Up-regulation of 12(S)-lipoxygenase induces a migratory phenotype in colorectal cancer cells.

12(S)-Lipoxygenase (LOX) and its product 12(S)-hydroxyeicosatetraenic (HETE) acid have been implicated in angiogenesis and tumour invasion in several tumour types while their role in colorectal cancer progression has not yet been studied. We have analysed 12(S)-LOX expression in colorectal tumours and found gene expression up-regulated in colorectal cancer specimens for which the pathology report described involvement of inflammation. Using cell line models exposed to 12(S)-HETE or over-expressing 12(S)-LOX malignant cell growth as well as tumour cell migration was found to be stimulated. Specifically, Caco2 and SW480 cells over-expressing 12(S)-LOX formed fewer colonies from sparse cultures, but migrated better in filter-migration assays. SW480 LOX cells also had higher anchorage-independent growth capacity and a higher tendency to metastasise in vivo. Knock-down or inhibition of 12(S)-LOX inhibited cell migration and anchorage-independent growth in both 12(S)-LOX transfectants and SW620 cells that express high endogenous levels of 12(S)-LOX. On the cell surface E-cadherin and integrin-ß1 expression were down-regulated in a 12(S)-LOX-dependent manner disturbing cell-cell interactions. The results demonstrate that 12(S)-LOX expression in inflammatory areas of colorectal tumours has the capacity to induce an invasive phenotype in colorectal cancer cells and could be targeted for therapy.

Reliability of lower extremity alignment measurement using radiographs and PACS.

PURPOSE: Lower extremity alignment is an important consideration prior to cartilage surgery and/or osteotomy about the knee. This is measured on full length standing hip to ankle radiographs, which has traditionally been done using hard copy radiographs. However, the advent of PACS (Picture Archiving and Communication Systems) has allowed these measurements to be done on computer based digital radiographs. The objectives of this study were to evaluate the intra- and inter-observer reliability of lower limb alignment measures manually obtained from hard copy radiographs versus using the Philips Easy Vision system, and to assess the subjective ease of use for the two methods. METHODS: Forty-two patients who underwent surgery and who had a standing hip to ankle radiograph on file were identified. Four raters, including two radiologists and two orthopaedic surgeons, measured each hard copy radiograph and computer image on two separate occasions. Three measurements were recorded for each hard copy radiograph and computer image-width of tibial plateau, the distance from the medial aspect of the tibial plateau to the weight-bearing line, and the mechanical axis. RESULTS: All correlations for this study were high. For tibial plateau data, the hard copy radiographs compared to PACS demonstrated intra-class correlation coefficients (ICC) ranging from 0.93 to 0.99 for inter-rater reliability for the four raters. The ICC for intra-rater reliability for hard copies ranged from 0.90 to 0.99 and for PACS from 0.94 to 0.99. The inter-rater data comparing raters ranged from 0.87 to 0.98 for hard copy radiographs and from 0.98 to 0.99 for PACS. For mechanical axis data, the ICC for hard copy radiograph compared to PACS ranged from 0.93 to 0.97 for the intra-rater reliability for the four raters. The intra-rater reliability for mechanical axis data on hard copy radiograph ranged from an ICC of 0.86 to 0.96, and for PACS the ICC ranged from 0.93 to 0.99. The inter-observer data for hard copy radiographs using the mechanical axis ranged from 0.88 to 0.94 and for PACS ranged from 0.93 to 0.97. The physicians rated PACS as statistically significantly easier to use when compared to hard copy (P = 0.03). CONCLUSION: Evaluation of lower extremity alignment using two techniques prior to knee surgery was found to have higher inter- and intra-observer reliability using PACS software. PACS is now used prior to cartilage surgery and/or osteotomy to measure both alignment and the location of the weight bearing line on the tibial plateau both before and after surgery. LEVEL OF EVIDENCE: Diagnostic study, Level I.

Bridging the gap--biocompatibility of microelectronic materials.

There is an increasing interest in cell-based microelectronic biosensors for high-throughput screening of new products from the biotech pipeline. This requires fundamental knowledge of the biocompatibility of the materials used as the growing support for the cells. Using monolayer-forming Caco-2 cells of human origin, the biocompatibility of silicon wafers coated with various metals, dielectrics and semiconductors was assessed. Besides microscopic inspection, proliferation of cells indicating viability as well as brush border enzyme activity indicating differentiation of adherent growing cells were chosen as parameters to estimate biocompatibility. The type of wafer used for deposition of the coating initially influences the biocompatibility of the final product. Whereas p-doped silicon was fully biocompatible, n-doped silicon reduced the proliferation of cells. Among the different coatings, Al and Ti even increased the cell growth as compared to glass. Culturing the cells for 6 days on coated wafers demonstrated that the differentiation of adhering cells on Ti- and ZrO2-coated wafers was comparable to glass, whereas coatings with Si3N4, Au, Al, and ITO reduced differentiation to 15-35%. In the cases of Au and Si3N4 this effect equilibrated with prolonged culturing. These results demonstrate the importance of a careful selection of the materials used for the production of cell-based biosensors.

Targeting of the gene product encoded by ORF UL56 of human cytomegalovirus into viral replication centers.

The highly conserved DNA-binding protein pUL56 of human cytomegalovirus (HCMV) was found to be predominantly localized throughout the nucleus as well as in viral replication centers of infected cells. The latter localization was abolished by phosphono acetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL56 co-localized in replication centers alongside pUL112-113 and pUL44 at late times of infection. By co-immunoprecipitations, a direct interaction with pUL44, a protein of the replication fork, was detected. These results showed for the first time that HCMV pUL56 is localized in viral replication centers, implicating that DNA replication is coupled with packaging.

Multimerization potential of the cytoplasmic domain of the human cytomegalovirus glycoprotein B.

In the present study the coding sequence of the cytoplasmic tail of the human cytomegalovirus glycoprotein B (gB) was expressed. The secondary structure of the purified recombinant protein was analyzed by circular dichroism, and the quaternary structure was investigated by gel permeation chromatography, and electron microscopy. Our data indicate that the cytoplasmic gB domain contains alpha-helix structures and assembles into tetramers, suggesting that the authentic gB may represent a homotetramer.

Analysis of frozen sections of intraoperative specimens obtained at the time of reoperation after hip or knee resection arthroplasty for the treatment of infection.

BACKGROUND: Despite the effectiveness of a two-stage exchange protocol for the treatment of deep periprosthetic infection, infection can persist after resection arthroplasty and treatment with antibiotics, leading to a failed second-stage reconstruction. Intraoperative analysis of frozen sections has been shown to have a high sensitivity and specificity for the identification of infection at the time of revision arthroplasty; however, the usefulness of this test at the time of reoperation after resection arthroplasty and treatment with antibiotics is, to our knowledge, unknown. METHODS: The medical records of sixty-four consecutive patients who had had a resection arthroplasty of either the knee (thirty-three patients) or the hip (thirty-one patients) and had had intraoperative analysis of frozen sections of periprosthetic tissue obtained at the time of a second-stage operation were reviewed. The mean interval between the resection arthroplasty and the attempted reimplantation was nineteen weeks. The results of the intraoperative analysis of the frozen sections were compared with those of analysis of permanent histological sections of the same tissues and with those of intraoperative cultures of specimens obtained from within the joint. The findings of the analyses of the frozen sections and the permanent histological sections were considered to be consistent with acute inflammation and infection if a mean of ten polymorphonuclear leukocytes or more per high-power field (forty times magnification) were seen in the five most cellular areas. RESULTS: The intraoperative frozen sections of the specimens from two patients (one of whom was considered to have a persistent infection) met the criteria for acute inflammation. Four patients were considered to have a persistent infection on the basis of positive intraoperative cultures or permanent histological sections. Overall, intraoperative analysis of frozen sections at the time of reimplantation after resection arthroplasty had a sensitivity of 25 percent (detection of one of four persistent infections), a specificity of 98 percent, a positive predictive value of 50 percent (one of two), a negative predictive value of 95 percent, and an accuracy of 94 percent. CONCLUSIONS: A negative finding on intraoperative analysis of frozen sections has a high predictive value with regard to ruling out the presence of infection; however, the sensitivity of the test for the detection of persistent infection is poor.

The human cytomegalovirus glycoprotein B gene (ORF UL55) is expressed early in the infectious cycle.

Northern hybridizations were carried out using mRNA preparations of human cytomegalovirus (HCMV)-infected cultures and gene-specific antisense RNA probes for transcriptional analysis of the gene cluster composed of genes for DNA polymerase, glycoprotein B (gB), herpes simplex virus-infected cell protein 18.5 homologue p130 and a major DNA-binding protein corresponding to open reading frames (ORFs) UL54-UL57, respectively. Monocistronic transcripts of 5 kb and 3.7 kb were found for ORFs UL54 and UL55, respectively, and five additional high molecular mass overlapping transcripts of 14 kb, 10 kb, 10 kb, 8 kb and 6 kb were found. Mapping of 5' ends showed that transcription was initiated at the expected distance downstream of predicted TATA elements; in the case of a UL56-specific transcript two potential initiation sites were identified. Transcription was found to terminate at the expected distance downstream of either of two prominent polyadenylation consensus motifs in the region of UL54. All transcripts were identified early in the infectious cycle, except for the UL55 (gB)-specific transcript of 3.7 kb which was not synthesized until late post-infection. However, specific immunoreactions demonstrated the presence of a gB-specific polypeptide early after infection in the absence of viral DNA synthesis. It is suggested that a bicistronic transcript of 8 kb encoded by ORFs UL55 and UL54 is involved in biosynthesis of early HCMV gB.

A 15-kilobase-pair region of the human cytomegalovirus genome which includes US1 through US13 is dispensable for growth in cell culture.

The genome of a temperature-sensitive, DNA-negative mutant of human cytomegalovirus was cloned in cosmids and analyzed by restriction endonuclease mapping and Southern blotting. The data presented show that in the mutant genome, nearly half of the short segment was deleted (14.3 to 15.1 kb; map position, 0.83 to 0.9), including the genes for a potential immediate early protein (US3) and a structural glycoprotein of 47 to 52 kDa (US6 through US11). The deleted DNA region was replaced by a 20.8- to 21.6-kb fragment that represented an inverted repetition of the retained portion of the short segment (map position, 0.92 to 1.0), suggesting that US20 through US36 were duplicated in the mutant. Northern (RNA) blots with appropriate probes of total cell RNA extracted from mutant-infected cells confirmed the absence of mRNAs originating from US3 or from US8 through US11. It is concluded that the deleted genes are dispensable for human cytomegalovirus replication in cell culture.

The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity.

Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in these cis-acting motifs, 5'-TAAAAA-3' (pac 1) and 5'-TTTTAT-3' (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.

Identification of the gene product encoded by ORF UL56 of the human cytomegalovirus genome.

Experiments were undertaken to identify the product of open reading frame (orf) UL56 of human cytomegalovirus (HCMV), the putative homolog of infected cell protein (ICP) 18.5 of herpes simplex virus type 1 (HSV-1) that is thought to be involved in viral nucleocapsid maturation. Northern blotting using a unique fragment of orf UL56 revealed a specific transcript of about 3.0 kb in HCMV-infected fibroblasts early and late postinfection (p.i.). Two overlapping fragments of UL56 were subcloned for procaryotic expression and the recombinant proteins were used for affinity purification of an antibody fraction from human convalescent serum (pab UL56). The affinity purified antibody recognized a polypeptide with an apparent molecular weight of about 130 kDa (p 130) in immunoblots of virions and of infected cell extracts late p.i. Upon treatment of isolated virions with nonionic detergent the p 130 polypeptide separated with the nucleocapsid/tegument fraction. The amino acid sequence of p 130 deduced from the nucleotide sequence exhibited a high homology to ICP 18.5 of HSV-1.

Stable constitutive expression of glycoprotein B (gpUL55) of human cytomegalovirus in permissive astrocytoma cells.

Permanent cell lines showing homogeneous constitutive expression of glycoprotein B (gpUL55; gB) of human cytomegalovirus (HCMV) were selected, in the presence of geneticin, from human astrocytoma cells (U373) after transfection with recombinant pRC/CMV-gB carrying the complete coding sequence for HCMV gB and for aminoglycoside phosphotransferase. The biosynthesis and processing including specific proteolytic cleavage, formation of disulphide-linked oligomers as well as transport of recombinant gB in three of four established transformed cell lines essentially resembled that found in infected parental U373 except for eventual degradation after 2 h of gB synthesis. Analysis of the fourth transformant expressing uncleaved gB suggested that proteolytic cleavage is not required for normal intracellular transport. The stable transformants retained permissiveness for productive superinfection with HCMV. The application of cell lines transformed with mutagenized HCMV gB for the rescue of genetically engineered HCMV mutants is discussed.

Structural analysis of the US-segment of a viable temperature sensitive human cytomegalovirus mutant.

Structural analysis of the US-segment of a viable temperature sensitive human cytomegalovirus mutant (ts9) by the use of restriction enzymes, specific amplifications by the polymerase chain reaction and DNA sequencing, revealed deletions of open reading frames (orf) US14 and part of US15 in addition to that concerning US1 through US13 as reported previously [9]. It was further verified that the Hind III H-fragment and a major portion of the Hind III W-fragment were duplicated in inverted orientation. By the use of a monospecific antibody prepared against a procaryotic recombinant US11-specific product it was shown that the ts9 mutant was in fact deficient in a 30 kDa polypeptide, the gene product of orf US11.

Nuclear translocation of mutagenized forms of human cytomegalovirus glycoprotein B (gpUL55).

To define structural elements involved in translocation of human cytomegalovirus (HCMV) glycoprotein B (gB) to the inner nuclear membrane (INM) compartment, mutagenized gB derivatives with deletions of the potential membrane anchor domains or of portions of the cytoplasmic tail were stably expressed in human astrocytoma cells. Subcellular localization examined by immunofluorescence and cell fractionation suggested that all gB derivatives reached the INM; however, reduced amounts were found after deletion of the extreme carboxy terminus [amino acids 856-906; gB(Del3)]. Pulse-chase analysis revealed accumulation in nuclear fractions of all gB derivatives during the chase, except for gB(Del3), which exhibited impaired nuclear retention. A carboxy-terminal nucleoplasmin-like signal localized within the respective deletion may thus be involved in nuclear transport and retention of HCMV gB. Immunoprecipitation after 32P-radiolabelling of the gB transfectants verified that the gB molecule is phosphorylated at a carboxy-terminal consensus motif for casein kinase II.

Identification of glycoprotein gpTRL10 as a structural component of human cytomegalovirus.

Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.

Recognition of compartmentalized intracellular analogs of glycoprotein H of human cytomegalovirus.

Infected cell proteins immunoprecipitated from human cytomegalovirus (HCMV)-infected fibroblasts with glycoprotein H (gH)-specific conformation-dependent monoclonal antibody (mab 14-4 b) were found to consist of three components of 86 kDa, 89 kDa, and 125 kDa (gp 86, 89, and 125). Affinity purified antibodies from human convalescent serum reactive with an NH2-terminal epitope of gH recognized three polypeptides of comparable size in immunoblots, suggesting antigenic relatedness of these three components of the gH-complex. Using subcellular fractions for immunoblotting, gp 86 was identified as an endoglycosidase H (endo H)-sensitive gH-form present in the nuclear fraction whereas gp 89 and gp 125 were endo H-resistant and present in the membrane fraction or in virions. Incomplete endo H-digestion suggested that four of six predicted N-glycosylation sites of the gH molecule were occupied by carbohydrate side chains. Analysis under nonreducing conditions revealed that the compartmentalized as well as virion-associated gH analogs form high molecular weight complexes. The relation of the recognized gH analogs to the processing pathway of gH is discussed.

Inhibition of human cytomegalovirus maturation by brefeldin A.

Brefeldin A (BFA) was found to interfere with specific events of human cytomegalovirus (HCMV) maturation in human fibroblasts. Ultrastructural as well as biochemical studies suggested that short-term exposure of infected cultures to BFA during the late infectious cycle primarily prevented Golgi-dependent processes, e.g. envelopment of naked cytoplasmic nucleocapsids in the trans-Golgi network (TGN) and normal processing of glycoprotein B. In contrast, the nuclear phase of viral morphogenesis, e.g. transport budding at the nuclear envelope, was not impaired. These observations were compatible with the interpretation that HCMV morphogenesis may involve sequential budding events at the nuclear envelope and at cisternae of the TGN. BFA treatment during the early infectious cycle efficiently inhibited HCMV-DNA synthesis and thus late viral functions, preventing production of viral progeny. Cytotoxicity was excluded as a cause for these findings.

Thyroid function after surgery for autonomous and non-autonomous nodular endemic goitre--effect of iodide-substitution.

The aim of this study was to evaluate the influence of postoperative iodide-substitution on the function of thyroid remnants of different quality and quantity in order to define the appropriate prophylaxis (iodide or thyroid hormone) to prevent recurrent goitre. In a prospective, randomized clinical trial, the following patients were examined: group I: simple, non-autonomous nodular goitre, bilateral thyroidectomy (n = 40); group II: simple, non-autonomous nodular goitre, "selective" (unilateral) thyroidectomy (n = 40); group III: autonomous nodular goitre, bilateral thyroidectomy (n = 40); group IV: autonomous nodular goitre, "selective" (unilateral) thyroidectomy (n = 35). The following parameters were measured 6 and 12 weeks postoperatively. Serum-total-T4, -T3, -TSH, TRH-test, 99mTc-Thyroid-Uptake (TcTU). Six weeks postoperatively the 4 groups were separately randomized into controls and treatment groups, who received 200 micrograms iodide/day orally. Six weeks postoperatively, patients in group I had lower T4 levels and both basal and stimulated TSH were higher than in the other groups, however no significant differences were observed in T3, T4/T3 ratio and TcTU. Twelve weeks postoperatively patients from groups I, II and III, who had been treated with iodide, had lower T3 and TcTU values but higher T4 and T4/T3 than the appropriate controls. Basal and stimulated TSH showed no differences between controls and iodide-treated patients in these groups. In group IV, T4 and T3 showed a tendency to elevation (n.s.), and basal and stimulated TSH as well as TcTU were lower in patients with iodide.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrieval of human cytomegalovirus glycoprotein B from the infected cell surface for virus envelopment.

Surface biotinylation of human cytomegalovirus (HCMV)-infected fibroblasts under pulse-chase conditions was used to define the cellular route of the dominant viral envelope glycoprotein gB into the cytoplasmic compartment of viral maturational envelopment. The results showed that a major fraction of gB was re-internalized from the infected cell surface prior to incorporation into the viral envelope. Viral particles carrying biotinylated gB were subsequently released into the culture medium. Viral release appeared to be inhibited in the presence of gB-specific antibody or when infected cultures were incubated at room temperature, but was not reduced by inhibitors of cellular glycoprotein transport. To our knowledge this is the first report describing that HCMV gB is retrieved from the infected cell surface prior to viral envelopment.

Wear rates of ceramic-on-ceramic bearing surfaces in total hip implants: a 12-year follow-up study.

A retrospective clinical and radiographic analysis was performed on 58 patients (60 hips; mean age at time of surgery, 45.2 years) at a minimum of 10-year follow-up (mean, 12.7 years) after total hip replacement using a ceramic-on-ceramic hearing total hip implant (Autophor, Smith and Nephew, Memphis, TN). Mean wear rate at final follow-up was 0.21 mim, averaging 0.016 mm/y. There were no cases of periprosthetic osteolysis in the acetabuulum or femur. For the unrevised components, there were 3 (5%) cases of protrusio acetabuli and 4 (7%) cases of acetabular component loosening. On the femoral side, 78.3% had distal pedestal formation, and 83% had greater than 2 mm implant-bone radiolucencies in more than 5 Gruen zones as a result of gross motion of the stem. Despite radiographic evidence of implant loosening, this hard bearing articulation functioned well in vivo for more than 12 years with remarkably low wear--approximately one tenth the rate reported for metal-on-polyethylene total hip bearings.