A realistic in silico model for structure/function studies of molybdenum–copper CO dehydrogenase

CO dehydrogenase (CODH) is an environmentally crucial bacterial enzyme that oxidizes CO to CO₂ at a Mo–Cu active site. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the protonation state of the water-derived equatorial ligand coordinated at the Mo-center, as well as the nature of intermediates formed during the catalytic cycle. To address the protonation state of the equatorial ligand, we have developed a realistic in silico QM model (~179 atoms) containing structurally essential residues surrounding the active site. Using our QM model, we examined each plausible combination of redox states (Mo^VI–Cu^I, Mo^V–Cu^II, Mo^V–Cu^I, and Mo^IV–Cu^I) and Mo-coordinated equatorial ligands (O^2?, OH^?, H₂O), as well as the effects of second-sphere residues surrounding the active site. Herein, we present a refined computational model for the Mo(VI) state in which Glu763 acts as an active site base, leading to a MoO₂-like core and a protonated Glu763. Calculated structural and spectroscopic data (hyperfine couplings) are in support of a MoO₂-like core in agreement with XRD data. The calculated two-electron reduction potential (E = ?467 mV vs. SHE) is in reasonable agreement with the experimental value (E = ?558 mV vs. SHE) for the redox couple comprising an equatorial oxo ligand and protonated Glu763 in the Mo^VI–Cu^I state and an equatorial water in the Mo^IV–Cu^I state. We also suggest a potential role of second-sphere residues (e.g., Glu763, Phe390) based on geometric changes observed upon exclusion of these residues in the most plausible oxidized states.     

The structure and function of the chloroplast photosynthetic membrane — a model for the domain organization

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14–18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.Abbreviations PS I and PS II Photosystem I and II - P 700 reaction center of PS I - LHC II light-harvesting complex II     

Radioimmunoscintigraphic approach for the in vivo detection of tuberculomas—A preliminary study in a rabbit model

Radioimmunoscintigraphic approach could provide a viable non-invasive alternative for the diagnosis of deeply situated tuberculomas. We have evaluated this in a rabbit model constructed to give a characteristic localized tubercular lesion, with complimentary morphological, histological and antigenic profiles. ¹³¹I-anti Myobacterium bovis (BCG) antibody was shown to localize at the lesion, where as ¹³¹I-bovine serum albumin and ^99mTc-red blood cell scans were negative. The clearance of intradermally-injected tuberculous antigen could be traced into ascending lymph nodes using ¹³¹I-anti M. bovis (BCG) antibody.     

Mouse models to study polycystic ovary syndrome: A possible link between metabolism and ovarian function?

Polycystic ovary syndrome (PCOS) is the most common cause of female infertility affecting 6–8% of women worldwide. PCOS is characterized by two of the following three criteria: clinical or biochemical hyperandrogenism, oligo- or amenorrhea, and polycystic ovaries (PCO). In addition, women with PCOS are often obese and insulin resistant, and are at risk for type 2 diabetes and cardiovascular disease. The etiology of PCOS remains unknown. Therefore, several animal models for PCOS have been generated to gain insight into the etiology and development of the PCOS-associated phenotypes. Androgens are considered the main culprit of PCOS, and therefore, androgenization of animals is the most frequently used approach to induce symptoms that resemble PCOS. Prenatal or prepubertal androgen treatment results in many characteristics of human PCOS, including anovulation, cyst-like follicles, elevated luteinizing hormone (LH) levels, increased adiposity, and insulin insensitivity. However, PCOS has a heterogeneous presentation, and therefore it is difficult to generate a model that exactly reproduces the reproductive and metabolic phenotypes observed in women with PCOS. In this review, we discuss several mouse models for PCOS, and compare the reproductive and/or metabolic phenotypes observed in several androgen-induced models as well as in several genetic models.     

In vitro culturing of ciliary respiratory cells—a model for studies of genetic diseases

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by the impaired functioning of ciliated cells. Its diagnosis is based on the analysis of the structure and functioning of cilia present in the respiratory epithelium (RE) of the patient. Abnormalities of cilia caused by hereditary mutations closely resemble and often overlap with defects induced by the environmental factors. As a result, proper diagnosis of PCD is difficult and may require repeated sampling of patients’ tissue, which is not always possible. The culturing of differentiated cells and tissues derived from the human RE seems to be the best way to diagnose PCD, to study genotype–phenotype relations of genes involved in ciliary dysfunction, as well as other aspects related to the functioning of the RE. In this review, different methods of culturing differentiated cells and tissues derived from the human RE, along with their potential and limitations, are summarized. Several considerations with respect to the factors influencing the process of in vitro differentiation (cell-to-cell interactions, medium composition, cell-support substrate) are also discussed.     

The effect of saccades on threshold perception — A model study

The effect of saccadic eye movements on threshold perception is investigated theoretically. The proposed model considers eye movements by taking into account the shifting of the stimulus pattern on the retina during the occurrence of an eye movement. Saccades are characterized by high velocity and short duration. These motions cause overshoots in the response of linear filters to certain stimulus patterns. Therefore, the model predicts facilitation effects of saccades in the perception of low spatial frequency patterns and patterns flickering with high temporal frequencies. These results agree with experimentally obtained data presented in a subsequent paper. A simple approach is formulated which approximates the complex shifting function of a saccade by a switching of the pattern.     

A nonlinear kinetic model of γ-aminobutyric acid metabolism in a rabbit model of acute liver failure

To investigate the mechanisms by which serum levels of γ-aminobutyric acid (GABA) become elevated in experimental acute liver failure, a multicompartmental model of GABA metabolism has been constructed and used to simulate previously generated data on the kinetics of ³H-GABA uptake by isolated hepatocytes from normal rats and the kinetics of ³H-GABA in the plasma of normal rabbits, rabbits with galactosamine-induced acute liver failure, and rabbits with divascularized livers. Modeling analysis revealed that acute liver failure was associated with values for the mean fractional catabolic rate of GABA, plasma volume, and hepatic extraction of GABA that were 29%, 12%, and 49% less, respectively, than the corresponding control values. The defect in hepatic tissue extraction of GABA was sufficient to account for only 60% of the 10-fold increase in serum GABA levels that occurs in acute liver failure. Furthermore the 10-fold increase in serum GABA levels occured in acute liver failure before the onset of overt hepatic encephalopathy when hepatic extraction of GABA was not appreciably different from that found in normal rabbits. Thus the increase in serum GABA levels that occurs in acute liver failure cannot be attributed to a defect in hepatic extraction of GABA alone. Indeed, the modeling analysis indicated that in acute liver failure there is a 3—8-fold increase in the rate of delivery of GABA to the systemic circulation, but did not indicate its source.     

The prospects for biogas—A European point of view

In many parts of the world, alternative energy sources are already needed or will be needed to augment fossil fuel supplies. ‘Biogas’, methanol, ethanol and hydrogen are all potentially useful in this respect, as they can be obtained (biologically or thermochemically) from renewable resources like biomass (trees, crops, etc.) and organic wastes (sewage, domestic refuse, animal manure). Some aspects of biofuel research and development in Member States of the European Community are highlighted, and estimates of the contributions that these fuels might make to current and future energy demands are reviewed. Some of the potential uses of biogases and liquid biofuels in a gas industry context are described. In the author's opinion, production of biogases and other biofuels will ultimately play a significant but relatively small rôle in conserving supplies of natural gas and other fossil fuels, and in alleviating or preventing environmental pollution.     

The conversion of orotic acid into uridine 5′-monophosphate by isolated perfused normal and regenerating rat livers

The release of (14)CO(2) from [7-(14)C]orotic acid was measured in isolated perfused normal and regenerating rat livers. With some limitations, the release of (14)CO(2) from [7-(14)C]orotic acid can be used to estimate UMP synthesis in perfused livers. Isolated perfused livers rapidly pick up labelled orotic acid added to perfusate and convert most of it into UMP. Perfused regenerating livers produce approx. 2.5 times as much UMP/g of liver as do perfused normal livers. However, the absolute amount of orotic acid converted into UMP is higher in perfused normal livers than in perfused regenerating livers. Perfused regenerating livers do not differ in their orotic acid uptake and UMP synthesis from livers of comparable size in which regeneration is not taking place. The total amount of orotic acid taken up by the liver (rather than the rate of uptake) and the size of the liver appear to be the determining factors in UMP production. The results suggest that the decrease in liver size caused by partial hepatectomy may be in itself sufficient to account for an increase in the flow of metabolites in the pyrimidine pathway at the early stages of liver regeneration.     

External detection of β-adrenoreceptors with 125I-hydroxybenzylpindolol in isolated perfused hearts

To assess the feasibility of characterizing β-adrenoreceptors externally with ligands labeled with γ-emitting radionuclides, we infused ¹²⁵I-hydroxybenzylpindolol (¹²⁵I-HYP) a potent, high affinity, β-adrenoreceptor ligand into isolated perfused hearts and monitored uptake and release of the label externally with a gamma probe. Perfusion with specific β-adrenergic displacing significantly increased the turnover rate constant of ¹²⁵I-HYP in comparison to that obtained with non-specific displacing agents. After modifying β-adrenoreceptor density by pretreatment of animals with thyroid hormone, an increase in ¹²⁵I-HYP uptake was observed. Thus, β-adrenoreceptor occupancy and increases in β-adrenoreceptor density can be detected externally with an approach which may offer promise for their characterization in vivo with radiolabeled adrenergic ligands.     

Terminal-sequence studies of high-molecular-weight ribonucleic acid. The 3′-termini of rabbit reticulocyte ribosomal ribonucleic acid

Sequences of the polynucleotide chains of RNA found in the large and small ribosomal subunits of rabbit reticulocytes have been determined from the 3'-end by use of periodate oxidation and condensation with [(3)H]isoniazid and by stepwise degradation. By these methods the hexanucleotide sequences have been found as -pGpUpUpUpGpU for the 28S RNA and -pGpUpCpGpCpU for the 6S RNA of the large ribosomal subunit and the octanucleotide sequence -pGpApUpCpApUpUpA for the 18S rRNA of the small ribosomal subunit. These sequences are present in at least 70% of all the RNA molecules and are discussed in relation to the specific cleavage of rRNA from its precursors and the role of multiple cistrons for rRNA in the DNA of higher organisms. The feasibility of using the method for longer sequence determinations is discussed.     

TGFβ signalling in the development of ovarian function

Ovarian development begins back in the embryo with the formation of primordial germ cells and their subsequent migration and colonisation of the genital ridges. Once the ovary has been defined structurally, the primordial germ cells transform into oocytes and become housed in structures called follicles (in this case, primordial follicles), a procedure that, in most mammals, occurs either shortly before or during the first few days after birth. The growth and differentiation of follicles from the primordial population is termed folliculogenesis. Primordial follicles give rise to primary follicles that transform into preantral follicles, then antral follicles (secondary follicles) and, finally (preovulatory) Graafian follicles (tertiary follicles) in a co-ordinated series of transitions regulated by hormones and local intraovarian factors. Members of the transforming growth factor-β (TGFβ) superfamily have been shown to play important roles in this developmental process starting with the specification of primordial germ cells by the bone morphogenetic proteins through to the recruitment of primordial follicles by anti-Mullerian hormone and, potentially, growth and differentiation factor-9 (GDF9) and, finally, their transformation into preantral and antral follicles in response to activin and TGF-β. Developmental and mutant mouse models have been used to show the importance of this family of growth factors in establishing the first wave of folliculogenesis.The author thanks the NHMRC of Australia for funding (Regkey 241000).     

A new model for interacting populations—II: Principle of competitive exclusion

The principle of competitive exclusion is investigated within the framework of the solvable model proposed earlier for two-species systems. The results elucidate the recent controversy over the interpretation of experimental data onDrosophila equilibrium. It is shown that the necessary and sufficient conditions for stable coexistence of competing species is that the product of intraspecific rate constants be greater than the product of interspecific rate constants. Inequalities between rate constants for the occurrence of stable equilibriumbelow andabove the line joining single species equilibria are derived. The availability of larger domain of coexistence suggests that the model presented here has the potential to accommodate a wider class of phenomena than the Gause—Volterra model according to which coexistence is possible only above the line of single species equilibrium.     

A study of staining for electron microscopy using collagen as a model system—VII. The effect of formaldehyde fixation

Exposure to formaldehyde brings about small but readily detectable changes in the staining behaviour of collagen fibrils. These changes can be interpreted in chemical terms by comparing fibril staining patterns with artificial patterns computer-generated from sequence data. Positive staining with phosphotung-state (where heavy metal is confined to anions), shows that most of the lysyl and hydroxylysyl side-chains lose their charge character as a result of formaldehyde treatment and cease to take up staining ions. The charge character of arginyl (and probably histidyl) residues is unaltered and these residues continue to react with stain. Acidic residues are also unaffected. These results accord with biochemical evidence that the initial reaction between proteins and formaldehyde leading to subsequent cross-linking involves modification of ε-amino (and α-amino) groups. They show too that the secondary condensation producing the actual cross-link does not alter the charge character of the second group, at least when it is on an arginyl (or histidyl) side-chain.Formaldehyde-induced changes in stain deposition can also be detected after negative staining, although they are slight compared with those brought about by glutaraldehyde. Unlike glutaraldehyde, formaldehyde introduces no bulky polymeric adducts into the fibril structure, and the conspicuous stain-excluding bands seen in negative staining patterns following glutaraldehyde fixation are absent after exposure to formaldehyde. For this reason, where chemical fixation is used to stabilize macromolecules and supramolecular aggregates prior to negative staining and high resolution electron optical imaging, formaldehyde would seem to be preferable to glutaraldehyde. Data from fibril staining patterns and from thermal stability measurements (made on collagen gels) show that formaldehyde fixation does not preclude a subsequent reaction with glutaraldehyde.As with other fixatives, there is reduced accessibility to stain after formaldehyde treatment. Accessibility is least in the overlap zone where the denser packing of collagen molecules provides greater opportunities for intermolecular cross-linking. Gel electrophoresis confirms that formaldehyde-induced cross-links in fibrils are predominantly intermolecular.     

A fluorescent carbapenem for structure function studies of penicillin-binding proteins, β-lactamases,and β-lactam sensors

By reacting fluorescein isothiocyanate with meropenem, we have prepared a carbapenem-based fluorescent β-lactam. Fluorescein–meropenem binds both penicillin-binding proteins and β-lactam sensors and undergoes a typical acylation reaction in the active site of these proteins. The probe binds the class D carbapenemase OXA-24/40 with close to the same affinity as meropenem and undergoes a complete catalytic hydrolysis reaction. The visible light excitation and strong emission of fluorescein render this molecule a useful structure–function probe through its application in sodium dodecyl sulfate–polyacrylamide gel electrophoresis assays as well as solution-based kinetic anisotropy assays. Its classification as a carbapenem β-lactam and the position of its fluorescent modification render it a useful complement to other fluorescent β-lactams, most notably Bocillin FL. In this study, we show the utility of fluorescein–meropenem by using it to detect mutants of OXA-24/40 that arrest at the acyl-intermediate state with carbapenem substrates but maintain catalytic competency with penicillin substrates.     

Terminal sequence studies of high-molecular-weight ribonucleic acid. The 3′-termini of rabbit globin messenger ribonucleic acid

A study of the relative utilization of thymine and thymidine as precursors for DNA synthesis during normal growth in Bacillus subtilis showed that thymine serves preferentially as a precursor for ;repair' synthesis, whereas thymidine is used preferentially for ;replicative' synthesis. Further, evidence was obtained which suggests that during normal growth both ;replicative' and ;repair' DNA syntheses occur simultaneously. ;Repair' synthesis is distinguished not only on the basis of its preferential utilization of thymine but also by its selective inhibition by caffeine. ;Replicative' synthesis, however, is selectively inhibited by 6-(p-hydroxyphenylazo)-uracil. ;Repair' synthesis would seem to be a ;pre-fork' phenomenon and its inhibition is highly lethal to the cell.     

A study of positive staining for electron microscopy using collagen as a model system—III. The effect of suberimidate fixation

Collagen is used as a model system to study the structural effects of the fixative dimethylsuberimidate (DMS) on a protein. Using reconstituted fibrils of type I calf skin collagen(of known amino acid sequence), electron-optical data from positive staining patterns were compared with chemical data by a computer-aided correlation procedure. The results show that, under the conditions used, the reaction between DMS and collagen is specific for the ϵ-amino groups of lysyl and hydroxylysyl residues and that no reaction takes place between DMS and arginyl residues. Other evidence suggests that (unlike glutaraldehyde) no polymerization of the DMS occurs and that a cross-link between the two ϵ-amino groups can only form when they are sufficiently close to be bridged by the DMS monomer.