Simple method for developing percentile growth curves for height and weight

The present paper demonstrates the ease of use of method I by Preece and Baine ([1978] Ann Hum Biol 5:1-24) in generating smoothed growth curves for both height and weight. Using the National Center for Health Statistics (NCHS) growth curve data, smoothed curves were developed and compared to those produced using the least-squares-cubic-spline method. Based on the lower sum of squares and better fit of shape as indicated by residual examination, it was concluded that the method I curve fitting procedure by Preece and Baine ([1978] Ann Hum Biol 5:1-24) fit centile growth curves for height and weight in 2-18-year-old male and female children as well as, if not better than, the least-squares-cubic-spline method used in developing the 1979 NCHS growth curves. Further, as this paper demonstrates, smoothed curves can be generated on a desktop computer using readily available software (the SOLVER function within Microsoft EXCEL).     

The mechanism of dienoyl-CoA reduction by 2,4-dienoyl-CoA reductase is stepwise: observation of a dienolate intermediate

The chemical mechanism of the 2,4-dienoyl-CoA reductase (EC 1.3.1.34) from rat liver mitochondria has been investigated. This enzyme catalyzes the NADPH-dependent reduction of 2,4-dienoyl-coenzyme A (CoA) thiolesters to the resulting trans-3-enoyl-CoA. Steady-state kinetic parameters for trans-2,trans-4-hexadienoyl-CoA and 5-phenyl-trans-2,trans-4-pentadienoyl-CoA were determined and demonstrated that the dienoyl-CoA and NADPH bind to the 2,4-dienoyl-CoA reductase via a sequential kinetic mechanism. Kinetic isotope effect studies and the transient kinetics of substrate binding support a random order of nucleotide and dienoyl-CoA addition. The large normal solvent isotope effects on V/K ((D)(2)(O)V/K) and V ((D)(2)(O)V) for trans-2,trans-4-hexadienoyl-CoA reduction indicate that a proton transfer step is rate limiting for this substrate. The stability gained by conjugating the phenyl ring to the diene in PPD-CoA results in the reversal of the rate-determining step, as evidenced by the normal isotope effects on V/K(CoA) ((D)V/K(CoA)) and V/K(NADPH) ((D)V/K(NADPH)). The reversal of the rate-determining step was supported by transient kinetics where a burst was observed for the reduction of trans-2,trans-4-hexadienoyl-CoA but not for 5-phenyl-trans-2,trans-4-pentadienoyl-CoA reduction. The chemical mechanism is stepwise where hydride transfer from NADPH occurs followed by protonation of the observable dienolate intermediate, which has an absorbance maximum at 286 nm. The exchange of the C alpha protons of trans-3-decenoyl-CoA, catalyzed by the 2,4-dienoyl-CoA reductase, in the presence of NADP(+) suggests that formation of the dienolate is catalyzed by the enzyme active site.     

Catalytic protein film voltammetry from a respiratory nitrate reductase provides evidence for complex electrochemical modulation of enzyme activity

The first step in the respiratory reduction of nitrate to dinitrogen in Paracoccus pantotrophus is catalyzed by the quinol-nitrate oxidoreductase NarGHI. This membrane-anchored protein directs electrons from quinol oxidation at the membrane anchor, NarI, to the site of nitrate reduction in the membrane extrinsic [Fe-S] cluster and Mo-bis-MGD containing dimer, NarGH. Liberated from the membrane, NarGH retains its nitrate reductase activity and forms films on graphite and gold electrodes within which direct and facile exchange of electrons between the electrode and the enzyme occurs. Protein film voltammetry has been used to define the catalytic behavior of NarGH in the potential domain and a complex pattern of reversible, nitrate concentration dependent modulation of activity has been resolved. At low nitrate concentrations the local maximum observed in the catalytic current-potential profile reveals how NarGH can catalyze nitrate reduction via two pathways having distinct specificity constants, k(obs)(cat)/K(obs)(M). Catalysis is directed to occur via one of the pathways by an electrochemical event within NarGH. On increasing the nitrate concentration, the local maximum in the catalytic current becomes less distinct, and the catalytic waveform adopts an increasingly sigmoidal form. A pattern of voltammetry similar to that observed during nitrate reduction is observed during reduction of the stereochemically distinct substrate chlorate. Centers whose change of oxidation state may define the novel catalytic voltammetry of NarGH have been identified by EPR-monitored potentiometric titrations and mechanisms by which the electrochemistry of Mo-bis-MGD or [Fe-S] clusters can account for the observed behavior are discussed.     

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata

A two-domain portion of the proteinase inhibitor precursor from Nicotiana alata (NaProPI) has been expressed and its structure determined by NMR spectroscopy. NaProPI contains six almost identical 53 amino acid repeats that fold into six highly similar domains; however, the sequence repeats do not coincide with the structural domains. Five of the structural domains comprise the C-terminal portion of one repeat and the N-terminal portion of the next. The sixth domain contains the C-terminal portion of the sixth repeat and the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceivable that adjacent domains in NaProPI interact to generate the circular "bracelet" with the N and C termini in close enough proximity to facilitate formation of the disulphide bonds that form the "clasp". The expressed protein, examined in the current study, comprises residues 25-135 of NaProPI and encompasses the first two contiguous structural domains, namely the chymotrypsin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue linker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no nuclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of interactions between the domains indicates that adjacent domains do not specifically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from other members of the Solanaceae having two, three, or four repeats. The lack of strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor.     

Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-psi[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY-3-psi[CH2NH2+]-Asp19I

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi[COO]-Leu18I). Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi[CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.     

OXYGEN CONSUMPTION ASSOCIATED WITH FERRIC REDUCTASE ACTIVITY AND IRON UPTAKE BY IRON-LIMITED CELLS OF CHLORELLA KESSLERI (CHLOROPHYCEAE)

Plasma membrane ferric reductase activity was enhanced 5-fold under iron limitation in the unicellular green alga Chlorella kessleri Fott et Nováková. Furthermore, ferric reductase activity in iron-limited cells was approximately 50% higher in the light than in the dark. In contrast, iron uptake rates of iron-limited cells were unaffected by light versus dark treatments. Rates of iron uptake were much lower than rates of ferric reduction, averaging approximately 2% of the dark ferric reduction rate. Ferric reduction was associated with an increased rate of O₂ consumption in both light and dark, the increase in the light being approximately 1.5 times as large as in the dark. The increased rate of O₂ consumption could be decreased by half by the addition of catalase, indicating that H₂O₂ is the product of the O₂ consumption and that the increased O₂ consumption is nonrespiratory. The stimulation of O₂ consumption was almost completely abolished by the addition of bathophenanthroline disulfonate, a strong chelator of Fe^{2 +} . Anaerobic conditions or the presence of exogenous superoxide dismutase affected neither ferric reduction nor iron uptake. We suggest that the O₂ consumption associated with ferric reductase activity resulted from superoxide formation from the aerobic oxidation of Fe^{2 +} , which is the product of ferric reductase activity. At saturating concentrations of Fe^{3 +} chelates, ferric reductase activity is much greater than the iron uptake rate, leading to rapid oxidation of Fe^{2 +} and superoxide generation. Therefore, O₂ consumption is not an integral part of the iron assimilation process.     

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.     

Infectious entry by amphotropic as well as ecotropic murine leukemia viruses occurs through an endocytic pathway

Infectious entry of enveloped viruses is thought to proceed by one of two mechanisms. pH-dependent viruses enter the cells by receptor-mediated endocytosis and are inhibited by transient treatment with agents that prevent acidification of vesicles in the endocytic pathway, while pH-independent viruses are not inhibited by such agents and are thought to enter the cell by direct fusion with the plasma membrane. Nearly all retroviruses, including amphotropic murine leukemia virus (MuLV) and human immunodeficiency virus type 1, are classified as pH independent. However, ecotropic MuLV is considered to be a pH-dependent virus. We have examined the infectious entry of ecotropic and amphotropic MuLVs and found that they were equally inhibited by NH4Cl and bafilomycin A. These agents inhibited both viruses only partially over the course of the experiments. Agents that block the acidification of endocytic vesicles also arrest vesicular trafficking. Thus, partial inhibition of the MuLVs could be the result of virus inactivation during arrest in this pathway. In support of this contention, we found that that the loss of infectivity of the MuLVs during treatment of target cells with the drugs closely corresponded to the loss of activity due to spontaneous inactivation at 37 degrees C in the same period of time. Furthermore, the drugs had no effect on the efficiency of infection under conditions in which the duration of infection was held to a very short period to minimize the effects of spontaneous inactivation. These results indicate that the infectious processes of both ecotropic and amphotropic MuLVs were arrested rather than aborted by transient treatment of the cells with the drugs. We also found that infectious viruses were efficiently internalized during treatment. This indicated that the arrest occurred in an intracellular compartment and that the infectious process of both the amphotropic and ecotropic MuLVs very likely involved endocytosis. An important aspect of this study pertains to the interpretation of experiments in which agents that block endocytic acidification inhibit infectivity. As we have found with the MuLVs, inhibition of infectivity may be secondary to the block of endocytic acidification. While this strongly suggests the involvement of an endocytic pathway, it does not necessarily indicate a requirement for an acidic compartment during the infectious process. Likewise, a lack of inhibition during transient treatment with the drugs would not preclude an endocytic pathway for viruses that are stable during the course of the treatment.