Surgical considerations in the management of malignant melanoma of the ear

Malignant melanomas of the external ear are rare and are difficult lesions to treat because of the cosmetic importance and the reconstructive difficulty of their location. The literature suggests that these lesions have a worse prognosis than melanomas occurring elsewhere and that radical resection is the "correct" treatment. To clarify this issue, we examined 21 consecutive patients (19 male, 2 female) with malignant melanoma of the ear seen at the Yale-New Haven Hospital over the last 10 years. Nineteen patients had a diagnosis of primary malignant melanoma of the ear, one had a local recurrence, and one had an in-transit melanoma from an unknown primary site. The mean thickness of the lesions was 2.7 mm. Two patients had palpable nodes, which in both cases turned out to be histologically positive for tumor. All patients underwent local excision and reconstruction using chondrocutaneous or fasciocutaneous flaps or skin grafts. There was one local recurrence (0.5 mm original thickness); there were two patients with regional recurrences, both of whom died within a year with disseminated disease. Forty-three percent have been followed for 5 or more years and all are alive and free of disease. This suggests that malignant melanoma of the ear may be safely treated by conservative excision and reconstruction.     

Cardiac myocyte adenosine A2a receptor activation fails to alter cAMP or contractility: role of receptor localization

Adenosine A2a receptors are found in coronary vascular tissue although, their presence in myocardium is subject to investigation. Although there have been numerous studies on adenosine A2a receptor agonist effects on contractility and cAMP levels in ventricular myocytes, these have yielded conflicting results. Negative pharmacological studies have even led to the conclusion that A2a receptors are not present in cardiac myocytes. The purpose of this study was to determine whether A2a receptors are expressed in rat ventricular myocytes and what physiological effects are mediated via activation of these receptors. Western blot analysis with a polyclonal antibody raised against a peptide sequence specific to the carboxy terminus of the A2a receptor revealed the presence of a band at approximately 45 kDa. However, the immunoreactivity was located in the nonmembrane fraction of the cell lysate. The membrane fraction only exhibited an immunoreactive band > or = 50 kDa. Treatment of isolated myocytes with the adenosine A2a agonist 2-[4-[(2-carboxyethyl)-phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680) exerted no effects on cAMP levels or myocyte twitch amplitude. These results indicate that although rat ventricular myocytes appear to express adenosine A2a receptors, stimulation with an A2a agonist exerts no functional effects, possibly because of the subcellular localization of the A2a receptor.     

T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA

DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.     

SN2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression

p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.     

Syntheses of novel substituted-boranophosphate nucleosides

A number of substituted (borano) nucleic acids, 3'-[diethylphosphite(cyano, carboxy, or carbamoyl) borano] deoxynucleosides (3a-4c) and 5'-[diethylphosphite(cyano or carboxy) borano] deoxynucleosides (6a-7d) were prepared by a variety of synthetic procedures. The syntheses of the pyrophosphates (2a-2c), as precursors for 3a-4c, are also described.     

Yoked complexes of human choriogonadotropin and the lutropin receptor: evidence that monomeric individual subunits are inactive

Human choriogonadotropin (hCG) contains an alpha-subunit, common to other members of the glycoprotein hormone family, and a unique beta-subunit that determines hormone specificity. It is generally thought that heterodimer formation is obligatory for full hormonal activity, although other studies have indicated that individual subunits and homodimeric hCGbeta were capable of low affinity binding to the LH receptor (LHR) and subsequent activation. Previously, we constructed two yoked hormone (hCG)-LHR complexes, where the two hormone subunits and the heptahelical receptor were engineered to form single polypeptide chains, i.e. N-beta-alpha-LHR-C and N-alpha-beta-LHR-C. Expression of both complexes led to constitutive stimulation of cAMP production. In the present study, we investigated whether the human alpha-subunit and hCGbeta can act as functional agonists when covalently attached to or coexpressed with the LH receptor. Our initial results showed that hCGbeta, but not alpha, was able to activate LHR with an increase in intracellular cAMP in human embryonic kidney 293 cells but not in Chinese hamster ovary or COS-7 cells. Further examination of this apparent cell-specific agonist activity of hCGbeta revealed that low levels of endogenous alpha-subunit were expressed in human embryonic kidney 293 cells, thus enabling sufficient amounts of active heterodimer to form with the transfected hCGbeta to activate LHR. The studies in Chinese hamster ovary and COS-7 cells clearly demonstrate that, even under experimental conditions where hormone-receptor interactions are maximized, individual subunits of hCG can not act as functional agonists, at least in their monomeric form.     

Oxidative and calcium stress regulate DSCR1 (Adapt78/MCIP1) protein

DSCR1 (adapt78) is a stress-inducible gene and cytoprotectant. Its protein product, DSCR1 (Adapt78), also referred to as MCIP1, inhibits intracellular calcineurin, a phosphatase that mediates many cellular responses to calcium. Exposure of human U251 and HeLa cells to hydrogen peroxide led to a rapid hyperphosphorylation of DSCR1 (Adapt78). Inhibitor and agonist studies revealed that a broad range of kinases were not responsible for DSCR1 (Adapt78) hyperphosphorylation, including ERK1/2, although parallel activation of the latter was observed. Phosphorylation of both DSCR1 (Adapt78) and ERK1/2 was attenuated by inhibitors of tyrosine phosphatase, suggesting the common upstream involvement of tyrosine dephosphorylation. The hyperphosphorylation electrophoretic shift in DSCR1 (Adapt78) mobility was also observed with other oxidizing agents (peroxynitrite and menadione) but not nonoxidants. Calcium ionophores strongly induced the levels of both hypo- and hyper-phosphorylated DSCR1 (Adapt78) but did not alter phosphorylation status. Calcium-dependent growth factor- and angiotensin II-stimulation also induced both DSCR1 (Adapt78) species. Phosphorylation of either or both serines in a 13-amino acid peptide made to a calcineurin-interacting conserved region of DSCR1 (Adapt78) attenuated inhibition of calcineurin. These data indicate that DSCR1 (Adapt78) protein is a novel, early stage oxidative stress-activated phosphorylation target and newly identified calcium-inducible protein, and suggest that these response mechanisms may contribute to the known cytoprotective and calcineurin-inhibitory activities of DSCR1 (Adapt78).     

A comparison of the long-term morbidity following deep circumflex iliac and fibula free flaps for reconstruction following head and neck cancer

Composite free tissue transfer has an established role in head and neck oncology for the reconstruction of the bony defect following tumor ablation, and while donor-site morbidity is variably reported, there is little consensus on the most favorable donor site. The fibula and deep circumflex iliac artery have distinct advantages in terms of the volume and length of bone in mandibular reconstruction. Few studies have compared their donor-site morbidity. The aim of this study was to compare the fibula and deep circumflex iliac artery flaps using a review of the case notes and cross-sectional review of patients attending a research clinic for validated orthopedic examination and completion of health-related quality-of-life questionnaires. Between February of 1993 and May of 2001, 44 fibula free flaps and 73 deep circumflex iliac artery free flaps were performed. Ninety-nine case notes and 36 patients were available for review of donor-site morbidity. Sixteen patients with fibula flaps and 20 patients with deep circumflex iliac artery flaps took part in the clinical examination component of the study, which was composed of a clinical examination by an orthopedic surgeon using the American Orthopedic Foot and Ankle Society ankle scoring system and the Harris hip scoring system, and two patient-completed questionnaires, the University of Washington Questionnaire and the Hospital Anxiety and Depression Scale. Subjective and objective markers of morbidity related to both flaps were similar in most parameters. However, fibula flaps were associated with more problems with donor-site healing, reduced power, and sensation. Poor orthopedic scores for both flaps were associated with notably poor scores on the University of Washington Questionnaire and the Hospital Anxiety and Depression Scale. The study would suggest that both deep circumflex iliac artery and fibula donor sites result in an acceptable and comparable morbidity for most patients, but in cases in which significant donor-site morbidity is encountered, health-related quality of life is significantly compromised.     

Eyeing endothelins: A cellular perspective

Endothelin is an endogenous vasoactive peptide that is considered among the most potent vasoconstrictor substances known. In addition to its vascular effects, endothelins and their receptors have been shown to be present in the eye and to have a number of ocular actions that may be important for ocular homeostasis, but, in excess can be a potential contributor to ocular neuropathy in glaucoma. The current review focuses on the cellular and molecular aspects of endothelins and its receptors in the eye with an emphasis on its relationship to ocular function and its potential role in the etiology of glaucoma pathophysiology.