A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.     

Use of diathermy for weeding heterogeneous tissue cultures

Summary Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelail cells. Primary cell cultures were observed at ×100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture. Dr. Marks receives a Medical Postgraduate Research Scholarship from the National Health and Medical Research Council of Australia. Financial support was received from the Leo Leukaemia and Cancer Research Trust and the Scleroderma Association of New South Wales.     

Trypanosoma brucei: analysis of relapsing populations in sensitive and resistant breeds of cattle

The clone DiTat 1.1 of Trypanosoma brucei brucei was injected into four bovids, and clones obtained from successive waves of parasitemia were used to study the expressed variant-specific surface glycoprotein repertoire. Twenty-four clones were obtained which could be classified into 12 different variable antigen types, in addition to the clone injected, using agglutination or immunofluorescence with monospecific antisera. The variable surface glycoproteins of the 25 clones were extracted using the detergent octyl-beta-D-glucopyranoside in the presence of the protease inhibitor, N-cbz-L-phenylalaninechloromethylketone. The molecular weights varied from 52,000 to 69,000 and the pI from 5.0 to 8.8. The virulence of 14 clones representing 13 variable antigen types was ascertained in mice. The mean survival time ranged from 20.5 to 43.0 days. Clones isolated from early peaks of parasitemia in the bovid were the most virulent while clones derived from later peaks were less virulent. It seems that organisms of diminishing virulence appear in bovids, leading to self-cure of the disease. All clones were sensitive to human serum in a blood infectivity inhibition test. Antibody against all virulent clones appeared in 20 cattle (10 Zebus, 10 Baoulés) which had been injected with T. brucei DiTat 1.1. There was no evidence for parasites of high or low virulence being preferentially expressed in resistant or sensitive hosts.     

Larval development and metamorphosis in the marine pulmonate Amphibola crenata (Mollusca: Pulmonata)

The development of the free-swimming veliger of Amphibola is followed from hatching to settlement, and the larval structures compared with those of post-metamorphic juveniles and adult snails. Observations of living specimens and light-microscope sections were combined with scanning electron microscopy to build up a composite picture of veliger structure.
Four stages in the development of veligers are recognized, each being characterized by the appearance of organ systems such as the mantle cavity, larval heart, adult heart and kidney, and larval pallial gland. At or after metamorphosis, the larval systems (heart, kidney and pallial gland) disappear, and the developing adult organs move to the positions characteristic of adult snails.
Organogenesis in Amphibola veligers is compared with that of prosobranch and opisthobranch larvae, and with that of pulmonate larvae with direct development. The closest similarity is seen to be with opisthobranch veligers.     

Isolation and Genetic Analysis of Caulobacter Mutants Defective in Cell Shape and Membrane Lipid Synthesis

In this paper we report the isolation, characterization and genetic analysis of several C. crescentus mutants altered in membrane lipid synthesis. One of these, a fatty acid bradytroph, AE6002, was shown to be due to a mutation in the fatA gene. In addition to the presence of the fatA506 mutation, this strain was found to contain two other mutations, one of which caused the production of a water-soluble brown-orange pigment (pigA) and another which caused formation of helical cells (hclA). Expression of the latter two phenotypes required complex media and both were repressed by glucose. However, the lesions were mapped to loci that are separated by a substantial distance. The hclA and the fatA genes mapped close together, possibly implying that comutation had occurred in AE6002. Data are presented that allow the unambiguous identification of a second Fat gene (fatB) in C. crescentus. The map position of another mutation in membrane lipid biogenesis, the glycerol-3-PO₄ auxotroph gpsA505, was also determined. During this study the flaZ gene was fine-mapped and the positions of proC and rif changed from the previously reported location.     

Immunoglobulin light chains in chronic lymphocytic leukemia and related diseases

The combination of two-dimensional mini gel electrophoresis with the 'western blot' technique proves to be a powerful tool in characterizing lymphoid cells. By testing for kappa and lambda immunoglobulin light chains we were able to differentiate between mono-, oligo-, and polyclonal B-cell diseases. The distribution of the lambda isotypes of 24 cases with chronic lymphocytic B-cell leukemia (B-CLL) tested was not random when compared to the distribution of the lambda light chains in B-lymphocytes of normal persons. This might implicate a genetic link between the lambda loci (chromosome 22) and the development of the lambda-CLL.     

Influence of the existence of a resting state on the decay of synchronization in cell culture

General relationships between the distribution of cell doubling times and the growth pattern of an initially synchronized cell population are applied to the model proposed by Smith and Martin (1973) in which the mitotic cycle or "B" phase is preceded by a random-exit resting "A" state. Results show that culture synchronization decays so rapidly as to be virtually unobservable unless the time spent by a cell in the B phase is at least equal to that spent in the A state. If synchronization persists over several mitotic cycles, the growth pattern is determined to a much greater extent by variation in the duration of the B phase than by the probability of exit from the A state. Accordingly the growth pattern of a cell population, like the doubling time distribution which governs the pattern, is of limited usefulness in detecting the existence of a resting state.     

Turbo Tree: a fast algorithm for minimal trees

A branch and bound algorithm is described for searching rapidlyfor minimal length trees from biological data. The algorithmadds characters one at a time, rather than adding taxa, as inprevious branch and bound methods. The algorithm has been programmedand is available from the authors. A worked example is givenwith 33 characters and 15 taxa. About 8 x 10¹² binary treesare possible with 15 taxa but the branch and bound program findsthe minimal tree in <5 min on an IBM PC. Received on January 15, 1987; accepted on February 23, 1987     

Construction of a Tn5 derivative determining resistance to gentamicin and spectinomycin using a fragment cloned from R1033

A physical and genetic map of the IncP plasmid R1033 was constructed: restriction fragments were subcloned and antibiotic resistance genes were located. The map is consistent with previous reports that R1033 is a derivative of RP4 carrying a 16-kb transposon Tn1696 which contains the antibiotic-resistance determinants present on R1033 but not on RP4. A BamHI fragment from R1033, determining resistance to gentamicin, spectinomycin and streptomycin, was cloned into Tn5, replacing the central Bg/II fragment that determined kanamycin resistance, producing a recombinant transposon Tn5-GmSpSm. This was shown to transpose in Rhizobium leguminosarum at a frequency similar to that of the parental Tn5.