In vitro and in vivo studies of Trypanosoma cruzi DNA polymerase

One major DNA polymerase has been purified and characterized from Trypanosoma cruzi. The enzyme has a sedimentation coefficient of 6.8 S corresponding to an approximate molecular weight of 180,000 assuming a globular shape. The enzyme recognizes activated DNA very efficiently, as well as synthetic polydeoxynucleotides, whereas poly rA-dT12 is very poorly utilized. Trypanosoma cruzi DNA polymerase is not inhibited at all by aphidicolin, while araCTP inhibits the enzyme very slightly. The purified enzyme is strongly inhibited by N-ethyl maleimide, dideoxyTTP, ethidium bromide and berenil. All our attempts to find a DNA polymerase sensitive to aphidicolin in vitro have failed, nor have we been able to find a low molecular weight DNA polymerase in this organism. However, when DNA synthesis was studied in whole trypanosomes, aphidicolin was shown to inhibit DNA synthesis more efficiently than ethidium bromide and berenil.     

High-resolution color video cytophotometry

Comparison was made between cytophotometric measurements obtained using two data acquisition systems, one a microphotometer and the other a rapid video camera system, to ascertain whether the degradation of data with the faster video acquisition system still results in recorded images of sufficient quality to permit computer discrimination between cells of very similar appearance. Normal-appearing intermediate cells from cases with normal cytology and those from patients with dysplasia or malignant disease, as well as the subvisual markers within these cells that have rendered them capable of cytophotometric discrimination, were used for the study. Comparison of the data recorded by the two systems indicates that the diagnostic information is preserved in the change-over to a full-field, video-rate scanning system, with differences in the data caused primarily by differences in the spectral response of the two systems. This was reflected in the substantial differences observed in the color-related features and the lesser differences seen in the textural features, while the morphometric features (outline and shape) were virtually unaffected. The differences were primarily expressed on a cell-to-cell basis; in sets of about 300 cells, which would be used in patient-to-patient comparisons, the feature values showed remarkable consistency between the two systems.     

Localization of hydrophobic sites in calmodulin and skeletal muscle troponin C studied using tryptic fragments: a simple method of their preparation

The exposure of hydrophobic sites on calmodulin, skeletal muscle troponin C and their tryptic fragments was investigated using Phenyl-Sepharose chromatography. A strong binding of both proteins and their fragments corresponding to the NH2-terminal halves of polypeptide chain of respective proteins in the presence of calcium ions was observed. Only a weak interaction with Phenyl-Sepharose or its lack was observed under these conditions for fragments corresponding to the COOH-terminal halves of calmodulin and troponin C, respectively. The elution of the samples from Phenyl-Sepharose column with ethylene glycol gradient allowed to compare relative hydrophobicity of both proteins and their fragments. The results show that hydrophobic properties of calmodulin and troponin C are virtually preserved in their fragments obtained as a result of their cleavage by trypsin in half. They also indicated that the exposure of hydrophobic residues caused by the binding of calcium ions takes place mainly in the NH2-terminal halves of polypeptide chains of both proteins. A simple method of purification of tryptic fragments of both proteins based on the difference in the strength of their interactions with Phenyl-Sepharose is described.     

Transitory hematologic effects of moderate exercise are not influenced by iron supplementation

A young women's exercise/fitness class tested the idea that administration of supplemental iron would prevent "sports anemia" that may develop during exercise and training and improve iron status of exercising females of menstrual age. Fifteen women (aged 18-37) were selected for each of three treatment groups: (1) no supplemental iron; (2) 9 mg X d-1 of Fe; and (3) 18 mg X d-1 of Fe (1 US Recommended Daily Allowance). Women exercised at approximately 85% of maximal heartrate for progressively increasing lengths of time in a jogging program and worked up to 45 min of exercise 4 d X week-1 for 8 weeks. Hematologic analysis was performed in weeks 1, 5, and 8. A significant decline in hemoglobin (Hb) concentration and hematocrit (Hct) was observed at week 5 when all data were examined without regard for iron intake; these red cell indices returned to pre-exercise levels by week 8. Reduction of mean cell hemoglobin concentration (MCHC) indicated that the midpoint decline was not caused by simple hemodilution during exercise. Serum ferritin (SF) concentration changed in parallel with Hb and Hct. Although the midpoint decline in SF was not statistically significant, it ruled out the possibility that turnover of red cell iron was directed to storage. Lowered MCHC and SF suggested lower availability of iron during the synthesis of a new generation of red cells. Few iron treatment effects of magnitude were observed. Iron did not prevent the midpoint decline in Hb concentration. Iron intake did not affect SF, serum iron, transferrin saturation, or final Hb, and Hct.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism of biological membranes: I. Mitochondria and red blood cell ghosts

Improved circular dichroism curves are reported for mitochondria and red blood cell ghosts. The red shift and low amplitudes of the suspension spectra are shown to be due to distortions arising from the particulate nature of the samples. Previously proposed details of membrane structure based on the “red shift” are not supported by the corrected spectra.     

Stimulation by epinephrine of adenyl cyclase activity, cyclic AMP formation, DNA synthesis and cell proliferation in populations of rat thymic lymphocytes

During the first ten minutes after the beginning of a continuous exposure of rat thymocyte populations (maintained in vitro) to epinephrine, there is an increase in the cellular concentration of cyclic AMP. The hormone also increases the activity of a crude preparation of the thymocyte's cyclic AMP-forming enzyme, adenyl cyclase. Between 30 and 45 minutes after the beginning of exposure to epinephrine, an additional part of the cell population begins to synthesize deoxyribonucleic acid (DNA). These changes are finally followed two to four hours later by an increase of the flow of cells into mitosis. Since cyclic AMP itself is known to stimulate both the initiation of DNA synthesis and thymocyte proliferation, and since the mitogenic action of epinephrine is shown to be potentiated by caffeine and inhibited by imidazole, it is concluded that the mitogenic action of this hormone is mediated by the cyclic nucleotide.     

Three-dimensional structure of abnormal human haemoglobins Chesapeake and J Capetown

The replacement of the invariant residue, arginine FG4(92)α, by a leucine in the mutant human haemoglobin Chesapeake causes drastically abnormal functional properties. When the arginine is replaced by a glutamine in haemoglobin J Capetown the mutant protein is almost normal. Crystallographic studies at 5.5 Å resolution show that the deoxy form of these two mutants have no significant structural distortions. In contrast, the structure of oxyhaemoglobin Chesapeake is considerably distorted. It appears that in the oxy form, the leucine side chain introduces impermissibly close van der Waal's contacts which disrupt the structure. This disturbance of the oxy structure is probably responsible for the abnormal properties displayed by haemoglobin Chesapeake. The structural basis for the milder abnormalities of haemoglobin J Capetown is as yet unknown.