The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. This study reveals phenotypic consequences of whole-animal tetraploidy that make C. elegans an excellent model for ploidy differences.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. Overall, this study reveals the phenotypic consequences of whole-animal tetraploidy in C. elegans.

Two distinct types of murine blast colony-forming cells are multipotential hematopoietic precursors.

Two types of blast colonies can be stimulated to develop in semisolid agar cultures of murine bone marrow cells. Typically, these are either multicentric colonies stimulated by stem cell factor (SCF) plus interleukin-6 (IL-6) or dispersed colonies stimulated by Flt3 ligand (FL) plus IL-6. Both types of blast colony-forming cells (BL-CFCs) can generate large numbers of lineage-committed granulocyte-macrophage progenitor cells and exhibit some capacity for self-generation and the formation of eosinophil and megakaryocyte progenitor cells. However, the two populations of BL-CFCs are largely distinct and partially separable by fluorescence-activated cell sorting and are distinguished by differing capacity to form granulocyte-committed progeny. Both types of BL-CFCs can generate dendritic cells and small numbers of lymphocytes but the FL-responsive BL-CFCs have a greater capacity to form both B and T lymphocytes. Both types of blast colonies offer remarkable opportunities to analyze multilineage commitment at a clonal level in vitro.

Nucleic acid technology screening of Australian blood donors for hepatitis C and human immunodeficiency virus-1 RNA: comparison of two high-throughput testing strategies.

BACKGROUND AND OBJECTIVES: The aim of this study was to compare the performance of two high-throughput strategies for hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) RNA nucleic acid technology (NAT) screening in a volunteer blood-donor population. MATERIALS AND METHODS: The semiautomated Chiron Procleix HIV-1/HCV transcription mediated amplification (TMA) assay was used to screen 1 439 765 donations in two different testing configurations. Three sites (termed PDT sites) performed a mixture of individual donation (ID) and minipool (MP) testing, where 1 113 288 donations were screened as pools of 24 and an additional 32 003 donations were screened in ID format. A further two sites (termed SDT sites) screened 294 474 donations exclusively in ID format. RESULTS: A significantly higher proportion of initial NAT reactives that failed to react on follow-up testing [termed non-repeatably reactive (NRR)] was observed for ID testing at SDT sites than at PDT sites (0.082 vs. 0.047%: P < 0.01). Within the PDT sites, however, there was no significant difference between the NRR rate for MP or ID samples (0.037 vs. 0.047%; not significant). There was a significant difference in failed run rates between PDT and SDT sites (P < 0.01), with PDT sites having a higher run failure rate owing to non-amplification of the internal control. The PDT sites also had a significantly higher overall invalid sample rate. However, the invalid sample rate, specifically caused by known equipment failure, was significantly higher in the SDT sites, possibly attributable to greater usage (P < 0.0001). Four HCV NAT-positive/antibody-negative samples were identified in the course of the study. CONCLUSIONS: The comparison of the performance of PDT with SDT sites identified only minor differences that did not adversely impact on the timely release of blood products. Although both ID and MP strategies showed excellent specificity, irrespective of site configuration, the significantly increased NRR rate, observed exclusively for ID testing performed at SDT sites, indicates a potential for contamination that may limit the number of samples that can optimally be processed using ID testing. The performance data for ID testing in particular should serve as a useful benchmark for evaluating candidate NAT systems that are fully automated.

Membrane interaction of Escherichia coli hemolysin: flotation and insertion-dependent labeling by phospholipid vesicles.

The 1,024-amino-acid acylated hemolysin of Escherichia coli subverts host cell functions and causes cell lysis. Both activities require insertion of the toxin into target mammalian cell membranes. To identify directly the principal toxin sequences dictating membrane binding and insertion, we assayed the lipid bilayer interaction of native protoxin, stably active toxin, and recombinant peptides. Binding was assessed by flotation of protein-liposome mixtures through density gradients, and insertion was assessed by labeling with a photoactivatable probe incorporated into the target lipid bilayer. Both the active acylated hemolysin and the inactive unacylated protoxin were able to bind and also insert. Ca(2+) binding, which is required for toxin activity, did not influence the in vitro interaction with liposomes. Three overlapping large peptides were expressed separately. A C-terminal peptide including residues 601 to 1024 did not interact in either assay. An internal peptide spanning residues 496 to 831, including the two acylation sites, bound to phospholipid vesicles and showed a low level of insertion-dependent labeling. In vitro acylation had no effect on the bilayer interaction of either this peptide or the full-length protoxin. An N-terminal peptide comprising residues 1 to 520 also bound to phospholipid vesicles and showed strong insertion-dependent labeling, ca. 5- to 25-fold that of the internal peptide. Generation of five smaller peptides from the N-terminal region identified the principal determinant of lipid insertion as the hydrophobic sequence encompassing residues 177 to 411, which is conserved among hemolysin-related toxins.

Exposure to GB virus type C or hepatitis G virus in selected Australian adult and children populations.

BACKGROUND: The epidemiology and disease association for the GB virus type C (GBV-C) or hepatitis G virus (HGV) are poorly understood. STUDY DESIGN AND METHODS: This study describes the exposure rates to GBV-C/HGV in diverse Australian population groups by testing for current infection and evidence of past infection with a reverse transcriptase polymerase chain reaction and an anti-E2 enzyme-linked immunosorbent assay, respectively. Subjects included volunteer blood donors, hepatitis C antibody (anti-HCV)-positive donors, children, hemodialysis patients, pregnant women attending a prenatal clinic, injecting drug users (IVDUs), and adult hemophiliacs. RESULTS: Combined GBV-C RNA and E2 antibody prevalence was 6.5 percent (6/93) in children, 13.3 percent (75/565) in blood donors, 14 percent (14/99) in pregnant women, 22.5 percent (18/80) in hemodialysis patients, 80 percent (56/70) in anti-HCV-positive donors, 88.6 percent (31/35) in IVDUs, and 85.7 percent (54/63) in adult hemophiliacs. Children had the lowest antibody rate, 1.1 percent, whereas the rate was 10.8 percent for blood donors and rose to 45.7 percent for IVDUs, 57.1 percent for anti-HCV-positive donors, and 74.6 percent for hemophiliacs. In contrast, current infection rates were comparable for children, blood donors, and pregnant women (5.4, 2.6, and 6%, respectively), rising to 11.1 percent for hemophiliacs, 24.3 percent for anti-HCV-positive donors, and 48.6 percent for IVDUs. Ten of 12 blood donors had persistent viremia, while 2 had recent infections, 1 with apparent resolution. CONCLUSION: Exposure to GBV-C can commence at an early age, although ongoing exposure may also occur among adults with no apparent risk factors. GBV-C RNA positivity was not associated with abnormal plasma alanine aminotransferase levels among blood donors.

An ELISA for the detection of antibody to the core antigen of hepatitis C virus: use in diagnosis and analysis of indeterminate samples.

AIMS: In order to examine more carefully the natural history of hepatitis C virus infection and to determine a role for anti-core in the discrimination of indeterminate samples, a solid phase ELISA to detect antibody of the immunoglobulin G class to the hepatitis C virus core antigen was developed using purified protein expressed in Escherichia coli from a major portion of the core antigen coding region. METHODS/RESULTS: In a study which examined 651 samples submitted for routine testing by a commercial ELISA (Ortho), only 11 samples showed discrepant results; of these, 10 were Ortho ELISA positive, anti-core negative and one was Ortho ELISA negative anticore positive. Supplemental tests showed that 5/10 of these samples were anti-HCV negative by RIBA and the reciprocal 5 were negative for anti-C22 but positive for anti-C100 and anti-C33. The Ortho ELISA negative, anticore positive sample was weakly positive for anti-C22. The anti-core ELISA was then used to examine 67 indeterminate samples from the blood bank; 11/11 samples which were HCV-RNA positive were anti-core positive and 7/56 samples which were HCV-RNA negative were anti-core positive. The anti-core titre was then examined in two groups of indeterminate samples; group 1, polymerase chain reaction-positive, anti-core positive and group 2, polymerase chain reaction-negative, anti-core positive. The geometric mean anti-core titres in these groups were 1 x 10(-3.6) and 1 x 10(-2.3), respectively. Thus in this group of indeterminate samples, all samples (except one) with an anti-core titre > or = 1/200 were polymerase chain reaction-positive, confirming a close correlation between anti-core levels and hepatitis C viraemia. Anti-core was detected with equal efficiency in patients infected with genotypes which differed to that used to express the recombinant core antigen.

The c-cbl proto-oncogene is preferentially expressed in thymus and testis tissue and encodes a nuclear protein.

Cas NS-1 is an acutely transforming murine retrovirus that induces early B-lineage lymphomas and occasional myeloid leukemias. The transforming sequence of this virus, v-cbl, shows no homology to known oncogenes but has some similarities to the yeast transcriptional factor GCN4. In this study we used a v-cbl probe to analyze mRNAs from a wide range of murine and human hemopoietic tumor cell lines and detected an 11-kilobase mRNA in all lineages. In normal mouse tissues the expression of c-cbl was highest in testis and thymus tissues, the predominant species in testis tissue being a 3.5-kilobase mRNA. The v-cbl oncogene was inserted into a bacterial expression vector to produce protein for the immunization of rabbits. Affinity-purified v-cbl antibodies identified abundant levels of p100gag-cbl in Cas NS-1-transformed fibroblasts and lower levels of a 135-kilodalton protein (p135c-cbl) in both normal and transformed cells. Subcellular fractionation showed that p100gag-cbl and p135c-cbl are both located in the nucleus and retained following 420 mM salt extraction. These results indicate that the translational product of a c-cbl is a 135-kilodalton nuclear protein.

Frozen section diagnosis of pancreatic carcinoma: a prospective study of 64 biopsies.

Useful morphologic criteria for frozen section diagnosis of pancreatic and periampullary carcinoma were established by prospective review of 64 frozen sections in this region, with permanent section correlation and patient follow-up. These were divided into three major and five minor criteria based on frequency of occurrence and reproducibility. Major criteria were: 1) nuclear size variation of 4:1 or greater between ductal epithelial cells, 2) incomplete ductal lumens, and 3) disorganized duct distribution. Minor criteria, less frequently and reproducibly observed but valuable diagnostic aids, included: 1) huge, irregular epithelial nucleoli; 2) necrotic glandular debris; 3) glandular mitoses; 4) glands unaccompanied by connective tissue stroma within smooth muscle bundles (periampullary biopsies); and 5) perineural invasion. Combined application of both major and minor criteria is especially helpful in cases complicated by chronic pancreatitis.

Effectiveness of once daily oral iron in the elderly.

The response to once daily oral iron was measured in 12 elderly patients with iron-deficiency anaemia over a six-week period. The average haemoglobin response over six weeks was comparable to the results in younger age groups. We concluded that a once daily iron tablet containing 105 mg of elemental iron is as effective as conventional treatment with multiple doses. The increased cost of a once daily tablet should be balanced by the benefits of improvement in drug compliance.

Rhabdoid tumor of the kidney: complete remission induced by cis-platinum and adriamycin.

A 4-month-old male infant with Stage I rhabdoid tumor of the kidney at presentation subsequently developed pulmonary metastatic disease shortly after diagnosis and initiation of Vincristine and Actinomycin D chemotherapy. The patient was then treated with cis-platinum and Adriamycin. Within 28 days he achieved a complete remission which was maintained for 5 months. Subsequent recurrent pulmonary lesions failed to regress with radiotherapy and high-dose cyclophosphamide when used as single sequential agents. Further clinical trials with cis-platinum and Adriamycin seem warranted since prognosis with this tumor is poor and successful chemotherapy after metastatic dissemination has not been previously reported.

Evolution of reactive lymphadenopathy into lymphoma over a nine-year period. A clinicopathologic study.

Reported is a case of lymphoma which was preceded for nine years by an apparently reactive lymphadenopathy. Original slides of the multiple lymphoid tissue samples are reviewed, with emphasis on the gradually increasing numbers of mitoses, atypical histiocytes, and eosinophils, suggestive of malignant transformation. The scarcity of such cases in the literature prompts this report.