NMR simulation analysis of statistical effects on quantifying cerebrovascular parameters

Determining tissue structure and composition from the behavior of the NMR transverse relaxation during free induction decay and spin echo formation has seen significant advances in recent years. In particular, the ability to quantify cerebrovascular network parameters such as blood volume and deoxyhemoglobin concentration from the NMR signal dephasing has seen intense focus. Analytical models have been described, based on statistical averaging of randomly oriented cylinders in both the static and slow diffusion regimes. However, the error in estimates obtained from these models when applied to systems in which the statistical assumptions of many, randomly oriented perturbers are violated has not been systematically investigated. Using a deterministic simulation that can include diffusion, we find that the error in estimated venous blood volume fraction and deoxyhemoglobin concentration obtained using a static dephasing regime statistical model is inversely related to the square root of number of blood vessels. The most important implication of this is that the minimum imaging resolution for accurate deoxyhemoglobin and blood volume estimation is not bound by hardware limitations, but rather by the underlying tissue structure.     

Change in dysphagia and laryngeal function after radical radiotherapy in laryngo pharyngeal malignancies — a prospective observational study

BackgroundIntensity modulated radiotherapy (IMRT) has the perceived advantage of function preservation by reduction of toxicities in the treatment of laryngo-pharyngeal malignancies. The aim of the study was to assess changes in dysphagia from baseline (i.e. prior to start of treatment) at three and six months post treatment in patients with laryngo-pharyngeal malignancies treated with radical radiotherapy ± chemotherapy. Functional assessment of other structures involved in swallowing was also studied.Materials and methods40 patients were sampled consecutively. 33 were available for final analysis. Dysphagia, laryngeal edema, xerostomia and voice of patients were assessed at baseline and at three and six months after treatment. Radiation was delivered with simultaneous integrated boost (SIB) using volumetric modulated radiation therapy (VMAT). Concurrent chemotherapy was three weekly cisplatin 100 mg/m².ResultsProportion of patients with dysphagia rose significantly from 45.5% before the start of treatment to 57.6% at three months and 60.6% at six months post treatment (p = 0.019). 67% patients received chemotherapy and addition of chemotherapy had a significant correlation with dysphagia (p = 0.05, r = −0.336). Severity of dysphagia at three and six months correlated significantly with the mean dose received by the superior constrictors (p = 0.003, r = 0.508 and p = 0.024, r = 0.391) and oral cavity (p = 0.001, r = 0.558 and p = 0.003, r = 0.501). There was a significant worsening in laryngeal edema at three and six months post treatment (p < 0.01) when compared to the pre-treatment examination findings with 60.6% of patients having grade two edema at six months. Significant fall in the mean spoken fundamental frequency from baseline was seen at 6 months (p = 0.04), mean fall was 21.3 Hz (95% CI: 1.5–41 Hz) with significant increase in roughness of voice post treatment (p = 0.01).ConclusionThere was progressive worsening in dysphagia, laryngeal edema and voice in laryngo-pharyngeal malignancies post radical radiotherapy ± chemotherapy.     

Fragile X mental retardation protein interactions with the microtubule associated protein 1B RNA

Fragile X mental retardation syndrome, the most common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine (RGG) box to bind to a subset of RNA targets that form a G quadruplex structure. We performed a detailed analysis of the interactions between the FMRP RGG box and the microtubule associated protein 1B (MAP1B) mRNA, a relevant in vivo FMRP target. We show that MAP1B RNA forms an intramolecular G quadruplex structure, which is bound with high affinity and specificity by the FMRP RGG box. We determined that hydrophobic interactions are important in the FMRP RGG box-MAP1B RNA association, with minor contributions from electrostatic interactions. Our findings that at low protein:RNA ratios the RNA G quadruplex structure is slightly stabilized, whereas at high ratios is unfolded, suggest a mechanism by which the FMRP concentration variation in response to a neurotransmitter stimulation event could act as a regulatory switch for the protein function, from translation repressor to translation activator.     

The sensitization of glioma cells to cisplatin and tamoxifen by the use of catechin

Telomerase expression strongly correlates with the grade of malignancy in glioma with inhibition illustrating a definite increase in chemosensitivity. This study was designed to investigate the effects of a green tea derivative, epigallocatechin-3-gallate (EGCG); together with either cisplatin or tamoxifen in glioma, and to investigate whether these effects are mediated through telomerase suppression. EGCG showed a significant cytotoxic effect on 1321N1 cells after 24 h and on U87-MG cells after 72 h (P < 0.001) without significantly affecting the normal astrocytes. Treatment with EGCG inhibited telomerase expression significantly (P < 0.01) and enhanced the effect of cisplatin and tamoxifen in both 1321N1 (P < 0.01) and U87-MG (P < 0.001) cells. EGCG, as a natural product has enormous potential to be an anti-cancer agent capable of enhancing tumour cell sensitivity to therapy.     

Yeast oxysterol-binding proteins: sterol transporters or regulators of cell polarization?

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) are a conserved family of soluble cytoplasmic proteins that can bind sterols, translocate between membrane compartments, and affect sterol trafficking. These properties make ORPs attractive candidates for lipid transfer proteins (LTPs) that directly mediate nonvesicular sterol transfer to the plasma membrane. To test whether yeast ORPs (the Osh proteins) are sterol LTPs, we studied endoplasmic reticulum (ER)-to-plasma membrane (PM) sterol transport in OSH deletion mutants lacking one, several, or all Osh proteins. In conditional OSH mutants, ER-PM ergosterol transport slowed ~20-fold compared with cells expressing a full complement of Osh proteins. Although this initial finding suggested that Osh proteins act as sterol LTPs, the situation is far more complex. Osh proteins have established roles in Rho small GTPase signaling. Osh proteins reinforce cell polarization and they specifically affect the localization of proteins involved in polarized cell growth such as septins, and the GTPases Cdc42p, Rho1p, and Sec4p. In addition, Osh proteins are required for a specific pathway of polarized secretion to sites of membrane growth, suggesting that this is how Osh proteins affect Cdc42p- and Rho1p-dependent polarization. Our findings suggest that Osh proteins integrate sterol trafficking and sterol-dependent cell signaling with the control of cell polarization.     

Use of CHROMagar in the Differentiation of Common Species of <Emphasis Type="Italic">Candida</Emphasis>

CHROMagar has been reported to be useful for the rapid and accurate identification of Candida species. We tested 135 isolates of Candida species isolated from oropharyngeal candidiasis in HIV patients and found that it was useful in the presumptive identification of Candida albicans and Candida krusei. Occasional strains of C. tropicalis produced colonies with a greenish tinge making it difficult to differentiate from C. albicans.     

Cryogenic and Laser Photoexcitation Studies Identify Multiple Roles for Active Site Residues in the Light-driven Enzyme Protochlorophyllide Oxidoreductase

The light-activated enzyme NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the trans addition of hydrogen across the C-17–C-18 double bond of protochlorophyllide (Pchlide), a key step in chlorophyll biosynthesis. Similar to other members of the short chain alcohol dehydrogenase/reductase family of enzymes, POR contains a conserved Tyr and Lys residue in the enzyme active site, which are implicated in a proposed reaction mechanism involving proton transfer from the Tyr hydoxyl group to Pchlide. We have analyzed a number of POR variant enzymes altered in these conserved residues using a combination of steady-state turnover, laser photoexcitation studies, and low temperature fluorescence spectroscopy. None of the mutations completely abolished catalytic activity. We demonstrate their importance to catalysis by defining multiple roles in the overall reaction pathway. Mutation of either residue impairs formation of the ground state ternary enzyme-substrate complex, pointing to a key role in substrate binding. By analyzing the most active variant (Y193F), we show that Tyr-193 participates in proton transfer to Pchlide and stabilizes the Pchlide excited state, enabling hydride transfer from NADPH to Pchilde. Thus, in addition to confirming the probable identity of the proton donor in Pchlide reduction, our work defines additional roles for these residues in facilitating hydride transfer through stabilization of the ground and excited states of the ternary enzyme complex.The light-driven enzyme protochlorophyllide oxidoreductase (POR)³ (EC 1.3.1.33) catalyzes the trans addition of hydrogen across the C-17–C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide) (see Fig. 1A) (1). This reaction is a key step in the synthesis of chlorophyll and leads to profound changes in the morphological development of photosynthetic organisms through modification and reorganization of plastid membranes (2, 3). In addition to POR, nonflowering land plants, algae, and cyanobacteria possess a light-independent Pchlide reductase, which consists of three separate subunits and allows these organisms to produce chlorophyllide in the dark (4). Together with DNA photolyase (5), POR is one of only two enzymes studied so far that exhibit a direct, natural requirement for light and because mixing strategies are no longer required to initiate the reaction, it is possible to trigger catalysis on very fast time scales and at cryogenic temperatures. Consequently, POR has proven to be an excellent model system for studying the role of protein dynamics in driving enzyme catalysis (1).Open in a separate windowFIGURE 1.The light-driven reduction of Pchlide. A, the trans addition of hydrogen across the C-17–C-18 double bond of Pchlide to form chlorophyllide (Chlide) in the chlorophyll biosynthesis pathway is catalyzed by the light-driven enzyme POR. B, shown is a three-dimensional model of the POR-catalyzed reaction based on the structural homology model of POR (26) and the proposed mechanism of hydride and proton transfer (8). Upon activation by light, a hydride is transferred to the C-17 position of Pchlide from the pro-S face of NADPH (shown in yellow), and the proton at the C-18 position is derived from Tyr-193 (shown in cyan). The conserved Lys-197 residue (shown in magenta) is proposed to decrease the pK_a of the Tyr to facilitate the proton transfer reaction.In the POR catalytic cycle, a ternary enzyme-NADPH-Pchlide complex is formed. Following light activation of this complex, a hydride ion is transferred from the pro-S face of NADPH to the C-17 atom of Pchlide (6, 7). The valence of the C-18 atom is satisfied by proton transfer, which is suggested to originate from an active site tyrosine residue (8). The catalytic cycle of POR has been analyzed through the trapping of intermediates at cryogenic temperatures. Following the initial light-driven reaction (9), there are a series of subsequent (slower) dark reactions (10, 11). The light-driven step involves hydride transfer from NADPH to form a charge transfer complex, which then facilitates protonation of the pigment intermediate during the first of the “dark” reactions (12). Moreover, through laser activation of catalysis, we have shown that both of these H-transfer reactions proceed by quantum mechanical tunneling coupled to motions in the enzyme-substrate complex on the submicrosecond time scale (13). The final dark steps in the reaction cycle involve a series of ordered product release and cofactor binding steps linked to conformational changes in the enzyme (10, 11, 14). Ultrafast measurements have uncovered spectral changes on the picosecond timescale that are likely to represent conformational changes prior to Pchlide reduction (15–19). Previous excitation of POR with a laser pulse leads to a more efficient conformation of the active site and an enhancement in the catalytic efficiency of the enzyme (18).POR is a member of a large family of enzymes known as short chain dehydrogenases/reductases (SDR). These are single domain NAD(P)⁺- or NAD(P)H-binding oxidoreductases that exist generally as dimers or tetramers (20). A number of SDR enzymes (e.g. carbonyl reductase, alcohol dehydrogenase, and dihydrofolate reductase) have been good model systems for studying the dynamics linked to enzyme catalysis (21–23). This family of enzymes has been amenable to studies of biological H-tunneling (24–26), and in particular the unique light-activated properties of POR make it an excellent system for studying mechanisms of H-transfer and dynamics in this family of enzymes.Structures of several SDR family members are available, and these have enabled the construction of a homology model of POR from Synechocystis (27). This model comprises a central parallel β-sheet of seven β-strands, surrounded by nine α-helices, with an additional unique 33-residue insertion between the fifth and sixth β-sheets. The NADPH cofactor binds within the N-terminal region of the enzyme, which contains a common nucleotide-binding motif with a tight βαβ fold, termed the Rossmann fold (27). Importantly, a Tyr and a Lys residue are both absolutely conserved throughout all members of the SDR family and are critical for catalysis in a number of enzymes (28–31). A common mechanism has been proposed for this group of enzymes, involving a Tyr-X-X-X-Lys motif. The Lys residue in this motif is presumed to facilitate proton donation from the Tyr hydroxyl group to substrate through favorable perturbation of the hydroxyl group pK_a (8, 31). In POR, multiple turnover assays have also indicated that these Tyr and Lys residues are important for activity (8, 32, 33), leading to a proposed mechanism that involves proton transfer from the conserved Tyr residue to the C-18 position of Pchlide (8) (Fig. 1B). The close proximity of the Lys residue is thought to allow the deprotonation step to occur at physiological pH by lowering the apparent pK_a of the phenolic group of the Tyr (8). However, confirmation of the exact role of these conserved residues has been compromised by the limited levels of activity observed in previous studies of the variant enzymes (8, 32, 33), and a detailed evaluation of the role of the active site Tyr and Lys residues on the chemical steps (i.e. hydride and proton transfer) has not been reported. We address this deficiency in the current work by analyzing a number of site-specific mutant forms altered at Tyr-193 and Lys-197 in a thermophilic POR from Thermosynechococcus elongatus BP-1. This was achieved using steady-state (multiple turnover) and laser photoexcitation (single turnover) methods and by trapping transient reaction intermediates by fluorescence spectroscopy performed at cryogenic temperatures.     

Kinetic Analysis of the Guanine Nucleotide Exchange Activity of TRAPP, a Multimeric Ypt1p Exchange Factor

TRAPP complexes, which are large multimeric assemblies that function in membrane traffic, are guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1p. Here we measured rate and equilibrium constants that define the interaction of Ypt1p with guanine nucleotide (guanosine 5'-diphosphate and guanosine 5'-triphosphate/guanosine 5′-(β,γ-imido)triphosphate) and the core TRAPP subunits required for GEF activity. These parameters allowed us to identify the kinetic and thermodynamic bases by which TRAPP catalyzes nucleotide exchange from Ypt1p. Nucleotide dissociation from Ypt1p is slow (∼ 10^− 4 s^− 1) and accelerated > 1000-fold by TRAPP. Acceleration of nucleotide exchange by TRAPP occurs via a predominantly Mg²⁺-independent pathway. Thermodynamic linkage analysis indicates that TRAPP weakens nucleotide affinity by < 80-fold and vice versa, in contrast to most other characterized GEF systems that weaken nucleotide binding affinities by 4-6 orders of magnitude. The overall net changes in nucleotide binding affinities are small because TRAPP accelerates both nucleotide binding and dissociation from Ypt1p. Weak thermodynamic coupling allows TRAPP, Ypt1p, and nucleotide to exist as a stable ternary complex, analogous to strain-sensing cytoskeleton motors. These results illustrate a novel strategy of guanine nucleotide exchange by TRAPP that is particularly suited for a multifunctional GEF involved in membrane traffic.