Susan A. Jackson

Effects of carbohydrates on membrane stability at low water activities

The relative effectiveness of a variety of carbohydrates in preserving the structural and functional integrity of membranes at low water activities was studied, using Ca-transporting microsomes from muscle as a model membrane. The order of effectiveness (greatest to lowest) was: trehalose, lactose, maltose, cellobiose, sucrose, glucose, fructose, sorbitol, raffinose, myo-inositol, glycerol. At the highest concentrations of the most effective sugars tested, microsomes were obtained upon rehydration that were similar structurally and functionally to fresh membranes. The least effective carbohydrates, alcohol sugars, all appear to be fusogenic. A structural explanation for relative effectiveness of the sugars was sought, but no clear relationship was found, except that effectiveness does not appear to be related to the number of position of hydroxyl groups available for hydrogen bonding.

Preservation of structural and functional activity in lyophilized sarcoplasmic reticulum.

Ca-transporting microsomes isolated from abdominal muscle of lobster were lyophilized in the presence and absence of trehalose. The dry membranes appeared with freeze fracture to collapse into cup-shaped structures embedded in a matrix of trehalose when lyophilized in the presence of trehalose. Upon rehydration, the dry membranes yielded vesicles that were morphologically indistinguishable from freshly prepared ones. These rehydrated vesicles also showed ATPase activity and Ca transport only slightly different from those activities in freshly prepared vesicles. The concentration of trehalose required to achieve this degree of stabilization is about 0.3 g trehalose/g membrane. When the membranes were dried at lower trehalose concentrations extensive fusion occurred between vesicles, along with lateral phase separations of membrane proteins and lipids. The rehydrated vesicles showed poor Ca uptake and coupling between ATPase activity and Ca uptake. The membranes may also be stabilized in the dry state by lyophilizing them in sucrose, but about three times as much sucrose is required as trehalose.

Ontogeny of low molecular weight stress protein p26 during early development of the brine shrimp, Artemia franciscana

Embryogenesis in the brine shrimp, Artemia sp., occurs by one of two pathways: (i) the direct, uninterrupted development of nauplius larvae within the female or (ii) the production of embryos that arrest development at the gastrula stage and enter diapause. Diapause embryos are released from females into the aqueous environment where they remain in diapause until activated by appropriate environmental cues and resume development. These encysted embryos possess at least one low molecular weight stress protein, which we refer to as p26 and which has been implicated previously in the stress response of activated embryos. We investigated the appearance of p26 in developing diapause embryos in utero and looked for its presence in embryos developing directly into nauplii. We found p26 to be specific to diapause‐destined embryos; it was not detected in direct‐developing embryos. We conclude that p26 is not required for the basic developmental program that produces the nauplius. In diapause‐destined embryos, p26 was first detectable after 3 days of development, at which time the embryos were late gastrulae. This protein continues to increase in amount until the encysted embryos are released, approximately 5 days after fertilization. At the time of release almost all p26 is located in the low speed supernatant fraction, but as released embryos continue diapause, p26 transfers to the pelleted nuclear fraction in increasing amounts. Our working hypothesis views p26 as a molecular chaperone preventing protein denaturation and aggregation under conditions associated with metabolic arrest and other stressful states, which these encysted embryos encounter.

Thermal resistance, developmental rate and heat shock proteins in Artemia franciscana, from San Francisco Bay and southern Vietnam.

Cysts (encysted gastrula embryos) of Artemia franciscana collected from salterns in San Francisco Bay, California, USA (SF) were inoculated into much warmer growth ponds in the Mekong Delta region of Vietnam (V) in 1996. V adults arising directly from these cysts during 17 April to 15 May produced their own cysts, which were collected, processed and stored until shipped to the USA for study. Adults grown in the laboratory from SF cysts (those used for the inoculation) were less resistant to high temperature than adults cultured from V cysts. V cysts produced heat-resistant adults, even though cultured under the same laboratory conditions as SF animals, at much lower temperatures than they ever experienced in Vietnam. Differences in thermal performance between SF and V adults were retained in the second generation, cultured from cysts produced in the laboratory by first generation adults, suggesting a genetic basis for the better heat resistance of V adults. We propose that the operation of natural selection in the Vietnam growth ponds produced adults with improved thermal tolerance, and that the basis for this tolerance was incorporated into the developmental program of their cysts. Surprisingly, differences in heat resistance of laboratory reared animals were not reflected in constitutive levels of the hsp70 family which were similar in first generation SF and V adults. A conditioning heat shock (HS, 37 degrees C, 30 min) led to the same level of induced thermotolerance in SF and V first generation adults when evaluated 24 h post-HS. Levels of hsp70 were also up-regulated at that time, but to about the same extent in SF and V adults. Developmental rates of SF cysts used for the inoculation were faster than those of cysts produced in Vietnam when both were incubated at 21+/-1 degrees C, suggesting that V cysts have become adapted to develop at higher temperatures.

Aerobic heat shock activates trehalose synthesis in embryos of Artemia franciscana

Encysted embryos (cysts) of the brine shrimp, Artemia franciscana, contain large amount of trehalose which they use as a major substrate for energy metabolism and biosynthesis for Development under aerobic conditions at 25°C. When cysts are placed at 42°C (heat shock) these pathways stop, and the cysts re‐synthesize the trehalose that was utilized during the previous incubation at 25°C, Glycogen and glycerol, produced from trehalose at 25°C, appear to be substrates for trehalose synthesis during heat shock. Anoxia prevents trehalose synthesis in cysts undergoing heat shock. These results are consistent with the view that trehalose may play a protective role in cells exposed to heat shock, and other environmental insults, in addition to being a storage form of energy and organic carbon for development.

Glycolysis compared in intact, permeabilized and sonicated L-929 cells

The effects of incubation time and cell density on glycolytic rate were examined in suspensions of intact, permeabilized and sonicated L‐929 cells. Sonicates exhibited strong dependence on cell density and a distinct lag in glycolytic rate, while intact cells showed no cell density dependence and linear glycolytic rates. Permeabilized cells exhibited linear glycolytic rates, but sometimes showed dependence on cell density. Rates of lactate production (nmol at 30 min/106 cells) were highest in sonicates and lowest in intact cells. These results are interpreted as support for the previously proposed hypothesis that enzymes of the glycolytic pathway are highly organized in intact L‐929 cells.

Significance of cyst fragments ofArtemia sp. recovered from a 27,000 year old core taken under the Great Salt Lake, Utah, USA

We present electron microscopic observations on fragments of encysted embryos of the crustacean,Artemia sp., recovered from a 27,000 year-old core section taken under the Great Salt Lake (GSL), Utah. The significance of these results comes from a consideration of the geological history of the GSL and the extraordinary properties and abundance ofArtemia cysts at this site. We make the case that this unique system, and probably other salt lakes as well, provides novel opportunities for the study of “ancient DNA” and related matters. The analysis provides the major focus of this paper, which we hope will stimulate interest in this aspect of salt lake research.