High-resolution crystal structures of human hemoglobin with mutations at tryptophan 37beta: structural basis for a high-affinity T-state,

The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37(C3)beta is replaced with Tyr (betaW37Y), Ala (betaW37A), Glu (betaW37E), or Gly (betaW37G) have been refined and analyzed with superposition methods that partition mutation-induced perturbations into quaternary structure changes and tertiary structure changes. In addition, a new cross-validation statistic that is sensitive to local changes in structure (a "local Rfree" parameter) was used as an objective measure of the significance of the tertiary structure changes. No significant mutation-induced changes in tertiary structure are detected at the mutation site itself for any of the four mutants studied. Instead, disruption of the intersubunit contacts associated with Trp37(C3)beta results in (1) a change in quaternary structure at the alpha1beta2 interface, (2) alpha subunit tertiary structure changes that are centered at Asp94(G1)alpha-Pro95(G2)alpha, (3) beta subunit tertiary structure changes that are located between residues Asp99(G1)beta and Asn102(G4)beta, (4) increased mobility of the alpha subunit COOH-terminal dipeptide, and (5) shortening of the Fe-Nepsilon2His(F8) bond in the alpha and beta subunits of the betaW37G and betaW37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order betaW37Y < betaW37A < betaW37E approximately betaW37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.     

Cryo-FIB-nanotomography for quantitative analysis of particle structures in cement suspensions

Cryo‐FIB‐nanotomography is a novel high‐resolution 3D‐microscopy technique, which opens new possibilities for the quantitative microstructural analysis of complex suspensions. In this paper, we describe the microstructural changes associated with dissolution and precipitation processes occurring in a fresh cement paste, which has high alumina and sulphate contents. During the first 6 min, precipitation of ettringite leads to a general decrease of the particle size distribution. In the unhydrated cement paste almost no particles smaller than 500 nm are present, whereas after 6 min this size class already represents 9 vol%. The precipitation of ettringite also leads to a significant increase of the particle number density from 0.294*10⁹/mm³ at t_0min to 20.55*10⁹/mm³ at t_6min. Correspondingly the surface area increases from 0.75 m²/g at t_0min to 2.13 m²/g at t_6min. The small ettringite particles tend to form agglomerates, which strongly influence the rheological properties. The particular strength of cryo‐FIB‐nt is the potential to quantify particle structures in suspension and thereby also to describe higher‐order topological features such as the particle–particle interfaces, which is important for the study of agglomeration processes.     

Results of irradiation treatment in patients with non-resectable oesophageal cancer

Forty-eight patients with non-resectable cancer of the oesophagus and oesophagogastric junction (Group A: Stage I/II, 32; Group B: Stage III/IV, 16) underwent intraluminal Iridium-192 high dose-rate afterloading therapy (5-7 Gy/session, total dose: 5-21 Gy, mean: 12.4 Gy) and external beam irradiation (Karnofsky > or = 80% 50-60 Gy/2 Gy per day; Karnofsky 60-79%: 30 Gy/3 Gy per day). Durable satisfactory palliation (intake of at least semi-solid food) was demonstrated in 96% of patients. The mean survival for group A was 19.1 months and that for group B, 6.9 months, with a 12-month survival rate of 66% (group A) and 0% (group B) (P < 0.001). Local tumour response and complication rate were significantly dose-related with a predicted response rate of 70.5%, and a complication rate of 50% at ERD 129.3 Gy.     

Surfactant nebulization versus instillation during high frequency ventilation in surfactant-deficient rabbits

Glutamate-mediated excitotoxicity plays an important role in the degeneration of nigrostriatal dopamine (DA) neurons induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), although the role of the N-methyl D-aspartate (NMDA) receptor subtype in this process is still uncertain. We studied glutamate receptor subtype agonist-induced ionic currents in acutely dissociated DAergic neurons from the rat substantia nigra zona compacta (SNc) using the nystatin-perforated patch-clamp whole-cell recording technique. The results fall into four main categories. First, single neurons, freshly isolated from SNc, exhibited a large soma and multipolar morphology, responded to DA, and stained positively for tyrosine hydroxylase (TH). Second, rapid application of L-glutamate (> 10(-5) M) induced an inward current with minimal desensitization at a clamp voltage of -60 mV. Third, kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-isoxazole (AMPA) induced an inward current that was similar to the glutamate-induced current while, in the same neuron, NMDA (10(-4) M) failed to induce any current response in Mg2+-free solution that contained 10(-5) M glycine at a clamp voltage of -60 mV. Under the same experimental conditions, NMDA induced a clear current response in isolated substantia nigra reticulata (SNr) neurons. Fourth, the specific NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV, 10(-4) M) failed to block 10(-4) M glutamate-induced inward current, while the specific KA/AMPA receptor antagonist 6-cyano-7-nitroguinoxaline-2, 3-dione (CNQX, 10(-5) M) completely blocked the glutamate-induced current. These results indicate that in single SNc DAergic neurons of 2-week-old rats, L-glutamate-induced inward current is mediated by non-NMDA receptors rather than by NMDA receptors.     

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.     

Potentiation of the time-dependent, antidepressant-induced changes in the agonistic behaviour of resident rats by the 5-HT1A receptor antagonist, WAY-100635

Acute and chronic antidepressant drug treatments respectively decrease and increase the aggressive behaviour of resident rats during encounters with unfamiliar conspecifics. We have now examined the effect of the 5-hydroxytryptamine1A receptor antagonist, WAY-100635, on fluoxetine-, paroxetine- or venlafaxine-induced changes in aggression. WAY-100635 (0.1 mg/kg), which did not modify behaviour when given alone, potentiated the venlafaxine (5.54 mg/kg)-induced reduction in aggression after acute treatment and, during chronic treatment, accelerated the fluoxetine (0.34 mg/kg/day)-induced increase in aggression, from day 5 to day 2. A similar change in time course was seen with paroxetine (0.33 mg/kg/day), although the increase in aggression was smaller. Venlafaxine (5.54 mg/kg/day, alone or co-administered with WAY-100635) increased aggression by day 2. During chronic treatment, therefore, venlafaxine, at the dose used, had a more rapid onset of action than either fluoxetine or paroxetine, whereas the fluoxetine- and paroxetine-, but not the venlafaxine-, induced increase in aggression was accelerated by WAY-100635. These studies further support the hypothesis that selective blockade of the 5-hydroxytryptamine1A receptor augments the effects of antidepressant drugs in an animal model predictive of antidepressant activity, presumably by concomitant blockade of the somatodendritic 5-hydroxytryptamine1A autoreceptor-mediated negative feedback system of serotonergic neurones.     

Differential expression of acidic and basic fibroblast growth factors in benign prostatic hyperplasia identified by immunohistochemistry

OBJECTIVES: Transitional cell carcinomas of upper urinary tract (uttTCC) constitute 5% to 6% of all urothelial tumors. Ureteropyeloscopy has become the standard for clinical evaluation of uutTCC. Moreover, endoscopic treatments have been advocated as a conservative approach for low grade tumors or patients with intermediate grade tumors whose renal function is compromised. Therefore, grading has become the most predictive variable in defining therapeutic approach. In addition to morphologic evaluation, a series of biologic markers may be used to increase the accuracy of grading such as DNA analysis and p53 protein expression. In this study, we have evaluated these markers by means of cell image analysis with the SAMBA 400 system. METHODS: Thirteen cases of uttTCC were studied with cytologic smear, cell block, and histologic confirmation. DNA analysis was performed on cytologic smear. Immunostaining was performed on cell blocks. A grade was assigned on the basis of DNA evaluation and p53 expression quantitation. These grades were combined for each case and compared with the initial cytologic grading and the final histologic grading. RESULTS: Cytology alone diagnosed TCC in all but 1 case that was diagnosed atypical. Discrepancies were found in primary grading: cytologic grading concurred with histologic grading in 6 of the 13 cases. CONCLUSIONS: These results, although in a limited but selected number of cases, show the potential of computerized evaluation of biologic markers as parameters for a more objective grading of tumors.     

U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells

We investigated the effects of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside on basal and K+-evoked release of [3H]noradrenaline from superfused synaptosomes from the rat cerebral cortex. Both substances produced concentration-dependent increases in the release of the labeled transmitter under basal and depolarized conditions. The effects of the donors on basal release were Ca2+-independent but were not inhibited by the carrier-uptake blocker, desipramine; the effects were abolished by hemoglobin (an NO scavenger). Thirty-five minutes after stimulation with sodium nitroprusside, the synaptosomes were still responsive to KCl stimulation, indicating that the donor's effects were not caused by damage to the synaptosome membrane. The cGMP analogue, 8-bromo-cGMP, had no effect on basal release, and the enhanced release produced by sodium nitroprusside was not inhibited by the specific inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, indicating that NO's effects on basal release of the neurotransmitter are guanylate cyclase-independent. Both of the NO donors had more marked effects on release of [3H]noradrenaline during K+-stimulated depolarization. The NO-mediated increase in this case was partially antagonized by 10 microM LH-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, and 8-Br-cGMP was also capable of producing concentration-dependent increases in the K+-stimulated release of the transmitter. These findings indicate that the effects of the NO donors on [3H]noradrenaline release during depolarization are partially mediated by the activation of guanylate cyclase.     

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation

DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.