Allen G. Hirsh

Kinetics of medfly mortality

A series of theoretical analyses of mortality were performed on recent data for Mediterranean fruit flies (Ceratitus capitata). Best fit computations were performed using the Marquardt-Levenburg technique on: 1) the classic Gompertz model, 2) the Johnson-Mehl-Avrami (JMA) model from chemical kinetics theory, which is formally a Weibull model, and 3) a hyperexponential gamma distribution model that we developed to help explain the relative success of the JMA model when compared with the Gompertz model. It was found that both the JMA and gamma models deviated from the data by about an order of magnitude less than the Gompertz model for the first few mean fly lifetimes. We discuss likely underlying causes for the success of these models vs. the classic model.

The high-order kinetics of cytolysis in stressed red cells

The rate of cytolysis in osmotically stressed unfertilized sea urchin eggs was analysed using a version of the Johnson-Mehl-Avrami equation, and fit with high precision (r2>0.90) if the data sets were divided into two sectors. The slow process was first-order but the Avrami coefficient,n, for the initial fast reaction was 7. Suspecting that this might be a peculiarity of cells which are primed for climactic behaviour, we examined the process in red cells, whose decay is known to be of first or lower order at low temperatures if protected from excessive osmotic stress. Human red cells subjected to ‘thermal shock’, in which osmotically stressed cells are cooled below +12°C, show a pattern almost identical to the stressed sea urchin eggs except thatn of the rapid process exceeded 10. Based on the geometrical implications of such a highn, we believe that this phenomenon reflects a stress failure in the cytoskeleton and has important ramifications in cryopreservation.ZusammenfassungUnter Anwendung einer Variante der Johnson-Mehl-Avrami Gleichung wurde die Cytolysegeschwindigkeit von osmotisch gespannten, unbefruchteten Seeigeleiern untersucht, wobei sich eine hohe Genauigkeit ergab (r2>0.90), wenn die Datensets in zwei Sektoren unterteilt werden. Der langsame Prozeß war erster Ordnung, jedoch betrug der Avrami-Koeffizient n für die anfängliche schnelle Reaktion 7. In der Vermutung, daß dies eine Eigenheit von Zellen ist, die für klimaktisches Verhalten vorbereitet sind, untersuchten wir den Prozeß in roten Zellen, für deren Abnahme bei niedrigen Temperaturen die erste oder eine niedrigere Ordnung bekannt ist, wenn sie von überschüssigem osmotischem Einfluß geschützt sind. Werden menschliche rote Zellen einem “Thermoschock” unterworfen, bei dem osmotisch angespannte Zellen unter +12°C abgekühlt werden, so zeigen sie einen Kurvenverlauf, der mit dem von gespannten Seeigeleiern fast identisch ist, jedoch mit der Ausnahme, daßn des schnellen Prozesses über 10 liegt. Ausgehend von den geometrischen Folgerungen eines so hohenn Wertes, glauben wir, daß diese Erscheinung einen Spannungsfehler im Zellgerüst widerspiegelt und eine wichtige Bedeutung bei der Kryopräservation besitzt.

Localization and characterization of intracellular liquid-liquid phase separations in deeply frozen populus using electron microscopy, dynamic mechanical analysis and differential scanning calorimetry

Abstract Populus balsamifera var. virginiana (Sargent), balsam poplar, is capable of withstanding liquid nitrogen temperatures when cooling is slow (less than 6°C h −1 ) and the twigs are winter dormant. Cooling to −50°C at rates as high as 42°C h −1 is also tolerated. The mechanical behavior during warming of dormant twigs cooled at rates varying from 3°C h −1 to 600 °C h −1 has been investigated using resonance mode dynamic mechanical analysis (DMA). The results were correlated with those of freeze-etch electron microscopy investigations of the samples as well as differential scanning calorimetry (DSC) of model solutions. The results of these investigations were compared with mortality data on the twigs. This has led us to hypothesize that at critical cooling rates below 0° C plasma membranes tear, leading to the rapid growth of intracellular ice. This in turn prevents the intracellular liquid-liquid phase separations seen by DMA in samples cooled at sublethal rates. DSC analysis of salt-sugar-protein model solutions supports the idea that when liquid-liquid phase separation does occur, some domains are sugar-depleted but rich in protein, while others are sugar-rich but low in protein. This may have important implications regarding the organization of extreme freezing resistance mechanisms in this natural system.

The kinetics of autolysis in osmotically stressed sea urchin eggs

Abstract We have measured the kinetics of death from hyperosmotic stress in unfertilized sea urchin eggs in an attempt to dissect the two conflicting influences of temperature lowering below the freezing point: as more ice forms during temperature lowering, the rate of injury increases rather than decreases. Two models are examined. An Arrhenius model, in which the osmotic stress imposed by extracellular ice formation is envisaged as reducing the “activation energy”, provides a qualitative model but fails to fit the data in detail. In contrast, a Johnson—Mehl—Avrami nucleation model, in which the osmotic stress triggers an autolysis that is essentially independent of temperature or degree of osmotic stress above a threshold value, fits the data well. Two separate decay processes were seen, for which the time constants were 269s and 658s and the Avrami exponents were 7 and 1 respectively. The latter is an ordinary first-order attrition, though its independence from temperature distinguishes it from an Arrhenius model. The nucleation model is difficult to furnish with a simple physical representation.

On the possibility of the production of many rare proteins by higher eukaryotes.

Arguments are presented which support the idea that the large amount of single copy DNA in the haploid genome of higher eukaryotes is coding for many (approximately 10(6) rare proteins, that is, proteins present at the level of no more than a few molecules per cell. It is asserted that a large number of such rare protein species is consistent with (1) apparent slow rates of DNA sequence evolution as shown by DNA annealing experiments, (2) physiological complexity and the DNA content of organisms, and (3) evidence that there are many proteins already known whose primary purpose is the regulation of the function of other proteins. It is shown that the best available detection techniques would not uncover these proteins unless a concerted effort were made to do so. An outline of such an effort, based on immune precipitation and two-dimensional gel electrophoresis, is presented and discussed.

An alternative explanation for phase transitions observed in quick frozen calcium cardiolipin solution

It is demonstrated that the phase changes reported for a quick frozen calcium cardiolipin solution containing CaCl2 are virtually identical to those seen in pure CaCl2. This introduces uncertainty as to whether the data in fact reflect the behavior of cardiolipin or of the associated CaCl2.