Inspiratory Obstruction

Obstructing lesions of the trachea and larynx which cause a predominantly inspiratory obstruction can be satisfactorily diagnosed by measuring both F.I.V.₁ and F.E.V.₁. Chronic airways obstruction involving intrathoracic airways produces a much lower F.E.V.₁/F.I.V.₁ percentage than normal, whereas obstruction to larynx and trachea causes a raised F.E.V.₁/F.I.V.₁ percentage. If flow-volume measurements are not available the F.E.V.₁/F.I.V.₁ percentage should provide a simple and useful method for diagnosis of upper airways obstruction.In one of the patients reported a predominantly inspiratory obstruction caused CO₂ retention. In patients with airways obstruction the correlation between Pco₂ and F.I.V.₁ was found to be the same as between Pco₂ and F.E.V.₁. This suggests that respiratory failure can be caused by either inspiratory or expiratory airways obstruction and that neither is of greater importance in producing CO₂ retention.     

Effect of Alpha-receptor Blocking Drugs and Disodium Cromoglycate on Histamine Hypersensitivity in Bronchial Asthma

Twenty patients with extrinsic type bronchial asthma are shown to have a significant fall in vital capacity (V.C.) and forced expiratory volume in 1 second (F.E.V.₁) after an intravenous infusion of 50μg. of histamine dihydrochloride. In 10 of these subjects the fall in V.C. and F.E.V.₁ produced by intravenous histamine is inhibited by the alpha-receptor blocking drugs phentolamine and phenoxybenzamine injected before the histamine test. The inhalation of disodium cromoglycate in 10 subjects is also shown to inhibit the fall in V.C. and F.E.V.₁ produced by the intravenous infusion of histamine. It is suggested that bronchial smooth muscle in asthmatic subjects has alpha-adrenergic receptor sites, and that a possible mechanism for the action of disodium cromoglycate is that it stabilizes the cell membrane, thereby altering calcium ion transport.     

Effect on airways resistance of prostaglandin E1 given by aerosol to healthy and asthmatic volunteers

The forced expiratory volume in one second (F.E.V.₁) was measured in healthy and asthmatic volunteers and the inhalation of prostaglandin E₁ (PGE₁) was compared with that of isoprenaline, using metered aerosols.In healthy volunteers PGE₁, either as the free acid or the neutral triethanolamine salt, did not affect the F.E.V.₁; the free acid was irritant to the upper respiratory tract. In five out of six asthmatic volunteers with reversible airways obstruction, inhalation of 55 μg of PGE₁ (triethanolamine salt) produced an increase in F.E.V.₁ comparable in both degree and duration to that produced by an inhalation of 550 μg. of isoprenaline sulphate.Though the triethanolamine salt was well tolerated in most of the asthmatic subjects studied, in one asthmatic subject this preparation caused coughing and there was a progressive reduction in the F.E.V.₁ associated with bronchospasm.     

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. ¹H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α₁β₂ interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α₁β₁ interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P₅₀), cooperativity (n₅₀), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P₅₀ values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207–7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.     

Suppression of telomerase activity in leukemic cells by mutant forms of <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> L-asparaginase

Active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA) (RrA_{+N17, D60K, F61L}, RrA_{+N17, A64V, E67K}, RrA_{+N17, E149R, V150P}, Rr_{AE149R, V150P} and RrA_{E149R, V150P, F151T}) have been obtained by the method of site-directed mutagenesis. The variants RrA_{E149R, V150P, F151T} and RrА_{+N17, E149R, V150P} could decrease reduce expression of the hTERT telomerase subunit and, therefore, activity of telomeres in Jurkat cells, but not in cellular lysates. At the same time, L-asparaginasеs of Escherichia coli, Erwinia carotovora, and Wolinella succinogenes, mutant forms RrА_{+N17, D60K, F61L} and RrА_{+N17, A64V, E67K} did not suppress telomerase activity. It is suggested that some regions in the RrA structure (amino acids residues 146–164, 1–17, 60–67) are responsible for suppression of telomerase activity. The results obtained show that antineoplastic activity of some RrA variants is associated both with reduction of concentration of free L-asparagine, and with decreased expression of the hTERT telomerase subunit; this opens new prospects for antineoplastic therapy.     

Impact of JAK2 V617F Mutation on Hemogram Variation in Patients with Non-Reactive Elevated Platelet Counts

Background

Non-reactive platelet counts elevation occurs mainly in myeloproliferative disorders (MPDs), which have been reported to be closely associated with JAK2 V617F mutation. Complete blood cell count (CBC) is essential in diagnosis of MPDs, however, the impact of JAK2 V617F mutation on the patients’ hemogram variation remains not clear.

Methods

JAK2 V617F mutation was detected by allele specific real-time quantitative fluorescence PCR (AS-qPCR).

Results

Of the 402 non-reactive platelet elevating patients, JAK2 V617F mutation was detected in 222 (55.2%) patients. RBC counts, WBC counts, platelet-large contrast ratio (P-LCR), platelet distribution width (PDW) and mean platelet volume (MPV) were much higher in JAK2 V617F mutated patients, except platelet counts. In addition, when the patients were classified into subgroups by blood cell counts, it was found that JAK2 V617F mutation rate increased progressively with the increase of RBC counts and WBC counts, other than platelet counts. Furthermore, trilineage hyperplasia group showed highest JAK2 V617F mutation rate (93.26%), followed by the bilineage hyperplasia groups. Lastly, JAK2 V617F mutant allele burden was found much higher in polycythemia vera (PV) patients [median(P₂₅–P₇₅): 45.02%(35.12%–54.22%)] than in essential thrombocythemia (ET) patients [median(P₂₅–P₇₅): 28.23%(17.77%–41.66%)], and that it increased with WBC counts (r = 0.393, p = 0.000) and RBC counts(r = 0.215, p = 0.001), other than platelet counts (r = −0.051, p = 0.452). Further analysis revealed that in ET patients, JAK2 V617F mutant allele burden correlated with WBC counts and platelet counts positively, other than RBC counts, while in PV patients, it correlated with WBC counts and RBC counts positively, but not platelet counts.

Conclusions

JAK2 V617F mutation occurs frequently in patients with non-reactive elevated platelet counts. The presence of JAK2 V617F mutation has great impact on hemogram variation, including RBC counts, WBC counts, platelet parameters and lineage hyperplasia, but not on platelet counts. Besides, JAK2 V617F mutant allele burden affects the blood cell proliferation pattern.     

Inter‐subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1‐ATPase

V‐type ATPases (V‐ATPases) are categorized as rotary ATP synthase/ATPase complexes. The V‐ATPases are distinct from F‐ATPases in terms of their rotation scheme, architecture and subunit composition. However, there is no detailed structural information on V‐ATPases despite the abundant biochemical and biophysical research. Here, we report a crystallographic study of V₁‐ATPase, from Thermus thermophilus, which is a soluble component consisting of A, B, D and F subunits. The structure at 4.5 Å resolution reveals inter‐subunit interactions and nucleotide binding. In particular, the structure of the central stalk composed of D and F subunits was shown to be characteristic of V₁‐ATPases. Small conformational changes of respective subunits and significant rearrangement of the quaternary structure observed in the three AB pairs were related to the interaction with the straight central stalk. The rotation mechanism is discussed based on a structural comparison between V₁‐ATPases and F₁‐ATPases.     

“Pinching” the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage

Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either l-glutamine or exogenous NH₃ as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH₃ passes through a gate located at the end of a ~25-Å tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH₃ transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with “pinching” the tunnel, thereby revealing processes that synchronize the transfer of NH₃ from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a “bottleneck” effect. The inability of V60F to use exogenous NH₃ was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-l-norleucine. Use of NH₂OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH₃ gate of CTPS to mediate inter-domain NH₃ transport.     

Antirestriction activity of the monomeric and dimeric forms of T7 Ocr

The Ocr antirestriction protein, which is encoded by bacteriophage T7 0.3 (ocr), specifically inhibits type I restriction-modification enzymes. Ocr belongs to a family of DNA-mimicking proteins. Native Ocr forms homodimers both in solution and in crystal. Ocr mutants with two amino acid substitutions (Orc F53D A57E and Ocr F53R V77D) were constructed to occur as monomers in solution. The dissociation constant K _d for the Ocr complex with EcoKI (R₂M₂S) proved to differ by three orders of magnitude between the (Ocr)₂ dimer and Ocr F53D A57E and Ocr F53R V77D monomers (10^?10 M vs. 10^?7 M). Antimodification activity was substantially lower in the Ocr monomers. The dimeric form found to be essential for high inhibitory activity of Ocr.     

A genetically encoded sensor for H2O2 with expanded dynamic range

Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H₂O₂] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H₂O₂ due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator’s dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H₂O₂] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.     

Two forms of Ca2+-dependent cyclic 3':5'-nucleotide phosphodiesterase from human aorta and effect of free fatty acids.

Investigations were carried out on two DEAE-cellulose columnresolvable Ca²⁺-dependent nucleotide 3′:5′-phosphodiesterases from human aorta. An extract from human aorta when chromatographed on DEAE-cellulose yielded five active cyclic nucleotide 3′:5′-phosphodiesterase fractions designated as F I, F II, F III, F IV, and F V and these fractions eluted at about 0.02, 0.08, 0.18, 0.28, and 0.38 M sodium acetate. F 111 and F IV were found to be activated by a protein modulator and free fatty acids. The two Ca²⁺-dependent cyclic nucleotide phosphodiesterases (F III and FIV) were clearly separated by rechromatography on DEAE-cellulose column and were found to hydrolyze guanosine 3′:5′-monophosphate (cyclic GMP) preferentially. Fatty acids as well as a protein modulator increased the maximum velocity of one form (F III) without affecting the K_m values and decreased the K_m values of the other (F IV) without changing maximum velocity. The extent of maximum stimulation by behenic acid (C₂₂) and a protein modulator was similar at optimal conditions. Fatty acids did not require calcium for stimulation of the phosphodiesterases. Stimulating activity diminished as the hydrocarbon chain length of the fatty acid was shortened or when more than two unsaturated bonds were introduced. Behenic acid (C₂₂) and eruic acid (C_22:1) were the most potent stimulators among the saturated or unsaturated fatty acids tested. The other DEAE-cellulose-resolvable aortic phosphodiesterase forms (F I, FII, and F V) were neither activated by the protein modulator nor stimulated significantly by fatty acids.     

Multiple equilibria and exotic behaviour in excitable membranes

The excitation equation for an excitable membrane dV/dt=F(V) may have multiple equilibria where F(V)=0, and these may be stable or unstable. We demonstrate multiple equilibria in the Hodgkin-Huxley equations when either g_K or [Ca²⁺]₀ is lowered in the presence of a hyperpolarising current density. Under these conditions molluscan somata exhibit exotic behaviours-endogenous paroxysmal depolarising shifts and complex multiple spikes reminiscent of the normal complex activity of some mammalian central neurones. Complex discharge waveforms can be an expression of membrane (differential) properties, rather than electrotonic, geometric (partial differential) behaviour.     

Novel molecular insights into the anti‐oxidative stress response and structure–function of a salt‐inducible cyanobacterial Mn‐catalase

KatB, a salt‐inducible Mn‐catalase, protects the cyanobacterium Anabaena from salinity/oxidative stress. In this report, we provide distinctive insights into the biological–biochemical function of KatB at the molecular level. Anabaena overexpressing the wild‐type KatB protein (KatBWT) detoxified H₂O₂ efficiently, showing reduced burden of reactive oxygen species compared with the strain overproducing KatBF2V (wherein F‐2 is replaced by V). Correspondingly, the KatBWT protein also displayed several folds more activity than KatBF2V. Interestingly, the KatB variants with large hydrophobic amino acids (F/W/Y) were more compact, showed enhanced activity, and were resistant to thermal/chemical denaturation than variants with smaller residues (G/A/V) at the second position. X‐ray crystallography‐based analysis showed that F‐2 was required for appropriate interactions between two subunits. These contacts provided stability to the hexamer, making it more compact. F‐2, through its interaction with F‐66 and W‐43, formed the proper hydrophobic pocket that held the active site together. Consequently, only residues that supported activity (i.e., F/Y/W) were selected at the second position in Mn‐catalases during evolution. This study (a) demonstrates that modification of nonactive site residues can alter the response of catalases to environmental stress and (b) has expanded the scope of amino acids that can be targeted for rational protein engineering in plants.     

Complex of a protective antibody with its Ebola virus GP peptide epitope: unusual features of a V lambda x light chain

13F6-1-2 is a murine monoclonal antibody that recognizes the heavily glycosylated mucin-like domain of the Ebola virus virion-attached glycoprotein (GP) and protects animals against lethal viral challenge. Here we present the crystal structure, at 2.0 Å, of 13F6-1-2 in complex with its Ebola virus GP peptide epitope. The GP peptide binds in an extended conformation, anchored primarily by interactions with the heavy chain. Two GP residues, Gln P406 and Arg P409, make extensive side-chain hydrogen bond and electrostatic interactions with the antibody and are likely critical for recognition and affinity. The 13F6-1-2 antibody utilizes a rare Vλ_x light chain. The three light-chain complementarity-determining regions do not adopt canonical conformations and represent new classes of structures distinct from Vκ and other Vλ light chains. In addition, although Vλ_x had been thought to confer specificity, all light-chain contacts are mediated through germ-line-encoded residues. This structure of an antibody that protects against the Ebola virus now provides a framework for humanization and development of a postexposure immunotherapeutic.     

Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia pestis as Next Generation Plague Vaccines

Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH₂-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced T_H1 and T_H2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.     

Mechanistic basis for LQT1 caused by S3 mutations in the KCNQ1 subunit of IKs

Long QT interval syndrome (LQTS) type 1 (LQT1) has been reported to arise from mutations in the S3 domain of KCNQ1, but none of the seven S3 mutations in the literature have been characterized with respect to trafficking or biophysical deficiencies. Surface channel expression was studied using a proteinase K assay for KCNQ1 D202H/N, I204F/M, V205M, S209F, and V215M coexpressed with KCNE1 in mammalian cells. In each case, the majority of synthesized channel was found at the surface, but mutant I_Ks current density at +100 mV was reduced significantly for S209F, which showed ∼75% reduction over wild type (WT). All mutants except S209F showed positively shifted V_1/2’s of activation and slowed channel activation compared with WT (V_1/2 = +17.7 ± 2.4 mV and τ_activation of 729 ms at +20 mV; n = 18). Deactivation was also accelerated in all mutants versus WT (126 ± 8 ms at −50 mV; n = 27), and these changes led to marked loss of repolarizing currents during action potential clamps at 2 and 4 Hz, except again S209F. KCNQ1 models localize these naturally occurring S3 mutants to the surface of the helices facing the other voltage sensor transmembrane domains and highlight inter-residue interactions involved in activation gating. V207M, currently classified as a polymorphism and facing lipid in the model, was indistinguishable from WT I_Ks. We conclude that S3 mutants of KCNQ1 cause LQTS predominantly through biophysical effects on the gating of I_Ks, but some mutants also show protein stability/trafficking defects, which explains why the kinetic gain-of-function mutation S209F causes LQT1.     

The Extracellular Loop 2 (ECL2) of the Human Histamine H4 Receptor Substantially Contributes to Ligand Binding and Constitutive Activity

In contrast to the corresponding mouse and rat orthologs, the human histamine H₄ receptor (hH₄R) shows extraordinarily high constitutive activity. In the extracellular loop (ECL), replacement of F169 by V as in the mouse H₄R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH₄R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gα_i2 and Gβ₁γ₂, and the membranes were studied in [³H]histamine binding and functional [³⁵S]GTPγS assays. The potency of various ligands at the hH₄R-F168A mutant decreased compared to the wild-type hH₄R, for example by 30- and more than 100-fold in case of the H₄R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH₄R was completely lost in the hH₄R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH₄R. By analogy, JNJ7777120 was a partial inverse agonist at the hH₄R, but a partial agonist at the hH₄R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH₄R.     

Exercise-induced Asthma

Tests in three patients with asthma occurring only on exertion showed that F.E.V.₁ fell progressively on exercise, to reach a minimum after 10 minutes. All patients showed a striking metabolic acidosis, with an accumulation of the products of anaerobic metabolism.     

Origins of the diversity of cytochrome P450 CYP74 family based on the results of site-directed mutagenesis

Bioinformatic analysis and site-directed mutagenesis allowed identification of the determinants of catalysis for CYP74, which are located in the central part of the I-helix and ERR triad. Mutations K302S and T366Y in tomato allene oxide syntase LeAOS3 induced possession of hydroperoxide lyase activity. In contrast to the wild-type MtHPL enzyme that produces C₁₂-aldoacid, mutant forms F284I, F287V, G288I, N285A, and N285T of alfalfa hydroperoxide lyase MtHPL synthesized C₁₃- and C₁₁-fragments. Our data provide evidence that the CYP74 family originated from a common ancestor with hydroperoxide lyase activity.