XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly

BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.     

Compartmentalization of visual centers in the Drosophila brain requires Slit and Robo proteins

Brain morphogenesis depends on the maintenance of boundaries between populations of non-intermingling cells. We used molecular markers to characterize a boundary within the optic lobe of the Drosophila brain and found that Slit and the Robo family of receptors, well-known regulators of axon guidance and neuronal migration, inhibit the mixing of adjacent cell populations in the developing optic lobe. Our data suggest that Slit is needed in the lamina to prevent inappropriate invasion of Robo-expressing neurons from the lobula cortex. We show that Slit protein surrounds lamina glia, while the distal cell neurons in the lobula cortex express all three Drosophila Robos. We examine the function of these proteins in the visual system by isolating a novel allele of slit that preferentially disrupts visual system expression of Slit and by creating transgenic RNA interference flies to inhibit the function of each Drosophila Robo in a tissue-specific fashion. We find that loss of Slit or simultaneous knockdown of Robo, Robo2 and Robo3 causes distal cell neurons to invade the lamina, resulting in cell mixing across the lamina/lobula cortex boundary. This boundary disruption appears to lead to alterations in patterns of axon navigation in the visual system. We propose that Slit and Robo-family proteins act to maintain the distinct cellular composition of the lamina and the lobula cortex.     

Compressing DNA sequence databases with coil

Background  

Publicly available DNA sequence databases such as GenBank are large, and are growing at an exponential rate. The sheer volume of data being dealt with presents serious storage and data communications problems. Currently, sequence data is usually kept in large "flat files," which are then compressed using standard Lempel-Ziv (gzip) compression – an approach which rarely achieves good compression ratios. While much research has been done on compressing individual DNA sequences, surprisingly little has focused on the compression of entire databases of such sequences. In this study we introduce the sequence database compression software coil.     

Characterization of tRNA-dependent peptide bond formation by MurM in the synthesis of Streptococcus pneumoniae peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.     

Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases

Escherichiacoli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3′-to-5′ translocase, and RecD, a 5′-to-3′ translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt out” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg²⁺-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3′-(dT)₆ and 5′-(dT)₆ or 5′-(dT)₁₀] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3′-(dT)₆ and 5′-(dT)₆ noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk_U = 774 ± 16 bp s^− 1) and kinetic step size (m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k_C = 45 ± 2 s^− 1). However, when the noncomplementary 5′ ssDNA tail is extended to 10 nt [5′-(dT)₁₀ and 3′-(dT)₆], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk_U = 538 ± 24 bp s^− 1) is observed with a similar kinetic step size (m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5′-(dT)₆ and 3′-(dT)₆ tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k_C initiation steps, with a similar kinetic step size (m = 4.4 ± 1.7 bp) and unwinding rate (mk_obs = 396 ± 15 bp s^− 1). These results suggest that the additional kinetic steps with rate constant k_C required for RecBCD to initiate unwinding of blunt-ended and twin (dT)₆-tailed DNA reflect processes needed to engage the RecD motor with the 5′ ssDNA.     

Primary Cell Cultures for Understanding Rat Epididymal Inflammation

Treatment‐induced epididymal inflammation and granuloma formation is only an occasional problem in preclinical drug development, but it can effectively terminate the development of that candidate molecule. Screening for backup molecules without that toxicity must be performed in animals (generally rats) that requires at least 2 to 3 weeks of in vivo exposure, a great deal of specially synthesized candidate compound, and histologic examination of the target tissues. We instead hypothesized that these treatments induced proinflammatory gene expression, and so used mixed‐cell cultures from the rat epididymal tubule to monitor the induction of proinflammatory cytokines. Cells were exposed for 24 hr and then cytotoxicity was evaluated with the MTS assay and mRNA levels of Interleukin‐6 (IL‐6) and growth‐related oncogene (GRO) were measured. We found that compounds that were more toxic in vivo stimulated a greater induction of IL‐6 and GRO mRNA levels in vitro. By relating effective concentrations in vitro with the predicted C_eff, we could rank compounds by their propensity to induce inflammation in rats in vivo. This method allowed the identification of several compounds with very low inflammatory induction in vitro. When tested in rats, the compounds produced small degrees of inflammation at an acceptable margin (approximately 20×), and have progressed into further development     

Immunopathological features of human pulmonary tumors following low-dose interleukin-2

Summary We administered preoperative low-dose interleukin-2 (IL-2) to 10 patients undergoing thoracotomy for pulmonary tumors. The in vivo effect of IL-2 on tumor-associated lymphocyte activity was assessed in the resected specimens by immunohistochemistry and compared with observations in 45 patients who did not receive IL-2. H & E evaluation revealed an increase in intra- and peritumoral lymphocyte infiltration in the IL-2-treated patients. Immunopathological evaluation with monoclonal antibodies revealed that this lymphocyte infiltration was predominantly CD5-positive T cells. The amount of intra-and peritumoral lymphocyte activity correlated with the dose of IL-2 administered (6000–90 000 international units/kg every 8 h for 48 h. IL-2-treated patients showed increases in T-cell-associated activation markers (IL-2 -receptor, transferrin receptor and HLA-DR) on peritumoral lymphocytes, but not on intratumoral lymphocytes. We previously reported that low-dose IL-2 increases the intrinsic natural killer cell cytotoxicity of intratumoral lymphocytes and suggest that this lymphocyte infiltration is further evidence that low-dose IL-2 can augment in vivo lymphocyte activity at the tumor site.This work was supported in part by USPHS grants CA 44 352 (S. H. G.) and 43 658 (A. J. C.). S. G. S. was supported by NIH Surgical Oncology Training Grant CA 09 010     

Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.