uptake inhibition was not shown. Acute CO intoxication(at 60-70% of HbCO for 3-4 hrs) caused an increase in K+-stimulated DA release to 147% of the control value. In conclusion, the diminished uptake and increased release of striatal DA in a CO intoxicated brain would cause an extraneuronal accumulation of DA with depletion of intraneuronal DA level, which may play a role in CO-induced hypoxic cell damage.

Studies of Hepatic, Brain Monoamine Oxidase and Brain Serotonin in Rats

The effects of 7-ethyl-8-methylf1avin (7-Et) and 7-methyl-8-ethyl-flavin (8-Et) on rat hepatic monoamine oxidase (MAO), brain MAO activity and 5-hydroxytryptamine (5-HT or serotonin) in rat brain were investigated. In the study of hepatic MAO activity, kynur-amine a nonphysiological substrate for both A and B type MAO, was used for a spectro-photometric method, and [14C]-labeled amines were also used for a radiometric procedure for camparison with MAO activity determined by the spectrophotometric method. The rate of change in MAO activity of hepatic mitochondria from rats receiving Rb-def and 7-Et and 8-Et flavin showed the activity was severely reduced during 8 weeks. Rapid reduction of enzyme activity (50% in def-group, 35% in 7-Et group and 8% 8-Et flavin group) was observed at the end of 2 weeks. The enzyme activity lasted with slow decre-ment of enzyme level from 4 weeks to the end of 8 weeks as low as 16% in def, 18% in 7-Et and 3% in 8-Et flavin group. The trend of decrement of MAO activity when kynura-mine was used as a substrate appears to be similar with the small variation of MAO activity when [14C]-labelled tyramine, dopamine, serotonin and tryptamine respectively were used as substrate. The rate of decay of brain mitochondrial MAO activity in rats receiving each respective f1avin was not rapid and severely depressed as the MAO activity we have found in liver mitochondrial MAO of rats during the 8 week experimental time, but a similar tendency of decay of MAO in each group was observed. The potent inhibitory effect of 8-Et on brain MAO was confirmed by the study of the simultaneous measure-ment of MAO activity in each experimental group. when the reduction Of brain MAO activity in rats receiving 8-Et after 6 weeks was approximately 80% of normal and in the same rats the concentration of brain 5-HT showed a 60% increment of that of the normal mts. During the experimental period there is no absolute parallelism between the MAO inhibition and 5-HT increase. However when the reduction of MAO activity reached 80% of normal value, the concentration of 5-HT increased dramatically as much as 60% of normal value. The results so far suggest clearly that 8-Et produces a much more potent inhibitory effect on the hepatic MAO a s well as brain MAO in rats. Therefore our present and previous results suggest that 7-Et and 8-Et flavin should bind itself to hepatic, brain MAO apoenzyme in the condition of total absence of riboflavin in these animals, and the holenzyme is catalytically inactive.

Effect of Alloxan-diabetic Rat Fed with Different Diets on Ureogenesis in Isolated Perfused Liver

The effect of alloxan-diabetic rat fed with normal, high fat, low protein and high protein diets on the rate of urea production and the activities of enzymes associated with the urea cycle (ornithine transcarbamoylase, E.C. 2.1.3.3, OTC; arginase, E.C. 3.5.5.1) have been studied in intact and isolated perfused liver. The amount of urea excretion was the highest in the high protein diet group. When each diet group was treated with alloxan, total urea excretion showed little differences between each diet group and its corresponding control group with the exception being in the normal diet group. However, the enzyme activity of OTC was increased significantly by alloxan treatment in low and high protein diet groups as compared to corresponding control groups. Similar results were obtained in arginase activity, although the magnitude of the change was less marked. In liver perfusion experiments on rats treated with alloxan, the amount of urea production and changes in OTC and arginase activity were very similar with those in the intact liver. These results suggest that alloxan treatment in normal diet group causes an increase in urea excretion both in intact and perfused liver regardless of changes in enzyme activities and total urea excretion, and enzyme activities are affected by changes in dietary components but the changes of enzyme activities may not correlate with total urea excretion.