An analysis of the inactivation of the frog sperm nucleus by toluidine blue

Toluidine blue, applied to frog sperm under appropriate conditions, inactivates specifically the sperm nucleus, leaving the extranuclear parts of the cell undamaged. Thus, the dye-treated spermatozoa stimulate eggs to cleave normally, but contribute no chromosomes to the resulting embryos, which develop as typical gynogenetic haploids. The concentration of dye required to produce this inactivation varies with pH. Measurements made over the entire pH range which can be tolerated by sperm cells showed that in the lower part of the range (5 to 7) the effective dye concentration was about 5 x 10(-6)M; in the intermediate range (7 to 8.5) it was 1 x (-6) to 1 x (-7)M; and for the higher pH values (8.5 to 10.0) it was about 5 x (-8)M. Using sperm suspensions containing 1500 cells per c. mm. these concentrations of dye produced specific inactivation of the sperm nuclei within 7 to 60 minutes at 18 degrees C. Tests of the reversibility of the inactivation were made by transferring the sperm from the dye to a dilute Ringer's solution after a known degree of inactivation had been produced. Following removal of the dye the sperm cells were tested on eggs over a period of 2 hours. During this time there was no indication of a reversal of the inactivation. Microscopic observations of sperm treated with (-5)M or 5 X (-5)M dye show that the dye is taken up by the sperm nucleus, which is faintly but definitely stained. The dye appears to be uniformly distributed in the nucleus, while extranuclear structures remain unstained. Measurements of the amount of dye bound per sperm nucleus indicate that the minimal quantity required for complete inactivation is about 6.7 x (-18) mole, while the maximal amount which can be bound without injury to extranuclear structures is about 1.5 x (-16) mole. The value obtained for the minimal requirement (6.7 X (16) mole = 4 X (6) molecules) suggests that there are roughly 4 million binding sites in the nucleus which, when blocked by dye molecules, somehow prevent the sperm chromosomes from participating in the development of the egg.     

Mitochondrial DNA polymorphism in Tuvinian population of reindeer Rangifer tarandus L.

Mitochondrial DNA variation was examined in one of the southem most populations of domestic reindeer, inhabiting Tyva Republic (Tuva). In Tuvinian population sequence polymorphism of the mitochondrial DNA D-loop region was demonstrated. In a sample of 29 individuals 7 mitotypes were distinguished, pointing to the preservation of rather high level of genetic diversity in this population.     

Early conception factor lateral flow assays for pregnancy in the mare

The ECF™ lateral flow assay test is marketed to detect non-pregnancy in mares. The objectives of the present study were to determine the accuracy of the ECF test, the accuracy of the electronic reader accompanying the ECF test, and agreement between two human readers and the electronic reader. Serum samples were collected from anestrus, cycling but not inseminated, and inseminated mares, and were evaluated with the ECF™ test (EDP Biotech Company, Knoxville, TN, USA) at The Ohio State University and at the EDP Biotech Laboratory. Specificity ranged from 0.07 to 0.16, the negative predictive value ranged from 0.15 to 0.33, and accuracy ranged from 0.43 to 0.52. The electronic reader did not add improve the accuracy or predictive values of the test. Based on the electronic reader, 80.0% of the serum samples collected from the anestrus mares were false positives; Readers 1 and 2 had 60.0 and 33.3% false positives, respectively. For samples collected during the estrous cycle, 83.9% were false positives by the electronic reader, whereas Readers 1 and 2 had 43.7 and 26.4% false positives. We concluded that, regardless of whether the test strips were evaluated by a human or electronic reader, this assay was not accurate for determination of the non-pregnant mare.     

Regulation of voltage-gated Ca channels by lipids

Great skepticism has surrounded the question of whether modulation of voltage-gated Ca²⁺ channels (VGCCs) by the polyunsaturated free fatty acid arachidonic acid (AA) has any physiological basis. Here we synthesize findings from studies of both native and recombinant channels where micromolar concentrations of AA consistently inhibit both native and recombinant activity by stabilizing VGCCs in one or more closed states. Structural requirements for these inhibitory actions include a chain length of at least 18 carbons and multiple double bonds located near the fatty acid's carboxy terminus. Acting at a second site, AA increases the rate of VGCC activation kinetics, and in Ca_V2.2 channels, increases current amplitude. We present evidence that phosphatidylinositol 4,5-bisphosphate (PIP₂), a palmitoylated accessory subunit (β_2a) of VGCCs and AA appear to have overlapping sites of action giving rise to complex channel behavior. Their actions converge in a physiologically relevant manner during muscarinic modulation of VGCCs. We speculate that M₁ muscarinic receptors may stimulate multiple lipases to break down the PIP₂ associated with VGCCs and leave PIP₂'s freed fatty acid tails bound to the channels to confer modulation. This unexpectedly simple scheme gives rise to unanticipated predictions and redirects thinking about lipid regulation of VGCCs.     

Preventive and Curative Biological Treatments for Control of Botrytis cinerea Stem Canker of Greenhouse Tomatoes

The effects of biological treatments with PlantShield^®, Prestop^®, Quadra 136, RootShield^®, and S33 (Rhodosporidium diobovatum) and chemical treatment with Decree^® applied as a preventive or curative sprays on stem canker caused by Botrytis cinerea on tomato plants grown in sawdust were studied under near-commercial greenhouse conditions. Prestop^® and Decree^®, applied as preventive or curative sprays, PlantShield^® applied as curative spray, and S33 and Q-136 applied as preventive or preventive plus one spray to wounded surface provided season-long protection from B. cinerea stem canker. These treatments also increased fruit yield and decreased the number of dead plants compared with the inoculated control.     

Late Glacial and Holocene environmental and climatic changes from a limnological transect through Colombia, northern South America

We present a synthesis of the palaeolimnological and palaeoclimatic reconstructions of four sites in Colombia. The record from Lake El Caimito, the westernmost site on the Pacific Coast, dates from the Late Holocene and shows lacustrine sedimentation frequently interrupted by fluvial pulses. These pulses probably reflect periods of increased precipitation related to La Niña phases. East of El Caimito site is the Patía swamp, situated between the Western and Central Andean Cordilleras. The Patía records the dynamics of forest expansion/reduction and changes in water levels. Although the climatic signal of the Patia core is difficult to reconstruct, there is a clear increase in humidity in the Mid-Holocene. The Fúquene Lake record, on the Eastern Andean Cordillera, records dry and cold conditions during the Late Pleistocene, very humid conditions for the early Mid-Holocene, and dry conditions during the mid-Late Holocene. Las Margaritas site, on the eastern savannas, records dry conditions during the Early Holocene and overall humid conditions for the Mid- and Late Holocene. Climate conditions from the Fuquene and Las Margaritas sites seem to reflect the Holocene movements of the Intertropical Convergence Zone (ITCZ); the latter site being more affected by humidity coming from the Amazon region.     

Does vinblastine add to the potency of alpha interferon in the treatment of renal cell carcinoma?

The combination of alpha interferon and vinblastine has been reported to yield a response rate of 30-40% in previously untreated patients with metastatic renal cell carcinoma. This combination was given to nine patients with advanced metastatic renal carcinoma after they failed or relapsed on alpha-interferon alone, to attempt to evaluate the role of vinblastine in this combination. Neither complete nor partial response was observed. Two patients had disease stabilization for two and seven months. Our preliminary results suggest that vinblastine did not add to the efficacy of interferon in this group of patients.