Basal signaling activity of human dopamine D2L receptor demonstrated with an ecdysone-inducible mammalian expression system

Basal signaling activity of the dopamine D2L (long isoform) receptor was investigated using an ecdysone-inducible mammalian expression system. Whereas basal signaling activity had been demonstrated for the D1, D2S (short isoform), D3, and D5 receptor subtypes, this issue has yet to be clearly resolved for the D2L receptors. An ecdysone-inducible mammalian expression system was used to express the dopamine D2L receptor in human embryonic kidney cells (HEK293). The two ecdysone analogs, muristerone A and ponasterone A, induced D2L receptor expression dose-dependently from 120 (non-induced) to 2000 fmol/mg protein which is similar to physiological D2 receptor density in dopaminergic brain regions. With this approach, we demonstrate significant basal D2L receptor activity. Basal and agonist-stimulated signaling activity, determined with assays of cAMP levels and [35S]GTPgammaS binding to assess G protein coupling, correlated with the level of receptor expression. Furthermore, among several antagonists tested, only fluphenazine and trifluoperazine exhibited clearly detectable inverse agonist activity. This differs from the D2S receptors, where most antagonists were reported to display full inverse agonism. Differences between inverse agonist effects of D2L and D2S antagonists may be relevant to the treatment of diseases involving dopamine D2 receptors.     

Surgical planning of the treatment of cholesteatoma and postoperative follow-up

There is no single surgical treatment of choice for aural cholesteatoma. The extent of the cholesteatoma, the amount of preoperative destruction, and the size of the mastoid pneumatization should guide the surgeon in choosing the type of operation for a particular ear--which may range from a simple extraction of the cholesteatoma (delivery) all the way to a radical mastoidectomy. It is the clinical acumen that will determine the type of surgery for a given cholesteatomatous ear, algorithms being of little use in the complex situation of a pathological condition with infinite variables. However, even when the most suitable surgical modality is chosen by the best of surgeons, the operated ear will still be left with the innate cause of the cholesteatoma, namely, its physiopathologic background and its tendency to develop a negative gas balance in the middle ear. Because of this tendency to redevelop negative middle ear pressure, insertion and reinsertion of ventilation tubes are often necessary to prevent recurrent retractions and maintain aeration of the middle ear in posterior tympanotomies and modified radical mastoidectomies. Further, when the mastoid bowl is marsupialized, as in radical and modified radical mastoidectomies, the mastoid bowl is often not self-cleansing, thus periodically requiring the help of the otologist to keep it clean and dry. My study consisted of a long-term follow-up of 368 cholesteatomatous ears, which were operated on according to 6 different surgical modalities, ie, 112 radical mastoidectomies, 88 modified radicals, 72 posterior tympanotomies, 52 atticotomies, 36 deliveries, and 8 obliterations. Of the 368 ears, 11% did not require any postoperative toilet, whereas 89% required revisiting the surgeon periodically on an average of every 5 months, for cleansing of the mastoid cavity or securing the aeration of the middle ear by reinserting a ventilation tube.     

Growth of the eustachian tube lumen with age

We undertook measurements of the lumina of 115 eustachian tubes of various ages. Our results showed that, like any other body organ, the eustachian tube lumen grows with age. The cartilaginous part of the eustachian tube was found to grow considerably more than the bony part. These findings, which were statistically confirmed, appear to contradict the theory ascribing the high incidence of acute otitis media in children to an enhanced susceptibility to infection due to their having a wider eustachian tube than adults.     

Malignant external otitis

During the years 1972-1985, 50 patients with malignant external otitis (MEO) were seen in our department. All our patients complained of severe earache; they presented initially with an apparently simple external otitis, but failed to improve when the usual measures were adopted. They all presented with granulation tissue in the external ear canal, and five of our patients had multiple cranial nerve involvement. MEO is in effect a severe external otitis which, if untreated, proceeds towards an osteomyelitis of the skull base. MEO is more prevalent in the summer, when external otitis is rampant. In some years, a relatively large number of these patients appear; in others there are none. The reason for this is unknown. In Israel, the disease is more prevalent in Jews than in Arabs. Diabetes was present in 68 per cent of our patients-severe diabetes in 42 per cent, mild diabetes in 26 per cent but 32 per cent of our patients were diabetes-free. The only otological past history in our patients was of a recent traumatic insult to the external ear canal; this was the case in about 8 per cent of them. Today, the treatment of choice of this important disease is local debridement supplemented by appropriate antibiotic treatment for 6-8 weeks. This should include some semi-synthetic penicillin to which pseudomonas aeruginosa is sensitive, combined with an appropriate aminoglycoside. During the earlier years of our encounter with MEO, two of our 10 patients died of it; later on, when we learned better how to treat it, the mortality rate decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human proton/oligopeptide transporter (POT) genes: Identification of putative human genes using bioinformatics

Purpose: The proton-dependent oligopeptide transporters (POT) gene family currently consists of ～70 cloned cDNAs derived from diverse organisms. In mammals, two genes encoding peptide transporters, PepT1 and PepT2 have been cloned in several species including humans, in addition to a rat histidine/peptide transporter (rPHT1). Because the Candida elegans genome contains five putative POT genes, we searched the available protein and nucleic acid databases for additional mammalian/human POT genes, using iterative BLAST runs and the human expressed sequence tags (EST) database. The apparent human orthologue of rPHT1 (expression largely confined to rat brain and retina) was represented by numerous ESTs originating from many tissues. Assembly of these ESTs resulted in a contiguous sequence covering ～95% of the suspected coding region. The contig sequences and analyses revealed the presence of several possible splice variants of hPHT1. A second closely related human EST-contig displayed high identity to a recently cloned mouse cDNA encoding cyclic adenosine monophosphate (cAMP)-inducible 1 protein (gi:4580995). This contig served to identify a PAC clone containing deduced exons and introns of the likely human orthologue (termed hPHT2). Northern analyses with EST clones indicated that hPHT1 is primarily expressed in skeletal muscle and spleen, whereas hPHT2 is found in spleen, placenta, lung, leukocytes, and heart. These results suggest considerable complexity of the human POT gene family, with relevance to the absorption and distribution of cephalosporins and other peptoid drugs.     

Allosteric modulation of muscarinic receptor signaling: alcuronium-induced conversion of pilocarpine from an agonist into an antagonist

Previous studies on allosteric interactions at muscarinic receptors have often focused on ligand-receptor binding interactions, because ligand binding seemed to reflect functional consequences. The prototypal allosteric agent alcuronium is known to bind with similar affinity to the M(2) subtype of muscarinic acetylcholine receptors whether or not the receptors are occupied by the agonist pilocarpine. To determine allosteric modulation of receptor signaling by alcuronium, the effects of pilocarpine were measured in contracting guinea pig left atria and on G-protein coupling in M(2)-transfected Chinese hamster ovary (CHO) cell membranes. Alcuronium dose-dependently suppressed pilocarpine-induced reduction of isometric contraction force in atria (pIC(50, Alc) = 5.63) without any effect on the EC(50) of pilocarpine, consistent with an allosteric mechanism. In contrast, alcuronium shifted the concentration-effect curve of the agonist oxotremorine M to the right without affecting the maximal effect, in a formally competitive manner (pK(A, Alc) = 5.54). If pilocarpine remained receptor bound in the presence of alcuronium, this indicates that pilocarpine can no longer act as an agonist. In support of this hypothesis, pilocarpine acted as a competitive antagonist against oxotremorine M in the presence of 10 microM alcuronium. Measuring guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in CHO-M(2) membranes yielded similar results. Alcuronium suppressed pilocarpine-induced stimulation of [(35)S]GTPgammaS binding (pIC(50, Alc) = 5.47) without shift in EC(50), whereas it competitively shifted the response to oxotremorine M (pK(A, Alc) = 5.97). [(3)H]Oxotremorine M binding data corresponded with the functional findings. In conclusion, alcuronium converted the agonist pilocarpine into an antagonist-a novel type of functional allosteric interaction.     

Eustachian tube teratoma and its terminological correctness.

Teratomas are germ cell tumours usually found in the young and are characteristically composed of tissue foreign to the place where they arise. Two teratomas of the middle ear were first described in 1866 and since then, and to the best of our knowledge, 19 additional cases have been reported in the literature under different terms such as hairy congenital polyps, epidermoid cysts, dermoid cysts, hamartoma, cutaneous teratoma and teratomas. The difficulty in classifying germ cell tumours may explain the different terminologies encountered in the literature. The authors describe a case of teratoma of the eustachian tube presenting as a mass in the middle and external ear of a 10-week-old girl. Using this case, a review of the literature is performed in light of the new classification of germ cell tumours proposed by the World Health Organization (WHO). It is of note that most of the teratomas that present in the middle ear arise from, or involve, the eustachian tube.