Cysteine scanning mutagenesis of helix V in the lactose permease of Escherichia coli

Using a functional lactose permease mutant devoid of Cys (C-less permease), each amino acid residue in putative transmembrane helix V was replaced individually with Cys (from Met145 to Thr163). Of the 19 mutants, 13 are highly functional (60-125% of C-less permease activity), and 4 exhibit lower but significant lactose accumulation (15-45% of C-less permease). Cys replacement of Gly147 or Trp151 essentially inactivates the permease (< 10% of C-less); however, previous studies [Menezes, M. E., Roepe, P. D., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1638; Jung, K., Jung, H., et al. (1995) Biochemistry 34, 1030] demonstrate that neither of these residues is important for activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to C-less permease with the exception of Trp151-->Cys and single Cys154 permeases which are present in reduced amounts. Finally, only three of the single-Cys mutants are inactivated significantly by N-ethylmaleimide (Met145-->Cys, native Cys148, and Gly159-->Cys), and the positions of the three mutants fall on the same face of helix V.     

Performance evaluation of a reservation random access scheme for packetized wireless systems with call control and hand-off loading

We consider a packet switched wireless network where each cell's communication channel is shared among packet voice sources. In this paper, we present a method for the design and analysis of wireless cells using a reservation random access (RRA) scheme for packet access control. This scheme is integrated with a call admission control procedure. We model the state process of a single cell as a vector Markov chain. We compute the steady state distribution of the Markov chain. This result is used to calculate the packet dropping probability and the call blocking probability. By setting limits on maximum permissible levels for the call blocking probability and the packet dropping probability, we obtain the Erlang capacity of a single cell, with and without hand-off traffic. For an illustrative RRA scheme, the Erlang capacity of a single cell is shown to be about twice that attained by a comparable fixed assigned TDMA scheme. We show that a cellular network using this RRA scheme and which applies can be no blocking of hand-off calls, exhibits similar call capacity levels.This work is supported by a University of California MICRO and Pacific-Bell Grant No. 94-107.     

Influence of Membrane Stresses on Postbuckling of Rectangular Plates Using a Nonlinear Elastic 3-D Cosserat Brick Element

This paper has two main objectives. The first is to examine the influence of membrane stresses on postbuckled deformations of nonlinear elastic isotropic rectangular plates. The second is to further examine the accuracy of a new 3-D Cosserat eight noded brick element (Nadler and Rubin in Int J Solids Struct 40: 4585–4614, 2003) which was developed within the context of the theory of a Cosserat point. The equations of the Cosserat element include both material and geometric nonlinearities. A number of example problems are considered which examine predictions of the Cosserat element for beams and plates and comparison has been made with results from the commercial codes ANSYS and ADINA. Also, the approximate nonlinear postbuckling solution described in Timoshenko and Gere (Theory of elastic stability, Mc Graw-Hill, New York) is shown to be more limited than originally expected. These results suggest that the Cosserat element is robust, can perform well under extreme conditions and is capable of modeling combinations of three-dimensional bodies with attached thin structures.     

Recognizing, assessing, and intervening with problems of professional competence.

Working collaboratively, psychologist educators and trainers at the doctoral, internship, and postdoctoral levels; credentialers; practitioners; and students offer 8 proposals for psychologists to consider in recognizing, assessing, and intervening with problems of professional competence in students and practicing professionals. In the proposals, the authors address the following topics: definitions and categories; preparing the system; self-assessment; remediation; diversity; communication across various levels of the system; confidentiality; and ethical, regulatory, and legal underpinnings. They also propose future directions for the assessment of problems in professional competence in both students and practicing psychologists. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Novel sulfated oligosaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K. Unexpected degradation by chondroitinase ABC

We prepared a series of oligosaccharides from king crab cartilage chondroitin sulfate K after exhaustive digestion with testicular hyaluronidase, and determined the structures of four tetrasaccharides and a pentasaccharide by fast atom bombardment mass spectrometry, high performance liquid chromatography analysis of chondroitinase AC-II digests, and 500-MHz 1H NMR spectroscopy. The tetrasaccharides shared the common core structure GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3GalNAc with various sulfation profiles. One structure was GlcAbeta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S), whereas three of them have the following hitherto unreported structures including a novel glucuronate 3-O-sulfate: GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S), GlcAbeta1-3GalNAc(4S)beta1-4GlcA(3S)beta1-3GalNAc(4S), and GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcA(3S)beta1-3GalNAc(4S), where 3S or 4S represents 3-O- or 4-O-sulfate, respectively. The structure of the pentasaccharide was determined as GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcA(3S)beta1- 3GalNAc(4S)beta1-4GlcA. Chondroitinase ABC digestion of the tetrasaccharides with GlcA(3S) at the internal position destroyed the disaccharide unit containing GlcA(3S) derived from the reducing side and resulted in only the disaccharide unit from the non-reducing side. In contrast, these tetrasaccharides remained totally resistant to chondroitinase AC-II. The results indicated that it is necessary to reevaluate the disaccharide composition of chondroitin sulfate poly- or oligosaccharides purified from various biological sources, since they were usually determined after chondroitinase ABC digestion. It is probable that the structures containing GlcA(3S) would not have been detected.     

Genetics of programmed cell death in C. elegans: past, present and future

Genetic studies of the nematode Caenorhabditis elegans have defined a variety of single-gene mutations that have specific effects on programmed cell death. Analyses of the genes defined by these mutations have revealed that cell death is an active process that requires gene function in cells that die. Specific genes are required not only to cause cell death but also to protect cells from dying. Gene interaction studies have defined a genetic pathway for the execution phase of programmed cell death in C. elegans. Molecular and biochemical findings are consistent with the pathway proposed from these genetic studies and have also revealed that the protein products of certain cell-death genes interact directly. This pathway appears to be conserved among organisms as diverse as nematodes and humans. Important questions remain to be answered about programmed cell death in C. elegans. For example, how does a cell decide to die? How is cell death initiated? What are the mechanisms of action of the cell-death protector and killer genes? What genes lie downstream of the cell-death execution pathway? The conservation of the central cell-death pathway suggests that additional genetic analyses of programmed cell death in C. elegans will help answer these questions, not only for this nematode but also for other organisms, including ourselves.     

Pulmonary vasoconstrictor effects of prostacyclin in rats: potential role of thromboxane receptors

Endogenous prostacyclin (PGI2; epoprostenol) is a potent endothelium-derived pulmonary vasodilator. However, the effects of exogenous PGI2 on isolated arteries could be either relaxant or contractile, depending on the species and organ studied. The present study investigated the distal pathways involved in the PGI2-induced contraction in rat intrapulmonary artery (PA) and relaxation in lamb PA. When vessels were precontracted with 30 mM K+, PGI2 (1 microM) induced relaxation in lamb PA but caused contraction in rat PA. Use of 30 mM K+, phenylephrine, serotonin, angiotensin II, or hypoxia to precontract the vessels did not alter the contractile effect of PGI2 in rat PA. Nevertheless, PGI2 produced a mild relaxation in rat PA precontracted by U-46619, a thromboxane A2 (TxA2)-receptor agonist, whereas the TxA2-receptor blocker SQ-29548 (0.1-0.5 microM) abolished the contractile response in rat PA. These data suggest that PGI2-induced contraction is mediated by activation of TxA2 receptors. The PGI2-induced modest relaxation in rat PA, which was only observed when TxA2 receptors were blocked by SQ-29548, suggests that the PGI2-mediated vasorelaxant pathway is diminished in these vessels. Simultaneous application of forskolin, an adenylate cyclase activator, and rolipram, a phosphodiesterase inhibitor, caused similar relaxation in both rat and lamb PA. This suggests that the adenosine 3',5'-cyclic monophosphate-dependent relaxing pathway is intact in rat PA and is comparable to that in lamb PA. On the basis of these data, we conclude that the pathways responsible for the paradoxical effects of PGI2 on rat and lamb PA are located upstream of the adenosine 3',5'-cyclic monophosphate-dependent relaxing pathway and that a paucity of PGI2 receptors in rat PA may be responsible.     

Internal excitation of intermediate mass fragments from collisions of 36Ar+Ag nuclei at 35 MeV/nucleon

An extruding wire knife was used to give adult male CFHB rats a minimally traumatic unilateral mechanical lesion of the medial forebrain bundle. In addition, some rats received bilateral intrastriatal injections of one of three fluorescent retrograde tracers either eight days before or eight days after the lesion. Injections made after the lesion revealed that about half of the animals had complete lesions of the nigrostriatal tract, while the other half were incompletely lesioned, the mean proportion of non-axotomized neurons being 23%. Over the 10 weeks following the lesions, the number of tyrosine hydroxylase-immunoreactive cells in the lesioned substantia nigra fell linearly, reaching a mean of 29% of that of the control substantia nigra. In the animals which were completely lesioned, neuronal survival at 10 weeks varied between 6 and 12%. That the disappearance of tyrosine hydroxylase-immunoreactive neurons was due to cell death rather than the loss of tyrosine hydroxylase itself was confirmed by labelling the cells with Fluoro Gold before axotomy; the tracer was seen in survival neurons, microglia and in a few involuted neurons which continued to be tyrosine hydroxylase-immunoreactive. This percentage of neurons surviving axotomy corresponds to the proportion of substantia nigra neurons which project to the contralateral striatum, and these neurons were in the region of the substantia nigra from which the contralateral projection originated. It is concluded that following mechanical transection of the nigrostriatal tract, all truly axotomized substantia nigra neurons die over a period of about 10 weeks.     

Relation of follicular dendritic reticulum cells to Reed-Sternberg cells of Hodgkin's disease with emphasis on the expression of CD21 antigen

Based on observations of 66 cases, in which tissues were specially processed to optimize the simultaneous preservation of cell membrane antigens and morphology, we provide evidence in favor of a relationship between follicular dendritic reticulum cells (FDRC) and Reed-Sternberg (RS) cells of Hodgkin's disease (HD) other than the lymphocyte predominance subtype. RS cells were intimately related to the FDRC network (75% of cases), and the expression of CD21 antigen was frequent (41% of cases). Exclusive expression of CD21 antigen was found in 11 cases of HD, while the expression of other B-cell-associated markers (CD19, CD20, CD22) was both variable and inconsistent. The expression of T-cell antigens (CD3, CD4, CD8) was rare. Null phenotype of RS cells was observed in 27 of 66 cases (41%). Epstein-Barr virus (EBV) nucleic acids were found in 34 of 66 (51.5%) cases. Double labeling techniques showed the presence of EBV-positive RS cells within the FDRC network. A non-B-cell origin of RS cells was supported by the differential expression of EBV latent antigens in HD (latent membrane protein+, EB nuclear antigen 2-), which is unusual in EBV-driven lymphoblastoid cell lines and EBV-positive B-cell lymphomas. FDRC and RS cells are known to share morphological traits (binucleated cells), and both cell types possess Fc receptor for IgG. The hypothesis is further backed by the findings of CD15 antigen expression by occasional RS-like dysplastic FDRC in Castleman's disease (five cases), which is characterized by hyperplasia of FDRC. Whether FDRC might be the only cells involved in the conversion to RS cells by the loss or gain of antigens remains to be determined.     

Effects of sleeping in a chemical protective mask on sleep quality and cognitive performance

PURPOSE: We wanted to determine whether sleep is disrupted when soldiers sleep in a new chemical protective mask, the M40. Sleep quantity and quality, extent of protection provided by the mask during sleep, and next day performance were assessed. METHOD: After several days of training, 9 male soldiers slept with and without the M40 mask on four occasions. RESULTS: Soldiers were able to tolerate the mask for most or all of the night. However, sleep, as assessed by wrist-worn activity monitors, was significantly disturbed. Minutes (mean +/- SEM) of waking significantly increased, from 25 +/- 2.1 to 86 +/- 8.5 per night (p < 0.001), and number of awakenings rose from 8 +/- 0.6 to 20 +/- 0.9 (p < 0.0001). Soldiers reported that it took longer and was more difficult to fall asleep when wearing the mask. Errors on a choice reaction time task increased significantly and subjects reported greater fatigue and sleepiness the day after sleeping in the mask. Protection provided by the masks varied substantially among subjects and declined over the course of the study. Some soldiers were protected throughout the night but others were only protected intermittently. CONCLUSION: We conclude that sleeping in the chemical protective mask should only be done when necessary, given the adverse effects on sleep and daytime function, as well as the variability of protection, of the mask.