Stabilization of supercooled fluids by thermal hysteresis proteins.

It has been reported that thermal hysteresis proteins found in many cold-hardy, freeze-avoiding arthropods stabilize their supercooled body fluids. We give evidence that fish antifreeze proteins, which also produce thermal hysteresis, bind to and reduce the efficiency of heterogenous nucleation sites, rather than binding to embryonic ice nuclei. We discuss both possible mechanisms for stabilization of supercooled body fluids and also describe a new method for measuring and defining the supercooling point of small volumes of liquid.     

A sensitive analytical method for the detection and quantitation of adenosine in biological samples

A new method for the measurement of adenosine in biological materials has been developed. The method is based on the combined principles of isotope dilution and enzymatic catalysis using a highly specific adenosine kinase isolated from rat heart. By differential centrifugation and gel filtration, this adenosine kinase was obtained free of adenosine deaminase and other enzymes which would have been a source of error in the use of this enzyme in the adenosine assay. The cardiac adenosine kinase was shown to be highly specific and to exhibit an apparent K_m for adenosine of 0.35 μM. Using this enzyme, unknown quantities of adenosine could be detected by measuring the effect of their addition on the conversion of radioisotopic adenosine to 5′-AMP in the kinase reaction. In this procedure, as little as 20 pmoles of adenosine could be detected. To test the applicability of the assay, measurements of the tissue content of this nucleoside were made in samples of dog and rat hearts frozen in situ under control, hypoxic, or ischemic conditions. The assay has several advantageous features when compared to other existing methods used to measure adenosine: a minimum of sample preparation is required before the actual assay procedure; many samples can be processed per day by a single operator; single determinations can be done on as little as 5 μl of sample, and the specificity of the assay can be readily checked by treatment of samples with adenosine deaminase.     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

The phosphorylation of ribosomal protein S6 in baby hamster kidney fibroblasts

Ribosomal protein S6 was extensively phosphorylated in pre-confluent but not in post-confluent baby hamster kidney fibroblasts. This appears to be the first example of increased phosphorylation of S6 under physiological conditions where the cellular concentration of cyclic AMP is not elevated. The extent of the phosphorylation of S6 was also independent of alterations in the protein synthetic activity of the cells, suggesting that the biological role of this phosphorylation may be unrelated to the functional ability of the ribosomes.     

The variable location of phosphoproteins on eukaryotic ribosomal subunits

A pair of phosphoproteins of low molecular weight detected on the 60S subunit of ribosomes isolated from Krebs II ascites cells are on the 40S subunit of ribosomes isolated from BHK-21/C13 cells. The possibility that these proteins may be located at the interface between the two subunits is discussed.     

Therapeutic donor insemination with frozen semen.

Although it is now accepted that cryopreserved semen must, on ethical and medicolegal grounds, be used for donor insemination many clinicians still believe that it has an unacceptably reduced fecundability rate as compared with fresh semen. We studied the outcome of 81 recipients who started therapeutic donor insemination (TDI) treatment during 1986 in a program that used exclusively cryopreserved semen; 55 had never undergone TDI and were receiving the first series (six cycles), 6 were receiving the second series (also six cycles), and 20 had achieved pregnancy through TDI previously and were starting the treatment again. Insemination with semen stored in 0.5-ml French straws was performed daily during the periovulatory period while the modified Insler score was 10 or greater out of 15. A total of 42 (52%) of the recipients became pregnant within six TDI cycles; 4 (10%) had a spontaneous abortion. An average of 4.8 straws were used per cycle among those who became pregnant and 5.1 per cycle among those who did not. On average 2.6 cycles were required to achieve pregnancy. The overall fecundability rate was 14.6%. We conclude that a TDI program involving exclusively frozen semen can be operated with a success rate comparable to rates achieved with fresh semen if a simple, established cryopreservation method and an uncomplicated clinical management protocol are used.