Influence of various cooling and warming temperatures on survival after thawing of cryopreserved Plasmodium berghei

Heavy concentrations of viable P. berghei in the natural milieu [20% ($\text{v}\text{v}$) parasitized red blood cells, or 20% ($\text{w}\text{v}$) homogenate of splenic tissue in which malarial cells sequestered wer suspended in a serum-free, protective medium. Various rates of cooling are designated as low (1.3 °C/min) and intermediate (4 °C/ min) on exposure in cold gas evolving from liquid nitrogen refrigerant to ?70 °C, and this followed by direct immersion in the low temperature refrigerant (?196 °C). Cooling designated high was accomplished by abrupt immersion of the sealed vials with the live malaria-bearing tissue in the liquid nitrogen refrigerant. Rates of warming and thawing were designated low (after slow rewarming of frozen tissue in air at 25.5 °C) and high (after rapid rewarming and thawing in a water bath at 40 °C). Strip chart recordings were made of the complete cooling and freezing wave patterns of the suspending medium to ?70 ° C. The functional survivals of the freeze-thaw P. berghei malaria were measured by a special infectivity titration method.None of the cooling and freezing treatments adversely influenced the parasite survivals. Our data showed the optimum cooling velocity that maximally protected this highly lethal P. berghei strain within the host erythrocyte matrix was 1.3 ° C/min to ?70 to ?196 ° C. The functional survivals of two RBC stabilates with P. berghei, after retrieval from 25 days storage in the liquid nitrogen refrigerant, excelled by more than 100-fold the infectivity titer found by viability assay in the pool of the 0-days nonfrozen infected RBC.The precise factors favoring the maximal survivals of the freeze-thaw P. berghei are unclear. Several factors, singly or in combination, may have played key roles in protecting the living P. berghei from the freeze-thaw damage. These factors are: The composition of the suspending medium fortified by additions of bicarbonate, glucose, lactalbumin hydrolysate and yeastolate; the presence of naturally occurring peptide-containing materials surrounding the parasites in the host red cell milieu; and the protective glycerol agent. Any of these constituents singly or combined possess potential for reducing freeze-thaw injury to the parasites to produce maximal survivals.     

Immobilization of free-ranging African lions (Panthera leo) with a combination of xylazine hydrochloride and ketamine hydrochloride

The combination of 55 mg/ml xylazine hydrochloride and 200 mg/ml ketamine hydrochloride was effective for immobilizing African lions in Tanzania. Nineteen adult females were given between 55 and 110 mg xylazine hydrochloride in the first dart. Initial doses of 110 mg xylazine hydrochloride and 450 mg ketamine hydrochloride equivalent to greater than 0.9 mg/kg xylazine hydrochloride were most effective in achieving rapid immobilization. Lower doses of xylazine hydrochloride required supplementation with ketamine hydrochloride. These doses could be delivered easily in 3-ml darts. The use of lightweight darts and a blowgun was found to be useful as a supplement to longer range dart projector systems since many animals could be approached at short range.     

Carfentanil and xylazine for immobilization of moose (Alces alces) on Isle Royale

Twenty-one moose were captured on Isle Royale between 28 May and 2 June 1984 at licks on the western end of the island. The animals were darted, at ranges of 10 to 35 m, with 3-cc dart syringes containing 3 or 4 mg of carfentanil and 100 or 175 mg of xylazine. Three animals were drugged with carfentanil alone. Immobilization time ranged from 2.5 to 6 min. There was no excitement phase evident in 18 of the animals and they rarely moved more than 30 m after darting. Reversal was begun at 30 to 90 min after darting using naloxone and diprenorphine given intramuscularly (i.m.) and intravenously (i.v.). Recovery time varied from 10 min to 3 hr with the longer times occurring at the higher doses of immobilizing drugs. Two animals died within 30 hr, one as the result of aspiration of rumen contents and the other was unable to get on its feet and was euthanized. Analysis of blood samples from the 18 moose immobilized with the drug combination yielded hemoglobin values of 13.1 +/- 0.3 g/dl, hematocrit: 37.6 +/- 0.7%, red blood cells: 5.46 +/- 0.1 million/microliter, leucocytes: 6.1 +/- 0.4 thousand/microliter, and serum urea nitrogen: 29.3 +/- 1.6 mg/dl. Our experience indicated that quiet, undisturbed moose can be immobilized with 3 mg carfentanil and 100 mg or less of xylazine.     

Scaling of body weight and fat content in fungus-gardening ant queens: does this explain why leaf-cutting ants found claustrally?

Offspring traits are among the most important life history traits, yet we lack an adequate understanding of their role in social insect life history evolution. Colony founding in the fungus-gardening ants (Tribe Attini) is different from most other ant species because the queens forage during the founding phase. Queens of the most derived genus, Atta, are the only attines that exhibit the more typical claustral founding, where the queens seal themselves in a below-ground chamber and produce their first generation of workers with only body fat reserves. Here I report the dry weights, fat content and energetic value of newly mated queens of ten attine species. Published phylogenies were used to make inferences on the evolutionary transitions in this clade. It appears that the evolution of fungus-gardening was associated with the manufacture of smaller, leaner queens as basal taxa are characterized by small bodies that contain relatively less fat than derived taxa. Moreover, there appears to be an allometric function between fat, energetic content and dry weight, which means that for fatter and claustral queens to develop, they also must become larger.     

Significantly Enhanced Heme Retention Ability of Myoglobin Engineered to Mimic the Third Covalent Linkage by Nonaxial Histidine to Heme (Vinyl) in Synechocystis Hemoglobin

Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the “hemoglobin (Hb) superfamily.” The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the nonaxial His¹¹⁷ and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date, and His¹¹⁷ is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a nonaxial His into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, electrospray ionization-MS, and MALDI-TOF analyses confirmed the presence of covalent linkage in Mb I107H. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and auto-oxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb-based blood substitute.