Polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients.

The feasibility of DNA amplification by the polymerase chain reaction for specific detection of Trypanosoma cruzi in human blood was investigated. We have used primers flanking a 220-bp fragment of highly repetitive elements, the E13 element, in T. cruzi nuclear DNA. Only polymerase chain reaction products from blood samples of chronic chagasic patients showed several amplified fragments in 1.6% agarose gels stained with ethidium bromide. Southern blot analysis demonstrated that the 220-bp amplified fragment is specific for T. cruzi DNA and very useful to detect the presence of the parasite in blood from chronic chagasic patients.

Maternal coordination of the daily rhythm of malate dehydrogenase activity in testes from young rats: effect of maternal sympathetic denervation of the pineal gland and administration of melatonin.

Chronic sympathetic denervation of the pineal gland by bilateral removal of the superior cervical ganglia (SCG) was performed on female rats 30 days before impregnation. The offspring, maintained in the dark from birth, had disruption of the malate dehydrogenase circadian rhythm in the testes at 25 days of age. A daily injection of melatonin (1 mg/kg s.c. at 10:00 or 18:00 h) to denervated mothers from the 14th day of pregnancy up to the 10th day postpartum produced one daily phase in the enzyme activity of tests in the offspring. Entrainment of daily enzyme activity also was obtained when the hormone was administered orally to the pups during the postnatal period or when pups were reared by intact (not denervated) foster mothers. The results indicate the involvement of the maternal pineal gland in the maternal transfer of photoperiodic information necessary for the coordination of the circadian system in young rats.

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light.

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.

[Corneal reinnervation after lamellar refractive corneal surgery]. / Korneale Reinnervation nach lamellärer refraktiver Hornhautchirurgie.

In a retrospective study corneal sensitivity after refractive autokeratoplasty for correction of myopia was measured. Thirty-eight patients operated on for keratomileusis in situ and 18 patients for keratomileusis myopica were examined with the Draeger esthesiometer between 1 and 23 months after surgery. Three measurements points were on the refractive disc, two others on the untouched cornea. After keratomileusis myopica, the corneal reinnervation is significantly delayed compared to keratomileusis in situ. In the first group sensitivity is normal after 2 years and in the second group after 1 year. Even refraction and visual acuity depend on metabolism and reorganization of the tissue after surgery. Besides retarded cell repopulation and disturbed stromal metabolism--caused by toxic and cryopreservation effects--the dissection depth of the refractive ablation is responsible for these phenomena.

Alkaline phosphatase isoenzymes for the diagnosis of metastatic tumors and lymphomas of liver and bone.

The plasmatic activities of total alkaline phosphatase (ALP) (E.C. 3.1.3.1), high molecular weight-ALP (high Mr-ALP) and bone-ALP isoenzymes, were determined in healthy individuals and in patients with: neoplasia without metastases, hepatic metastases, bone metastases and mixed metastases (hepatic and bone). Variables were individually used to assess incidence of metastases and percentages of false negative and false positive results were calculated. The three values were then used together to assess metastases incidence and sensitivity, specificity, positive predictive value, negative predictive value and predictive capacity were estimated. We conclude that none of the variables per se are reliable for the diagnosis of metastases. On the other hand, the three values show high percentages of sensitivity, specificity, positive predictive value, negative predictive value and a high probability (0.93) of accurate diagnosis when applied to a larger population, with similar prevalence values.

Placental alkaline phosphatase of high molecular weight in plasma of pregnant women in the last trimester of gestation.

Human blood plasma contains alkaline phosphatase (EC 3.1.3.1) isoenzymes from bone, liver and intestine. Blood of pregnant women in the third trimester of gestation also contains an isoenzyme of alkaline phosphatase from placenta, placental-AP (PLAP). Serum from pregnant women in the last trimester of gestation shows activity of soluble placental alkaline phosphatase (sol-PLAP). It is also known that a membrane bound high molecular weight placental-AP (high Mr-PLAP) is present in butanol extracts from placental tissue. Nevertheless, up to now, high Mr-PLAP has not been founded in human plasma. A method developed in this laboratory allows detection of membrane-bound alkaline phosphatase in pellet of plasma centrifuged at 100,000x g. By applying this method we have detected high molecular weight placental alkaline phosphatase in plasma of healthy pregnant women in the third trimester of pregnancy.

Determination of alkaline phosphatase isozymes in amniotic fluid.

A simple method for the determination of the three isozymes of alkaline phosphatase (EC 3.1.3.1) contained in amniotic fluid (fetal intestinal, placental, and liver-bone-kidney) is presented. Total alkaline phosphatase activity was assayed in 10,000 g supernatants of amniotic fluid from 30 normal women between the 16th and 20th week of pregnancy. Electrophoretic patterns and inhibition by L-phenylalanine and L-homoarginine studies showed that all the fetal intestinal isozyme was precipitated in the pellet after centrifugation at 100,000 g for 90 min. Thus, the difference between total alkaline phosphatase activity and activity in the 100,000 g supernatant corresponds to fetal intestinal alkaline phosphatase. Placental isozyme can be determined by assaying alkaline phosphatase in the 100,000 g supernatant after heating at 56 degrees C for 90 min. Liver-bone-kidney isozyme activity is obtained by subtracting placental alkaline phosphatase activity from that of the 100,000 g supernatant. Mean percentages of the total alkaline phosphatase for each of the isozymes in amniotic fluid were 81% for fetal intestinal alkaline phosphatase, 7.5% for placental alkaline phosphatase and 12.0% for liver-bone-kidney alkaline phosphatase. Determination of fetal intestinal alkaline phosphatase by this method could be applied to the diagnosis of cystic fibrosis in fetuses having a 1:4 risk of being affected.

Circadian rhythm of lactate dehydrogenase in rat testis.

Activity of total lactate dehydrogenase (LDH) and of the isozyme X (LDH X or C4) have been determined at 2 hr intervals during 24 hr cycles in testis of adult rats maintained since birth in a photoperiod of 14 hr light: 10 hr dark. LDH X activity of epididymal sections (caput, corpus and cauda) from the same animals was also determined. Total LDH and LDH X activities in testis exhibited circadian rhythms with different timing. LDH X in the three portions of epididymis showed diurnal variations similar to those in testis. Rats subjected to constant light or constant dark presented marked modifications of LDH X profiles, indicating that the photoperiod plays a synchronizer role. While total soluble proteins did not show variations in testis of rats exposed to the photoperiod, a circadian rhythm was demonstrated in animals maintained in constant light or dark.

Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit.

Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda.

[Corneal reinnervation after keratomileusis in situ and keratomileusis myopica--a comparison]. / Hornhautreinnervation nach Keratomileusis in situ und Keratomileusis myopica--Ein Vergleich.

BACKGROUND: In a retrospective study corneal sensitivity after keratomileusis in situ and keratomileusis myopica was measured. METHOD: 5 points on the cornea were measured with the Draeger aesthesiometer at 18 eyes after keratomileusis myopica and at 66 eyes after keratomileusis in situ. RESULTS: The ingrowth of corneal nerve fibres is delayed after keratomileusis myopica compared to keratomileusis in situ because of delayed stromal repopulation of keratocytes and disturbances of stromal metabolism due to toxic effects of the cryopreservation solution.

Placental alkaline phosphatase of high molecular weight in plasma of pregnant women in the last trimester of gestation.

Human blood plasma contains alkaline phosphatase (EC 3.1.3.1) isoenzymes from bone, liver and intestine. Blood of pregnant women in the third trimester of gestation also contains an isoenzyme of alkaline phosphatase from placenta, placental-AP (PLAP). Serum from pregnant women in the last trimester of gestation shows activity of soluble placental alkaline phosphatase (sol-PLAP). It is also known that a membrane bound high molecular weight placental-AP (high Mr-PLAP) is present in butanol extracts from placental tissue. Nevertheless, up to now, high Mr-PLAP has not been founded in human plasma. A method developed in this laboratory allows detection of membrane-bound alkaline phosphatase in pellet of plasma centrifuged at 100,000x g. By applying this method we have detected high molecular weight placental alkaline phosphatase in plasma of healthy pregnant women in the third trimester of pregnancy.

Alkaline phosphatase isoenzymes for the diagnosis of metastatic tumors and lymphomas of liver and bone.

The plasmatic activities of total alkaline phosphatase (ALP) (E.C. 3.1.3.1), high molecular weight-ALP (high Mr-ALP) and bone-ALP isoenzymes, were determined in healthy individuals and in patients with: neoplasia without metastases, hepatic metastases, bone metastases and mixed metastases (hepatic and bone). Variables were individually used to assess incidence of metastases and percentages of false negative and false positive results were calculated. The three values were then used together to assess metastases incidence and sensitivity, specificity, positive predictive value, negative predictive value and predictive capacity were estimated. We conclude that none of the variables per se are reliable for the diagnosis of metastases. On the other hand, the three values show high percentages of sensitivity, specificity, positive predictive value, negative predictive value and a high probability (0.93) of accurate diagnosis when applied to a larger population, with similar prevalence values.