Comparison of the enthalpy state of vesicles of different size by their interaction with α-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with α-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with α-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of α-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, $\text{ΔH}$, of the interaction of the three vesicle types with α-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24°C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic $\text{ΔH}$ values around 24°C for large vesicles approximates the transition enthalpy of the pure phospholipid     

Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.     

Fast and accurate side-chain topology and energy refinement (FASTER) as a new method for protein structure optimization

We have developed an original method for global optimization of protein side-chain conformations, called the Fast and Accurate Side-Chain Topology and Energy Refinement (FASTER) method. The method operates by systematically overcoming local minima of increasing order. Comparison of the FASTER results with those of the dead-end elimination (DEE) algorithm showed that both methods produce nearly identical results, but the FASTER algorithm is 100-1000 times faster than the DEE method and scales in a stable and favorable way as a function of protein size. We also show that low-order local minima may be almost as accurate as the global minimum when evaluated against experimentally determined structures. In addition, the new algorithm provides significant information about the conformational flexibility of individual side-chains. We observed that strictly rigid side-chains are concentrated mainly in the core of the protein, whereas highly flexible side-chains are found almost exclusively among solvent-oriented residues.     

Leaf gas exchange and water status responses of a native and non-native grass to precipitation across contrasting soil surfaces in the Sonoran Desert

Arid and semi-arid ecosystems of the southwestern US are undergoing changes in vegetation composition and are predicted to experience shifts in climate. To understand implications of these current and predicted changes, we conducted a precipitation manipulation experiment on the Santa Rita Experimental Range in southeastern Arizona. The objectives of our study were to determine how soil surface and seasonal timing of rainfall events mediate the dynamics of leaf-level photosynthesis and plant water status of a native and non-native grass species in response to precipitation pulse events. We followed a simulated precipitation event (pulse) that occurred prior to the onset of the North American monsoon (in June) and at the peak of the monsoon (in August) for 2002 and 2003. We measured responses of pre-dawn water potential, photosynthetic rate, and stomatal conductance of native (Heteropogon contortus) and non-native (Eragrostis lehmanniana) C₄ bunchgrasses on sandy and clay-rich soil surfaces. Soil surface did not always amplify differences in plant response to a pulse event. A June pulse event lead to an increase in plant water status and photosynthesis. Whereas the August pulse did not lead to an increase in plant water status and photosynthesis, due to favorable soil moisture conditions facilitating high plant performance during this period. E. lehmanniana did not demonstrate heightened photosynthetic performance over the native species in response to pulses across both soil surfaces. Overall accumulated leaf-level CO₂ response to a pulse event was dependent on antecedent soil moisture during the August pulse event, but not during the June pulse event. This work highlights the need to understand how desert species respond to pulse events across contrasting soil surfaces in water-limited systems that are predicted to experience changes in climate.     

Losing the desire: selection can promote obligate asexuality

Whilst parthenogenesis has evolved multiple times from sexual invertebrate and vertebrate lineages, the drivers and consequences of the sex-asex transition remain mostly uncertain. A model by Stouthamer et al. recently published in BMC Evolutionary Biology shows a pathway by which obligate asexuality could be selected for following endosymbiont infection.     

Functional polymorphisms in the TERT promoter are associated with risk of serous epithelial ovarian and breast cancers

Genetic variation at the TERT-CLPTM1L locus at 5p15.33 is associated with susceptibility to several cancers, including epithelial ovarian cancer (EOC). We have carried out fine-mapping of this region in EOC which implicates an association with a single nucleotide polymorphism (SNP) within the TERT promoter. We demonstrate that the minor alleles at rs2736109, and at an additional TERT promoter SNP, rs2736108, are associated with decreased breast cancer risk, and that the combination of both SNPs substantially reduces TERT promoter activity.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Effects of an increase in summer precipitation on leaf, soil, and ecosystem fluxes of CO2 and H2O in a sotol grassland in Big Bend National Park, Texas

Global climate models predict that in the next century precipitation in desert regions of the USA will increase, which is anticipated to affect biosphere/atmosphere exchanges of both CO₂ and H₂O. In a sotol grassland ecosystem in the Chihuahuan Desert at Big Bend National Park, we measured the response of leaf-level fluxes of CO₂ and H₂O 1 day before and up to 7 days after three supplemental precipitation pulses in the summer (June, July, and August 2004). In addition, the responses of leaf, soil, and ecosystem fluxes of CO₂ and H₂O to these precipitation pulses were also evaluated in September, 1 month after the final seasonal supplemental watering event. We found that plant carbon fixation responded positively to supplemental precipitation throughout the summer. Both shrubs and grasses in watered plots had increased rates of photosynthesis following pulses in June and July. In September, only grasses in watered plots had higher rates of photosynthesis than plants in the control plots. Soil respiration decreased in supplementally watered plots at the end of the summer. Due to these increased rates of photosynthesis in grasses and decreased rates of daytime soil respiration, watered ecosystems were a sink for carbon in September, assimilating on average 31 mmol CO₂ m⁻² s⁻¹ ground area day⁻¹. As a result of a 25% increase in summer precipitation, watered plots fixed eightfold more CO₂ during a 24-h period than control plots. In June and July, there were greater rates of transpiration for both grasses and shrubs in the watered plots. In September, similar rates of transpiration and soil water evaporation led to no observed treatment differences in ecosystem evapotranspiration, even though grasses transpired significantly more than shrubs. In summary, greater amounts of summer precipitation may lead to short-term increased carbon uptake by this sotol grassland ecosystem.     

Cyclic peptide models of the Ca2+-binding loop of alpha-lactalbumin

A series of cyclic peptides with different linkers were designed and synthesized to model the elbow-type Ca2+-binding loop of alpha-lactalbumin (LA). All amino acids of the Ca2+-binding loop are strikingly well conserved among LAs of different species with the sequence Lys79-Phe-Leu-Asp82-Asp-Asp-Leu-Thr- Asp87-Asp88, where three carboxylates of Asp82, Asp87, and Asp88 and the amide carbonyl oxygen atoms of Lys79 and Asp84 participate in Ca2+ binding. Alanine-containing models were also prepared for monitoring the role of the binding (82, 87-88) and nonbinding Asp residues (83-84) in coordinating the cation. The structural features of synthetic peptides and their Ca2+-binding properties were investigated in solution by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. In water, the CD curves show a strong negative band below 200 nm as a sign of the presence of unfolded conformers. In TFE, all cyclic peptides were found to have a CD spectrum, reflecting the presence of folded (turn) conformers. The effect of Ca2+ was dependent on the structure and concentration of the model and the Ca2+ to peptide ratio (r(cat)). A surprising time dependence of the FTIR spectra of Ca2+ complexes of the Ala-containing peptides was observed. The shape of the broad amide I band showed no more change after approximately 60 min. Contrary to this, the deprotonation of the side chain COOH group(s) and formation of the final coordination sphere of Ca2+ took more time. Infrared spectra showed that in the Ca2+ complex of model comprising the binding Asp residues of LA, the cation is coordinated to the COO- groups of all three Asps, while in the complex of model comprising nonbinding Asp residues of LA, the two neighboring Asp side chains form a bridged Ca2+-binding system.     

SABmark--a benchmark for sequence alignment that covers the entire known fold space

The Sequence Alignment Benchmark (SABmark) provides sets of multiple alignment problems derived from the SCOP classification. These sets, Twilight Zone and Superfamilies, both cover the entire known fold space using sequences with very low to low, and low to intermediate similarity, respectively. In addition, each set has an alternate version in which unalignable but apparently similar sequences are added to each problem.