High Throughput Primer Walking of cDNA Clones

Primer walking of cloned DNA is a standard research tool. It has been used in the past to determine the sequence of individual clones of interest. With the expansion of DNA sequencing capacity the need to be able to walk larger numbers of clones has become necessary. Our laboratory is a mid-sized genomics facility. In conjunction with the Advanced Biomedical Computing Center (ABCC) we have developed methods for automating the primer selection, DNA sequencing, contig assembly and sequence analysis for clones arrayed in microtiter format. This approach has allowed us to walk 475 clones (five microtiter plates) selected from a cDNA library.     

Single-Threaded vs. Multithreaded: Where Should We Focus?

Today, with the increasing popularity of multicore processors, one approach to optimizing the processor's performance is to reduce the execution times of individual applications running on each core by designing and implementing more powerful cores. Another approach, which is the polar opposite of the first, optimizes the processor's performance by running a larger number of applications on a correspondingly larger number of cores, albeit simpler ones. The difference between these two approaches is that the former focuses on reducing the latency of individual applications or threads (it optimizes the processor's single-threaded performance), whereas the latter focuses on reducing the latency of the applications' threads taken as a group (it optimizes the processor's multithreaded performance). The panel, from the 2007 Workshop on Computer Architecture Research Directions, discusses the relevant issues.     

Intermittent Phenomena in Switched Systems With High Coupling Strengths

In this paper, one kind of intermittency generated by a discontinuous system is studied. Although this system, which is composed of two switched subsystems coupled with a high strength, is nonsmooth, the mechanism of this kind of intermittency can be analyzed with several explicit relations between the intermittency characteristics and the system control parameters. In particular, estimates of "steady-state" values of the system (in the laminar phases) and a critical value for this intermittency can be derived, which are helpful in relevant control systems design. Moreover, some power laws for the observed intermittency are obtained and discussed     

Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.     

Cloning and expression analysis of a novel salicylate suppressible gene, Hs-CUL-3, a member of cullin/Cdc53 family

By using a mRNA differential display technique to search for salicylate suppressible genes, we identified a cDNA in human foreskin fibroblasts, which by GenBankTM DNA data base search shows sequence homology to the recently reported cullin/Cdc53 (CUL) family genes, especially CUL-3. We have cloned the full-length human CUL-3 (Hs-CUL-3) cDNA. It encodes a 768-amino acid polypeptide and has a predicted molecular weight of 88,939. The amino acid sequence of Hs-CUL-3 shows 46% homology to that of its Caenorhabditis elegans ortholog, Ce-CUL-3, and 27 and 23% to that of Hs-CUL-1 and Hs-CUL-2, respectively. Northern blot analysis showed that phorbol 12-myristate 13-acetate increased the expression of Hs-CUL-3 mRNA in a concentration- and time-dependent manner, and this increase was inhibited by sodium salicylate. Hs-CUL-3 widely expressed in human tissues and its expression in cultured COLO205 colon cancer cells was increased when compared with that in normal colon cells. It is likely that Hs-CUL-3 is involved in cell proliferation control.     

Pyrolysis of tertiarybutylphosphine at low pressure

The pyrolysis of tertiarybutylphosphine (TBP) has been studied in the low pressure conditions used for chemical beam epitaxy (CBE). The pyrolysis studies were carried out in low pressure reactors of two different configurations, one of which is a cracker cell designed for use in a CBE system. The reaction products were studied using a quadrupole mass spectrometer. The products observed are accounted for by a reaction mechanism involving homolysis of the parent TBP molecule to produce PH₂ and C₄H₉ radicals. These undergo subsequent reactions to form the stable products C₄H₈, PH₃ and H₂, with smaller amounts of P and P₂ being produced. The production of the sub-hydride PH₂ using this cracker cell design indicates that the use of partially cracked TBP may be a promising technique for reducing the amount of carbon incorporated into the growing epitaxial layer.     

Bioavailability of phenytoin acid and phenytoin sodium with enteral feedings

In the absence of E1B, the 289-amino acid product of human adenovirus type 5 13S E1A induces p53-independent apoptosis by a mechanism that requires viral E4 gene products (Marcellus, R.C., J.C. Teodoro, T. Wu, D.E. Brough, G. Ketner, G.C. Shore, and P.E. Branton. 1996. J. Virol. 70:6207-6215) and involves a mechanism that includes activation of caspases (Boulakia, C.A., G. Chen, F.W. Ng, J. G. Teodoro, P.E. Branton, D.W. Nicholson, G.G. Poirier, and G.C. Shore. 1996. Oncogene. 12:529-535). Here, we show that one of the E4 products, E4orf4, is highly toxic upon expression in rodent cells regardless of the p53 status, and that this cytotoxicity is significantly overcome by coexpression with either Bcl-2 or Bcl-XL. Conditional expression of E4orf4 induces a cell death process that is characterized by apoptotic hallmark features, such as externalization of phosphatidylserine, loss of mitochondrial membrane potential, cytoplasmic vacuolation, condensation of chromatin, and internucleosomal DNA degradation. However, the wide-spectrum inhibitor of caspases, tetrapeptide zVAD-fmk, does not affect any of these apoptogenic manifestations, and does not alter the kinetics of E4orf4-induced cell death. Moreover, E4orf4 expression does not result in activation of the downstream effector caspase common to most apoptosis-inducing events, caspase-3 (CPP32). We conclude, therefore, that in the absence of E1A, E4orf4 is sufficient by itself to trigger a p53-independent apoptosis pathway that may operate independently of the known zVAD-inhibitable caspases, and that may involve an as yet uncharacterized mechanism.     

Percutaneous transhepatic drainage of the nondilated biliary system

Although reticulocyte counts can be reliably performed for up to 48 h after storage in EDTA, it is unclear whether this is applicable to the pediatric age group. In order to evaluate this, manual reticulocyte counts were performed on 20 specimens from pediatric patients stored at 4 degrees C for up to 24 h post collection. Samples were evaluated at 1-3, 6, 12, 18, and 24 h after storage in EDTA vacutainer tubes at 4 degrees C. The age of the subjects ranged from 1 day to 9 years with a median age of 3 years. Patients' reticulocyte counts ranged from 0 to 27% (5.89 +/- 7.21). No clinically significant changes were evident in the reticulocyte count over 24 h after specimen collection. The mean of the 20 specimens at 1-3 h was 5.50 and at 24 h was 5.40 (P > .05). The standard deviation of the mean values ranged from 7.03 to 7.26 (P > .05). The results indicate that reticulocyte counts may be performed on previously drawn blood held at 4 degrees C for up to 24 h post collection in a pediatric population without significant difference from baseline values.     

Effective index drift from molecular hydrogen diffusion inhydrogen-loaded optical fibres and its effect on Bragg gratingfabrication

When hydrogen loading is used to enhance the photosensitivity of silica-based optical waveguides and fibres, the presence of molecular hydrogen dissolved in the glass matrix changes the effective index of propagation of guided optical modes by as much as 0.05%. Real-time monitoring of the reflectivity spectrum of Bragg gratings written in such conditions shows that the centre wavelength follows the changes in hydrogen concentration due to diffusion and reaction with glass defects