Proteinase yscD. Purification and characterization of a new yeast peptidase

A newly recognized peptidase, designated proteinase yscD, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the Pro-Phe bond of the synthetic peptide substrate Bz-Pro-Phe-Arg-4-nitroanilide and the Ala-Ala bond of Ac-Ala-Ala-Pro-Ala-4-nitroanilide, Ac-Ala-Ala-Pro-Phe-4-nitroanilide, and MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide with high efficiency (Bz-, Ac-, and MeO-Suc are defined as benzoyl, acetyl, and methoxy-succinyl, respectively). [3H]Methylcasein does not serve as a substrate. Optimum pH for cleavage of Bz-Pro-Phe-Arg-4-nitroanilide is in the range of 6.5 to 7; for Ac-Ala-Ala-Pro-Ala-4-nitroanilide the range is between 5.75 and 6. For MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide the pH optimum was found to be 5.5. The purified enzyme has an apparent Stokes radius of Rs = 37.9 A as judged by gel chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a molecular weight of approximately 83,000 for the enzyme. Mercurials and EDTA were found to be potent inhibitors of proteinase yscD activity.     

Mouse mammary cells ind-valine medium

Summary Cells of a mouse mammary epithelial cell line as well as fibroblasts from a mouse mammary explant were severely inhibited from proliferating in a medium in whichd-valine was substituted forl-valine. After the first few days ind-valine medium, the number of epithelial cells did not increase despite the fact that a few percent continued to synthesize DNA. The cells did recognize the presence of thed-valine in the medium because cells ind-valine increased in volume and their numbers remained stationary, whereas cells without valine shrank and the cell numbers decreased with time. The NMuMG cells were obtained from Mr. Robert Owens and were produced with support from the National Cancer Institute, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention under the auspices of the Office of Naval Research and the Regents of the University of California. This project was funded by the National Cancer Institute, Bethesda, MD, Contract N01-CB-74094.     

Feeder cells impart a growth stimulus to recipient epithelial cells by direct contact.

Mouse mammary epithelial cells have been shown to proliferate when cultured in the same vessel with lethally irradiated cells of the LA7 rat mammary tumor line. Presented here are experiments that indicate that the LA7 feeder cells stimulate growth of the normal mouse mammary cells by a mechanism that involves direct contact between the two cell types. It is possible that the LA7 feeder cells stimulate proliferation by secretion of a labile growth factor, by secretion of a soluble growth factor in such low concentrations that dilution by travel over a distance makes it less effective, that the stimulus is transduced directly through membrane receptors on the recipient epithelial cells, or that a growth message is sent through gap junctions between cells. This feeder cell system is proposed as an in vitro model for epithelial wound healing.     

Defective recovery of semi-conservative DNA synthesis in xeroderma pigmentosum cells following split-dose ultraviolet irradiation.

In normal human fibroblasts we observe an enhancement of the recovery of the rate of semi-conservative DNA synthesis after split-dose UV-irradiation relative to a single total UV dose. The enhanced recovery is totally absent in both a xeroderma pigmentosum variant line and two xeroderma pigmentosum lines belonging to complementation groups A and C.     

Instantaneous benthic response to different organic matter quality: In situ experiments in the Benguela Upwelling System

The benthic community in continental slope and deep-sea sediments of the Benguela Upwelling System was supplied with ¹³C-labelled organic matter (OM) of two different qualities using a benthic chamber lander. Freeze-dried cultures of Skeletonema costatum served as 'fresh' OM. 'Altered' OM of the same material had been additionally dialysed to remove low-molecular weight compounds. In order to investigate the benthic response pattern, mineralization of labelled OM, uptake by macrofauna and incorporation into bacteria were followed over 18-36 h. Total oxygen uptake was not affected beyond natural variation by the OM addition. Mineralization dominated the ¹³C-labelled phytodetritus processing, constituting 71-95% of the total processed OM. Bacterial incorporation of phytodetrital carbon exceeded macrofaunal uptake at all stations. Stations situated in a major centre of OM deposition showed phytodetritus processing rates on average twice as high as outside the depocentre. Phytodetritus processing was 1.5, 2.5 and 4.3 times higher for fresh than for altered OM at 605, 1019 and 1335 m water depth, respectively. Our observations clearly indicate the importance of OM quality on mineralization rates.     

Long-term culture of porcine bladder epithelial cells

Epithelial cells from normal pig bladders proliferated when cocultured with lethally irradiated feeder cells of the LA7 rat mammary tumor line. When the bladder cells and feeders were plated together at a confluent density, the bladder cells proliferated as the feeder cells died, resulting in a confluent culture of bladder cells. The bladder cells were successfully subcultured by plating with freshly irradiated LA7 feeder cells. In this way, bladder cells from five pigs were carried to confluency in passages 1, 4, 7, 7, and 13, amounting to at least 6, 18, 24, 26, and 45 doublings in culture, respectively, and none showed signs of slowed proliferation at the time of culture termination. Fibroblasts never became a prominent feature of these cultures, and their frequency was determined to be about 26 fibroblasts per 10(5) cells in passage 9. Pig bladder cells in 0.5% serum doubled in number in slightly over 3 d, whereas cells in 5.0% serum doubled in about 6 d. In fresh medium without feeder cells only minimal proliferation of bladder cells occurred. In LA7-conditioned medium the bladder cell numbers decreased, leading to the conclusion that the stimulus from LA7 cells is mechanically or physically transmitted. The bladder cells reacted with antibodies to keratins 7 and 18 but not to keratin 14 or vimentin. Tight junctions, visualized with an antibody to the ZO1 protein, connected all the cells to their neighbors. Most cells in passage 9 carried the diploid chromosome number of 38.