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.     

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.     

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     

Net positives: conservative approach to measurement of proportions positive in substerilization process studies.

A method (designated the net positive approach) is described for scoring results of sterility tests done with more than one recovery condition on product items treated with inactivating agents at levels less than those used in sterilization processes proper (subprocess treatments). Three tests of sterility, one per recovery condition, constitute a single sample. A growth in one or more recovery conditions is scored as a net positive for that sample. The net positive approach reduces the occurrence of false-negatives and assigns a value to the proportion of items reported as nonsterile (proportions positive) that is conservative.     

Protein Patterns of Growing and Starved Cells of a Marine Vibrio sp.

Fingerprint protein patterns were produced by two-dimensional polyacrylamide electrophoresis on lysed cells of a Vibrio sp., Ant-300, which were prepared from growing and starved cultures. The cells were labeled with [³⁵S]methionine during growth and subsequently starved for up to 30 days. Samples were taken at selected time points representing stages in the starvation-survival process. Unlabeled starved cells were allowed to recover in the presence of [³⁵S]methionine to determine protein changes associated with the recovery from starvation. All growth conditions produced similar protein fingerprints; however, some protein spots disappeared, whereas others were seen only during starvation.     

Acrosomal enzymes of human spermatozoa before and after in vitro capacitation

The effect of in vitro capacitation (events that occur before the acrosome reaction) on the acrosomal enzymes of human spermatozoa was determined. Capacitation of human spermatozoa was assessed by their ability to penetrate denuded hamster oocytes. The activities of a number of enzymes commonly associated with the sperm acrosome, including nonzymogen acrosin, proacrosin, inhibitor-bound acrosin, hyaluronidase, acid phosphatase, beta-glucuronidase, beta-glucosidase, beta-N-acetylglucosaminidase, beta-galactosidase and beta-N-acetylgalactosaminidase were assessed. With the exception of acid phosphatase, no alteration in enzyme activity occurred after 4 h of incubating the spermatozoa under capacitation conditions although gamete fusion took place. The acid phosphatase levels decreased twofold, presumably due to the loss of seminal (prostatic acid phosphatase that loosely adheres to spermatozoa. After 8 h of capacitation, a large decrease in sperm enzyme levels took place only in the case of hyaluronidase, although small decreases were also noted in total acrosin, proacrosin and inhibited acrosin. No new electrophoretically migrating forms of acrosin were observed. Decreases in total acrosin and proacrosin, but not in inhibited acrosin, also occurred when spermatozoa were incubated under noncapacitating conditions for 8 h, indicating that capacitation may specifically cause the release of some acrosin inhibitor from human spermatozoa. It is concluded that, with the possible exception of hyaluronidase, the in vitro capacitation of human spermatozoa does not cause a major change in its acrosomal enzyme content so that these hydrolases are fully present before the acrosome reaction takes place during gamete fusion. Serum albumin appears to protect against the loss of some of these enzymes since the activity of several glycosidases was significantly reduced when the spermatozoa were incubated for 8 h in human serum albumin-free medium.     

Initial Step in Catabolism of Glucose by the Meningopneumonitis Agent

The relative rates of catabolism of glucose and glucose-6-phosphate by intact-cell suspensions of the meningopneumonitis agent, a member of the psittacosis group (Chlamydia), and the properties of the hexokinase and glucose-6-phosphate dehydrogenase of these suspensions were investigated. It is proposed that the hexokinase is a host enzyme bound to the surface of the meningopneumonitis cell and that glucose-6-phosphate is the first substrate in the conversion of hexose to pentose to be attacked by enzymes synthesized by the meningopneumonitis agent.     

The influence of pH and temperature on the properties of myosin

1. The rate of denaturation of myosin solutions at temperatures between 32 degrees and 45 degrees and at pH values between 5.3 and 6.2 has been studied, by using adenosine-triphosphatase activity and solubility in m-potassium chloride at pH6.1 as criteria. 2. Myosin, when heated, loses its adenosine-triphosphatase activity before it becomes insoluble. 3. The loss of adenosine-triphosphatase activity and solubility are both first-order and pH-dependent reactions. Myosin, however, becomes insoluble only when heated within a narrow range of pH values. 4. The thermodynamic functions found for the two processes of denaturation are compared and discussed. 5. The possibility is discussed that, in muscle undergoing rigor, conditions may obtain that would denature myosin.