Microsequence analysis of peptides and proteins: trimethylsilylisothiocyanate as a reagent for COOH-terminal sequence analysis

A reinvestigation of the isothiocyanate-based chemistry for cyclic degradations of peptides and proteins revealed that the reagent trimethylsilylisothiocyanate (TMS-ITC) gives superior results in terms of coupling efficiency and lack of complicating side reactions. Acetic anhydride (10 min at various temperatures) was used to activate the carboxyl terminus, and 6 N HCl (30 min at room temperature) was used for cleavage as originally described by G.R. Stark (Biochemistry 8, 4735, 1968). Reaction conditions for efficient coupling were explored using subtractive chemistry on bradykinin, a nonapeptide, and separation of the reaction products by reverse-phase high-performance liquid chromatography. The products were analyzed by fast atom bombardment-mass spectrometry and shown to be the N-acetylated starting material and the N-acetylated des-Arg9 derivative of bradykinin. The pseudo-first-order rate constants measured at 50, 70, and 90 degrees C were 5.6 X 10(-5), 5.1 X 10(-4), and 8.6 X 10(-4) s-1, respectively. In order to obtain complete couplings within 30-40 min at 50 degrees C, the effect of pyridine catalysis was studied. The addition of 0.225 M pyridine resulted in roughly doubling the rates at 50 and 70 degrees C. In the case of bradykinin, the reaction with TMS-ITC in the presence of the pyridine catalyst at 50 degrees C was complete in 15 min. In order to apply this methodology to the analysis of proteins, the thiohydantoin derivatives of amino acids were synthesized and separated by reverse-phase HPLC. The derivatives were also characterized by mass spectrometry. The above reaction conditions were tested on 3 nmol of sperm whale apomyoglobin for three cycles of degradation. The sample was first coupled to p-phenylene diisothiocyanate-derivatized aminopropyl glass with a 90% yield. The approximate initial yield of glycine at cycle one was 30%. The first three cycles corresponded exactly to the predicted carboxy-terminal sequence of myoglobin. These results demonstrate the feasibility of a new Stark reagent for automated carboxy-terminal chemistry.     

Submarine foraging behavior of alcids in an artificial environment

We used an artificial environment at Sea World, Inc, San Diego, to study underwater foraging behavior of alcids. Larger birds dove longer and had greater wingbeat frequencies. The pigeon guillemot Cepphus columba was the only species to use both feet and wings for propulsion; all others used just wings. Aggressive interactions underwater were common. Competition among alcids in the wild may occur primarily underwater, and artificial environments may be the best means to study such interactions.     

The nuclear DNA content of human breast carcinoma. Associations with clinical stage, axillary lymph node status, estrogen receptor status and outcome

Using Feulgen-DNA cytophotometry, the nuclear DNA content was determined in specimens from 169 female patients with unilateral primary carcinoma of the breast. The tumors were classified as either diploid (73 cases: 43%) or hyperdiploid (96 cases), according to the ploidy of the tumor cells. Statistically significant associations were found between the DNA content and other characteristics of the patients and their tumors. (1) In postmenopausal women, inoperable tumors were more likely to be hyperdiploid (P less than .005). (2) In patients with operable disease, diploid tumors were less likely to have metastasized to the axillary lymph nodes (P less than .005) and were also less likely to have four or more positive nodes (P = .0044). (3) Overall, 71% of the diploid tumors and 52% of the hyperdiploid tumors were estrogen-receptor (ER) positive. This difference in proportions was statistically significant (P less than .05), but when the patients were divided into premenopausal and post-menopausal groups, the proportions of ER-positive tumors were not significantly different in either group. (4) In 113 patients considered suitable for studies on outcome (mean length of follow-up of 27 months, with a range from 0 to 71 months), the rates of relapse were 3 of 55 diploid cases and 17 of 58 hyperdiploid cases. The rate of relapse was higher in the hyperdiploid group, irrespective of lymph node status.     

Growth determinations for unattached bacteria in a contaminated aquifer.

Growth rates of unattached bacteria in groundwater contaminated with treated sewage and collected at various distances from the source of contamination were estimated by using frequency of dividing cells and tritiated-thymidine uptake and compared with growth rates obtained with unsupplemented, closed-bottle incubations. Estimates of bacterial generation times [(In 2)/mu] along a 3-km-long transect in oxygen-depleted (0.1 to 0.7 mg of dissolved oxygen liter-1) groundwater ranged from 16 h at 0.26 km downgradient from an on-land, treated-sewage outfall to 139 h at 1.6 km and correlated with bacterial abundance (r2 = 0.88 at P less than 0.001). Partitioning of assimilated thymidine into nucleic acid generally decreased with distance from the contaminant source, and one population in heavily contaminated groundwater assimilated little thymidine during a 20-h incubation. Several assumptions commonly made when frequency of dividing cells and tritiated-thymidine uptake are used were not applicable to the groundwater samples.     

Aerobic and anaerobic swimming speeds of spermatozoa investigated by twin beam laser velocimetry.

The motility of bovine and ovine spermatozoa has been studied under aerobic and anaerobic conditions, using a dual beam laser velocimeter. Cells swimming under aerobic conditions were found to be characterized by a translational swimming speed and a rotation rate that were approximately double those of cells swimming in an anaerobic environment. Both types of spermatozoa have been found to exhibit a sudden coordinated transition between fast and slow swimming states when the available oxygen is exhausted. This transition from aerobic to anaerobic swimming states has also been shown to be reversible. Studies of the duration of aerobic motility using the same apparatus have shown that the cells have a constant motile efficiency over the temperature range 32 degrees-42 degrees C.     

Monotonic Change of the Mean Phenotype in Two-Locus Models

It is shown that the mean phenotype monotonically approaches the optimum in a class of symmetric, two-locus, two-allele models with stabilizing selection. In this model, mean fitness does not change monotonically. Thus, Fisher's fundamental theorem does not hold, even though another quantity of evolutionary interest, the mean phenotype, can be shown to change monotonically. Using this quantity, it is proven that global stability results for this model.     

Cycles in cannibalistic egg-larval interactions

A model of a cannibalistic larval-egg interaction such as occurs in Tribolium is developed which leads to a system of nonlinear Volterra integral equations. I determine the local stability properties of the unique equilibrium point of the model. A Hopf bifurcation analysis shows that the model always undergoes a subcritical bifurcation when stability is lost. Numerical solutions confirm the presence of multiple attractors over a range of parameter values. The form of the cycles observed in the numerical solutions is analogous to that observed in laboratory populations of Tribolium.     

Population biology of two land snails (Mesomphix spp.): variation among six southern appalachian sites with differing disturbance histories

Summary Ecological and genetic properties of two North American terrestrial gastropods (Mesomphix spp.) were characterized in paired control and previously logged watersheds in two North Carolina forests (Coweeta and the Great Smoky Mountains National Park) of the Southern Appalachian Biosphere Reserve Cluster. Shell growth was greater in the control sites, but density and mortality were largely independent of prior logging history and forest reserve. Based on starch gel electrophoresis data, both species showed their highest levels of genetic diversity in the Coweeta forest, the component of the reserve cluster which had the most extensive and variable history of logging disturbance. M. subplanus also exhibited higher levels of heterozygosity in logged than in control watersheds, and M. andrewsae showed over twice as many rare alleles in disturbed sites as in control sites. F-statistic analysis depicted both excess levels of homozygosity and moderate genetic differentiation among the populations, reflecting the effects of small population size and perhaps drift and inbreeding. Estimated gene flow was relatively low. These results correspond to the recent finding by Bryant et al. (1987) and others on the effects of bottlenecks, and to the contrasting history of habitat instability of the two major study forests.