Assay of cell attachment and spreading factors

Summary Methods are presented for the quantitative assay of proteins influencing cell attachment and spreading. These include assays in which cell-substratum interactions are quantitated directly and a serum-free assay in which cell growth is dependent on attachment under substratum-limited conditions. Procedures are also presented for the identification of active sites of substratum molecules through the use of antibodies and synthetic peptides that are inhibitory for cell attachment.     

The interaction of conflicting retinal motion stimuli in oculomotor control

Summary Oculomotor response has been assessed in humans during the presentation of conflicting retinal motion stimuli. In the majority of experiments a background stimulus was made to move with a constant velocity ramp in one direction followed by rapid resets at regular intervals. In the absence of an adequate fixation target this ramp-reset stimulus induced a nystagmus with a slow-phase velocity and saccadic frequency which remained almost constant as reset frequency was increased from 2 to 5 Hz. Moreover, the induced eye velocity could be considerably increased if the subject attempted active matching of display velocity. During both active and passive responses eye velocity gain reached a peak when display velocity was between 2°/s and 5°/s. The presence of small stationary targets induced a suppression of the passive ramp-reset response which was modified by target eccentricity and by tachistoscopic target illumination. When subjects pursued a sinusoidally oscillating target against a stationary structured background, eye velocity gain was significantly less than for pursuit against a blank background. The degree of interaction between conflicting stimuli was found to be dependent on their relative size, peripheral location and velocity. However, it appears that the human observer is able selectively to enhance feedback gain from one particular source in order to dominate stimuli from other unwanted sources.     

Patterns of cytokine production by mycobacterium-reactive human T-cell clones.

To gain insight into the functional capacity of human T cells in the immune response against Mycobacterium tuberculosis, we evaluated the spectrum of cytokines produced by mycobacterium-reactive human T-cell clones. Nine of 11 T-cell clones bearing alpha beta or gamma delta T-cell receptors produced both Th1 and Th2 cytokines, a pattern resembling that of murine Th0 clones. The most frequent pattern was secretion of gamma interferon, tumor necrosis factor alpha (TNF), and interleukin-10 (IL-10), in combination with IL-2, IL-5, or both. Two clones produced only Th1 cytokines, and none produced exclusively Th2 cytokines. Although IL-4 was not detected in cell culture supernatants, IL-4 mRNA was detected by polymerase chain reaction amplification in two of six clones. There were no differences between the cytokine profiles of alpha beta and gamma delta T cells. A striking finding was the markedly elevated concentrations of TNF in clone supernatants, independent of the other cytokines produced. Supernatants from mycobacterium-stimulated T-cell clones, in combination with granulocyte-macrophage colony-stimulating factor, induced aggregation of bone-marrow-derived macrophages, and this effect was abrogated by antibodies to TNF. The addition of recombinant TNF to granulocyte-macrophage colony-stimulating factor markedly enhanced macrophage aggregation, indicating that TNF produced by T cells may be an important costimulus for the granulomatous host response to mycobacteria. The cytokines produced by T cells may exert immunoregulatory and immunopathologic effects and thus mediate some of the clinical manifestations of tuberculosis.     

Regional mapping of the gene encoding dihydroorotate dehydrogenase,an enzyme involved in UMP synthesis,electron transport,and superoxide generation,to human chromosome region 16q22

De novo UMP synthesis is a critical metabolic pathway for nucleic acid synthesis and for a variety of metabolic pathways. The pathway is a target for many widely used cancer chemotherapy agents, several of which are pyrimidine analogs. Humans and cattle have been described with mutations in UMP synthesis that lead to serious inborn errors of metabolism. Dihydroorotate dehydrogenase (EC 1.3.3.1) (DHODH) carries out the fourth committed step in the pathway and may also be important for mitochondrial electron transport and oxygen radical metabolism. We report here that the gene encoding this enzyme in humans is located in the chromosomal region 16q22. With the mapping of DHODH, the mapping of all the steps of UMP synthesis is complete. All three genes involved map to different human chromosomes. This information is important in consideration of regulation of UMP synthesis in mammals, including humans.     

Anticipatory control of hand and eye movements in humans during oculo-manual tracking

Anticipatory activity of hand and eye has been examined during oculo-manual tracking of a constant velocity visual target with a hand cursor. Both target and cursor were presented briefly (< 480 ms), but repeatedly, at regular inter-stimulus intervals (ISI). In Expt 1, the build-up of hand and eye responses was examined for target velocities varying from 10–40 deg s⁻¹ with an ISI of 2.4 s. The velocity 100 ms after target onset (i.e. prior to visual feedback) for both hand and eye ( V ₁₀₀) progressively increased over the first four presentations but then attained a steady state (SS). SS V ₁₀₀ values for eye and hand increased in proportion to target velocity and were thus predictive of forthcoming movement. Hand velocity exceeded eye velocity but both exhibited similar anticipatory trajectories. In Expt 2, target velocity was constant (40 deg s⁻¹) but ISI varied from 0.48–3.74 s. Subjects made anticipatory eye movements for all ISIs but hand movements were often reactive at the longest ISI. If the target failed to appear as expected, subjects initiated predictive hand and eye responses with timing appropriate for the prevailing ISI. In Expt 3, predictive responses were compared with responses to randomised presentation. Peak hand velocity was greater in the randomised mode than in the predictive condition, whereas the converse was true for peak eye velocity. This difference is discussed in terms of the mechanisms of positional error correction in hand and eye. Results provide evidence of similar anticipatory mechanisms in hand and eye, using storage of velocity and timing to achieve rapid prediction of target motion.     

Cryptococcus neoformans var. gattii in the koala (Phascolarctos cinereus): serological evidence for subclinical cryptococcosis.

Cryptococcus neoformans var. gattii has been shown to have a strong association with eucalypts frequently used by koalas and, not surprisingly, it has been shown to colonize the nasal cavities of koalas. The progression from nasal colonization to tissue invasion is critical to understanding the pathogenesis of cryptococcosis in this species and provides a model for pathogenesis of cryptococcosis in other species. Cryptococcal antigenaemia was detected in twenty-eight healthy koalas from three different regions. This was interpreted as representing limited subclinical disease. One koala developed cryptococcal pneumonia 6 months after leaving the study, whereas another developed cryptococcal meningoencephalitis during the course of the study. Opportunistic necropsies on ten antigen-positive koalas resulted in discovery of small cryptococcal lesions in two (paranasal sinus and lung, respectively). Our data suggest that cryptococcal antigenaemia occurs commonly in koalas, especially in areas with a high environmental presence of C n. var. gattii. Subclinical disease appears most likely to manifest as a small focal lesion in the respiratory tract. Possible outcomes include elimination by an effective immune response, quiescence with possibility of later re-activation or direct progression to overt disease. Symptomatic and subclinical cases showed differences in levels of antigenaemia. The data presented have significant implications for koalas in captivity.     

Proton modulation of M-like potassium current (IKx) in rod photoreceptors

The effect of extracellular pH (pH_e 6.9–8.1) and intracellular pH (pH_i 6.4–8.1) on the non-inactivating voltage-sensitive M-like potassium current (I_Kx) was studied in patch-clamped salamander rod photoreceptors. The midpoint of the I_Kx activation curve shifted by 6.6 mV per pH_e unit, with acidification producing positive shifts and alkalinization producing negative shifts. The time constant of I_Kx activation shifted with pH_e in a manner consistent with the shifts in the activation curve. Maximum conductance and gating charge were unaffected by changes in pH_e. I_Kx did not depend on pH_i. Given the importance of I_Kx in rod function, these results suggest that pH_e could affect the signal transmitted from rods by changing I_Kx activation parameters.     

Nitrosative stress in the bronchial mucosa of severe chronic obstructive pulmonary disease

BACKGROUND: Reactive nitrogen species, formed via the reaction of nitric oxide (NO) with superoxide anion and via (myelo)peroxidase-dependent oxidation of NO(2)(-), have potent proinflammatory and oxidizing actions. Reactive nitrogen species formation and nitrosative stress are potentially involved in chronic obstructive pulmonary disease (COPD) pathogenesis. OBJECTIVES: To investigate the expression of markers of nitrosative stress, including nitrotyrosine (NT), inducible NO synthase (iNOS), endothelial NO synthase (eNOS), myeloperoxidase (MPO), and xanthine oxidase (XO) in bronchial biopsies and bronchoalveolar lavage from patients with mild to severe stable COPD compared with control groups (smokers with normal lung function and nonsmokers). METHODS: The expression of NT, iNOS, eNOS, MPO and XO in the bronchial mucosa and bronchoalveolar lavage of patients was measured by using immunohistochemistry, Western blotting, and ELISA and correlated with the inflammatory cell profile. RESULTS: Patients with severe COPD in stable phase had higher numbers of NT(+) and MPO(+) cells in their bronchial submucosa compared with mild/moderate COPD, smokers with normal lung function, and nonsmokers (P < .01). iNOS(+) and eNOS(+) but not XO(+) cells were significantly increased in smokers with COPD or normal lung function compared with nonsmokers (P < .05 and P < .01, respectively). In patients with COPD, the number of MPO(+) cells was significantly correlated with the number of neutrophils (r = +0.61; P < .0025) in the bronchial submucosa. Furthermore, the number of NT(+) and MPO(+) cells was negatively correlated with postbronchodilator FEV(1). CONCLUSION: These data suggest that nitrosative stress, mainly mediated by MPO and neutrophilic inflammation, may contribute to the pathogenesis of severe COPD.     

Effect of topical capsaicin on the cutaneous responses to inflammatory mediators and to antigen in man

Topical capsaicin pretreatment is known to deplete cutaneous sensory nerves of neuropeptides. We have assessed the effect of topical capsaicin pretreatment on the responses to intradermal injections of histamine and platelet-activating factor (PAF) in six normal subjects, and of prostaglandin E2, histamine, and antigen in 10 atopic subjects. Capsaicin pretreatment caused significant inhibition of the immediate flare response to histamine in both normal (19.8 +/- 2.6 to 7.3 +/- 2.9 cm2 at 5 minutes; p less than 0.01) and atopic subjects (16.5 +/- 1.4 to 10.3 +/- 1.9 cm2 at 5 minutes; p less than 0.01). The PAF-induced flare was also inhibited from 12.2 +/- 2.9 to 2.7 +/- 1.6 cm2 at 5 minutes after injection (p less than 0.01). In contrast, capsaicin pretreatment did not significantly alter the flare responses to prostaglandin E2 or antigen in atopic subjects. The acute wheal responses to all stimuli were unchanged, as was the late-phase response to antigen. These results support the hypothesis that the cutaneous vasodilator effect of histamine and PAF may be mediated by a local axon reflex involving the release of neuropeptides from sensory nerves. A consistent effect of capsaicin pretreatment on the flare response induced by endogenous mediators released during a cutaneous IgE-mediated response was not observed. Increases in microvascular permeability and the late-phase response to antigen are independent of neuropeptide release from cutaneous nerves.