Loss of PRP4K drives anoikis resistance in part by dysregulation of epidermal growth factor receptor endosomal trafficking.

Anoikis acts as a critical barrier to metastasis by inducing cell death upon cancer cell detachment from the extracellular matrix (ECM), thereby preventing tumor cell dissemination to secondary sites. The induction of anoikis requires the lysosomal-mediated downregulation of epidermal growth factor receptors (EGFRs) leading to termination of pro-survival signaling. In this study, we demonstrate that depletion of pre-mRNA splicing factor 4 kinase (PRP4K; also known as PRPF4B) causes dysregulation of EGFR trafficking and anoikis resistance. We also report a novel cytoplasmic localization of PRP4K at the late endosome, and demonstrate both nuclear and cytoplasmic localization in breast, lung and ovarian cancer tissue. Mechanistically, depletion of PRP4K leads to reduced EGFR degradation following cell detachment from the ECM and correlates with increased TrkB, vimentin and Zeb1 expression. As a result, PRP4K loss promotes sustained growth factor signaling and increased cellular resistance to anoikis in vitro and in a novel zebrafish xenotransplantation model of anoikis sensitivity, as well as increased metastasis in a mouse model of ovarian cancer. Thus, PRP4K may serve as a potential biomarker of anoikis sensitivity in ovarian and other epithelial cancers.

Splenic abscess.

The character and management of splenic abscess has changed in the past decade. The condition is more frequent, diagnosis is more easily established, and survival is more likely. Seven patients with splenic abscess from 1981-1992 were retrospectively reviewed. These patients had different etiologies for their splenic abscess, including hematogenous bacteria spread, contiguous spread, and previous history of trauma to the spleen or left upper quadrant. Most patients presented clinically with fever, left upper quadrant tenderness, and leukocytosis. All patients underwent CT scanning that was reliably diagnostic. All seven patients underwent splenectomy. Six of the seven patients were discharged from the hospital. We conclude that CT scan remains the gold standard for definitive diagnosis of splenic abscess, and splenectomy is very effective therapy.

Enteritis necroticans with midgut necrosis caused by Clostridium perfringens.

Enteritis necroticans is a necrotizing process manifesting as segmental gangrene of the bowel, triggered by Clostridium perfringens toxins under specific dietary conditions. It is a rare disease in developed countries and is probably underdiagnosed. A case of enteritis necroticans presenting with midgut necrosis with sepsis and hemolysis is reported herein. Bacteriologic culture of blood and peritoneal content revealed C perfringens. Dietary history, including the ingestion of meat together with sweet potatoes, should increase clinical suspicion of enteritis necroticans. Early recognition and timely surgical intervention are required for successful treatment. Clinicians are encouraged to be aware of this clinically fulminant yet rarely recognized surgical entity.

Sequence of the gene encoding an immunodominant microneme protein of Eimeria tenella.

A heterodisperse family of antigens, previously detected on sporozoites and merozoites of Eimeria tenella, has been localised to the microneme organelles within the sporozoite. Sequencing of genomic and cDNA clones shows that the gene for this antigen family contains 4 exons separated by 3 short (519, 226 and 156 nucleotides) intervening sequences and that the predicted polypeptide from the longest open reading frame has 4 structural domains. One of these contains 5 copies of the thrombospondin-like motif, previously identified in the partial sequence of the gene, which is conserved in a variety of molecules which have been demonstrated to have adhesive properties. A second domain of the polypeptide has strong similarity to a conserved region that occurs in another group of molecules which have adhesive properties, including the alpha subunits of several integrins, complement factor Bb and a number of extracellular matrix glycoproteins. Overall the antigen resembles the thrombospondin-related anonymous protein identified in the erythrocytic stage of Plasmodium falciparum. The structure of the gene supports a role for this microneme antigen in cell-cell or cell-matrix interactions.

Regions of an Eimeria tenella antigen contain sequences which are conserved in circumsporozoite proteins from Plasmodium spp. and which are related to the thrombospondin gene family.

Coccidiosis, caused by Eimeria spp., is a major disease of economic importance to the poultry industry. The cloning and characterisation of genes coding for antigens of those species infecting chickens is an initial step in the identification of protective antigens suitable for the development of a genetically engineered vaccine. This report describes the molecular characterisation of an antigen of E. tenella produced by the recombinant lambda amp3 bacteriophage EtHL6. Three native polypeptides corresponding to the EtHL6 antigen, with sizes between 110 and 94 kDa, have been identified on both sporozoites and second generation merozoites of E. tenella by mouse antisera raised against the EtHL6 fusion protein. The DNA insert is a 722-bp EcoRI fragment encoding a polypeptide comprising three tandem blocks of amino acids which are highly homologous to each other. Each region, A, B and C, contains a strongly hydrophilic domain and two pairs of cysteine residues. Computer analysis has identified similarities with a group of proteins which include the circumsporozoite antigen and thrombospondin-related anonymous protein (TRAP) of malaria parasites, human thrombospondin, mouse properdin and the terminal components of the complement pathway.

Isolation of lambda amp3 genomic recombinants coding for antigens of Eimeria tenella.

Eimeria are the causative agents of coccidosis, a disease which is of world wide economic importance in the poultry industry. Immunity resulting from infection is species-specific and both antibody and cell-mediated responses have been implicated. As an initial step in the development of a genetically-engineered vaccine against coccidiosis, libraries of EcoRI-digested genomic DNA from E. tenella have been constructed in Escherichia coli using the expression vector lambda amp3. Screening of the libraries with serum from chickens immunized by infection has identified at least 24 different recombinant phage which produce eimeria antigens fused to beta-galactosidase. A significant proportion of the Eimeria DNA inserts cross-hybridise with each other and contain sequences which are highly represented in the genome. The identification of these clones will enable the isolation of intact genes from E. tenella DNA and facilitate detailed analysis of the antigens and immune responses.

Acute appendicitis in women of childbearing age.

To find a way to decrease the incidence of laparotomies negative for appendicitis, we studied 108 female patients between the ages of 15 and 45 years who had undergone appendectomy with the diagnosis of acute appendicitis. Of these 108 patients, 56 had acute appendicitis and 52 had normal appendixes. The patients between the ages of 15 and 25 years had a 59% incidence of negative laparotomies, in comparison with those patients between 36 and 45 years old, who had an incidence of 22%. In patients with normal appendixes, 18 had no intra-abdominal pathologic findings. Twenty had pelvic inflammatory disease, and nine had ovarian abnormalities. There were no differences in the clinical symptoms, vital signs, roentgenographic findings, or other laboratory studies between the two groups. There were no in-hospital perforated appendixes in the patients who were operated on within 48 hours of admission. To decrease the incidence of negative appendectomies, we recommend in-house observation and simultaneous examination of the patient by the surgeon and a gynecologist.

The use of pNJ5000 as an intermediate vector for the genetic manipulation of Agrobacterium Ti-plasmids.

The use of broad-host-range plasmids derived from RP4 as intermediate vectors for the transfer of narrow-host-range recombinant plasmids from Escherichia coli to Agrobacterium tumefaciens as a preliminary to marker exchange is described. Recombinant plasmids having a ColE1 type origin were linked to the RP4 derivative. Cointegrate formation appeared to take place by RecA-independent, homologous recombination within a short piece of DNA derived from the beta-lactamase gene of Tn1/Tn3 carried by both vector components, so that it never disrupted the recombinant portion of the construction. pNJ5000 provides an unstable intermediate vector for use in marker exchange experiments, while its stable relative pNJ1020 provides a carrier for use in binary vector systems.

Expression of the Pseudomonas gene coding for carboxypeptidase G2 in Saccharomyces cerevisiae.

The Pseudomonas gene coding for carboxypeptidase G2 was introduced into Saccharomyces cerevisiae on an Escherichia coli/yeast shuttle vector pROG5. The level of enzyme activity obtained was independent of the orientation of the gene within the pBR322-derived tetracycline resistance gene of the vector, indicating that expression can occur from a Pseudomonas promoter in yeast.

Identification of the promoter of the Pseudomonas gene coding for carboxypeptidase G2.

A 213-bp region of noncoding DNA upstream of the ATG start codon of the Pseudomonas carboxypeptidase G2 (CPG2) structural gene has been shown to contain the CPG2 promoter. The mRNA start point (+1) on the DNA sequence has been identified by mapping the 5' end of the CPG2 transcript. The identified promoter region contains a -10 region (TATAAG) that closely resembles the Escherichia coli consensus sequence (TATAAT), but has no easily recognisable -35 region. The lack of homology in the -35 region explains why this particular pseudomonad gene is poorly expressed in E. coli. Similar sequence differences may turn out to be the cause of the generally observed inefficient expression of Pseudomonas genes in E. coli. The promoter region also carries a sequence (CTGGCACTCGAATTGCT) that closely matches the consensus nif promoter sequence (CTGGPyAPyPuNNNNTTGCA) of Klebsiella pneumoniae and Rhizobium, and a similar sequence (ATGGCATGGCGGTTGCT) found in the promoter region of the xylABC operon of the TOL plasmid of Pseudomonas putida.

The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor.

The FT37/1 tumor line induced by Agrobacterium tumefaciens on flax epicotyls contains 22-24 copies of the T-DNA encoded nopaline synthase gene per cell. All the gene copies are methylated to some extent but the methylation is not uniform, nor does it reflect the methylation level of the flanking plant DNA. This extensive methylation correlates with an extremely low level of expression of the nopaline synthase gene. Treatment of the tumor line with the in vivo demethylating drug 5-azacytidine at a concentration of 3 X 10(-5) M, resulted in the demethylation of, on average, one copy of the nopaline synthase gene per cell. This demethylation was paralleled by an increase in the transcription of the gene and indicates that cytosine methylation is capable of suppressing the expression of plant genes in vivo.

Nopaline Ti-plasmid, pTiT37, T-DNA insertions into a flax genome.

The FT37/1 plant tumor line induced on flax epicotyls by T37, a nopaline strain of Agrobacterium tumefaciens, contains multiple copies of the pTiT37, T-DNA. There are three to four distinct full-length insertions and one tandem insertion. Allowing for the different copy numbers of the inserts, this amounts to seven to eight T-DNA copies per genome unit. The genome unit in this case is the haploid DNA value (7 X 10(8) bp) which predicts a T-DNA copy number of 14-16 per diploid cell. Three novel types of abnormal insertion are also present. FIL (one copy per basic genome) comprises 3.04-4.47 kb of T-DNA derived from the left end, with a normal left border and an abnormal right border. FIR (four copies per basic genome) comprises 5.88-6.47 kb of T-DNA derived from the right end, with a normal right border and an abnormal left border. The third abnormality is represented by fragment "X," a HindIII fragment of 4.90 kb which contains homology with several noncontiguous regions of the T-DNA and which may derive from a tandem insertion. Of the two possible left-border sites (primary and secondary) in which fusion with plant DNA sequences has been observed, only the primary is used.

The distribution of rabbit alpha-foetoprotein (RAFP) and its molecular variants throughout gestation.

The levels of rabbit alpha-foetoprotein (RAFP) in foetal serum, neonatal serum and amniotic fluid during gestation have been evaluated by quantitative radial immunodiffusion. RAFP attained its peak concentration on the 24th day of pregnancy in foetal serum, while in amniotic fluid peaks were observed on ;the 14th and 26th day. Temporal effects on the heterogeneity of RAFP were analysed by concanavalin A affinity chromatography and agarose electrophoresis. The proportion of concanavalin A non-reactive RAFP decreased during pregnancy in foetal serum, amniotic fluid and yolk sac extracts and these findings were confirmed by crossed affino-immunoelectrophoresis. On ordinary crossed immunoelectrophoresis 14-day foetal serum migrated as a single peak, but by 24 days a second peak was evident. No similar change was detected in amniotic fluid, yolk sac or liver extract. The findings are compared with those from similar studies on other species and a model is proposed to explain the observed heterogeneity of RAFP.