Molecular characterization of the canine HMGB1.

Due to the close similarities of numerous canine diseases to their human counterparts, the dog could join the mouse as the species of choice to unravel the genetic background of complex diseases as e.g. cancer and metabolic diseases. Accordingly, the role of the dog as a model for therapeutic approaches is strongly increasing. However, prerequisite for such studies is the characterization of the corresponding canine genes. Recently, the human high mobility group protein B1 (HMGB1) has attracted considerable interest of oncologists because of what is called its "double life". Besides its function as an architectural transcription factor HMGB1 can also be secreted by certain cells and then acts as a ligand for the receptor for advanced glycation end products (RAGE). The binding of HMGB1 to RAGE can activate key cell signaling pathways, such as p38(MAPK), JNK, and p42/p44(MAPK) emphasizing the important role of HMGB1 in inflammation and tumor metastasis. These results make HMGB1 a very interesting target for therapeutic studies done in model organisms like the dog. In this study we characterized the molecular structure of the canine HMGB1 gene on genomic and cDNA levels, its predicted protein, the gene locus and a basic expression pattern.

High mobility group protein HMGA1 expression in breast cancer reveals a positive correlation with tumour grade.

Members of the HMGA protein (high mobility group protein A) family act as master switches of the chromatin structure by bending DNA and thus modulating the formation of transcription factor complexes of a number of target genes. Accordingly, HMGA proteins have been shown to be associated with the development and/or progression of a variety of benign and malignant tumours. Nevertheless, the HMGA1 expression studies published so far have not included primary breast cancer samples. In this study we have investigated the HMGA1 expression patterns in a series of 170 breast cancer samples by immunohistochemistry. We have found a strong variation in HMGA1 expression between the tumours. Based on an immunoreactive score (IRS) 14.1% of the tumour samples were scored to IRS 8-12 (strong positivity for HMGA1), 24.7% were scored to IRS 4-6 (moderate positivity), 25.3% were scored to IRS 1-3 (weak positivity), and 35.9% showed no positivity at all. Immunoreaction could be detected in all histological types of breast cancers analysed with the exception of invasive papillary and cribriform carcinoma. Statistical analysis revealed a strong correlation between tumour grade and HMGA1 expression (rs=0.3516, p<0.0001). Thus, the HMGA1 expression level can be considered a potential prognostic marker for breast cancer.

Variation of HMGB1 expression in breast cancer.

The amount of steroid hormone receptor proteins does not always correlate with the response of breast cancers to endocrine therapy. This may partly be due to the fact that binding of the estrogen receptor (ER) to estrogen responsive elements (ERE) of its target genes is mediated by additional cellular proteins. One of these is the high mobility group protein HMGB1, known to interact with ER thus dramatically increasing its binding to ERE. This is the first report analysing the expression patterns of HMGB1 in breast cancer cells. Northern blot analyses of the 1.4 kb and the 2.4 kb transcripts of HMGB1 in 13 breast cancer samples revealed a strong intertumoural variation by a factor of 8.5 and 14.5, respectively. This variation may contribute to the different response, of estrogen receptor-positive breast tumours to endocrine therapy, making HMGB1 a marker of considerable clinical interest.

Back to the roots of a new exon--the molecular archaeology of a SP100 splice variant.

Retropseudogenes are intronless DNA sequences sharing a high degree of homology with the cDNA of their corresponding active genes. They are thought to have originated by reverse transcription of messenger RNA and reintegration of the cDNA into the genome. Usually considered a type of evolutionary waste, they melt into the background of their surrounding DNA by the loss of similarity to the active gene or disappear from the genome by the accumulation of deletions. On the other hand, in this paper we describe the evolutionary recycling of this genomic waste. Recently, a splice variant of the gene encoding the nuclear protein SP100 was identified in which the 3' part of the cDNA is replaced by an alternative exon apparently encoding an HMG1-DNA-binding domain. We were able to show that this HMG box is contributed by a new exon arising from an HMG1 retropseudogene that we have molecularly characterized in detail. In addition to being found in human cells, corresponding fusion transcripts were shown in Pan troglodytes, Gorilla gorilla, and Hylobates lar, but not in Macaca mulatta. Genomic DNA from M. mulatta enabled us to amplify by PCR the 5' part but not the 3' part of the HMG1 retropseudogene. From our data we thus can date the underlying retrotransposition to more than 35 million years ago. Our findings offer a model as to how new exons may evolve during evolution. To our knowledge this is the first example of a retropseudogene becoming part of an active gene in which both parental parts are well characterized and remain in-frame with their cDNA.

alpha-Functionalized phosphonylphosphinates: synthesis and evaluation as transcarbamoylase inhibitors.

Diverse alpha-methyl-substituted phosphonylphosphinates (P-C-P-C-X) are accessible from a protected, pentafluorophenylsulfonated phosphonylphosphinate via nucleophilic displacement. The utility of this route is demonstrated with several nitrogen nucleophiles. The resulting amine and amino acid phosphonylphosphinate derivatives were evaluated as inhibitors of Streptococcus faecalis ornithine transcarbamoylase (OTC). Compared with the structurally related phosphonoacetyl-L-ornithine (L-PALO), a known inhibitor of OTCs from various sources, the phosphonylphosphinates are surprisingly poor inhibitors, binding several orders of magnitude less tightly to the enzyme. These results suggest that the tetrahedral intermediate formed in the normal transcarbamoylase reaction is poorly mimicked by a tetrahedral and anionic phosphonate, either because of directly unfavorable interactions with a hydrogen-bond acceptor within the active site or because transition-state analogues are unable to induce the protein conformation changes that normally accompany reaction.

Transsylvian approach to the tentorial hiatus--anatomical remarks on the microsurgical exposure.

A. The transsylvian approach: 1. Splitting of the arachnoid frontally to the medial superficial cerebral vein. 2. Incision of the arachnoid from laterally to medially. 3. Deep dissection frontally to the branches of the middle cerebral artery. 4. Cutting of medial and lateral bridging veins. 5. Spreading of the sylvian fissure. 6. Depending on the length of the intraarachnoidal part of the internal carotid artery and of the posterior communicating artery the alternatives are either subfrontal or subtemporal. B. Through this approach, access to the rostral half of the tentorial hiatus is possible. Also structures which are contralateral to the craniotomy can be reached.

Percutaneous aspiration and drainage for suspected abdominal infection.

Percutaneous drainage (PCD) of abdominal infection is a therapeutic modality whose role is not well defined. Surgical literature on abdominal infection cites a cumulative mortality rate in the range of 20% to 30%, markedly dissimilar from the 80% to 90% cure rates reported in the literature on PCD. We reviewed the PCD experience at a tertiary teaching hospital from 1981 to 1983. Fifty-five patients were suspected to have localized abdominal infection and underwent 66 procedures. PCD was attempted after percutaneous needle aspiration produced drainable fluid. Cure is defined by complete resolution of the abdominal process without any surgical intervention. Palliation is defined as acute decompression of the abdominal process permitting an elective corrective procedure to be performed. Failure is defined as false diagnosis, unsuccessful drainage requiring operation, or recurrence of infection. Diagnosis of the abdominal process was successfully made by aspiration in 59/66 (89%) attempts. PCD was curative in 31/66 (47%) attempts and failed or was palliative in 35/66 (53%). Simple nonfungal, nonfistulous abdominal abscesses were cured with PCD in 25/26 attempts (96%). PCD failure was encountered in 10 infected organized hematomas or thick phlegmons, nine fungal infections, nine abscesses with enteric communication, and five infected necrotic tumors. Abscesses with an underlying enteric communication were cured in 28%, were palliated in 32% and failed in 32% of PCD attempts. Abscesses with yeast as a major component or with necrotic tumor were never cured with PCD. PCD is a valuable diagnostic and therapeutic tool that is curative in simple abdominal abscesses. Its therapeutic role in complex abdominal infections seems to be limited.

'Breakthrough' enterococcal septicemia in surgical patients. 19 cases and a review of the literature.

We studied 19 surgical patients with 24 postoperative episodes of enterococcal septicemia not arising from the biliary or urinary tracts or from infected heart valves. Fifteen episodes occurred despite the administration of broad-spectrum antibiotics; in only one patient were these drugs effective against enterococcus. There were 14 episodes of enterococcemia in 11 patients following which the patient survived for at least one week. Thirteen (93%) of those episodes were treated with either ampicillin or drainage, or both. Five of the six long-term survivors received ampicillin therapy. Overall mortality was 68%. The data suggest that the enterococcus may emerge as a blood-borne pathogen in immunodepressed, postoperative patients receiving antibiotics for other infections of enteric origin. Antibiotic therapy specifically directed against this organism (and surgical drainage, if necessary) may be indicated during polymicrobial sepsis of enteric or mixed origin. If the spectrum of antibiotics does not include enterococcus, this organism can cause "breakthrough" sepsis, as can many other opportunistic organisms.

Indications for therapy for fungemia in postoperative patients.

We reviewed the clinical courses of 63 surgical patients who had experienced one or more days of fungemia, to determine the clinical setting for such infections and to define indications for systemic therapy. Fifty-one patients experienced fungemia as a late complication of intraperitoneal infection. Candida was identified as part of a polymicrobial flora in 70%. If untreated, the mortality was 83% (30 of 36). No untreated patients with fungemia for more than one day survived. Adequate therapy with amphotericin B (total dose, greater than 3 mg/kg) improved survival to 67% (ten of 15). Autopsies performed in 20 cases revealed visceral Candida microabscesses in seven, with the gastrointestinal tract (12) and intraabdominal abscess (five) as the most common sources of fungi. These data support the concept of Candida as an important participant in polymicrobial infection and recommend therapy with amphotericin B for patients with intraperitoneal infection experiencing fungemia.

Candida infections in surgical patients. Dose requirements and toxicity of amphotericin B.

The natural history of candidiasis in general surgical patients has been poorly documented, and the toxicity of amphotericin B is widely heralded. For these reasons therapy for candidiasis is frequently withheld in situations where antimicrobial treatment seems indicated on clinical grounds. The clinical courses of 47 general surgical patients who received amphotericin therapy for presumed Candida infection were reviewed. Nineteen patients had had solid tumors, but 12 were either localized or resected tumors. Only nine patients had received prior cancer themotherapy. Twenty-one patients were treated for fungemic disease, 10 for Candida in peritonitis fluid, and 16 for apparent colonization associated with fever and organ failure syndromes. Pre-existing renal or other organ failure was the primary determinant of survival with 4/22 survivors (18%) in patients with renal failure compared with 17/25 (78%) survivors in patients without such organ failure. In patients with serum creatinine values less than 2.5 mg/dl, amphotericin therapy was associated with a transient 30% fall in creatinine clearance and a proportionate rise in serum creatinine. Dose response curves were determined and revealed substantial sterilization of cultures in both fungemic and nonfungemic patients receiving greater than or equal to 6 mg/kg. This was confirmed by autopsy material. We suggest that in this acutely ill patient popoulation uncontrolled infection is the primary determinant of organ failure. Short-term limited dosing with amphotericin B (6-8 mg/kg total dose) in conjunction with appraisal of clinical response is adequate therapy for most presumed Candida infections. Long-term high dose therapy, such as that recommended in immuodepressed patients, is not a routine necessity.