Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

Background

The CXCR4 chemokine receptor regulates migration and homing of cancer cells to specific metastatic sites. Determination of the CXCR4 receptor status will provide predictive information for disease prognosis and possible therapeutic intervention. However, previous attempts to localize CXCR4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have produced predominant nuclear and occasional cytoplasmic staining but did not result in the identification of bona fide cell surface receptors.

Methodology/Principal Findings

In the present study, we extensively characterized the novel rabbit monoclonal anti-CXCR4 antibody (clone UMB-2) using transfected cells and tissues from CXCR4-deficient mice. Specificity of UMB-2 was demonstrated by cell surface staining of CXCR4-transfected cells; translocation of CXCR4 immunostaining after agonist exposure; detection of a broad band migrating at M _r 38,000–43,000 in Western blots of homogenates from CXCR4-expressing cells; selective detection of the receptor in tissues from CXCR4+/+ but not from CXCR4−/− mice; and abolition of tissue immunostaining by preadsorption of UMB-2 with its immunizing peptide. In formalin-fixed, paraffin-embedded human tumor tissues, UMB-2 yielded highly effective plasma membrane staining of a subpopulation of tumor cells, which were often heterogeneously distributed throughout the tumor. A comparative analysis of the mouse monoclonal antibody 12G5 and other frequently used commercially available antibodies revealed that none of these was able to detect CXCR4 under otherwise identical conditions.

Conclusions/Significance

Thus, the rabbit monoclonal antibody UMB-2 may prove of great value in the assessment of the CXCR4 receptor status in a variety of human tumors during routine histopathological examination.     

Support for the allotonic frequency hypothesis in an insectivorous bat community

The allotonic frequency hypothesis proposes that certain insectivorous bat species can prey upon moths that can hear bat echolocation calls by using echolocation frequencies outside the sensitivity range of moth ears. The hypothesis predicts that the peak frequencies of bat echolocation calls are correlated with the incidence of moths in the diets of these bats. The aim of this study was to test this prediction on a bat community dominated by bats using low duty cycle echolocation calls, i.e. aerial foraging, insectivorous species using frequency modulated calls. The community consisted of nine species, two molossids, Sauromys petrophillus and Tadarida aegyptiaca, five vespertilionids, Eptesicus capensis, Eptesicus hottentotus, Miniopteris schreibersii, Myotis tricolor, and Myotis lesueuri, one rhinolophid, Rhinolophus clivosus, and one nycterid, Nycteris thebaica. The insect fauna in the habitat used by the bat community was suited to the testing of the allotonic frequency hypothesis because more than 90% of the moths comprising the insect fauna were tympanate. These included Pyralidae (3.8%), Geometridae (44.9%), Notodontidae (3.8%), Arctiidae (4.6%), Lymantriidae (0.8%) and Noctuidae (32.4%). As predicted, peak echolocation frequency was correlated with the incidence of moths in the diets of these nine species (r=0.98, df=7, P<0.01). Furthermore, multivariate analysis revealed that echolocation frequency (t=9.91, n=129, P<0.001) was a better predictor of diet than forearm length (t=5.51, n=129, P<0.001) or wing area (t=-3.41, n=129, P<0.001). This suggests that the selection pressure exerted by moth hearing might have acted directly on call frequency and secondarily on body size and wing morphology, as part of the same adaptive complex. It is unlikely that dietary differences were due to temporal and spatial differences in the availability of prey because the pattern of differences in skull morphology of the nine species supported our dietary analyses. The skull morphology of a bat represents a historical record of the kind of diet it has become adapted to over its evolutionary history. These results suggest that prey defences may mediate other factors structuring bat communities, e.g. competition. Competition may be reduced for those species of bats that can circumvent prey defences.     

A fluorescence-based homogeneous assay for measuring activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is one of the key enzymes of bacterial lipid A biosynthesis, catalyzing the removal of the N-acetyl group of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. The lpxC gene is essential in Gram-negative bacteria but absent from mammalian genomes, making it an attractive target for antibacterial drug discovery. Current assay methods for LpxC are not suitable for high throughput screening, since they require multiple product separation steps and the use of radioactively labeled material that is difficult to prepare. A homogeneous fluorescence-based assay was developed that uses UDP-3-O-(N-hexyl-propionamide)-N-acetylglucosamine as a surrogate substrate. This surrogate can be prepared from commercially available UDP-GlcNAc by enzymatic conversion to UDP-MurNAc, which is then chemically coupled to n-hexylamine. Following the LpxC reaction, the free amine of the deacetylation product can be derivatized by fluorescamine, thus generating a fluorescent signal. This surrogate substrate has a K(m) of 367 microM and k(cat) of 0.36 s(-1), compared to 2 microM and 1.5 s(-1) for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC(50)s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.     

IFN-beta pretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis

All human melanoma cell lines (assessed by annexin V and TUNEL assays) were resistant to apoptosis induction by TRAIL/Apo2L protein. TRAIL/Apo2L activated caspase-8 and caspase-3, but subsequent apoptotic events such as poly(ADP-ribose) polymerase cleavage and DNA fragmentation were not observed. To probe the molecular mechanisms of cellular resistance to apoptosis, melanoma cell lines were analyzed for expression of apoptosis regulators (apoptotic protease-associated factor-1, FLIP, caspase-8, caspase-9, caspase-3, cellular inhibitor of apoptosis, Bcl-2, or Bax); no correlation was observed. TRAIL/Apo2L was induced in melanoma cell lines by IFN-beta and had been correlated with apoptosis induction. Because IFN-beta induced other gene products that have been associated with apoptosis, it was postulated that one or more IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma cell lines were treated with IFN-beta for 16-24 h before treatment with TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone, >30% of cells underwent apoptosis in response to the combined treatment. Induction of apoptosis by IFN-beta and TRAIL/Apo2L in combination correlated with synergistic activation of caspase-9, a decrease in mitochondrial potential, and cleavage of poly(ADP-ribose) polymerase. Cleavage of X-linked inhibitor of apoptosis following IFN-beta and TRAIL/Apo2L treatment was observed in sensitive WM9, A375, or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in vitro IFN-beta and TRAIL/Apo2L combination treatment had more potent apoptotic and anti-growth effects when compared with either cytokine alone in melanoma cells lines.     

How might intensification of farming influence dung beetle diversity (Coleoptera: Scarabaeidae) in Maputo Special Reserve (Mozambique)?

There are concerns over the increasing encroachment of humans, domestic livestock, and farming onto Maputo Special Reserve because of the potential for habitat modification. Therefore, differences between an undisturbed area of the reserve and a neighbouring farming area are assessed using dung beetle as indicators. In each of the two areas, pig-dung-baited pitfall traps were used to sample dung beetle assemblages in two contrasting habitats, grassland and forest. Distributional analysis of the 57 species and 36 942 individuals that were captured, showed that species richness, species turnover, relative abundance patterns, and biogeographical composition differed strongly between both habitats and areas under different land usage. However, in analyses that apportion variation, the greatest amounts were related to habitat rather than land usage. Even so, in both habitats, the total and mean number of species per trap was higher in the farmed area than in the reserve although this was a significant trend only in grassland. Furthermore, in grassland, widespread species were better represented in the farmed area than in the reserve whereas in forest, widespread species were poorly represented compared to grassland. Also in forest, Maputaland endemics were better represented in the reserve than in the farmed area. Further work is necessary to separate the different geographical, ecological, and land usage factors responsible for the patterns detected in this preliminary study. Even so, there are clearly differences between the Maputo Special Reserve and the farmed area.     

Role of beta-alanine in cuticular tanning,sclerotization, and temperature regulation in Drosophila melanogaster

Injection of 20 nl of 1.0 M beta-alanine, about the minimal amount needed to produce wild-type tanned phenomenocopies from newly eclosed mutant black Drosophila melanogaster, increases stiffness and puncture-resistance of the wing cuticle. Increasing the concentration of beta-alanine to 2.0 M increases puncture-resistance further. Injection of 1.0 M of the beta-alanine analogue beta-aminoisobutyric acid, does not induce tanning or puncture-resistance, nor does injection of 1.0 M dopamine. However, injection of 1.0 M beta-alanine and 1.0 M dopamine increases puncture-resistance more than an injection of 1.0 M beta-alanine, though not more than an injection of 2.0 M beta-alanine alone. Within 10 min after injection of [3-³H]beta-alanine into newly eclosed normal flies, ³H becomes 8.7 times more concentrated in the cuticle than in an equal area of underlying epidermis. ³H is excluded from the epidermis or cuticle of ebony strains. Ebony strains show a deficiency of cuticular electron-absorbing material, and the cuticular lamellae show a tendency to separate from each other. Compaction of the chitinous lamellae is induced in alkali-detanned pupal sheaths by exposure to nascent quinones of N-acetyldopamine or N-beta-alanyldopamine. Glucosamine, but not N-acetylglucosamine, reacts with such quinones in tanning reactions. Under an infrared beam, black cuticular pigmentation induces more rapid heating of haemocoel fluids than does tan pigmentation. A theory of pigmentation and sclerotization relative to environmental adaptation is presented.     

Psychiatric Diagnosis Revisited: Towards a System of Staging and Profiling Combining Nomothetic and Idiographic Parameters of Momentary Mental States

Background

Mental disorders may be reducible to sets of symptoms, connected through systems of causal relations. A clinical staging model predicts that in earlier stages of illness, symptom expression is both non-specific and diffuse. With illness progression, more specific syndromes emerge. This paper addressed the hypothesis that connection strength and connection variability between mental states differ in the hypothesized direction across different stages of psychopathology.

Methods

In a general population sample of female siblings (mostly twins), the Experience Sampling Method was used to collect repeated measures of three momentary mental states (positive affect, negative affect and paranoia). Staging was operationalized across four levels of increasing severity of psychopathology, based on the total score of the Symptom Check List. Multilevel random regression was used to calculate inter- and intra-mental state connection strength and connection variability over time by modelling each momentary mental state at t as a function of the three momentary states at t-1, and by examining moderation by SCL-severity.

Results

Mental states impacted dynamically on each other over time, in interaction with SCL-severity groups. Thus, SCL-90 severity groups were characterized by progressively greater inter- and intra-mental state connection strength, and greater inter- and intra-mental state connection variability.

Conclusion

Diagnosis in psychiatry can be described as stages of growing dynamic causal impact of mental states over time. This system achieves a mode of psychiatric diagnosis that combines nomothetic (group-based classification across stages) and idiographic (individual-specific psychopathological profiles) components of psychopathology at the level of momentary mental states impacting on each other over time.     

Immunohistochemical localization of prostaglandin H synthase in the sheep placenta from early pregnancy to term.

Prostaglandin H synthase (PGHS) activity within intrauterine tissues is considered to catalyze a critical step in prostaglandin (PG) biosynthesis at parturition. In sheep, the placenta is a major site of PG production throughout pregnancy, but little information is available concerning the cells that are responsible. Therefore we determined the distribution of immunoreactive (IR-) PGHS in ovine placental tissue obtained at different times of pregnancy using immunohistochemical techniques. In placentomes from early pregnancy (Days 30-54), IR-PGHS was present in maternal epithelial syncytium, but was not detectable in trophoblast cells. Between Day 54 and Day 100, the number of cells that stained positive for PGHS declined in the maternal epithelial layer in the body of the placenta, but IR-PGHS was present in maternal epithelial cells overlying the vascular cones of the placental hemophagous zone. It was also present in the chorionic fibroblasts, but remained undetectable from all classes of trophoblast cells. IR-PGHS was first detectable in the trophoblastic epithelium by Day 114. Between Day 119 and term the trophoblast mononuclear epithelial cells were intensely immunopositive for PGHS, although immunonegative binucleate cells were present. The maternal epithelium was immunonegative except during the last 7-10 days of pregnancy when PGHS immunostaining appeared in both basal and apical regions of the placenta. Thus, the cellular localization of IR-PGHS changes during ovine pregnancy, from predominantly maternal during the first half of gestation to undetectable and then to predominantly trophoblastic between Day 114 and term, suggesting a gestation-dependent change in sites of PG production during ovine pregnancy. Appearance of IR-PGHS in the trophoblast precedes activation of the fetal hypothalamic-pituitary-adrenal axis, generally considered to provide the trigger to the onset of parturition in sheep, and would therefore appear to be regulated through alternative pathways or mechanisms.