Pharmacological evidence for complex and multiple site interaction of CXCR4 with SDF-1alpha: implications for development of selective CXCR4 antagonists

The C-X-C chemokine SDF-1 and its receptor CXCR4, mediate a pivotal role in the pathophysiology of HIV-1 infection and vascular inflammatory diseases. In this study, we investigated the pharmacological properties of SDF-1alpha interaction with CXCR4 in human leukemia cell lines. Our data, based on [125I]-SDF-1alpha radioligand binding, SDF-1alpha-induced [35S]-GTPgammaS binding and use of specific CXCR4 antagonist AMD3100 reveals the complex nature of SDF-1alpha-CXCR4 interaction. Firstly, homologous competition with cold SDF-1alpha revealed a bimodal ligand displacement curve and secondly, although AMD3100 inhibited both SDF-1alpha-mediated chemotaxis (IC(50)=4.7 nM) and [35S]-GTPgammaS binding (IC(50)=7.4 nM) with high affinity, it was intriguingly up to 3000-fold less potent (IC(50)=15.2 microM) in the radioligand binding assay. These results provide pharmacological evidence for the recently described two-site model for SDF-1alpha-CXCR4 interaction. Accordingly, inhibition of SDF-1alpha binding to one of the receptor sites is sufficient to antagonize function, without causing its complete displacement from the receptor. Furthermore, these findings have important implications in the development and evaluation of CXCR4-selective small molecule antagonists for therapeutic use.     

Vitamin B12 responsive homocystinuria and megaloblastic anemia: heterogeneity in methylcobalamin deficiency

A male infant with methyl-B12 deficiency (cblE) presented at age 6 weeks with lethargy, staring spells, and vomiting. He later became hypotonic and unresponsive to stimuli and required intubation and ventilation. He had homocystinuria and hypomethioninemia with megaloblastic anemia but normal serum folate and vitamin B12 concentrations. No methylmalonic aciduria was detected. Fibroblasts, cultured from the patient, were unable to grow in medium in which homocysteine replaced methionine and incorporated abnormally small amounts of [14C]-methyl-tetrahydrofolate but normal amounts of [14C]-propionate into protein. Methyl-B12 content of fibroblasts was low, while the adenosyl-B12 content was normal. Methionine synthase activity was decreased when the assay was performed under both optimal and suboptimal reducing conditions, suggesting heterogeneity in the cblE disease. The patient responded dramatically to hydroxocobalamin treatment. Homocystinuria disappeared after 10 days of therapy, and methionine was normalized after 3 weeks. Psychometric testing at age 15 months showed a developmental age of 9 months.     

GJB2: the spectrum of deafness-causing allele variants and their phenotype

Genetic testing was completed on 1,294 persons with deafness referred to the Molecular Otolaryngology Research Laboratories to establish a diagnosis of DFNB1. Exon 2 of GJB2 was screened for coding sequence allele variants by denaturing high-performance liquid chromatography (DHPLC) complemented by bidirectional sequencing. If two deafness-causing mutations of GJB2 (encoding Connexin 26) were identified, further screening was not performed. If only a single deafness-causing mutation was identified, we screened for the g.1777179_2085947del (hereafter called del(GJB6-D13S1830); GenBank NT_024524.13) and mutations in the noncoding region of GJB2. Phenotype-genotype correlations were evaluated by categorizing mutations as either protein truncating or nontruncating. A total of 205 persons carried two GJB2 exon 2 mutations and were diagnosed as having DFNB1; 100 persons carried only a single deafness-causing allele variant of exon 2. A total of 37 of these persons were c.35delG carriers, and 51 carried other allele variants of GJB2. Persons diagnosed with DFNB1 segregating two truncating/nonsense mutations had a more severe phenotype than persons carrying two missense mutations, with mean hearing impairments being 88 and 37%, respectively (P < 0.05). The number of deaf c.35delG carriers was greater than expected when compared to the c.35delG carrier frequency in normal-hearing controls (P < 0.05), suggesting the existence of at least one other mutation outside the GJB2 coding region that does not complement GJB2 deafness-causing allele variants.     

Cation channels,cell volume and the death of an erythrocyte

Similar to a variety of nucleated cells, human erythrocytes activate a non-selective cation channel upon osmotic cell shrinkage. Further stimuli of channel activation include oxidative stress, energy depletion and extracellular removal of Cl^–. The channel is permeable to Ca²⁺ and opening of the channel increases cytosolic [Ca²⁺]. Intriguing evidence points to a role of this channel in the elimination of erythrocytes by apoptosis. Ca²⁺ entering through the cation channel stimulates a scramblase, leading to breakdown of cell membrane phosphatidylserine asymmetry, and stimulates Ca²⁺-sensitive K⁺ channels, thus leading to KCl loss and (further) cell shrinkage. The breakdown of phosphatidylserine asymmetry is evidenced by annexin binding, a typical feature of apoptotic cells. The effects of osmotic shock, oxidative stress and energy depletion on annexin binding are mimicked by the Ca²⁺ ionophore ionomycin (1 µM) and blunted in the nominal absence of extracellular Ca²⁺. Nevertheless, the residual annexin binding points to additional mechanisms involved in the triggering of the scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Susceptibility to annexin binding is enhanced in several genetic disorders affecting erythrocyte function, such as thalassaemia, sickle-cell disease and glucose-6-phosphate dehydrogenase deficiency. The enhanced vulnerability presumably contributes to the shortened life span of the affected erythrocytes. Beyond their role in the limitation of erythrocyte survival, cation channels may contribute to the triggering of apoptosis in nucleated cells exposed to osmotic shock and/or oxidative stress.     

Sensation Seeking and Cortical Augmenting-Reducing

The experiment was designed to establish the relationship between the Sensation Seeking Scales (SSS) and cortical augmenting-reducing. Forty-nine male undergraduate Ss were used. Ss were presented with five intensities of light flashes in randomly presented blocks of trials at each intensity. Averaged evoked response (AER) amplitudes were measured at each intensity of light. Augmenting-reducing was measured for each S as the slope of the relationship between stimulus intensity and amplitude of response. This slope measure correlated very significantly (r= .59) with the Disinhibition subscale of the SSS and positively, but not significantly, with other subscales. Comparing the low and high scorers on the Disinhibition scale, a significant interaction between groups and stimulus intensities was found but no main effects of stimulus intensity or groups were found. The high Disinhibitors did not differ from the lows at the low stimulus intensities but did differ significantly at the highest intensity where the lows showed a marked reducing tendency. The results show an interesting convergence between the Disinhibition type of sensation seeking, manic tendencies, and the AER.     

Direct screening of clinical specimens for multiple respiratory pathogens using the Genaco Respiratory Panels 1 and 2.

We report here on the results of a pilot study comparing our clinical diagnostic virology laboratory's current methods of respiratory pathogen detection against the Genaco Respiratory Infections Panels 1 and 2. These assays employ xMap (Luminex) liquid phase bead conjugated array technology to facilitate automated detection of PCR and RT-PCR products, which provides potential for levels of assay multiplexing above those currently practical with either conventional gel-resolved or real-time methods. In the study presented here we used the Genaco panels to simultaneously screen previously analyzed clinical specimens (nasopharyngeal washings) for twenty-one important pathogens. Our results indicate the Genaco panels met or exceeded our current methods' sensitivity and specificity although allowing for detection of a wider range of infectious agents than practical by current diagnostic laboratory practices. In addition, the Genaco panels provided data on the presence of multiple respiratory pathogens in single specimens, which would otherwise be missed in most instances. To our knowledge, this study represents the first trial of these panels on standard clinical specimens in a routine diagnostic setting.     

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase

BACKGROUND: Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. OBJECTIVE: To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. METHODS: Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. RESULTS: The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). CONCLUSION: The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.     

Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein

Specific mutations in some tumor suppressor genes such as p53 can act in a dominant fashion. We tested whether this mechanism may also apply for the neurofibromatosis type-2 gene (NF2) which, when mutated, leads to schwannoma development. Transgenic mice were generated that express, in Schwann cells, mutant NF2 proteins prototypic of natural mutants observed in humans. Mice expressing a NF2 protein with an interstitial deletion in the amino-terminal domain showed high prevalence of Schwann cell-derived tumors and Schwann cell hyperplasia, whereas those expressing a carboxy-terminally truncated protein were normal. Our results indicate that a subset of mutant NF2 alleles observed in patients may encode products with dominant properties when overexpressed in specific cell lineages.     

Selection of cytotoxic T lymphocytes against autologous human melanoma from lymph nodes with metastatic melanoma using repeatedin vitro sensitization

Our goal was to determine the cytotoxic activity of effector cells in lymph nodes with metastatic melanoma. Lymphocytes contained within tumor cells from metastatic lymph nodes of two patients were allowed to proliferate in recombinant IL-2 (rIL-2, 100-1,000 units/ml) after 14–21 days of culture. Each set of lymphocytes showed cytotoxicity against autologous melanoma (AM, mean 72%) at effector to target ratio of 201 and K562 cells (mean 60%) using 4-h chromium-51 release assay. Using unlabeled AM and K562, each AM could partially block the activity against K562, but K562 could not block the activity against AM. These activated lymphocytes underwentin vitro sensitization (IVS) with irradiated AM cells and rIL-2 at 2-week intervals. After repeated IVS over about 50 days, each patient's lymphocytes showed cytotoxicity against AM (mean 54%) but not K562 (mean 5%,P < 0.001). These results indicate that different cytotoxic effector cells were present in the early and late phase of lymphocyte tumor culture. Repeated IVS resulted in the selection of specific cytotoxic T lymphocytes. Cold target inhibition assay demonstrated that melanoma cells contained common and individual AM-associated antigen in addition to K562-associated antigens.This work was supported by Biomedical Research Support Grant of the University of Arizona (no. 2S07 RR05675-20), the Elsa U. Pardee Foundation Grant, partly by the Arizona Chronic Disease Research Commission and partly by CA23074 from the National Institutes of Health, Bethesda, 20892, U.S.A.Recipient of the American Cancer Society Clinical Oncology Career Award, 1987–90.     

Viewer-centred and object-centred coding of heads in the macaque temporal cortex

Summary An investigation was made into the sensitivity of cells in the macaque superior temporal sulcus (STS) to the sight of different perspective views of the head. This allowed assessment of (a) whether coding was viewer-centred (view specific) or object-centred (view invariant) and (b) whether viewer-centred cells were preferentially tuned to characteristic views of the head. The majority of cells (110) were found to be viewer-centred and exhibited unimodal tuning to one view. 5 cells displayed object-centred coding responding equally to all views of the head. A further 5 cells showed mixed properties, responding to all views of the head but also discriminating between views. 6 out of 56 viewer and object-centred cells exhibited selectivity for face identity or species. Tuning to view varied in sharpness. For most (54/73) cells the angle of perspective rotation reducing response to half maximal was 45–70° but for 19/73 it was >90°. More cells were optimally tuned to characteristic views of the head (the full face or profile) than to other views. Some cells were, however, found tuned to intermediate views throughout the full 360 degree range. This coding of many distinct head views may have a role in the analysis of social signals based on the interpretation of the direction of other individuals' attention.