Understanding liver regeneration to bring new insights to the mechanisms driving cholangiocarcinoma.

Cancer frequently arises in epithelial tissues subjected to repeated cycles of injury and repair. Improving our understanding of tissue regeneration is, therefore, likely to reveal novel processes with inherent potential for aberration that can lead to carcinoma. These highly conserved regenerative mechanisms are increasingly understood and in the liver are associated with special characteristics that underlie the organ's legendary capacity for restoration of size and function following even severe or chronic injury. The nature of the injury can determine the cellular source of epithelial regeneration and the signalling mechanisms brought to play. These observations are shaping how we understand and experimentally investigate primary liver cancer, in particular cholangiocarcinoma; a highly invasive malignancy of the bile ducts, resistant to chemotherapy and whose pathogenesis has hitherto been poorly understood. Interestingly, signals that drive liver development become activated in the formation of cholangiocarcinoma, such as Notch and Wnt and may be potential future therapeutic targets. In this review, we summarise the work which has led to the current understanding of the cellular source of cholangiocarcinoma, how the tumour recruits, sustains and is educated by its supporting stromal environment, and the tumour-derived signals that drive the progression and invasion of the cancer. With few current treatments of any true efficacy, advances that will improve our understanding of the mechanisms driving this aggressive malignancy are welcome and may help drive therapeutic developments.

An evaluation of hernia education in surgical residency programs.

PURPOSE: The purpose of this study was to evaluate surgical residents' educational experience related to ventral hernias. METHODS: A 16-question survey was sent to all program coordinators to distribute to their residents. Consent was obtained following a short introduction of the purpose of the survey. Comparisons based on training level were made using χ(2) test of independence, Fisher's exact, and Fisher's exact with Monte Carlo estimate as appropriate. A p value <0.05 was considered significant. RESULTS: The survey was returned by 183 residents from 250 surgical programs. Resident postgraduate year (PG-Y) level was equivalent among groups. Preferred techniques for open ventral hernia varied; the most common (32 %) was intra-abdominal placement of mesh with defect closure. Twenty-two percent of residents had not heard of the retrorectus technique for hernia repair, 48 % had not performed the operation, and 60 % were somewhat comfortable with and knew the general categories of mesh prosthetics products. Mesh choices, biologic and synthetic, varied among the different products. The most common type of hernia education was teaching in the operating room in 87 %, didactic lecture 69 %, and discussion at journal club 45 %. Number of procedures, comfort level with open and laparoscopic techniques, indications for mesh use and technique, familiarity and use of retrorectus repair, and type of hernia education varied significantly based on resident level (p < 0.05). CONCLUSION: Exposure to hernia techniques and mesh prosthetics in surgical residency programs appears to vary. Further evaluation is needed and may help in standardizing curriculums for hernia repair for surgical residents.

Mutational analysis of the Cy motif from p21 reveals sequence degeneracy and specificity for different cyclin-dependent kinases.

Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.

Expression of antisense hsp70 is a major determining factor in heat-induced cell death of P-19 carcinoma cells.

Overexpressed heat shock protein 70 (Hsp70) is known to be associated with thermoprotection in a number of cell lines and transgenic animals. We hypothesized that because overexpression of Hsp70 protects cells from lethal heat stress, inhibition of expression should make cells susceptible to heat stress. The model used for this study was a stably transfected P-19 carcinoma cell line expressing antisense hsp70 under the control of the hsp70b promoter. The results showed marked inhibition of Hsp70 expression after heat shock correlated with heat-induced cell death. Hsp90 and Hsc70 protein expression were not affected by the antisense construct. Unexpectedly, heme oxygenase (HO-1), another highly inducible heat shock protein, was not induced after heat shock in the antisense hsp70 cell line. Heat shock transcription factor-1 (HSF-1) was in a highly phosphorylated state in the antisense cell line before and after heat shock. This was in contrast to the untransfected control P-19 cells where HSF-1 was primarily highly phosphorylated after heat shock. A control cell line expressing only the vector, pMAMneo, without the antisense construct also showed partial loss of Hsp70 induction but not increased cell death after heat shock. The findings support the role of Hsp70 in thermoresistance.

Overexpression of heme oxygenase in neuronal cells, the possible interaction with Tau.

Increased expression of heme oxygenase-1 (HO-1) is a common feature in a number of neurodegenerative diseases. Interestingly, the spatial distribution of HO-1 expression in diseased brain is essentially identical to that of pathological expression of tau. In this study, we explored the relationship between HO-1 and tau, using neuroblastoma cells stably transfected with sense and antisense HO-1 constructs as well as with the vector alone. In transfected cells overexpressing HO-1, the activity of heme oxygenase was increased, and conversely, the level of tau protein was dramatically decreased when compared with antisense HO-1 or CEP transfected cells. The suppression of tau protein expression was almost completely reversed by zinc-deuteroporphyrin, a specific inhibitor of heme oxygenase activity. The activated forms of ERKs (extracellular signal-regulated kinases) were also decreased in cells overexpressing HO-1 although no changes in the expression of total ERK-1/2 proteins were observed. These data are in agreement with the finding that the expression of tau is regulated through signal cascades including the ERKs, whose activities are modulated by oxidative stresses. The expression of tau and HO-1 may be regulated by oxidative stresses in a coordinated manner and play a pivotal role in the cytoprotection of neuronal cells.

Chromoblastomycosis: a retrospective study of six cases at the Royal Darwin Hospital from 1989 to 1994.

Chromoblastomycosis, a localized chronic cutaneous and subcutaneous infection of the skin caused by pigmented fungi, is most common in the world's tropical and subtropical zones. The condition rarely occurs in Australia. We present 6 cases of chromoblastomycosis seen at the Royal Darwin Hospital, Northern Territory, from 1989 to 1994 and affecting predominantly male Caucasians ranging from 38 to 71 yrs of age. Clinically the lesions were verrucous or nodular. They mimicked basal or squamous cell carcinoma, nevi or solar keratoses. Histopathologic findings were nonspecific. The only pathognomonic finding was the presence of brown spores or sclerotic bodies within granulomata or within microabscesses in the skin.

Cytotoxin production by Helicobacter pylori from patients with upper gastrointestinal tract diseases.

A cytotoxin produced by some Helicobacter pylori strains has recently been identified. The cytotoxin induces intracellular vacuolization of cultured cells. The aim of the present study was to examine the frequency of occurrence of cytotoxin-producing strains of H. pylori from subjects with upper gastrointestinal disease including nonulcer dyspepsia, gastric and duodenal ulcer disease, gastroesophageal reflux disease, and gastric cancer. Broth culture filtrates of clinical isolates of H. pylori recovered from 175 patients were used to inoculate Vero and HeLa cell monolayers for the detection of vacuolating cytotoxin activity. The results obtained demonstrated that the highest percentage of strains producing cytotoxin were found in subjects with peptic ulcer disease (gastric ulcer, 65%; duodenal ulcer, 66%; P < 0.01 compared with nonulcer dyspepsia, 38%). Of the 11 patients with gastroesophageal reflux disease, 4 of 5 patients in this group who had esophageal ulcers, were found to be infected with strains that produced cytotoxin. Three of the four patients with carcinoma of the stomach were also found to be infected with cytotoxic strains of H. pylori. With increasing severity of mucosal damage in subjects with a normal upper gastrointestinal tract, macroscopic gastritis, duodenitis, and peptic ulceration, there were corresponding increase in the proportion of strains producing cytotoxin; these increases were 32, 46, 50, and 66%, respectively. H. pylori strains from subjects with ulcer disease commonly produced vacuolating cytotoxin, suggesting that it may be a virulence factor in the pathogenesis of peptic ulcer disease.

Comparative evaluation of detection assays for Chlamydia trachomatis.

The performances of a commercial nucleic acid hybridization test (Gen-Probe Pace 2 Chlamydia trachomatis) and two commercial enzyme immunoassays (EIAs) (Abbott Chlamydiazyme and Pharmacia Chlamydia EIA) were evaluated against cell culture for the detection of C. trachomatis infection, with cervical swabs obtained from 1,037 women visiting a public sexual health center. The positivity rate by cell culture was 4.7%. Sensitivity and specificity for each test were as follows: Gen-Probe, 95.8 and 98.3%; Chlamydiazyme, 80.4 and 99.3%; Pharmacia EIA, 80.8 and 99.1%. Analysis of discrepant results with probe confirmation assay (Gen-Probe) and direct immunofluorescence (Syva Microtrak) revealed 12 cases of C. trachomatis infection for which culture was negative, resulting in the definition of a true-positive case as opposed to a culture positive. The positivity rate by true-positive definition was 5.9%, and sensitivity and specificity for each test were as follows: Gen-Probe, 96.7 and 99.6%; Chlamydiazyme, 77.5 and 100%; Pharmacia EIA, 77.0 and 100%; cell culture, 80.0 and 100%. We conclude that the Gen-Probe Pace 2 C. trachomatis test is a sensitive and specific alternative to cell culture for the detection of C. trachomatis.

Ribotyping of Helicobacter pylori from clinical specimens.

Ribotyping is a method used to type strains of bacteria by analyzing the restriction enzyme digestion patterns of the rRNA genes. This method was applied to 126 strains of Helicobacter pylori from 100 unrelated symptomatic patients who had endoscopies done and to 15 strains from 15 infected subjects from seven families. Analysis of the rRNA gene patterns revealed 77 distinct ribotypes from the 100 patients. From 15 of these subjects, isolates were recovered from antral mucosal biopsies at follow-up endoscopy. All follow-up isolates from the same patient, with one exception, yielded identical digest patterns. This patient had strains with two distinct digest patterns obtained from a set of three isolates cultured from biopsy specimens taken at different times. Five patients who had isolates recovered from different sites in the stomach (antrum, gastric body, duodenum, and pyloric channel) showed ribotyping patterns which were identical for each patient yet distinct between patients. In seven family groups studied, identical digest patterns were detected in members of two families, with variability in strains detected among members of the remaining families. This study demonstrates that ribotyping provides a useful, reliable, reproducible, and highly discriminatory typing scheme for the study of H. pylori infection.

Comparative evaluation of three selective media and a nonselective medium for the culture of Helicobacter pylori from gastric biopsies.

Plating on solid media is the standard technique used in most laboratories for the isolation of Helicobacter pylori from gastric biopsies. Recently, various selective media were developed for this purpose. We compared and evaluated three selective media, Skirrow's, Dent's CP, and modified Glupczynski's Brussels campylobacter charcoal media, and chocolate agar medium for the isolation of H. pylori. Gastric biopsies taken from a total of 203 patients were plated in parallel on all four media. An isolation rate of 51% (104 of 203) was obtained with a combination of all four media. Of the 104, 92 (88%) were positive with Dent's medium and with modified Glupczynski's medium. Skirrow's medium gave the highest isolation rate, 96% (100 of 104). However, growth of H. pylori was scant (only one to five colonies) when growth occurred on Skirrow's medium alone. Overall, modified Glupczynski's medium provided significantly heavier growth. Chocolate agar medium yielded a 76% (79 of 104) positivity rate. We recommend the use of a combination of two selective media for the maximum recovery of H. pylori from antral biopsies.

Hypothyroidism due to destruction of the thyroid by Kaposi's sarcoma.

A case of hypothyroidism that manifested as depression and deteriorating functional status and that ultimately resulted in the death of a 41-year-old patient with AIDS is described. Postmortem examination revealed destruction of the thyroid gland by Kaposi's sarcoma. Analysis of stored serum samples revealed that the patient had become profoundly hypothyroid during his terminal illness. Practitioners are reminded of the need to exclude metabolic causes when treating encephalopathy in patients infected with human immunodeficiency virus.

Topological dispositions of lysine alpha 380 and lysine gamma 486 in the acetylcholine receptor from Torpedo californica.

The locations have been determined, with respect to the plasma membrane, of lysine alpha 380 and lysine gamma 486 in the alpha subunit and the gamma subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the alpha subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the gamma subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [3H]-borohydride. Saponin was added to a portion of the vesicles prior to labeling to render them permeable to pyridoxal phosphate. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine alpha 380 and lysine gamma 486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The peptides bound and released by the immunoadsorbents were positively identified and quantified by high-pressure liquid chromatography. Modification of lysine alpha 380 in the native acetylcholine receptor in sealed vesicles increased 5-fold in the presence of saponin, while modification of lysine gamma 486 was unaffected by the presence of saponin. The conclusions that follow from these results are that lysine alpha 380 is on the inside surface of a vesicle and lysine gamma 486 is on the outside surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Legionella fairfieldensis sp. nov. isolated from cooling tower waters in Australia.

Three Legionella-like organisms were isolated from water from the cooling towers of two Australian institutions. The strains grew on buffered charcoal-yeast extract (BCYE) agar but not on BCYE agar in the absence of L-cysteine. Gas-liquid chromatography profiles of the isolates were consistent with those for Legionella spp. They were serologically distinct from other legionellae in a slide agglutination test. DNA hybridization studies showed that the three isolates belong to a new species of Legionella, Legionella fairfieldensis (ATCC 49588).

Magnetic resonance imaging of limbic encephalitis.

In two patients with limbic encephalitis serial magnetic resonance (MR) imaging showed evolution of abnormal high-signal intensity in both hippocampal formations on T2-weighted images.

[The demonstration of an extramembranous location for the putative amphipathic helix of the acetylcholine receptor]. / Dokazatel'stvo vnemembrannogo raspolozheniia predpolagaemoi amfipaticheskoi spirali atsetilkholinovogo retseptora.

It has been demonstrated that the lysyl residues at the centers of the potential amphipathic helices of the alpha- and beta-subunits could be readily labeled in native acetylcholine receptor with anionic electrophiles. This result indicates that the regions in the sequences of the alpha- and beta-polypeptides which patterns of hydropathy are those of amphipathic helices are fully exposed on the surface of the native protein and do not span the membrane.

Evidence for the extramembranous location of the putative amphipathic helix of acetylcholine receptor.

Evidence has been obtained demonstrating that the peptides GVKYIAE and AIKYIAE found in the potential amphipathic helices of the alpha and beta subunits, respectively, of acetylcholine receptor are not buried in the membrane. The peptide KYIAE was synthesized, and polyclonal antibodies were prepared against a conjugate of bovine serum albumin and synthetic peptide. An immunoadsorbent capable of binding and subsequently releasing peptides ending with the sequence-YIAE was produced by attaching these specific antibodies to agarose. Native acetylcholine receptor was labeled with pyridoxal phosphate and Na[3H]BH4. The labeled protein was stripped of phospholipid and digested with the protease from Staphylococcus aureus strain V8. The digest was submitted to immunoadsorption to isolate the labeled indigenous peptides. As a control, alpha and beta polypeptides prepared by gel filtration of a solution of acetylcholine receptor in detergent were stripped of detergent and labeled with pyridoxal phosphate and Na[3H]BH4 in the presence of 8 M urea. The labeled alpha and beta polypeptides were digested and submitted to immunoadsorption. The specific radioactivities of the indigenous peptides from the alpha and beta subunits labeled under native and denaturing conditions were nearly equal. In similar experiments using isethionyl (2', 4'-dinitrophenyl)-3-amino-propionimidate as the labeling agent, the indigenous peptides from native and denatured receptor were also labeled to the same extent. Since these peptides are labeled to the same extent whether or not the protein is denatured, they cannot be buried in the membrane.

Generalized seizures deplete brain energy reserves in normoxemic newborn monkeys.

The cerebral metabolic response to bicuculline (BC)-induced status epilepticus (SE) was studied in two-week-old ketamine-anesthetized marmoset monkeys. During 30-min clonic seizures, mean blood pressure, plasma glucose and paO2 did not decrease and plasma lactate doubled. Brains were funnel-frozen and punch biopsies of frontoparietal cortex, temporal cortex and thalamus were analyzed for ATP, phosphocreatine (PCr), glucose and lactate. There were marked reductions of ATP (to 56-77% of controls), PCr (to 23-28% of controls) and glucose (to 1-4% of controls), and lactate increased 3- to 6-fold in seizure animals. NADH fluorescence increased during seizures in cerebral cortex, thalamus, amygdaloid nuclei, hippocampus, posterior striatum and hemispheric white matter. This suggests a reduced tissue redox state in these regions and is correlated with the high energy phosphate depletion and elevated lactate in cortex and thalamus. Our results demonstrate a significant depletion of energy reserves and glucose in cerebral cortex and thalamus during neonatal seizures in the absence of adverse systemic factors. These seizure-induced metabolic changes in brain could have adverse long-term effects on brain development and function.

Parasitic infections of refugees.

The Victorian refugee screening programme, in contrast to similar programmes elsewhere in Australia, includes an examination for faecal parasites. The results of such screening at Fairfield Infectious Diseases Hospital for the 16-month period December 1, 1984-March 31, 1986 are presented. For most of the population groups that were studied, there was a high prevalence of intestinal parasites, which often warranted treatment. Additionally, marked differences were noted among the nationalities in the range of parasites that was detected. This information is of particular importance for those who are delivering health services to members of these different ethnic communities, especially in other states where screening is less comprehensive.

L-fucose labeling of brain tumors in rats.

These studies used L-[14C]fucose to identify 9L brain tumors in rats. Ten days after intracranial implantation of 9L tumor cells, labeled L-fucose was injected intravenously. Autoradiography demonstrated high levels of radioactive L-fucose in the resultant 9L tumors but very little L-fucose in normal brain tissue. In vitro studies comparing uptake of labeled fucose into 9L cells, normal rat fibroblasts and transformed rat fibroblasts, rat astrocytes, and human brain tissue suggest that fucose is not preferentially transported into the 9L cells. These data imply that a permissive blood-brain barrier rather than differences in fucose metabolism underlie 9L tumor labeling.