Genotyping of alcohol dehydrogenase type 2 and 3 using a two-buffer polyacrylamide gel electrophoresis system.

Genetic polymorphisms in the alcohol dehydrogenase genes, ADH2 and ADH3, have been shown to affect individual susceptibility to diseases such as alcoholism and oesophageal cancer. Although several PCR-based methods for genotyping these enzymes have been previously developed, the two-buffer polyacrylamide gel electrophoresis system has the ability to rapidly resolve all classes of point mutations within 2-3 hours instead of the conventional overnight separation. The success of this technique is partly attributable to a discontinuous two-phase buffer system and horizontal flatbed gel electrophoresis rather than conventional vertical gels. Using a modification of this system, we were able to detect all of the known polymorphisms within ADH2 exons 3 and 9 and ADH3 exon 8, as well as a rare variant within ADH2 exon 9. This method is rapid, cost-effective, and is ideal for large scale screening programmes.     

p.R254Q mutation in the aquaporin‐2 water channel causing dominant nephrogenic diabetes insipidus is due to a lack of arginine vasopressin‐induced phosphorylation

Vasopressin regulates human water homeostasis by re‐distributing homotetrameric aquaporin‐2 (AQP2) water channels from intracellular vesicles to the apical membrane of renal principal cells, a process in which phosphorylation of AQP2 at S256 by cAMP‐dependent protein kinase A (PKA) is thought to be essential. Dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, is caused by AQP2 gene mutations. Here, we investigated a reported patient case of dominant NDI caused by a novel p.R254Q mutation. Expressed in oocytes, AQP2‐p.R254Q appeared to be a functional water channel, but was impaired in its transport to the cell surface to the same degree as AQP2‐p.S256A, which mimics non‐phosphorylated AQP2. In polarized MDCK cells, AQP2‐p.R254Q was retained and was distributed similarly to that of unstimulated wt‐AQP2 or AQP2‐p.S256A. Upon co‐expression, AQP2‐p.R254Q interacted with, and retained wt‐AQP2 in intracellular vesicles. In contrast to wild‐type AQP2, forskolin did not increase AQP2‐p.R254Q phosphorylation at S256 or its translocation to the apical membrane. Mimicking constitutive phosphorylation in AQP2‐p.R254Q with the p.S256D mutation, however, rescued its apical membrane expression. These date indicate that a lack of S256 phosphorylation is the sole cause of dominant NDI here, and thereby, p.R254Q is a loss of function instead of a gain of function mutation in dominant NDI. © 2009 Wiley‐Liss, Inc.     

Liposarcoma of the head and neck: The University of Texas M. D. Anderson Cancer Center experience

Background.

Our aim was to review our experience with liposarcoma of the head and neck region.

Methods.

This is a retrospective case series at a comprehensive cancer center (1945–2005).

Results.

Of 30 patients, 10 (33%) were initially misdiagnosed. Local recurrences were common (overall rate = 53%), and 4 patients (13%) developed distant metastases. Decreased crude disease‐specific survival rates were significantly associated with recurrence (especially distant recurrence [0%]), age less than 38 years (40%), and pleomorphic subtype (45%); however, in Kaplan‐Meier analyses, only larger tumor size, negative margins, round cell subtype, and pleomorphic subtype were associated with significantly decreased disease‐specific survival (log‐rank test p = .048, .041, .021, and .012, respectively).

Conclusions.

Based on this limited experience and existing literature, we continue to recommend surgery with negative margins as the treatment of choice and that adjuvant therapies should be considered in patients with high‐grade histology, large tumors, positive margins, or certain subsites. © 2008 Wiley Periodicals, Inc. Head Neck, 2009     

Right-sided heart failure in carcinoid syndrome

We reported the case of a 63-year-old man with a history of carcinoid syndrome evaluated for an exertional dyspnoea. Two-dimensional echocardiogram showed the characteristic right-sided cardiac valves involvement associated with this uncommon enterochromaffin malignancy.     

Late mitral annuloplasty failure: the loss of the ring

An 81-year-old man complained with increasing dyspnoea and asthenia few months after a successful double mitral and tricuspid valvuloplasty intervention. The echocardiogram revealed a partial detachment of the mitral annuloplasty ring due to dehiscence determining a severe regurgitation. Curiously, at Color-Doppler analysis of mitral inflow, the prolapse of the detached ring produced a fascinating circular line inside the regurgitation signal, mimicking the shape of a gemstone ring. We described a case of a relatively uncommon cause of late mitral valvuloplasty failure characterized by a really unusual echocardiographic finding.     

Relative contribution of ecto-ATPase and ecto-ATPDase pathways to the biphasic effect of ATP on acetylcholine release from myenteric motoneurons

Background and purpose:

The relative contribution of distinct ecto-nucleotidases to the modulation of purinergic signalling may depend on differential tissue distribution and substrate preference.

Experimental approach:

Extracellular ATP catabolism (assessed by high-performance liquid chromatography) and its influence on [³H]acetylcholine ([³H]ACh) release were investigated in the myenteric plexus of rat ileum in vitro.

Key results:

ATP was primarily metabolized via ecto-ATPDase (adenosine 5′-triphosphate diphosphohydrolase) into AMP, which was then dephosphorylated into adenosine by ecto-5′-nucleotidase. Alternative conversion of ATP into ADP by ecto-ATPase (adenosine 5′-triphosphatase) was more relevant at high ATP concentrations. ATP transiently increased basal [³H]ACh outflow in a 2′,3′-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP)-dependent, tetrodotoxin-independent manner. ATP and ATPγS (adenosine 5′-[γ-thio]triphosphate), but not α,β-methyleneATP, decreased [³H]ACh release induced by electrical stimulation. ADP and ADPβS (adenosine 5′[β-thio]diphosphate) only decreased evoked [³H]ACh release. Inhibition by ADPβS was prevented by MRS 2179 (2′-deoxy-N⁶-methyl adenosine 3′,5′-diphosphate diammonium salt, a selective P2Y₁ antagonist); blockade of ADP inhibition required co-application of MRS 2179 plus adenosine deaminase (which inactivates endogenous adenosine). Blockade of adenosine A₁ receptors with 1,3-dipropyl-8-cyclopentyl xanthine enhanced ADPβS inhibition, indicating that P2Y₁ stimulation is cut short by tonic adenosine A₁ receptor activation. MRS 2179 facilitated evoked [³H]ACh release, an effect reversed by the ecto-ATPase inhibitor, {"type":"entrez-protein","attrs":{"text":"ARL67156","term_id":"1186396857","term_text":"ARL67156"}}ARL67156, which delayed ATP conversion into ADP without affecting adenosine levels.

Conclusions and implications:

ATP transiently facilitated [³H]ACh release from non-stimulated nerve terminals via prejunctional P2X (probably P2X₂) receptors. Hydrolysis of ATP directly into AMP by ecto-ATPDase and subsequent formation of adenosine by ecto-5′-nucleotidase reduced [³H]ACh release via inhibitory adenosine A₁ receptors. Stimulation of inhibitory P2Y₁ receptors by ADP generated alternatively via ecto-ATPase might be relevant in restraining ACh exocytosis when ATP saturates ecto-ATPDase activity.     

Staged reconstruction for resection wounds in sarcoma patients treated with brachytherapy

Adjuvant brachytherapy reduces local recurrences following wide local excision of large, high-grade sarcomas, but its use with immediate flap reconstruction is associated with a high wound complication rate following previous radiotherapy. To avoid flap irradiation and reduce wound-healing morbidity, a treatment strategy using negative-pressure wound therapy (NPWT) for temporary wound coverage during brachytherapy followed by delayed flap reconstruction was used in 3 previously irradiated sarcoma patients. NPWT was continued after brachytherapy catheter removal to stimulate vascularization, granulation, and wound contraction. Flap reconstructions were performed after the adequacy of the resection margins was pathologically confirmed and the wound bed appeared grossly vascularized. Prior to reconstruction, 2 patients required additional excision of positive or close permanent-section surgical margins. There were no major wound-healing complications during 9-18 months' follow-up. Staged closure using this approach may have advantages over immediate flap reconstruction in some sarcoma patients. Potential advantages include avoiding flap irradiation, reducing wound size and magnitude of the reconstructive procedure, and ensuring tumor-free surgical margins before definitive reconstruction.     

Retroperitoneal soft tissue sarcoma: an analysis of radiation and surgical treatment

PURPOSE: To evaluate the clinical outcomes of patients with localized retroperitoneal soft tissue sarcoma (STS) treated with complete surgical resection and radiation. METHODS AND MATERIALS: The medical records of 83 patients were reviewed retrospectively. Sixty patients presented with primary disease and the remaining 23 had recurrence after previous surgical resection. RESULTS: With a median follow-up of 47 months, the actuarial overall disease-specific survival (DSS), distant metastasis-free survival, and local control (LC) rates were 44%, 67%, and 40%, respectively. Of the 38 patients dying of disease, local disease progression was the sole site of recurrence for 16 patients and was a component of progression for another 11 patients. Multivariate analysis indicated that histologic grade was associated with the 5-year rates of DSS (low-grade, 92%; intermediate-grade, 51%; and high-grade, 41%, p = 0.006). Multivariate analysis also indicated an inferior 5-year LC rate for patients presenting with recurrent disease, positive or uncertain resection margins, and age greater than 65 years. The data did not suggest an improved local control with higher doses of external-beam radiation (EBRT) or with the specific use of intraoperative radiotherapy (IORT). Radiation-related complications (10% at 5 years) developed in 5 patients; all had received their EBRT postoperatively. CONCLUSIONS: Although preoperative radiation therapy and aggressive surgical resection is well tolerated in patients, local disease progression continues to be a significant component of disease death. In this small cohort of patients, the use of higher doses of EBRT or IORT did not result in clinically apparent improvements in outcomes.