Evidence of a role for the novel zinc-finger transcription factor ZKSCAN3 in modulating Cyclin D2 expression in multiple myeloma.

Dysregulation of cyclin D2 contributes to the pathogenesis of multiple myeloma, and can occur through translocations that activate MAF/MAFB or MMSET/FGFR3. However, cyclin D2 induction can also be seen in the absence of such translocations, such as in patients with hyperdiploid disease, through unknown mechanisms. In UniGene cluster data-mining and ECgene analysis, we found that zinc-finger with KRAB and SCAN domains 3 (ZKSCAN3), a novel transcription factor, is overrepresented in this malignancy, and three consensus ZKSCAN3 binding sites were found in the cyclin D2 promoter. Analysis of a panel of myeloma cell lines, primary patient samples and datasets from Oncomine and the Multiple Myeloma Genomics Portal (MMGP) revealed expression of ZKSCAN3 messenger RNA (mRNA) in a majority of samples. Studies of cell lines by western blotting, and of primary tissue microarrays by immunohistochemistry, showed ZKSCAN3 protein expression in a majority, and in a manner that paralleled messenger levels in cell lines. ZKSCAN3 overexpression was associated with increased gene copy number or genomic DNA gain/amplification in a subset based on analysis of data from the MMGP, and from fluorescence in situ hybridization studies of cell lines and primary samples. Overexpression of ZKSCAN3 induced cyclin D2 promoter activity in a MAF/MAFB-independent manner, and to an extent that was influenced by the number of consensus ZKSCAN3 binding sites. Moreover, ZKSCAN3 protein expression correlated with cyclin D2 levels in cell lines and primary samples, and its overexpression induced cyclin D2. Conversely, ZKSCAN3 suppression using small hairpin RNAs (shRNAs) reduced cyclin D2 levels, and, importantly, inhibited myeloma cell line proliferation. Finally, ZKSCAN3 was noted to specifically bind to oligonucleotides representing sequences from the cyclin D2 promoter, and to the endogenous promoter itself in myeloma cells. Taken together, the data support the conclusion that ZKSCAN3 induction represents a mechanism by which myeloma cells can induce cyclin D2 dysregulation, and contribute to disease pathogenesis.

Indications of proton-dominated cosmic-ray composition above 1.6 EeV.

We report studies of ultrahigh-energy cosmic-ray composition via analysis of depth of air shower maximum (X(max)), for air shower events collected by the High-Resolution Fly's Eye (HiRes) observatory. The HiRes data are consistent with a constant elongation rate d/d[log(E)] of 47.9+/-6.0(stat)+/-3.2(syst) g/cm2/decade for energies between 1.6 and 63 EeV, and are consistent with a predominantly protonic composition of cosmic rays when interpreted via the QGSJET01 and QGSJET-II high-energy hadronic interaction models. These measurements constrain models in which the galactic-to-extragalactic transition is the cause of the energy spectrum ankle at 4x10(18) eV.

Matrix metalloproteinase-10 is a critical effector of protein kinase Ciota-Par6alpha-mediated lung cancer.

Protein kinase Ciota (PKCiota) drives transformed growth of non-small cell lung cancer (NSCLC) cells through the Rho family GTPase Rac1. We show here that PKCiota activates Rac1 in NSCLC cells by formation of a PKCiota-Par6alpha complex that drives anchorage-independent growth and invasion through activation of matrix metalloproteinase-10 (MMP-10) expression. RNAi-mediated knockdown of PKCiota, Par6alpha or Rac1 expression inhibits NSCLC transformation and MMP-10 expression in vitro. Expression of wild-type Par6alpha in Par6alpha-deficient cells restores transformation and MMP-10 expression, whereas expression of Par6alpha mutants that either cannot bind PKCiota (Par6alpha-K19A) or couple to Rac1 (Par6alpha-DeltaCRIB) do not. Knockdown of MMP-10 expression blocks anchorage-independent growth and invasion of NSCLC cells and addition of catalytically active MMP-10 to PKCiota- or Par6alpha-deficient cells restores anchorage-independent growth and invasion. Dominant-negative PKCiota inhibits tumorigenicity and MMP-10 expression in subcutaneous NSCLC tumors. MMP-10 and PKCiota are coordinately overexpressed in primary NSCLC tumors, and tumor MMP-10 expression predicts poor survival in NSCLC patients. Our data define a PKCiota-Par6alpha-Rac1 signaling axis that drives anchorage-independent growth and invasion of NSCLC cells through induction of MMP-10 expression.

First observation of the Greisen-Zatsepin-Kuzmin suppression.

The High Resolution Fly's Eye (HiRes) experiment has observed the Greisen-Zatsepin-Kuzmin suppression (called the GZK cutoff) with a statistical significance of five standard deviations. HiRes' measurement of the flux of ultrahigh energy cosmic rays shows a sharp suppression at an energy of 6 x 10(19) eV, consistent with the expected cutoff energy. We observe the ankle of the cosmic-ray energy spectrum as well, at an energy of 4 x 10(18) eV. We describe the experiment, data collection, and analysis and estimate the systematic uncertainties. The results are presented and the calculation of the statistical significance of our observation is described.

Involvement of PKC-iota in glioma proliferation.

UNLABELLED: Atypical protein kinase C-iota (PKC-iota) protects cells against apoptosis and may play a role in cell proliferation. However, in vivo, the status and function of PKC-iota in human normal brain tissue, gliomas, benign and malignant meningiomas as well as its in vitro status in proliferating and confluent glioma cells, remains unknown. OBJECTIVES: The objectives of our research were to determine whether expression of PKC-iota is altered either in gliomas or in benign and malignant meningiomas, compared to normal brain. In addition, we wished to establish the expression of PKC-iota in proliferating plus in cell cycle-arrested glioma cell lines, as well as the relationship between PKC-iota siRNA on PKC-iota protein content and cell proliferation. MATERIALS AND METHODS: Western blot analyses for PKC-iota were performed on 12 normal brain biopsies, 15 benign meningiomas, three malignant meningiomas and three gliomas. RESULTS: Results demonstrated no (n = 9) or very weak (n = 3) detection of PKC-iota in normal brain tissue. In comparison, PKC-iota was robustly present in the majority of the benign meningiomas. Similarly, PKC-iota was abundant in all malignant meningiomas and gliomas. Western blotting for PKC-iota in confluent or proliferating glioma cell lines depicted substantial quantities of PKC-iota in proliferating T98G and U-138MG glioma cells. In contrast, confluent cells had either 71% (T98G) or 21% (U-138MG) less PKC-iota than proliferating cells. T98 and U-138 MG glioma cells treated with 100 nm PKC-iota siRNA had lower levels of cell proliferation compared to control siRNA-A and complete down-regulation of PKC-iota protein content. CONCLUSION: These results support the concept that presence of PKC-iota may be required for cell proliferation to take place.

Measurement of the flux of ultrahigh energy cosmic rays from monocular observations by the High Resolution Fly's Eye experiment.

We have measured the cosmic ray spectrum above 10(17.2) eV using the two air-fluorescence detectors of the High Resolution Fly's Eye observatory operating in monocular mode. We describe the detector, phototube, and atmospheric calibrations, as well as the analysis techniques for the two detectors. We fit the spectrum to a model consisting of galactic and extragalactic sources.

Memories of childhood abuse: dissociation, amnesia, and corroboration.

OBJECTIVE: This study investigated the relationship between self-reported childhood abuse and dissociative symptoms and amnesia. The presence or absence of corroboration of recovered memories of childhood abuse was also studied. METHOD: Participants were 90 female patients admitted to a unit specializing in the treatment of trauma-related disorders. Participants completed instruments that measured dissociative symptoms and elicited details concerning childhood physical abuse, sexual abuse, and witnessing abuse. Participants also underwent a structured interview that asked about amnesia for traumatic experiences, the circumstances of recovered memory, the role of suggestion in recovered memories, and independent corroboration of the memories. RESULTS: Participants reporting any type of childhood abuse demonstrated elevated levels of dissociative symptoms that were significantly higher than those in subjects not reporting abuse. Higher dissociative symptoms were correlated with early age at onset of physical and sexual abuse and more frequent sexual abuse. A substantial proportion of participants with all types of abuse reported partial or complete amnesia for abuse memories. For physical and sexual abuse, early age at onset was correlated with greater levels of amnesia. Participants who reported recovering memories of abuse generally recalled these experiences while at home, alone, or with family or friends. Although some participants were in treatment at the time, very few were in therapy sessions during their first memory recovery. Suggestion was generally denied as a factor in memory recovery. A majority of participants were able to find strong corroboration of their recovered memories. CONCLUSIONS: Childhood abuse, particularly chronic abuse beginning at early ages, is related to the development of high levels of dissociative symptoms including amnesia for abuse memories. This study strongly suggests that psychotherapy usually is not associated with memory recovery and that independent corroboration of recovered memories of abuse is often present.

Monoclonal antibodies to proteins from cocksfoot grass (Dactylis glomerata) pollen: isolation and N-terminal sequence of a major allergen.

Monoclonal antibodies (Mabs) raised to an aqueous extract of cocksfoot grass (Dactylis glomerata) pollen have been characterised. Mab 1B9 was demonstrated by SDS-PAGE and Western blotting to recognise a major allergen of an approximate molecular weight of 28 kD in this extract, termed DG3, and a component with a molecular weight of between 35 and 40 kD in an extract of Secale cereale (cultivated rye) pollen. The 28 kD component of cocksfoot grass pollen isolated by affinity chromatography using Mab 1B9 was recognised by IgE antibodies in 80% (8 of 10) atopic sera, but only weakly by 25% (1 of 4) non-atopic sera tested. N-terminal sequencing of DG3 purified by affinity chromatography, 2 D electrophoresis and electroblotting to polyvinylidenedifluoride revealed significant homology with a group-V allergen (Phl p V) from timothy grass (Phleum pratense).

Single-dose enoxacin compared with 3-day treatment for urinary tract infection.

Oral treatment of simple urinary tract infections generally involves 5 to 7 days of antibiotic therapy. This study with enoxacin, a new antibacterial agent of the quinolone-azaquinolone class, investigated the efficacy of a single dose compared with 3 days of treatment. A total of 154 outpatients with symptoms of simple cystitis were treated in an open randomized study with enoxacin, either one 600-mg dose or 200 mg twice a day for 3 days. A urine sample was collected for culture before treatment, 7 to 10 days after treatment, and 4 to 6 weeks after treatment. Seventy-three patients had positive bacterial cultures from the pretreatment urine sample; the predominant pathogen was Escherichia coli, along with a number of other gram-negative organisms and Staphylococcus spp. Of these patients, 33 received a single dose of enoxacin and 40 were treated for 3 days. Follow-up examination at 7 to 10 days showed negative urine cultures in 76% of patients from the single-dose group and 89% from the multiple-dose group, a difference that was not statistically significant (P = 0.665, Fisher's exact test). A number of patients were lost to follow-up at 4 to 6 weeks. However, of those who did attend, only three patients were shown to have relapsed or become reinfected (two in the multiple-dose group and one in the single-dose group). Enoxacin was well tolerated in both groups of patients; the few adverse events were mostly mild.

Cloning of cDNA coding for an allergen of Cocksfoot grass (Dactylis glomerata) pollen.

Messenger RNA isolated from Cocksfoot grass (Dactylis glomerata) anthers has been used to generate a cDNA library in lambda t11. Three cDNA clones (7.8, 8.1, and 8.3) were demonstrated to be recognized by human IgE antibodies in atopic serum and by rabbit polyclonal antiserum raised to a crude aqueous extract of Cocksfoot pollen. The size of the cDNA inserts was determined as approximately 700 bp, and restriction mapping demonstrated them to be identical sequences. Lysogens obtained in Escherichia coli Y1089 allowed expression of a 140 kD beta-galactosidase fusion protein containing 24 kD of cloned allergen protein. Fusion proteins were recognized by IgE antibodies in 75% (6/8) of atopic sera tested, but were not detected by nonatopic sera. On the basis of size and frequency of recognition in the atopic population, the cloned protein may present a major allergen. Monoclonal antibodies specific for the major allergen of Cocksfoot pollen were not reactive with the fusion proteins. Reactivity of human IgE antibodies with the fusion protein could be blocked by crude Cocksfoot pollen extract, but not by the major allergen DG3 purified from the extract by affinity chromatography. Human and rabbit antibodies affinity purified against fusion protein 7.8 did not allow identification of the native protein component in crude extract encoded for by the cDNA clones.

Hot air electric hand driers compared with paper towels for potential spread of airborne bacteria.

Hot air hand driers are increasingly used in both public areas and hospitals, but there is little literature on their bacteriology. Four units were examined by comparing the bacterial aerosols released from hands during use by sets of twelve subjects with those released by paper towels. Tests on two units also included hand imprints on agar plates for detection of residual bacteria. No significant difference between aerosols liberated by towels and driers were observed for two units, while the other two generated significantly fewer aerosols than towels. Impression plates revealed similar numbers of bacteria on the hands after drying by either method. Hot air hand driers appear safe from a bacteriological viewpoint.

Iatrogenic superior vena cava syndrome treated with streptokinase.

The development of an iatrogenic superior vena cava syndrome secondary to a thrombosis from an indwelling Hickman catheter in a patient with ovarian carcinoma is presented. The patient was treated with a combination of streptokinase and heparin with successful and dramatic results. Streptokinase appears to be highly effective in the treatment of iatrogenic superior vena cava syndrome from Hickman catheters. It appears that the Hickman catheter may be safely left in situ post-treatment.

A rapid enhanced chemiluminescent immunoassay for allergen-specific IgE antibodies.

An enhanced chemiluminescent immunoassay is described for the measurement of allergen-specific IgE antibodies. The assay is demonstrated for pollen from four grass species. A comparison was made between results obtained by this method and those obtained by the radioallergosorbent (RAST) procedure; a high degree of correlation (r = 0.95) was found for D. glomerata specific IgE. The assay is rapid and can be carried out in under 1 hour. The advantages of the luminescent assay as compared with the RAST procedure are discussed.

Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya).

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3' untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.