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Results show that during mitotic cell division Golgi membranes disperse and reform across endoplasm reticulum, maximally using constitutive recycling pathways. Data further indicate that endoplasm reticulum plays an essential role in the Golgi coplex's genesis and inheritance.

Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. Weinvestigated the role of TyrB26, a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, nonaromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakestbinding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the GluB26 analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although TyrB26 is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stabilityand self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences. [ABSTRACT FROM AUTHOR]

We discuss an on-line tool that facilitates access to the large collection of climate impacts on crop yields produced by the Agricultural Model Intercomparison and Improvement Project. This collection comprises the output of seven crop models which were run on a global grid using climate data from five different general circulation models under the current set of representative pathways. The output of this modeling endeavor consists of more than 36,000 publicly available global grids at a spatial resolution of one half degree. We offer flexible ways to aggregate these data while reducing the technical barriers implied by learning new download platforms and specialized formats. The tool is accessed trough any standard web browser without any special bandwidth requirement. [ABSTRACT FROM AUTHOR]

Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed LysB28, ProB29-insulin ("lispro") as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (TyrB26) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ±0.2 kcal/ mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. 1HNMRstudies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-TyrB26 modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-TyrB26-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages. [ABSTRACT FROM AUTHOR]

Proteins targeted to the plasma membrane (PM) of cells are degraded at different rates. Sorting motifs contained within the cytoplasmic domains of transmembrane proteins, post-translational modifications (e.g. ubiquitination), and assembly into multiprotein or protein-lipid complexes all may affect the efficiency of endocytosis and recycling and influence the delivery to degradative compartments. Using the SNAP-tag labeling system, we examined the turnover of a model PM protein, the α chain of the interleukin-2 receptor (Tac). The surface lifetimes of SNAP-Tac fusions were influenced by their mode of entry into cells (clathrin-dependent versus clathrin-independent), their orientation in the PM (transmembrane versus glycosylphosphatidylinositol- anchored), and ubiquitination in their cytosolic domains. In addition, shedding of SNAP-Tac into the medium was greatly influenced by its O-linked glycosylation status. For a number of PM proteins, delivery to lysosomes and ectodomain shedding represent distinct parallel mechanisms to determine protein half-life. [ABSTRACT FROM AUTHOR]

Dairy food wastewater disposal represents a major environmental problem. This review discusses micro-organisms associated with anaerobic digestion of dairy food wastewater, biochemistry of the process, factors affecting anaerobic digestion, and efforts to develop defined cultures. Anaerobic digestion of dairy food waste-water offers many advantages over other treatments in that a high level of waste stabilization is achieved with much lower levels of sludge. In addition, the process produces readily usable methane with low nutrient requirements and no oxygen. Anaerobic digestion is a series of complex reactions that broadly involve 2 groups of anaerobic or facultative anaerobic microorganisms: acidogens and methanogens. The first group of microorganisms breaks down organic compounds into CO2 and volatile fatty acids. Some of these organisms are acetogenic, which convert long-chain fatty acids to acetate, CO2, and hydrogen. Methanogens convert the acidogens' products to methane. The imbalance among the different microbial groups can lead not only to less methane production, but also to process failure. This is due to accumulation of intermediate compounds, such as volatile fatty acids, that inhibit methanogens. The criteria used for evaluation of the anaerobic digestion include levels of hydrogen and volatile fatty acids, methane:carbon ratio, and the gas production rate. A steady state is achieved in an anaerobic digester when the pH, chemical oxygen demand of the effluent, the suspended solids of the effluent, and the daily gas production remain constant. Factors affecting efficiency and stability of the process are types of microorganisms, feed C:N ratio, hydraulic retention time, reactor design, temperature, pH control, hydrogen pressure, and additives such as manure and surfactants. As anaerobic digesters become increasingly used in dairy plants, more research should be directed toward selecting the best cultures that maximize methane production from dairy food waste. [ABSTRACT FROM AUTHOR]

Abstract: It is well established that exposure of mice to perfluorooctanoate (PFOA) or perfluorooctane sulfonate (PFOS) exerts adverse effects on the thymus and spleen. Here, we characterize the effects of a 10-day dietary treatment with these compounds (0.001–0.02%, w/w) on the bone marrow (BM) of mice. At a dose of 0.02%, both compounds reduced food consumption and caused atrophy of the thymus and spleen. At this same dose, histopathological and flow cytometric analysis revealed that (i) the total numbers of BM as well as the numbers of myeloid, pro/pre B, immature B and early mature B cells were all reduced significantly; and (ii) these adverse effects were reversed either partially or completely 10days after withdrawal of these compounds. At the lower dose of 0.002%, only PFOA reduced the B-lymphoid cell population. Finally, mice fed an amount of diet equivalent to that consumed by the animals exposed to 0.02% PFOA also exhibited atrophy of the thymus and spleen, and a reduction in the number of B-lymphoid population, without affecting myeloid cells. Thus, in mice, immunotoxic doses of PFOA or PFOS induce adverse effects on the myeloid and B-lymphoid cells in the BM, in part as a consequence of reduced food consumption. [Copyright &y& Elsevier]

Mammalian testis-determining factor SRY contains a high mobility group box, a conserved eukaryotic motif of DNA bending. Mutations in SRY cause XY gonadal dysgenesis and somatic sex reversal. Although such mutations usually arise de novo in spermatogenesis, some are inherited and so specify male development in one genetic background (the father) but not another (the daughter). Here, we describe the biophysical properties of a representative inherited mutation, V60L, within the minor wing of the L-shaped domain (box position 5). Although the stability and DNA binding properties of the mutant domain are similar to those of wild type, studies of SRY-induced DNA bending by subnanosecond time-resolved fluorescence resonance energy transfer (FRET) revealed enhanced conformational fluctuations leading to long range variation in bend angle. 1H NMR studies of the variant protein-DNA complex demonstrated only local perturbations near the mutation site. Because the minor wing of SRY folds on DNA binding, the inherited mutation presumably hinders induced fit. Stopped-flow FRET studies indicated that such frustrated packing leads to accelerated dissociation of the bent complex. Studies of SRY-directed transcriptional regulation in an embryonic gonadal cell line demonstrated partial activation of downstream target Sox9. Our results have demonstrated a nonlocal coupling between DNA-directed protein folding and protein-directed DNA bending. Perturbation of this coupling is associated with a genetic switch poised at the threshold of activity. [ABSTRACT FROM AUTHOR]

Objectives: This study investigated changes in left atrial (LA) volumes and phasic atrial function, by deciles, with normal aging. Background: LA volume increase is a sensitive independent marker for cardiovascular disease and adverse outcomes. To use this variable more effectively as a marker of pathology and a gauge of outcome, physiological changes due to aging alone need to be quantitated. Methods: A detailed transthoracic echocardiogram was performed in 220 normal subjects; 89 (41%) were male and their age ranged from 20 to 80 years (mean 45 ± 17 years). Maximum (end-ventricular systole), minimum (end-ventricular diastole), and pre–a-wave volumes were measured using the biplane method of disks. LA filling, passive emptying, conduit and active emptying volumes, and fractions were calculated. Transmitral inflow, pulmonary vein flow, and pulsed-wave Doppler tissue imaging parameters were measured as expressions of left ventricular diastolic function. For purposes of analysis, subjects were divided by age deciles. Results: LA indexed maximum (0.05 ml/m2 per year) and minimum (0.06 ml/m2 per year) volume increased with age but only became significant in the eighth decade (26.0 ± 6.3 ml/m2, p = 0.02, and 13.5 ± 3.9 ml/m2, respectively; p < 0.001). Impaired left ventricular diastolic relaxation was apparent in decade 6 and was associated with a shift in phasic LA volumes so that LA expansion index and passive emptying decreased with increasing age, whereas active emptying volume increased. Conclusions: In normal healthy subjects, LA indexed volumes remain nearly stable until the eighth decade when they increase significantly. Therefore, an increase in LA size that occurs before the eighth decade is likely to represent a pathological change. Changes in phasic atrial volumes develop earlier consequent to age-related alteration in LV diastolic relaxation. [ABSTRACT FROM AUTHOR]

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system, which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. In mammals, one gone encodes two forms of the reductase, one targeted to the cytosol and the other to mitochondria. The cytosolic form displays faster mobility than the mitochondrial form, suggesting a lower molecular weight for the former. The apparent size difference and targeting to two cellular compartments had been proposed to result from differential splicing of mRNA. We now show that differential targeting is effected by use of two initiation sites, one of which includes a mitochondrial targeting sequence, whereas the other does not. We also demonstrate that the mass of the cytosolic form is not less than that of the mitochondrial form; the faster mobility of cytosolic form is due to its myristoylation. Lipidation of methionine sulfoxide reductase A occurs in the mouse, in transfected tissue culture cells, and even in a cell-free protein synthesis system. The physiologic role of myristoylation of MsrA remains to he elucidated. [ABSTRACT FROM AUTHOR]

Objectives: We sought to determine which transthoracic echocardiographic (TTE) measurements most strongly predict heart failure (HF) and to develop an index for risk stratification in outpatients with coronary artery disease (CAD). Background: Many TTE measurements have been shown to be predictive of HF, and they might be useful if aggregated into a risk-prediction index. Methods: We performed TTE in 1,024 outpatients with stable CAD enrolled in the Heart and Soul study and followed them for 4.4 years. With Cox proportional hazard models, we evaluated the association of 15 TTE measurements with subsequent HF hospital stay. Those measurements that independently predicted HF were combined into an index. Variables were defined as normal or abnormal on the basis of dichotomous cutoffs determined from the American Society of Echocardiography. Abnormal variables in each measurement were assigned points on the basis of strength of association with HF. Results: Of the 15 variables, 5 measurements were independent predictors of HF: left ventricular mass index (LVMI), left atrial volume index (LAVI), mitral regurgitation (MR), left ventricular outflow tract velocity-time integral (VTILVOT), and diastolic dysfunction (DD). In multivariate analysis, each of the 5 measurements independently predicted HF: LVMI >90 g/m2 (hazard ratio [HR]: 4.1; 95% confidence interval [CI]: 2.3 to 7.2, p < 0.0001); pseudo-normal or restrictive DD (HR: 2.9; 95% CI: 1.8 to 4.5, p < 0.0001); VTILVOT <22 mm (HR: 2.2; 95% CI: 1.4 to 3.5, p = 0.0004); mild, moderate, or severe MR (HR: 1.8; 95% CI: 1.2 to 2.8, p = 0.009); and LAVI >29 ml/m2 (HR: 1.6; 95% CI: 1.0 to 2.5, p < 0.06). Combining these measurements, the Heart Failure Index ranged from 0 to 8, representing risk as follows: 3 points for LVMI, 2 points for DD, and 1 point for VTILVOT, MR, and LAVI. Among participants with 0 to 2 points: 4% had HF hospital stays (reference); 3 to 4 points: 10% (HR: 2.4; 95% CI: 1.3 to 4.4, p = 0.003); 5 to 6 points: 24% (HR: 6.2; 95% CI: 3.6 to 10.6, p < 0.0001); 7 to 8 points: 48% (HR: 13.7; 95% CI: 7.2 to 25.9, p < 0.0001). Conclusions: We identified 5 TTE measurements that independently predict HF in patients with stable CAD and combined them as an index that might be useful for risk stratification and serial observations. [Copyright &y& Elsevier]

The carbohydrate 2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose (BacAc2) is found in a variety of eubacterial pathogens. In Campylobacter jejuni, PglD acetylates the C4 amino group on UDP-2-acetamido-4-amino-2,4,6-trideoxy-α-D-glucopyranose (UDP-4-amino-sugar) to form UDP-BacAc2. Sequence analysis predicts PglD to be a member of the left-handed β helix family of enzymes. However, poor sequence homology between PglD and left-handed β helix enzymes with existing structural data precludes unambiguous identification of the active site. The co-crystal structures of PglD in the presence of citrate, acetyl coenzyme A, or the UDP-4-amino-sugar were solved. The biological assembly is a trimer with one active site formed between two protomers. Residues lining the active site were identified, and results from functional assays on alanine mutants suggest His-125 is critical for catalysis, whereas His-15 and His-134 are involved in substrate binding. These results are discussed in the context of implications for proteins homologous to PglD in other pathogens. [ABSTRACT FROM AUTHOR]

Activation of cyclic nucleotide-modulated channels such as CNG and HCN channels is promoted by ligand-induced conformational changes in their C-terminal regions. The primary intersubunit interface of these C termini includes two salt bridges per subunit, formed between three residues (one positively charged and two negatively charged amino acids) that we term the SB triad. We previously hypothesized that the SB triad is formed in the closed channel and breaks when the channel opens. Here we tested this hypothesis by dynamically manipulating the SB triad in functioning CNGA1 channels. Reversing the charge at positions Arg-431 and Glu-462, two of the SB triad residues, by either mutation or application of charged reagents increased the favorability of channel opening. To determine how a charge reversal mutation in the SB triad structurally affects the channel, we solved the crystal structure of the HCN2 C-terminal region with the equivalent E462R mutation. The backbone structure of this mutant was very similar to that of wild type, but the SB triad was rearranged such that both salt bridges did not always form simultaneously, suggesting a mechanism for the increased ease of opening of the mutant channels. To prevent movement in the SB triad, we tethered two components of the SB triad region together with cysteine-reactive cross-linkers. Preventing normal movement of the SB triad region with short cross-linkers inhibited channel opening, whereas longer cross-linkers did not. These results support our hypothesis that the SB triad forms in the closed channel and indicate that this region expands as the channel opens. [ABSTRACT FROM AUTHOR]

Whey proteins in general and specifically β-lactoglobulin, α-lactalbumin, and immunoglobulins have been thought to decrease proteolysis in cheeses manufactured from concentrated retentates from ultrafiltration. The proteins found in whey are called whey proteins and are called milk serum proteins (SP) when they are in milk. The experiment included 3 treatments; low milk SP (0.18%), control (0.52%), and high milk SP (0.63%), and was replicated 3 times. The standardized milk for cheese making of the low milk SP treatment contained more casein as a percentage of true protein and more calcium as a percentage of crude protein, whereas the nonprotein nitrogen and total calcium content was not different from the control and high SP treatments. The nonprotein nitrogen and total calcium content of the milks did not differ because of the process used to remove the milk SP from skim milk. The low milk SP milk contained less free fatty acids (FFA) than the control and high milk SP treatment; however, no differences in FFA content of the cheeses was detected. Approximately 40 to 45% of the FFA found in the milk before cheese making was lost into the whey during cheese making. Decreasing the milk SP content of milk by 65% and increasing the content by 21% did not significantly influence general Cheddar cheese composition. Higher fat recovery and cheese yield were detected in the low milk SP treatment cheeses. There was more proteolysis in the low milk SP cheese and this may be due to the lower concentration of undenatured β-lactoglobulin, α-lactalbumin, and other high molecular weight SP retained in the cheeses made from milk with low milk SP content. [ABSTRACT FROM AUTHOR]

Microfiltration (MF) is a membrane process that can separate casein micelles from milk serum proteins (SP), mainly β-lactoglobulin and α-lactalbumin. Our objective was to develop a multistage MF process to remove a high percentage of SP from skim milk while producing a low concentration factor retentate from microfiltration (RMF) with concentrations of soluble minerals, nonprotein nitrogen (NPN), and lactose similar to the original skim milk. The RMF could be blended with cream to standardize milk for traditional Cheddar cheese making. Permeate from ultrafiltration (PUF) obtained from the ultrafiltration (UF) of permeate from MF (PMF) of skim milk was successfully used as a diafiltrant to remove SP from skim milk before cheese making, while maintaining the concentration of lactose, NPN, and nonmicellar calcium. About 95% of the SP originally in skim milk was removed by combining one 3x MF stage and two 3x PUF diafiltration stages. The final 3x RMF can be diluted with PUF to the desired concentration of casein for traditional cheese making. The PMF from the skim milk was concentrated in a UF system to yield an SP concentrate with protein content similar to a whey protein concentrate, but without residuals from cheese making (i.e., rennet, culture, color, and lactic acid) that can produce undesirable functional and sensory characteristics in whey products. Additional processing steps to this 3-stage MF process for SP removal are discussed to produce an MF skim retentate for a continuous cottage cheese manufacturing process. [ABSTRACT FROM AUTHOR]

Oxidative stress is implicated in a number of neurodegenerative diseases and is associated with the selecrive loss of dopaminergic neurons of the substantia nigra in Parkinson's disease. The role of α-synuclein as a potential target of intracellular oxidants has been demonstrated by the identification of posttranslational modifications of synuclein within intracellular aggregates that accumulate in Parkinson's disease brains, as well as the ability of a number of oxidative insults to induce synuclein oligomerization. The relationship between these relatively small soluble oligomers, potentially neurotoxic synuclein protofibrils, and synuclein filaments remains unclear. We have found that metal-catalyzed oxidation of α-synuclein inhibited formation of synuclein filaments with a concomitant accumulation of β sheet-rich oligomers that may represent synuclein protofibrils. Similar results with a number of oxidative and enzymatic treatments suggest that the covalent association of synuclein into higher molecular mass oligomers/protofibrils represents an alternate pathway from filament formation and renders synuclein less prone to proteasomal degradation. [ABSTRACT FROM AUTHOR]

To determine the influence of milk preacidification with CO2 on Cheddar cheese aging and proteolysis, cheese was manufactured from milk with and without added CO2. The experiment was replicated 3 times. Carbon dioxide (approximately 1600 ppm) was added to the cold milk, resulting in a milk pH of 5.9 at 31°C in the cheese vat. The starter and coagulant usage rates were equal for the control and CO2 treatment cheeses. The calcium content of the CO2 treatment cheese was lower, but no difference in moisture content was detected. The higher CO2 content of the treatment cheeses (337 vs. 124 ppm) was maintained throughout 6 mo of aging. In spite of having almost one and a half times the salt-in-moisture, proteolysis as measured by pH 4.6 and 12% trichloroacetic acid soluble nitrogen expressed as percentages of total nitrogen, was higher in the CO2 treatment cheeses throughout aging. The ratio of αs-casein (CN) to para-κ-CN decreased faster in the CO2 treatment cheeses than in the control cheeses, especially before refrigerated storage. No difference was detected in the ratio of β-CN to para-κ-CN between the control and CO2 treatment cheeses. Intact αs- and β-CN were found in the expressible serum (ES) from the CO2 treatment cheese as well as αs1-I-CN, but they were not detected in the ES from the control cheese. No CN was detected in the ES from the curd before the salting of either the control or CO2 treatment cheese. Higher proteolysis in the cheese made from milk preacidified with CO2 may have been due to increased substrate availability in the water phase or increased chymosin activity or retention in the cheese. [ABSTRACT FROM AUTHOR]

Preacidification of milk for cheese making may have a beneficial impact on increasing proteolysis during cheese aging. Unlike other acids, CO2 can easily be removed from whey. The objectives of this work were to determine the effect of milk preacidification on Cheddar cheese composition, the recovery of individual milk components, and yield. Carbon dioxide was injected inline after the cooling section of the pasteurizer. Cheeses with and without added CO2 were made simultaneously from the same batch of milk. This procedure was replicated 3 times. Carbon dioxide in the cheese milk was about 1600 ppm, which resulted in a milk pH of about 5.9 at 31°C. The starter culture and coagulant addition rates were the same for both the CO2 treatment and the control. The whey pH at draining of the CO2 treatment was lower than the control. Total make time was shorter for the CO2 treatment compared with the control. Cheese manufactured from milk acidified with CO2 retained less of the total calcium and fat than the control cheese. The higher fat loss was primarily in the whey at draining. Preacidification with CO2 did not alter the crude protein recovery in the cheese. The CO2 treatment resulted in a higher added salt recovery in the cheese and produced a cheese that contained too much salt. Considering the higher added salt retention, the salt application rate could be lowered to achieve a typical cheese salt content. Cheese yield efficiency of the CO2 treated milk was 4.4% lower than the control due to fat loss. Future work will focus on modifying the make procedure to achieve a normal fat loss into the whey when CO2 is added to milk. [ABSTRACT FROM AUTHOR]

Normally, reduced-fat Cheddar cheese is made by removal of fat from milk prior to cheese making. Typical aged flavor may not develop when 50% reduced-fat Cheddar cheese is produced by this approach. Moreover, the texture of the reduced-fat cheeses produced by the current method may often be hard and rubbery. Previous researchers have demonstrated that aged Cheddar cheese flavor intensity resides in the watersoluble fraction. Therefore, we investigated the feasibility of fat removal after the aging of Cheddar cheese. We hypothesized the typical aged cheese flavor would remain with the cheese following fat removal. A physical process for the removal of fat from full-fat aged Cheddar cheese was developed. The efficiency of fat removal at various temperatures, gravitational forces, and for various durations of applied forces was determined. Temperature had the greatest effect on the removal of fat. Gravitational force and the duration of applied force were less important at higher temperatures. A positive linear relationship between temperature and fat removal was observed from 20 to 33°C. Conditions of 30°C and 23,500 x g for 5 min removed 50% of the fat. The removed fat had some aroma but little or no taste. The fatty acid composition, triglyceride molecular weight distribution, and melting profile of the fat retained in the reduced-fat cheeses were all consistent with a slight increase in the proportion of saturated fat relative to the full-fat cheeses. The process of fat removal decreased the grams of saturated fat per serving of cheese from 6.30 to 3.11 g. The flavor intensity of the reduced-fat cheeses were at least as intense as the full-fat cheeses. [ABSTRACT FROM AUTHOR]

Thermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling in vivo, of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (ΔGU 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain. [ABSTRACT FROM AUTHOR]

Insulin, a protein critical for metabolic homeostasis, provides a classical model for protein design with application to human health. Recent efforts to improve its pharmaceutical formulation demonstrated that iodination of a conserved tyrosine (TyrB26) enhances key properties of a rapid-acting clinical analog. Moreover, the broad utility of halogens in medicinal chemistry has motivated the use of hybrid quantum- and molecular-mechanical methods to study proteins. Here, we (i) undertook quantitative atomistic simulations of 3-[iodo-TyrB26]insulin to predict its structural features, and (ii) tested these predictions by X-ray crystallography. Using an electrostatic model of the modified aromatic ring based on quantum chemistry, the calculations suggested that the analog, as a dimer and hexamer, exhibits subtle differences in aromatic-aromatic interactions at the dimer interface. Aromatic rings (TyrB16, PheB24, PheB25, 3-I-TyrB26, and their symmetry-related mates) at this interface adjust to enable packing of the hydrophobic iodine atoms within the core of each monomer. Strikingly, these features were observed in the crystal structure of a 3-[iodo-TyrB26]insulin analog (determined as an R6 zinc hexamer). Given that residues B24-B30 detach from the core on receptor binding, the environment of 3-I-TyrB26 in a receptor complex must differ from that in the free hormone. Based on the recent structure of a "micro-receptor" complex, we predict that 3-I-TyrB26 engages the receptor via directional halogen bonding and halogen-directed hydrogen bonding as follows: favorable electrostatic interactions exploiting, respectively, the halogen's electron-deficient σ-hole and electronegative equatorial band. Inspired by quantum chemistry and molecular dynamics, such "halogen engineering" promises to extend principles of medicinal chemistry to proteins. [ABSTRACT FROM AUTHOR]

Crystallographic studies of insulin bound to fragments of the insulin receptor have recently defined the topography of the primary hormone-receptor interface. Here, we have investigated the role of PheB24, an invariant aromatic anchor at this interface and site of a human mutation causing diabetes mellitus. An extensive set of B24 substitutions has been constructed and tested for effects on receptor binding. Although aromaticity has long been considered a key requirement at this position, MetB24 was found to confer essentially native affinity and bioactivity. Molecular modeling suggests that this linear side chain can serve as an alternative hydrophobic anchor at the hormone-receptor interface. These findings motivated further substitution of PheB24 by cyclohexanylalanine (Cha), which contains a nonplanar aliphatic ring. Contrary to expectations, [ChaB24]insulin likewise exhibited high activity. Furthermore, its resistance to fibrillation and the rapid rate of hexamer disassembly, properties of potential therapeutic advantage, were enhanced. The crystal structure of the ChaB24 analog, determined as an R6 zinc-stabilized hexamer at a resolution of 1.5A, closely resembles that of wild-type insulin. The nonplanar aliphatic ring exhibits two chair conformations with partial occupancies, each recapitulating the role of PheB24 at the dimer interface. Together, these studies have defined structural requirements of an anchor residue within the B24-binding pocket of the insulin receptor; similar molecular principles are likely to pertain to insulin-related growth factors. Our results highlight in particular the utility of nonaromatic side chains as probes of the B24 pocket and suggest that the nonstandard Cha side chain may have therapeutic utility. [ABSTRACT FROM AUTHOR]

Many consumers are concerned with fat intake. However, many reduced-fat foods, including reduced-fat cheese, lack robust flavors. The objectives of this study were to characterize the flavors found in full-fat cheese, cheese fat, and reduced-fat cheese made from aged Cheddar using a novel process to remove the fat (Nelson and Barbano, 2004). Two full-fat, aged cheeses (9 and 39 mo) were selected, and the fat was removed using the novel fat removal process. Fun-fat cheeses, shredded and reformed full-fat cheeses, corresponding reduced-fat cheeses, and cheese fats were then analyzed using descriptive sensory and instrumental analysis followed by consumer acceptance testing. Cheeses were extracted with diethyl ether followed by isolation of volatile material by high vacuum distillation. Volatile extracts were analyzed using gas chromatography/olfactometry with aroma extract dilution analysis. Selected compounds were quantified. The 39-mo cheese was characterized by fruity and sulfur notes, and the 9-mo-old cheese was characterized by a spicy/brothy flavor. Reduced-fat cheeses had similar flavor profiles with no difference in most sensory attributes to corresponding full-fat cheeses. Sensory profiles of the cheese fats were characterized by low intensities of the prominent flavors found in the full-fat cheeses. Instrumental analysis revealed similar trends. Consistent with sensory analysis, there were lower concentrations and log3 flavor dilution factors for most compounds in the cheese fats compared with both the reduced- and full-fat cheeses, regardless of compound polarity. Consumers found the intensity of flavor in the reduced-fat cheese to be equal to the full-fat cheeses. This study demonstrated that when fat was removed from aged full-fat Cheddar cheese, most of the flavor and flavor compounds remained in the cheese and were not removed with the fat. [ABSTRACT FROM AUTHOR]

Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-β assembly. Protection from fibrillation in β cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific β-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-β assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect β cells from toxic protein misfolding in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Misfolding of the protein α-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val³-Val37 and Lys45Thr92 form curved α-helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix. [ABSTRACT FROM AUTHOR]

Genetic investigations of psychiatric disease have historically relied on subjectively assessed disease diagnoses to define phenotypes. Recent developments in several areas have provided various new approaches to behavioral disorder phenotyping that promise to advance our understanding of the genetic and environmental etiologies of these traits. Such developments include re-evaluation of the boundaries between different psychiatric categories, implementation of quantitative neurobiological assessments that may serve as endophenotypes, generation of increasingly sophisticated animal behavioral models, and investigation of explicit environmental covariates. At the same time, movement toward large-scale, collaborative efforts is increasing the effectiveness of traditional genetic mapping approaches. [Copyright &y& Elsevier]

A detailed subfractionation of the non-pregnant porcine corpus luteum (CL) was performed employing differential centrifugation. Marker enzyme assays (i.e., lactate dehydrogenase for the cytosol, NADPH-cytochrome P450 reductase for the endoplasmatic reticulum, catalase (CAT) for peroxisomes, glutamate dehydrogenase for the mitochondrial matrix and acid phosphatase for lysosomes) in all subfractions obtained exhibited a pattern of distribution similar to that observed with rat liver. These subfractions should be useful in connection with many types of future studies.In disagreement with previous biochemical and morphological studies, peroxisomes (identified on the basis of catalase activity and by Western blotting of catalase and of the major peroxisomal membrane protein (PMP-70)) sedimented together with mitochondria (i.e., at 5000×gav for 10 min) and not in the post-mitochodrial fraction prepared at 30,000×gav for 20 min by Peterson and Stevensson [Biochim. Biophys. Acta 1135 (1992) 207]. No other classical peroxisomal enzymes were detectable in the porcine ovary, raising questions concerning the function of peroxisomes in this organ.Furthermore, UDP-glucuronosyltransferase (UGT), generally considered to be an integral membrane protein anchored in the endoplasmatic reticulum, was recovered in both the cytosolic (i.e., the supernatant after centrifugation at 50,000×gav for 1 h) and the microsomal fraction of the porcine corpus luteum, even upon further centrifugation of the former. In contrast, UGT sediments exclusively in the microsomal fraction upon subfractionation of the liver and ovary from rat. [Copyright &y& Elsevier]