Re-examination of the "3/4-law" of metabolism

We examine the scaling law B is proportional to M(alpha)which connects organismal resting metabolic rate B with organismal mass M, where alpha is commonly held to be 3/4. Since simple dimensional analysis suggests alpha = 2/3, we consider this to be a null hypothesis testable by empirical studies. We re-analyse data sets for mammals and birds compiled by Heusner, Bennett and Harvey, Bartels, Hemmingsen, Brody, and Kleiber, and find little evidence for rejecting alpha = 2/3 in favor of alpha = 3/4. For mammals, we find a possible breakdown in scaling for larger masses reflected in a systematic increase in alpha. We also review theoretical justifications of alpha = 3/4 based on dimensional analysis, nutrient-supply networks, and four-dimensional biology. We find that present theories for alpha = 3/4 require assumptions that render them unconvincing for rejecting the null hypothesis that alpha = 2/3.     

Hydroperoxy-10,12-octadecadienoic acid stimulates cytochrome P450 3A protein aggregation by a mechanism that is inhibited by substrate

We recently demonstrated that microsomes from nicardipine-treated rats will form cytochrome P450 3A (CYP3A) aggregates when incubated at 37 degrees C. CYP3A substrates inhibited the protein aggregation and subsequent degradation, suggesting that this process is important in substrate-mediated stabilization of CYP3A. In this paper, we demonstrate that oxidative stress is a key factor in the formation of CYP3A aggregates in incubated microsomes and in a reconstituted system with purified enzymes. Our data further suggest that the effects of oxidative stress are mediated by lipid hydroperoxides, which are efficiently metabolized by CYP3A. In the presence of substrate, the CYP3A-mediated lipid hydroperoxide metabolism is inhibited along with the associated protein aggregation. Therefore, these studies provide a mechanistic model of why CYP3A has a relatively short half-life and how substrates stabilize CYP3A.     

Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein

Vitamin D-binding protein (DBP), a multi-functional serum glycoprotein, has a triple-domain modular structure. Mutation of Trp145 (in Domain I) to Ser decreased 25-OH-D(3)-binding by 80%. Furthermore, recombinant Domain I (1-203) and Domain I + II (1-330) showed specific and strong binding for 25-OH-D(3), but Domain III (375-427) did not, suggesting that only Domains I and II might be required for vitamin D sterol-binding. Past studies have suggested that Domain III is independently capable of binding G-actin. We exploited this apparently independent ligand-binding property of DBP to purify DBP-actin complex from human serum and rabbit muscle actin by 25-OH-D(3) affinity chromatography. Competitive (3)H-25-OH-D(3) binding curves for native DBP and DBP-actin complex were almost identical, further suggesting that vitamin D sterol- and actin-binding activities by DBP might be largely independent of each other. Trypsin treatment of DBP produced a prominent 25 kDa band (Domain I, minus 5 amino acids in N-terminus), while actin was completely fragmented by such treatment. In contrast, tryptic digestion of purified DBP-actin complex showed two prominent bands, 52 (DBP, minus 5 amino acids in the N-terminus) and 34 kDa (actin, starting with amino acid position 69) indicating that DBP, particularly its Domains II and III were protected from trypsin cleavage upon actin-binding. Similarly, actin, except its N-terminus, was also protected from tryptic digestion when complexed with DBP. These results provided the basis for our studies to crystallize DBP-actin complex, which produced a 2.5 A crystal, primitive orthorhombic with unit cell dimensions a=80.2A, b=87.3A, and c=159.6A, P2(1)2(1)2(1) space group, V(m)=2.9. Soaking of crystals of actin-DBP in crystallization buffer containing various concentrations of 25-OH-D(3) resulted in cracking of the crystal, which was probably a reflection of a ligand-induced conformational change in the complex, disrupting crystal contacts. In conclusion, we have provided data to suggest that although binding of 25-OH-D(3) to DBP might result in discrete conformational changes in the holo-protein to influence actin-binding, these binding processes are largely independent of each other in solution.     

1,2,5-Trisubstituted 1,4-diazepine-3-one: A novel dipeptidomimetic molecular scaffold

Summary The continuing effort to transform bioactive peptides into non-peptide peptidomimetics of therapeutic potential requires a diversity of tools such as molecular scaffolds, pseudopeptide modifications, and conformation mimetics. To this end, a novel polyfunctional monoheterocyclic system, 1,2,5-trisubstituted hexahydro-3-oxo-1H-1,4-diazepine ring (DAP), was designed. The linear precursor for the DAP was generated through a reductive alkylation step including a modified side chain and an α-amino function of two amino acid derivatives. Structural analysis of model diastereomeric DAPs, employing¹H and¹³C NMR and computer simulation, revealed the conformational preferences of this system. The structural similarities to the 1,4-benzodiazepine, a common molecular scaffold for many non-peptidic peptidomimetic agents, and the pronounced dipeptidomimetic character of the DAP system offer a new powerful tool to medicinal chemists engaged in rational peptide-based drug design.     

Calmodulin controls the rod photoreceptor CNG channel through an unconventional binding site in the N-terminus of the beta-subunit.

Calmodulin (CaM) controls the activity of the rod cGMP-gated ion channel by decreasing the apparent cGMP affinity. We have examined the mechanism of this modulation using electrophysiological and biochemical techniques. Heteromeric channels, consisting of alpha- and beta-subunits, display a high CaM sensitivity (EC50 </=5 nM) similar to the native channel. Using surface plasmon resonance spectroscopy, we identified two unconventional CaM-binding sites (CaM1 and CaM2), one in each of the N- and the C-terminal regions of the beta-subunit. Ca2+ co-operatively stimulates binding of CaM to these sites exactly within the range of [Ca2+] occurring during a light response. Deletion of the N-terminal CaM1 site results in channels that are no longer CaM-sensitive, whereas deletion of CaM2 has only minor effects. We discuss different models to explain the high-affinity binding of CaM.     

Zero-sum allocational strategies determine the allometry of specific leaf area

? Premise of the study: Specific leaf area (SLA) is a critical component of the leaf economics spectrum, and many functional leaf traits have been empirically demonstrated to covary with SLA. However, a complete understanding of how change in leaf size influences SLA has not yet emerged. ? Methods: To help develop a more complete understanding of the determinants of variability in SLA, we present a covariation model of leaf allometry that predicts a zero-sum interdependence of leaf thickness, density, and surface area on leaf mass. We test the model's predictions on measurements of 900 leaves from 44 angiosperm species. ? Key results: We observe that "diminishing returns," the negative allometry (slope < 1) of surface area versus mass, does not hold universally across species. Rather, the scaling of SLA is linked to the relative allocation to thickness and density. Specifically, diminishing returns are observed when leaves grow thicker, more than their density decreases, with increasing mass. Finally, we confirm model predictions that the allometric dependence of area, thickness, and density on mass can be well approximated by a zero-sum allocational process. ? Conclusions: Our work adds to the growing body of evidence that allometric covariation is a hallmark of the scaling behavior of complex plant and leaf traits. Moreover, because our model makes predictions based on allocational constraints, it provides a foundation to understand how deviations from zero-sum tradeoffs in allocation to leaf thickness, density, or area determine the allometry of SLA and, ultimately, underlie adaptive strategies within and across plant species.     

A Blind Spot in Confocal Reflection Microscopy: The Dependence of Fiber Brightness on Fiber Orientation in Imaging Biopolymer Networks

We investigate the dependence of fiber brightness on three-dimensional fiber orientation when imaging biopolymer networks with confocal reflection microscopy (CRM) and confocal fluorescence microscopy (CFM). We compare image data of fluorescently labeled type I collagen networks concurrently acquired using each imaging modality. For CRM, fiber brightness decreases for more vertically oriented fibers, leaving fibers above ∼50° from the imaging plane entirely undetected. As a result, the three-dimensional network structure appears aligned with the imaging plane. In contrast, CFM data exhibit little variation of fiber brightness with fiber angle, thus revealing an isotropic collagen network. Consequently, we find that CFM detects almost twice as many fibers as are visible with CRM, thereby yielding more complete structural information for three-dimensional fiber networks. We offer a simple explanation that predicts the detected fiber brightness as a function of fiber orientation in CRM.     

Quantifying enzymatic lysis: estimating the combined effects of chemistry, physiology and physics

The number of microbial pathogens resistant to antibiotics continues to increase even as the rate of discovery and approval of new antibiotic therapeutics steadily decreases. Many researchers have begun to investigate the therapeutic potential of naturally occurring lytic enzymes as an alternative to traditional antibiotics. However, direct characterization of lytic enzymes using techniques based on synthetic substrates is often difficult because lytic enzymes bind to the complex superstructure of intact cell walls. Here we present a new standard for the analysis of lytic enzymes based on turbidity assays which allow us to probe the dynamics of lysis without preparing a synthetic substrate. The challenge in the analysis of these assays is to infer the microscopic details of lysis from macroscopic turbidity data. We propose a model of enzymatic lysis that integrates the chemistry responsible for bond cleavage with the physical mechanisms leading to cell wall failure. We then present a solution to an inverse problem in which we estimate reaction rate constants and the heterogeneous susceptibility to lysis among target cells. We validate our model given simulated and experimental turbidity assays. The ability to estimate reaction rate constants for lytic enzymes will facilitate their biochemical characterization and development as antimicrobial therapeutics.