Continuous-wave quantum yields of various cobalamins are influenced by competition between geminate recombination and cage escape

Quantum yields of photolysis of the cobalt-carbon bond for three cobalamin compounds were measured with a continuous-wave laser at 442 nm under both aerobic and anaerobic conditions. Aerobically, the initial homolysis product, Co(II) cobalamin, is trapped by oxygen to form aquocobalamin. Use of an excess of the radical trapping reagent 2,2,6,6-tetramethyl-1-piperidinyloxyl, under anaerobic conditions, scavenges the carbon radical and allows detection of the cobalt(II) photoproduct. Quantum yields measured under anaerobic conditions for 5'-deoxyadenosylcobalamin (phi (Co-C alpha),442 = 0.20 +/- 0.03) and methylcobalamin (phi (Co-C alpha),442 = 0.35 +/- 0.03) are in agreement with the values obtained under aerobic conditions (phi (Co-C alpha),442 = 0.19 +/- 0.04 and phi (Co-C alpha),442 = 0.36 +/- 0.04, respectively). Additionally, the quantum yield values for 5'-deoxyadenosylcobalamin and its base-off derivative (phi (Co-C alpha),442 = 0.045 +/- 0.015) match those obtained on a nanosecond time scale [Chen, E., & Chance, M. R. (1990) J. Biol. Chem. 256, 12987-12994]. A comparison of quantum yields obtained anaerobically for 5'-deoxyadenosylcobalamin and methylcobalamin in H2O versus ethylene glycol shows a 4-fold decrease for the former cobalamin and no change for the latter. These quantum yields are evaluated in terms of time-independent radical separation distances.     

Kinetic analysis of the coding properties of O6-methylguanine in DNA: the crucial role of the conformation of the phosphodiester bond

Production by N-nitroso compounds of O6-alkylguanine (O6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, leading to G:C to A:T transition mutations, despite the fact that DNA containing O6-alkylG:T base pairs is less stable than that containing O6-alkylG:C pairs. We have examined the kinetics of incorporation by Klenow fragment (KF) of Escherichia coli DNA polymerase I of thymine (T) and of cytosine (C) opposite O6-MeG in the template DNA strand. Both T and C were incorporated opposite O6-MeG much slower than nucleotides forming regular A:T or G:C base pairs. Using various concentrations of dTTP, dCTP, or their phosphorothioate (Sp)-dNTP alpha S analogues, or a mixture of dTTP and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed KF-DNA complex was measured (pre-steady-state kinetics). The results were consistent with the kinetic scheme (Kuchta, R. D., Benkovic, P., & Benkovic, S. J. (1988) Biochemistry 27, 6716-6725): (1) binding of dNTP to polymerase-DNA; (2) conformational change in polymerase; (3) formation of phosphodiester between the dNTP and the 3'-OH of the primer; (4) conformational change of polymerase; (5) release of pyrophosphate. The results were analyzed mathematically to identify the steps at which the rate constants differ significantly between the incorporation of T and C. The only significant difference was the 5-fold difference in the rates of formation of the phosphodiester bond (for dTTP, kforward = 3.9 s-1 and kback = 1.9 s-1; for dCTP, kforward = 0.7 s-1 and kback = 0.9 s-1). These pre-steady-state progress curves were biphasic with a rapid initial burst followed by an apparently steady-state rise. Deconvolution of these curves gave direct evidence for the importance of the conformational change after polymerization by showing that the curves represented the sum of the rapid accumulation of the product of step 3 followed by the slow conversion of that to the product of step 5 (because of the rapidity of the release of pyrophosphate there was no significant accumulation of the product of step 4). The equilibrium constants for each step suggest that the greatest change in the Gibbs free energy occurs at the conformational change after polymerization and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic.(ABSTRACT TRUNCATED AT 400 WORDS)     

A simple handling technique for mammalian oocytes and embryos during preparation for transmission electron microscopy

Due to their small size, mammalian oocytes and embryos pose unique problems during preparation for transmission electron microscopy. This paper outlines a method which combines protein embedding with centrifugation to locate the specimens on the face of a Beem capsule mould. This method facilitates both the processing of oocytes with minimal loss and rapid location of the specimens within the block for simultaneous sectioning, staining and examination.     

Instructional texts rewritten by five expert teams: Revisions and retention improvements.

700 undergraduates were tested in 3 experiments on original or rewritten versions of 52 instructional texts about Army job tasks, general science, philosophy, and history. Five experts had rewritten various sets of the texts and stated hypotheses about the efficacious features of their revisions. We tested their hypotheses and several others. Recall and recognition tests were given immediately and after a 24-hr delay. Results showed that revisions made by 3 of the 5 experts improved retention of text information. The kind and number of revisions and improvements varied across the text sets. Most expert hypotheses were not supported, and they made many revisions they were declaratively unaware of. Some of our hypotheses about the revision features were supported, but different features were effective for different sets of texts. It was concluded that some experts have effective knowledge about improving instructional text, but it exists primarily in procedural form. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Metastatic parathyroid carcinoma in the MEN2A syndrome

We report a patient with a metastatic parathyroid carcinoma and medullary carcinoma of the thyroid. This patient represents a variation of the multiple endocrine neoplasia syndrome (MEN) type 2A. There was no evidence of a phaeochromocytoma. The case illustrates the difficulties that may be encountered in localising the source of PTH secretion; the patient underwent four unsuccessful exploratory operations of the neck and mediastinum before further investigations revealed a single metastatic deposit of parathyroid carcinoma involving the first thoracic vertebra. PCR amplification and sequencing of the RET oncogene from the metastatic parathyroid carcinoma and genomic DNA revealed a heterozygous mutation (Cys634Tyr) in exon 11, as has previously been described to occur in MEN 2A. In addition, loss of tumour heterozygosity was demonstrated at loci from chromosomes 1, 2, 3p, 13q and 16p. This represents the first report of a parathyroid carcinoma in a MEN2A patient, in which the multiple allelic deletions are consistent with the generalised losses observed in aggressive tumours.     

Effects of anxiety arousal and mental stress on the vestibulo-ocular reflex

Although the subjective reports of patients suggest that anxiety may aggravate vertigo and imbalance, there has been little research into how anxiety might directly affect balance system functioning. We conducted two studies to examine the effect of anxiety and arousal on the vestibulo-ocular reflex (VOR). In the first study, pre-lest fear ratings were obtained from 20 normal subjects and 36 anxious subjects immediately prior to rotation and caloric testing. Fear ratings were significantly correlated with the maximum slow-phase velocity (SPV) of nystagmus induced by caloric testing. In the second study, we assessed the VOR response to rotation of 36 normal subjects under 3 task conditions: a) minimal alerting (counting backwards during rotation), b) physical arousal (induced by exertion prior to rotation); c) mental arousal (induced by performance of stressful mental tasks during rotation). Both the physical and mental tasks induced a significant increase in heart rate compared with the alerting condition. The maximum SPV of the nystagmus induced by rotation was significantly greater during performance of the mental task than in the other two conditions. These combined results indicate that anxiety may influence the gain of the VOR.     

Datum-Hierarchy Tree Method for Tolerance Analysis of Plating and Heat Treatment Operations

This paper presents a new method for the tolerance analysis of plating and heat treatment operations. It is suitable for both manual and computerised tolerance analysis. The new method uses a datum-hierarchy tree to identify tolerance stacks and a stock removal set matrix to calculate the positions of surfaces created during the manufacturing sequence. A tolerance chart is used to associate stock removal sets with the geometry of the workpiece, to show the manufacturing sequence, and to record the operation data and the tolerance analysis results. Plating and heat treatment operations are represented as edges in the tree, so that for each operation (edge) the input and output nodes represent consecutive surfaces in a stock removal set. Rules are given for calculating the tolerance stackups and the positions of the surfaces in the stock removal sets. The method is illustrated with an example.     

Modulation of postural tremors at the wrist by supramaximal electrical median nerve shocks in essential tremor, Parkinson's disease and normal subjects mimicking tremor

The response of postural wrist tremors to supramaximal median nerve stimulation was examined in patients with hereditary essential tremor (n = 10) and Parkinson's disease (n = 9), and in normal subjects mimicking wrist tremor (n = 8). The average frequency of on-going tremor was the same in all three groups. Supramaximal peripheral nerve shocks inhibited and then synchronised the rhythmic electromyographic (EMG) activity of all types of tremor. The duration of inhibition ranged from 90 to 210ms, varying inversely with the frequency of on-going tremor. There was no significant difference in mean duration of inhibition or in the timing of the first peak after stimulation on the average rectified EMG records between the three groups. The degree to which supramaximal peripheral nerve shocks could modulate the timing of rhythmic EMG bursts in the forearm flexor muscles was also quantified by deriving a resetting index. No significant difference in mean resetting index of the three groups was found. These results suggest that such studies cannot be used to differentiate between the common causes of postural wrist tremors.     

Time-dependent optical spectroscopy and imaging for biomedicalapplications

The application of optical spectroscopy and imaging in living tissue is complicated by multiple scattering of light. In spectroscopy, this scattering causes uncertainty in the pathlength traveled by photons in the tissue, while images suffer reduced resolution and contrast. Picosecond light sources and fast detectors have made it possible to address these problems by direct measurement of the photon time-of-flight. Diffusion models of light propagation can be used to relate the measured distribution of photon transit times to the scattering and absorption coefficients of the tissue. The advantages of absolute absorption measurement are demonstrated for two problems: determination of hemoglobin oxygenation in tissue and in vivo measurement of the uptake of an exogenous chromosphere such as photosensitizer. Optical imaging may also be improved by the elimination of multiply scattered photons or by selective detection of photons arriving from a given region of the tissue. The potential advantages of these techniques are discussed and illustrated with experimental data