Altitude-induced erythrocytic 2,3-DPG and hemoglobin changes in rats of various ages.

Rats of various ages (2, 12, 24, and 40 mo of age) were exposed for 4 wk to either a simulated high altitude of 23,000 ft or to a Peoria, Ill., altitude of 650 ft above sea level. Hematocrit ratios, hemoglobin, and erythrocytic 2,3-diphospho-glycerate (2,3-DPG) concentrations were measured. Hematocrit and hemoglobin determinations revealed a decrease in erythrocytic content with increasing age, and the augmented erythropoietic response was seen in all age groups of animals as a result of altitude exposure. The maximal erythrocytic content of hemoglobin in the 40-mo-old animals was significantly lower than that of all other age groups. Erythrocytic 2,3-DPG levels were significantly changed by aging alone. In the 40-mo-old group there was a 35% increase over the next highest sea-level value. However, while erythrocytic 2,3-DPG content increased significantly in all other age groups following altitude exposure, it decreased 46% in the 40-mo-old group.     

Cloning and expression of murine lymphotoxin cDNA

The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.     

A simplified method for passage and long-term growth of human mammary epithelial cells

Summary A method is described for culturing human mammary epithelial cells in primary culture and allowing more than 50 generations and a 1000-fold increase from starting inocula without need of enzymatic transfers. Organoids dissociated from breast tissue are plated in medium containing 1.05 mM Ca⁺⁺ to effect attachment and growth to monolayer density. Medium is then switched to one containing 0.06 mM Ca⁺⁺ to overcome “renewal inhibition” and to stimulate growth. In low Ca⁺⁺ media, primary cultures become a long-term, continuous source of free-floating viable cells free of fibroblasts. A fundamental requirement for extended growth in primary culture is maintaining calcium levels at approximately 0.06 mM. Above 0.06 mM Ca⁺⁺, cells divide only 3 to 4 times in primary cultures before terminal differentiation occurs. At 0.06 mM Ca⁺⁺, cells continue to divide for periods of time determined partly by feeding schedule, but up to 6 mo. and 50 generations of (linear) growth. Cells released from monolayer were greater than 90% viable and yielded 10⁵ cells/cm² of attached cells every 72 h. Free-floating single cells readily replated and cloned, when transferred, without need of trypsin for dissociation. Long-term free-floating cells were typical mammary epithelium: (a) they formed domes and exhibited renewal inhibition, (b) they produced ductlike formations in collagen gels, (c) they contained epithelium-specific keratin filaments, and (d) they were diploid.     

Hydrolysis of an orally active platelet inhibitory prostanoid amide in the plasma of several species

The prostanoid 3-oxa-4,5,6-trinor-3,7-inter-m-phenylene-PPGE₁-amide (OI-PGE₁-amide) has a prolonged duration of oral platelet aggregation inhibitory activity when compared to the parent free acid (OI-PGE₁) in the rat. When incubated in rat plasma at 1μg/ml for 30 seconds prior to addition of ADP, OI-PGE₁-amide inhibits in vitro rat platelet aggregation ≈50%. OI-PGE₁ inhibits at 1 ng/ml. Inhibition of platelet aggregation by plasma incubated with OI-PGE₁-amide (1μg/ml) increases with time and the rate of this increase differs with species. Incubation of OI-PGE₁ in plasma does not result in an increase of platelet inhibitory activity with time. The increase of platelet inhibitory activity was assumed to indicate hydrolysis of OI-PGE₁-amide to the more active OI-PGE₁. A compound, different from OI-PGE₁-amide, was isolated by an ion exchange/silica gel separation sequence from an incubation of OI-PGE₁-amide in rat plasma. It had potent platelet aggregation inhibitory activity. This material was shown to be OI-PGE₁ by thin-layer chromatography, gas chromatography and mass spectral analysis. Studies with [³H]-OI-PGE₁-amide confirmed the formation of OI-PGE₁ in plasma incubations. Amide hydrolytic activity was significantly different between species, the rank order being: rat > guinea pig > monkey human > dog. This relationship corresponded with that determined by measuring the increase in platelet inhibitory activity with time in plasma incubations of OI-PGE₁-amide reported above. Present data indicate that (a) OI-PGE₁-amide is hydrolyzed to the parent acid by plasma enzymes of several species and (b) hydrolytic activity of plasma varies widely between species.     

Limited dispersal and its effect on population structure in the milkweed beetle Tetraopes tetraophthalmus

Summary The movement patterns of adult milkweed beetles, Tetraopes tetraphthalmus, were monitored via a mark-recapture technique. Movement or dispersal patterns were studied in two natural populations, one in which the host plant, Asclepias syriaca, was nearly continuously distributed over a 250×90 m area and another where Asclepias was distributed in 17 small discrete patches. In both populations dispersal distances resulting from the flight patterns of the adult beetles were quite short, averaging less than 40 m from the point of first encounter 10 days after marking. Males were shown to be more vagile than females. The distribution of dispersal distances collected from one of the populations was fit to three statistical distributions cited in the literature as expected from dispersal by many small-scale movements or observed in other species. It was found that an equation describing an exponential decay gave the best statistical fit to the data collected here for milkweed beetles. The data is discussed in the context of the effects of the limited dispersal power of the beetles and the distribution of suitable habitat on the population structure of Tetraopes.     

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.     

Reconstruction of the lower lip and chin with the composite radial forearm-palmaris longus free flap

Four cases of total lip and chin reconstruction are presented. In three, the composite radial forearm-palmaris longus free flap was used for reconstruction. In the fourth case, the palmaris longus was separated from the flap but still used as a lower lip sling. In all cases, the entire lower lip and the soft tissue of the chin were reconstructed in one stage. All patients healed primarily, and the three who underwent radiotherapy tolerated it without complications. Lip seal and speech were good, and there was no problem with drooling. Postoperative results emphasize the importance of respecting the aesthetic unit of the lower lip and chin.