Acquisition of thymidylate by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

The pathway for the acquisition of thymidylate in the obligate bacterial parasite Rickettsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3H]thymidine added to the culture. In the thymidine kinase-negative host cells, the label available to the rickettsiae in the host cell cytoplasm would have been thymidine, and in the thymidine kinase-positive host cells, it would have been both thymidine and TMP. Further support for the inability to utilize thymidine was the lack of thymidine kinase activity in extracts of R. prowazekii. However, [3H]uridine incorporation into the DNA of R. prowazekii was demonstrable (973 +/- 57 dpm/3 x 10(8) rickettsiae). This labeling of rickettsial DNA suggests the transport of uracil, uridine, uridine phosphates (UXP), or 2'-deoxyuridine phosphates, the conversion of the labeled precursor to thymidylate, and subsequent incorporation into DNA. This is supported by the demonstration of thymidylate synthase activity in extracts of R. prowazekii. The enzyme was determined to have a specific activity of 310 +/- 40 pmol/min/mg of protein and was inhibited greater than or equal to 70% by 5-fluoro-dUMP. The inability of R. prowazekii to utilize uracil was suggested by undetectable uracil phosphoribosyltransferase activity and by its inability to grow (less than 10% of control) in a uridine-starved mutant cell line (Urd-A) supplemented with 50 microM to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 microM uridine (117% of control). However, no measurable uridine kinase activity could be measured in extracts of R. prowazekii. Normal rickettsial growth (92% of control) was observed when the host cell was blocked with thymidine so that the host cell's dUXP pool was depressed to a level inadequate for growth and DNA synthesis in the host cell. Taken together, these data strongly suggest that rickettsiae transport UXP from the host cell's cytoplasm and that they synthesize TTP from UXP.     

Purification and substrate specificity of two cysteine proteinases of Giardia lamblia.

The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of Mr = 95,000 and 35,000 +/- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (Mr = 95,000) and proteinase II (Mr = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.     

Parental investment decision rules in tree swallows: parental defense, abandonment, and the so-called Concorde Fallacy

I conducted an experiment to detect the importance of past investmentand expected benefits to reproductive "decision making" by treeswallows (Tachycineta bicolor). Three experimental groups, balancedfor measurable indicators of parental quality, were created:one had its clutch reduced in size near the beginning of incubation,the second had its brood reduced near chickfledging, and thethird had no brood or clutch reduction. All experimental groupswere tested for differences in 19 measures of parental defensebehavior at the same stage in the nesting cycle by exposingthem on successive days to two different predators, a ferretand a rat snake, at the nest box. There were no detectable effectsof experimental treatment on parental defense; however, therewere differences in abandonment frequency related to the severityof experimental clutch reduction. These results suggest thattree swallows respond to differences in expected benefits in"deciding" whether or not to abandon a nesting attempt. Becausepast investment can affect the prospective costs of abandonmentand renesting, the most natural currency for comparing costsand benefits is prospective. Adopting such a currency for parental"decisions" lessens concern over the so-called Concorde Fallacy,but it highlights our relative ignorance of how past reproductiveeffort influences the costs of future reproduction. [Behav Ecol1991,2:133–142]     

Chronobiologic approach to beat-to-beat variations of cultured murine myocardial cells

An earlier demonstration of a circadian rhythm in rat atria by others is complemented herein by observations in culture: A single murine myocardial cell and two sets of grouped cells beating in culture for several days reveal several features of an anticipated, presumably built-in spectrum of multifrequency rhythms and trends, the chronome. Circadian and about 12-h (circasemidian) components are modulated by an approximately 84-h (circasemiseptan) component, which cannot be separated from trends in view of the brevity of the series. The circumstance under which the culture is aging and in which fibroblasts proliferate is a further complication that limits the findings to a single cycle reproduced in three separate cultures. Whether it is a rhythm that repeats itself or a response to placement into culture, an approximately 3.5-d component in the beating of myocardial cells in culture is to be aligned with a very prominent similar component found in the incidence of 85,819 human myocardial infarctions.     

Ornithogalum macrum Speta (Hyacinthaceae), eine merkwürdige neue art aus der türkei

Ornitogalum macrum Speta, a new species, is described from the Çakilli-Pass on the road from Akseki to the Bey sehir-Lake in Turkey. It is characterized by its tenderness. Roots turning yellow and concrescente leaves of the bulb speak in favour of a membership to theO. umbellatum group. But the late appearance of the foliage leaves in spring and the lack of ridges on the gynoeceum have hitherto never been found in this connection. There is however an indisputable similarity as to characteristics of theO. undulatum group. The chromosome number is 2n=54. The interphase nuclei are chromomeric.     

Tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction by mycoplasmas.

We investigated 22 mycoplasma and acholeplasma species for their ability to reduce tetrazolium salts by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The test results were evaluated visually, as well as spectrophotometrically, by using an enzyme-linked immunosorbent assay reader. Our results were very similar to the results obtained when the tetrazolium salt reduction assay described by Aluotto et al. was used. However, the MTT reduction assay appeared to be better because it is faster, more objective and sensitive, easier to evaluate, and less expensive; in addition, it allows quantitative determinations. By using regression analysis a linear correlation between formazan production and the number of colony-forming units was demonstrated for all of the species investigated, indicating that the MTT assay can also be used for growth, toxicity, or chemosensitivity tests for the mycoplasma species that are capable of reducing tetrazolium salts.     

Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I.

The carbohydrate specificity of mistletoe toxic lectin-I (ML-I) was studied by haemagglutination-inhibition assay. The results indicated that ML-I has a broad range of affinity for Gal alpha,beta linked sequences. The galabiose (E, Gal alpha 1----4Gal) sequence, a receptor of the uropathogenic E. coli ligand, was one of the best disaccharide inhibitors tested. The lectin also exhibits affinity for Lac(Gal beta 1----4Glc), T(Gal beta 1----3GalNAc), I/II(Gal beta 1----3/4GlcNAc) and B(Gal alpha 1----3Gal) sequences. Gal alpha 1----4Gal and Gal beta 1----4Glc are frequently occurring sequences of many glycosphingolipids located at the mammalian cell membranes, such as intestinal and red blood cell membranes, for ligand binding and toxin attachment. This finding provides important information concerning the possible mechanism of intoxication of cells by the mistletoe preparation.