Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: interleukin-8, but not NAP-2 or GRO alpha, stimulates phospholipase D activity

Interleukin-8 (IL-8) and the structurally related cytokines neutrophil-activating peptide-2 (NAP-2) and GRO alpha are powerful chemotactic agents for human neutrophils. Although these three chemokines act by binding to overlapping but not identical receptor subsets, the data available to date have stressed the similarities in their mechanisms of action. The present studies were undertaken to further our understanding of the signal transduction mechanisms associated with these neutrophil agonists. IL-8, NAP-2, and GRO alpha stimulated similar increases in the level of cytoplasmic free calcium. They were also shown to stimulate qualitatively similar increases in the levels of protein tyrosine phosphorylation. In contrast, only IL-8 enhanced the formation of phosphatidylethanol (PEt), the product catalyzed by phospholipase D (PLD) in the presence of ethanol. The formation of PEt stimulated by IL-8 was inhibited by pertussis toxin and the tyrosine kinase inhibitors erbstatin and herbimycin A. The ability of IL-8 to stimulate the activity of PLD was additively enhanced, or primed, by cytochalasin B and by tumor necrosis factor alpha. Although all three chemokines increased the level of free cytoplasmic calcium to the same extent, IL-8 was significantly more potent than either NAP-2 or GRO alpha with respect to its ability to enhance CD11b expression and to stimulate chemotactic and oxidative responses. The differences between IL-8, NAP-2, and GRO alpha in their ability to stimulate PLD is likely to be related to their respective binding affinities for the two IL-8 receptors (IL-8R-A and IL-8R-B). These results suggest that the signalling pathways activated by IL-8R-A and IL-8R-B diverge at a step preceding the activation of PLD.     

Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique

Effects of cocaine on the muscle nicotinic acetylcholine receptor were investigated by using a chemical kinetic technique with a microsecond time resolution. This membrane-bound receptor regulates signal transmission between nerve and muscle cells, initiates muscle contraction, and is inhibited by cocaine, an abused drug. The inhibition mechanism is not well understood because of the lack of chemical kinetic techniques with the appropriate (microsecond) time resolution. Such a technique, utilizing laser-pulse photolysis, was recently developed; by using it the following results were obtained. (i) The apparent cocaine dissociation constant of the closed-channel receptor form is approximately 50 microM. High carbamoylcholine concentration and, therefore, increased concentrations of the open-channel receptor form, decrease receptor affinity for cocaine approximately 6-fold. (ii) The rate of the receptor reaction with cocaine is at least approximately 30-fold slower than the channel-opening rate, resulting in a cocaine-induced decrease in the concentration of open receptor channels without a concomitant decrease in the channel-opening or -closing rates. (iii) The channel-closing rate increases approximately 1.5-fold as the cocaine concentration is increased from 20 to 60 microM but then remains constant as the concentration is increased further. The results are consistent with a mechanism in which cocaine first binds rapidly to a regulatory site of the receptor, which can still form transmembrane channels. Subsequently, a slow step (t1/2 approximately 70 ms) leads to a receptor form that cannot form transmembrane channels, and acetylcholine receptor-mediated signal transmission is, therefore, blocked. Implications for the search for therapeutic agents that alleviate cocaine poisoning are mentioned.     

Inhibition of murine renal carcinoma pulmonary metastases by systemic administration of interferon gamma: mechanism of action and potential for combination with interleukin 4

We have previously demonstrated that IFN-gamma causes cell growth inhibition and up-regulation of MHC antigens in human renal cell carcinoma cell lines. In this study, we have investigated the therapeutic potential of IFN-gamma for the treatment of 5-day established pulmonary metastases induced by i.v. injection of Renca cells, a murine renal adenocarcinoma. We found that systemic injections of IFN-gamma significantly reduced the number of lung metastases in a dose-dependent manner and increased mouse survival. Histological evaluation of IFN-gamma-treated lungs showed residual small tumor nodules containing extensive necrosis and mononuclear infiltrates. Immunohistochemistry studies on lung sections showed macrophage infiltration into tumor nodules, and in vivo depletion of macrophages partially inhibited IFN-gamma antitumor effect, suggesting a role for the macrophages in tumor destruction. Lymphocyte depletion of either natural killer (NK) cells or CD4+ or CD8+ T-cell subsets or both T-cell subsets did not affect the IFN-gamma effect, whereas depletion of both NK and T cells decreased the antitumor activity of IFN-gamma. These data indicate that neither T cells nor NK cells are essential for this activity but that either lymphocyte population can contribute to the IFN-gamma effect. An optimal dose of IFN-gamma inhibited by 60% the growth of Renca cells treated for 3 days in vitro, but this effect was transient and less pronounced in a long-term colony assay, suggesting that IFN-gamma direct growth inhibition may play a role but may not be sufficient to mediate its antitumor effect in vivo. In vitro, IFN-gamma caused up-regulation of class I MHC antigens and induction of class II antigen expression in Renca cells, an effect that may enhance Renca immunogenicity but may be relevant only when a T-cell response is elicited. A sequential administration of IFN-gamma followed by interleukin 4 was therapeutically better than IFN-gamma alone for the treatment of advanced pulmonary metastases, probably due to different antitumor mechanisms induced by these two cytokines.     

Efficacy of one dose fluoroquinolone before prostate biopsy

OBJECTIVES: To demonstrate the efficacy of a simple preparation for prostate biopsy (PBX) and to determine its potential cost savings. METHODS: One hundred fifty consecutive PBXs were performed using a Fleet enema and a single oral dose (300 mg) of ofloxacin as the pre-PBX preparation. RESULTS: Of the 150 PBXs we performed, only 1 (0.67%) patient developed a urinary tract infection. CONCLUSIONS: A simple and inexpensive pre-PBX preparation proved to be successful in preventing infectious complications and is presented as a potential model for inclusion in clinical pathways for diagnosing adenocarcinoma of the prostate.     

Maturation of peroxisomes in differentiating human hepatoblastoma cells (HepG2): possible involvement of the peroxisome proliferator-activated receptor alpha (PPAR alpha)

Although several variant alleles at the human NAT1 gene locus have been reported, their relationship to phenotypic variations in NAT1 function remains unclear. We have used in-vivo and invitro phenotyping tests, along with PCR-based cloning and heterologous expression, to investigate the extent of variation in NAT1 function and to characterize novel allelic variants at the NAT1 gene locus. The NAT1-selective substrate p-aminosalicylic acid (PAS) was used as a probe for NAT1 function. In-vivo PAS acetylation rates were estimated by determining the ratio of PAS to N-acetylated PAS (AcPAS) in urine and plasma following the oral ingestion of Nemasol Sodium. Excluding outliers, a 65-fold variation in the urinary AcPAS:PAS ratio was observed (n = 144), while a 5.6-fold variation in the plasma AcPAS:PAS ratio was seen in a subset (n = 19) of this sample. Urinary and plasma ratios correlated moderately (r = 0.74, p < 0.0005). One individual (case 244) had a marked impairment of PAS N-acetylation, with 10-fold lower urinary and plasma AcPAS:PAS ratios compared with other subjects. Biochemical investigations in whole blood lysates from case 244 suggested a NAT1 kinetic defect, with a 20-fold increased apparent K(m) for PAS and a 90-fold decreased Vmax for AcPAS formation. We subcloned, sequenced and expressed the protein-coding regions of the NAT1 alleles from case 244 and from seven other selected probands. Sequence analysis revealed the presence of two new variant alleles, designated as NAT1 x 14 and NAT1 x 15, in case 244, as well as one variant, NAT1 x 11, which has been observed in previous investigations. NAT1 x 14 contained a missense mutation (G560-->A) that is predicted to change a single amino acid (Arg187-->Gln), as well as two 3' non-coding region mutations (T1088-->A and C1095-->A) that have previously been observed in the NAT1 x 10 allelic variant. NAT1 x 15 had a single nonsense mutation (C559-->T; Arg187-->stop) and, thus, encodes a truncated protein. The activity of recombinant NAT1 14 mirrored the defective enzyme function in whole blood lysates from case 244, while NAT1 15 was completely inactive. Expressed NAT1 11, on the other hand, had identical activity to the wild type NAT1 4 allele, suggesting that the coding region mutations in this variant are functionally silent. The frequencies of NAT1 x 11, NAT1 x 14 and NAT1 x 15 were 0.021, 0.028 and 0.014 (n = 288 alleles), respectively, suggesting that they are relatively rare in our predominantly Caucasian sample.     

The controls of fat intake

OBJECTIVE: To present evidence on the control of the size of fatty meals in the context of the result of interactions of orosensory positive-feedback and postingestive negative-feedback mechanisms activated by fat stimuli in rodent models of feeding and of obesity. METHOD: We examined the effects of orosensory stimuli and postingestive stimuli in Sprague-Dawley rats, and in the genetically obese Zucker (fa/fa) rat. We used the sham feeding rat preparation to isolate the orosensory stimulating and postingestive satiating properties of oils. The negative-feedback satiating properties of fats were elicited by intestinal infusions of fats. The Zucker rat is an animal model of obesity with abnormal control of meal size and increased intake of fats. Using this model we further examined the interaction of orosensory and postingestive stimuli in the control of meal size. RESULTS: The orosensory properties of fats are sufficient to drive sham feeding and are not dependent on the postabsorptive metabolic consequences of oils in normal and Zucker rats. The satiating action of fats must act at preabsorptive sites because reduction of intake occurs before absorption of fat. The satiating potency of fats is dependent upon their specific chemical conformation and is mediated by endogenous cholecystokinin and afferent fibers of the abdominal vagus. We have found that oils produce significantly more orosensory positive feedback in obese Zucker rats than in lean rats in experimental tests of preference. This is probably the major abnormal mechanism responsible for the increased preference for fats that is characteristic of obese rats because we have not identified any significant decrease in the postingestive satiating potency of fats in obese Zucker rats. CONCLUSIONS: Fat intake is controlled by both orosensory and postingestive stimuli in normal and genetically obese rodents. In the Zucker rat the investigation of this model of genetic obesity has produced data that is congruent with the preference for high fat foods in obese people and suggests further experiments directed toward a deeper understanding of the controls of fat intake and how they are disordered.     

Digital wavelet-encoded MRI: a new wavelet-encoding methodology

A new digital wavelet-encoding method for MRI is described. The method differs from previously described wavelet-encoding approaches, because the point-spread function is made independent of the wavelet basis used. This has a significant practical advantage, because wavelet bases can now be considered that would otherwise be excluded due to the difficulty of precisely exciting wavelet-shaped RF profiles. The method has been implemented on a clinical MRI system, and human images are presented.     

An intriguing controversy over protein structural class prediction

A recent report by Bahar et al. [(1997), Proteins 29, 172-185] indicates that the coupling effects among different amino acid components as originally formulated by K. C. Chou [(1995), Proteins 21, 319-344] are important for improving the prediction of protein structural classes. These authors have further proposed a compact lattice model to illuminate the physical insight contained in the component-coupled algorithm. However, a completely opposite result was concluded by Eisenhaber et al. [(1996), Proteins 25, 169 179], using a different dataset constructed according to their definition. To address such an intriguing controversy, tests were conducted by various approaches for the datasets from an objective database, the SCOP database [Murzin et al. (1995), J. Mol. Biol. 247, 536-540]. The results obtained by both self-consistency and jackknife tests indicate that the overall rates of correct prediction by the algorithm incorporating the coupling effect among different amino acid components are significantly higher than those by the algorithms without counting such an effect. This is fully consistent with the physical reality that the folding of a protein is the result of a collective interaction among its constituent amino acid residues, and hence the coupling effects of different amino acid components must be incorporated in order to improve the prediction quality. It was found by a revisiting the calculation procedures by Eisenhaber et al. that there was a conceptual mistake in constructing the structural class datasets and a systematic mistake in applying the component-coupled algorithm. These findings are informative for understanding and utilizing the component-coupled algorithm to study the structural classes of proteins.