Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Crystal structure of human immunoglobulin fragment Fab New refined at 2.0 A resolution.

The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies.     

Size-spectra as indicators of the effects of fishing on coral reef fish assemblages

The data requirements and resources needed to develop multispecies indicators of fishing impacts are often lacking and this is particularly true for coral reef fisheries. Size-spectra, relationships between abundance and body-size class, regardless of taxonomy, can be calculated from simple sizeabundance data. Both the slope and the mid-point height of the relationship can be compared at different fishing intensities. Here, we develop size-spectra for reef fish assemblages using body size- abundance data collected by underwater visual census in each of ten fishing grounds across a known gradient of fishing intensity in the Kadavu Island group, Fiji. Slopes of the size-spectra became steeper (F_9,69=3.20, p<0.01) and the height declined (F_9,69=15.78, p<0.001) with increasing fishing intensity. Regressions of numbers of individuals per size class across grounds were negative for all size classes, although the slope was almost zero for the smallest size class. Response to exploitation of each size class category was greatest for larger fish. Steepening of the slope with increasing fishing intensity largely resulted from reductions in the relative abundance of large fish and not from the ecological release of small fish following depletion of their predators. The slope and height of the size-spectrum appear to be good indicators of fishing effects on reef fish assemblages.     

Letter: Crystallographic study of an esteroproteolytic enzyme

The esteroproteolytic enzyme, a serine protease from porcine pancreas, has been crystallized and characterized by X-ray diffraction. Two closely related crystal forms were observed. The unit cell dimensions of the first form are $\text{a = 59.2}\text{A}\text{?}\text{, b = 96.4}\text{A}\text{?}\text{and}\text{c = 47.4}\text{A}\text{?}$, space group P2₁2₁2 with one molecule (mol. wt 30,000) per asymmetric unit. The unit cell dimensions of the second form are $\text{a = 59.2}\text{A}\text{?}\text{, b = 96.4}\text{A}\text{?}\text{and}\text{c = 94.8}\text{A}\text{?}$, space group P2₁2₁2₁. Mercury derivatives of enzyme inhibitors were used to obtain multiple isomorphous heavy-atom substituents suitable for phase determination.     

Defining the response of a microorganism to temperatures that span its complete growth temperature range (-2°C to 28°C) using multiplex quantitative proteomics

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses.     

SARs stimulate but do not confer position independent gene expression.

Two minimal scaffold-associated regions (SARs) from Drosophila were tested in stably transformed cells for their effects on the expression of reporter genes. The expression of genes bounded by two SARs is consistently stimulated by about 20- to 40-fold, if the average of a pool of cell transformants is analyzed. However, analysis of individual, stable cell transformants demonstrates that flanking SAR elements do not confer position-independent expression on the reporter gene and that the extent of position-dependent variegation is similarly large with or without the flanking SAR elements. The SAR stimulation of expression is observed in stable but not in transiently transfected cell lines. The Drosophila scs and scs' boundary elements, which do not bind to the nuclear matrix in vitro, are only about one-tenth as active as SARs in stimulating expression in stable transformants. Interestingly, the SAR stimulatory effect can be blocked by a fragment containing CpG islands (approximately 70% GC), if positioned between the SAR and the enhancer. In contrast, when inserted in the same position, control fragments, such as the scs/scs' elements, do not interfere with SAR function.     

Mass transfer from mothers to pups and mass recovery by mothers during the post-breeding foraging period in southern elephant seals (Mirounga leonina) at King George Island

Mass transfer from mother to pup during the lactation period, and mass recovery for the same females during the foraging period were measured in the southern elephant seal at King George Island, Antarctica. During the 19.2 ± 0.9-day lactation period measured (which represented 87% of the entire nursing), females lost a mean mass of 10.56 ± 1.76 kg/day (n = 27), while their pups gained a mean mass of 5.27 ± 1.1 kg/day. There was a correlation between daily body weight gain in pups and daily weight loss by their mothers. Pup weaning mass was positively related to maternal post-partum mass. Serial samples showed that weight losses by females and gains by their pups were not linear over lactation, but showed lower values at the beginning and at the end of lactation. During the 60.5 ± 6.2-day foraging phase between the end of lactation and molt, females gained 2.21 ± 0.65 kg/day (n = 12), or 54% of the mass lost during nursing. Growth rates reported here are higher than those reported in other breeding sites. However, the ratio of body mass loss by females to gain by their pups was similar, suggesting that higher growth rates and greater weaning mass at South Shetland are due to a higher mean weight of females on arrival at this breeding site. The foraging period was shorter and the mass gained greater than those measured at South Georgia; this could be related to relatively shorter distances to foraging areas. Received: 20 September 1996 / Accepted: 28 April 1997     

Oxidative damage to proteins in yeast cells exposed to adaptive levels of H(2)O(2)

When yeast cells are exposed to sublethal concentrations of oxidants, they adapt to tolerate subsequent lethal treatments. Here, we show that this adaptation involves tolerance of oxidative damage, rather than protection of cellular constituents. o- and m-tyrosine levels are used as a sensitive measure of protein oxidative damage and we show that such damage accumulates in yeast cells exposed to H(2)O(2) at low adaptive levels. Glutathione represents one of the main cellular protections against free radical attack and has a role in adaptation to oxidative stress. Yeast mutants defective in glutathione metabolism are shown to accumulate significant levels of o- and m-tyrosine during normal aerobic growth conditions.     

Ecology and Genetic Structure of Zoonotic Anisakis spp. from Adriatic Commercial Fish Species

Consumption of raw or thermally inadequately treated fishery products represents a public health risk, with the possibility of propagation of live Anisakis larvae, the causative agent of the zoonotic disease anisakidosis, or anisakiasis. We investigated the population dynamics of Anisakis spp. in commercially important fish—anchovies (Anisakis), sardines (Sardina pilchardus), European hake (Merluccius merluccius), whiting (Merlangius merlangus), chub mackerel (Scomber japonicus), and Atlantic bluefin tuna (Thunnus thynnus)—captured in the main Adriatic Sea fishing ground. We observed a significant difference in the numbers of parasite larvae (1 to 32) in individual hosts and between species, with most fish showing high or very high Anisakis population indices. Phylogenetic analysis confirmed that commercial fish in the Adriatic Sea are parasitized by Anisakis pegreffii (95.95%) and Anisakis simplex sensu stricto (4.05%). The genetic structure of A. pegreffii in demersal, pelagic, and top predator hosts was unstructured, and the highest frequency of haplotype sharing (n = 10) was between demersal and pelagic fish.