The tumor suppressor Caliban regulates DNA damage-induced apoptosis through p53-dependent and -independent activity.

We previously identified Caliban (Clbn) as the Drosophila homolog of human Serologically defined colon cancer antigen 1 gene and demonstrated that it could function as a tumor suppressor in human non-small-cell lung cancer (NSCLC) cells, although its mode of action was unknown. Herein, we identify roles for Clbn in DNA damage response. We generate clbn knockout flies using homologous recombination and demonstrate that they have a heightened sensitivity to irradiation. We show that normal Clbn function facilitates both p53-dependent and -independent DNA damage-induced apoptosis. Clbn coordinates different apoptosis pathways, showing a two-stage upregulation following DNA damage. Clbn has proapoptotic functions, working with both caspase and the proapoptotic gene Hid. Finally, ecotopic expression of clbn(+) in NSCLC cells suppresses tumor formation in athymic nude mice. We conclude that Caliban is a regulator of DNA damage-induced apoptosis, functioning as a tumor suppressor in both p53-dependent and -independent pathways.

decapentaplegic is a direct target of dTcf repression in the Drosophila visceral mesoderm.

Drosophila T cell factor (dTcf) mediates transcriptional activation in the presence of Wingless signalling and repression in its absence. Wingless signalling is required for the correct expression of decapentaplegic (dpp), a Transforming Growth Factor (beta) family member, in parasegments 3 and 7 of the Drosophila visceral mesoderm. Here we demonstrate that a dpp enhancer element, which directs expression of a reporter gene in the visceral mesoderm in a pattern indistinguishable from dpp, has two functional dTcf binding sites. Mutations that reduce or eliminate Wingless signalling abolish dpp reporter gene expression in parasegment 3 and reduce it in parasegment 7 while ectopic expression of Wingless signalling components expand reporter gene expression anteriorly in the visceral mesoderm. However, mutation of the dTcf binding sites in the dpp enhancer results in ectopic expression of reporter gene expression throughout the visceral mesoderm, with no diminution of expression in the endogenous sites of expression. These results demonstrate that the primary function of dTcf binding to the dpp enhancer is repression throughout the visceral mesoderm and that activation by Wingless signalling is probably not mediated via these dTcf binding sites to facilitate correct dpp expression in the visceral mesoderm.

Ultrabithorax protein is necessary but not sufficient for full activation of decapentaplegic expression in the visceral mesoderm.

To elucidate the mechanisms by which homeotic selector (HOM) genes specify the unique features of Drosophila segments, we have analyzed the regulation of decapentaplegic (dpp), a transforming growth factor (TGF)-beta superfamily member, and have found that the Ultrabithorax (Ubx) HOM protein directly activates dpp expression in parasegment 7 (PS7) of the embryonic visceral mesoderm. Other factors are also required, including one that appears to act through homeodomain protein binding sites and may be encoded by extradenticle (exd). The exd protein binds in a highly co-operative manner to regulatory sequences mediating PS7-specific dpp expression, consistent with a genetic requirement for exd function in normal visceral mesoderm expression of dpp. A second mechanism contributing to PS7 expression of dpp appears not to require Ubx protein directly, and involves a general visceral mesoderm enhancer coupled to a spatially specific repression element. Thus, even in an apparently simple case where visceral mesoderm expression of the dpp target gene mirrors that of the Ubx HOM protein, full activation by Ubx protein requires at least one additional factor. In addition, a distinct regulatory mode not directly involving Ubx protein also appears to contribute to PS7-specific expression.

Cross regulation of decapentaplegic and Ultrabithorax transcription in the embryonic visceral mesoderm of Drosophila.

The Drosophila decapentaplegic gene (dpp) encodes a TGF-beta family member involved in signal transduction during embryonic midgut formation. The shortvein (shv) class of cis-regulatory dpp mutants disrupt expression in parasegments 4 and 7 (ps4 and ps7) of the embryonic visceral mesoderm (VM) surrounding the gut and cause abnormalities in gut morphogenesis. We demonstrate that cis-regulatory elements directing expression in ps4 and ps7 are separable and identify DNA fragments that generate ps4 and ps7 expression patterns using reporter gene constructs. dpp reporter gene expression in both ps4 and ps7 is autoregulated as it requires endogenous dpp+ activity. Reporter gene ps7 expression requires the wild-type action of Ultra-bithorax (Ubx), and abdominal-A. Furthermore, the expression of certain Ubx reporter genes is coincident with dpp in the VM. Both the mis-expression of Ubx reporter genes in the developing gastric caecae at ps4 and its normal expression in ps7 are dependent upon endogenous dpp+ activity. We conclude that dpp both responds to and regulates Ubx in ps7 of the visceral mesoderm and that Ubx autoregulation within this tissue may be indirect as it requires more components than have previously been thought.

Evaluation of an automated immunodiagnostic assay system for direct detection of herpes simplex virus antigen in clinical specimens.

The Vitek ImmunoDiagnostic Assay System (VIDAS) is a 2 1/3-h automated qualitative enzyme-linked fluorescent immunoassay developed for the direct detection of herpes simplex virus (HSV) antigen in clinical specimens. A total of 356 clinical specimens submitted for HSV isolation were prospectively evaluated with the VIDAS, and the results of the technique were compared with those of both HSV isolation in cell culture and Herpchek, a nonautomated enzyme immunoassay. Compared to cell culture, VIDAS had a sensitivity of 91.6% and a specificity of 89.3%, with positive and negative predictive values of 82.6 and 95.0%, respectively. In comparison to Herpchek, VIDAS had a sensitivity of 93.7% and a specificity of 93.0%, with positive and negative predictive values of 89.4 and 95.9%, respectively. The results demonstrated that the VIDAS required minimal manipulation in order to produce results comparable to those of Herpchek and HSV isolation in cell culture.

Detection of human cytomegalovirus in clinical specimens by centrifugation culture with a nonhuman cell line.

The sensitivities of MRC-5 and mink lung (ML) cells in centrifugation culture were compared simultaneously for the detection of cytomegalovirus (CMV) IE antigen (immediate-early antigen) from clinical specimens. Of 413 samples assayed, 51 (12%) were positive for CMV by both centrifugation and standard cell culture. At 20 h postinoculation (p.i.), 46 of 51 (90.2%) CMV-positive specimens were detected in ML cells. At 40 h p.i., 50 of 51 (98.0%) CMV-positive specimens were detected in ML cells, compared with 48 of 51 (94.0%) in MRC-5 cells. There was no significant difference in the detection of CMV in either cell line by centrifugation culture. However, in 19 of 23 positive samples that had countable foci at 20 h p.i., there was a 25% increase in the number of positive foci observed for ML cells compared with MRC-5 cells. Less toxicity was also noted for ML cells than for MRC cells, particularly in viral blood specimens. These data suggest that ML cells are comparable to MRC-5 cells for the rapid detection of CMV by centrifugation culture.

Evaluation of a latex particle agglutination assay for the detection of cytomegalovirus antibody in patient serum.

The Virogen CMV Antibody Test is a simple and rapid latex agglutination assay for the detection of cytomegalovirus antibody in human serum and plasma. Evaluation of this assay with respect to enzyme immunoassay yielded a sensitivity of 98% with a specificity of 100%. In comparison to CMVScan, the Virogen CMV Antibody Test had a sensitivity of 98.4% and a specificity of 100%.

Structural analysis of the uEGF gene in the sea urchin strongylocentrotus purpuratus reveals more similarity to vertebrate than to invertebrate genes with EGF-like repeats.

The gene uEGF, a member of the epidermal growth factor family in the sea urchin Stronglyocentrotus purpuratus, is known to express two transcripts that are regulated developmentally in the embryo. We have partially sequenced several uEGF genomic and cDNA clones. We suggest that the smaller transcript is the result of splicing out an internal region present in the larger mRNA, probably with eight EGF-like repeats. The predicted two uEGF products have a signal peptide followed by an EGF-like repeat and a region with approximately 120 amino acids homologous to domain III in complement component C1s. Following these domains, the short product has 12 tandem EGF-like repeats, whereas the long product has approximately 20 tandem repeats. At the carboxy terminus both products have a region homologous to avidin. Unlike Notch and lin-12, no transmembrane domain was found in uEGF. We also show here that uEGF shares two characteristics with vertebrate members of the EGF family, but not with invertebrate members of the same family. (1) All the EGF-like domains sequenced are represented by single exons. (2) All the introns sequenced follow the first nucleotide of a codon. This supports the hypothesis that the organization of the EGF-like domains in vertebrates and in uEGF derived from a common ancestor. Thus, an alternative molecular datum is provided to support the hypothesis of echinoderm-chordate relationships.

Evaluation of a HSV specific monoclonal antibody reagent for laboratory diagnosis of herpes simplex virus infection.

A new FITC-conjugated HSV specific monoclonal antibody reagent (Syva Co., Palo Alto, Ca) was evaluated for the confirmation of HSV clinical isolates. The reagent was also compared to type-specific monoclonal antibodies for the pre-CPE detection of HSV from clinical specimens in centrifugation culture and by direct examination of specimens smears by direct immunofluorescent antibody staining (DFA). HSV was isolated from 75 of 232 specimens (32%). All 75 isolates were confirmed with both the type-specific antibodies and the HSV-specific reagent. In centrifugation culture HSV was detected in 36 of 105 (34%) specimens. The HSV specific reagent detected all 36 specimens that were positive with the type-specific reagents. HSV infection was diagnosed by DFA in 31 of 50 (62%) specimen smears. The HSV-specific reagent detected all 31 positive specimens. This reagent confirmed and detected all HSV positive specimens that were positive by the type-specific monoclonal antibody reagents. The reagent contains monoclonal antibodies specific for both HSV1 and HSV2 in a single mixture, which produces a highly sensitive HSV FA staining reagent.

Detection of herpes simplex virus by using A549 cells in centrifugation culture with a rapid membrane enzyme immunoassay.

The RAMP herpes simplex virus (HSV) culture confirmation test was compared with immunofluorescence (IF) staining with a specific HSV monoclonal antibody reagent for the detection of HSV in centrifugation culture. The RAMP test detected 47 of 57 IF-positive specimens (sensitivity, 88.6%) and agreed with 217 of 220 IF-negative specimens (specificity, 98.6%). The RAMP test can be performed in less than 15 min and gives an immediate visual result. However, the sensitivity and the false-positive and false-negative results need further investigation.

Detection of cytomegalovirus from clinical specimens in centrifugation culture by in situ DNA hybridization and monoclonal antibody staining.

An in situ DNA hybridization kit for cytomegalovirus (CMV) was evaluated for the detection of CMV in centrifugation culture. Of 61 clinical specimens, 17 (27.8%) were positive for CMV by monoclonal antibody staining following centrifugation. Of the 17 positive specimens, 15 were detected by DNA hybridization (24.5%). However, the earliest that CMV could be detected by DNA hybridization was 58 h as compared with 16 h with monoclonal antibodies following centrifugation. DNA hybridization remains of great interest for the study and detection of CMV infection. However, current DNA hybridization techniques are not sufficiently rapid to replace the use of monoclonal antibodies in centrifugation culture.

A sea urchin gene encodes a polypeptide homologous to epidermal growth factor.

A sea urchin DNA clone complementary to an embryonic messenger RNA whose protein product bears striking homology to the epidermal growth factor family of proteins has been identified and characterized. The structure of the protein is similar to that of previously identified regulatory genes in Drosophila and Caenorhabditis. RNA gel blot hybridization showed a unique temporal pattern of expression of this gene during embryogenesis and transcript enrichment in the embryonic ectoderm. These results suggest that this member of the epidermal growth factor gene family plays a role in early development decisions in sea urchin embryos.

Message-specific sequestration of maternal histone mRNA in the sea urchin egg.

Nucleate and anucleate fragments of sea urchin eggs were prepared by centrifugation on sucrose step gradients. The amount of total RNA, poly(A)+ RNA, histone mRNA, actin mRNA, alpha-tubulin mRNA, and mitochondrial rRNA was determined for each fragment. Total RNA, poly(A)+ RNA, actin mRNA, and alpha-tubulin mRNA all distributed in the same ratio as the volume of the fragments. In contrast, the mitochondrial rRNA was found preferentially distributed in the anucleate fragments, coinciding with the distribution of the mitochondria. Histone mRNAs did not follow the fragment volume ratios, but rather were always found associated with the fragment containing the nucleus. To distinguish between nuclear association and possible artifacts associated with centrifugation, eggs were manually cut into nucleate and anucleate fragments and the amount of histone mRNA was determined for each set. Again only the fragments containing the nucleus had detectable amounts of histone mRNA. Although histone mRNAs were always associated with the nucleate fragment, very little histone mRNA was found associated with isolated egg nuclei prepared under gentle isotonic isolation conditions. Furthermore, embryos that have had first nuclear breakdown blocked with 6-dimethylaminopurine still initiated the recruitment of histone mRNAs into polysomes at the same time as control embryos, thus indicating that nuclear breakdown is not necessary for normal histone message utilization. These results demonstrate a message-specific sequestration of maternal histone mRNA which is physically different from that of other maternal mRNAs and which may govern the timing of maternal histone synthesis in sea urchin embryos.