Two mutations in the M2 region of the human á4 neuronal nicotinic subunit -á4(S248F) and á4(776ins3) -have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) (Steinlein et al. 1995(Steinlein et al. , 1997. The á4(S248F) mutation is a serine to phenylalanine substitution at position 248 in the human á4 nicotinic subunit. The á4(776ins3) mutation is a 3-base pair insertion that adds a leucine at position 259 in the amino acid sequence of the human á4 subunit. Photo-affinity labelling and structurefunction experiments show that the M2 region of the nicotinic subunits forms the conducting pore of the receptor (reviewed in Karlin & Akabas, 1995). Thus, both ADNFLE mutations lie in the conducting pore of the nicotinic receptor. ADNFLE patients suffer from brief and occasionally violent nocturnal seizures . However, the physiological mechanism responsible for these seizures has not been established. Previous studies show that the predominant brain nicotinic receptor subtype is á4â2 (Whiting & Lindstrom, 1987;Flores et al. 1991;Whiting et al. 1991). Receptors formed by co-expressing á4(S248F) or á4(776ins3) subunits with wild-type (WT) â2 subunits in Xenopus oocytes (Weiland et al. 1996; Steinlein et al. 1997;Kuryatov et al. 1997) differ from the WT receptor in several ways but no common effects of the two mutations on the acetylcholine (ACh) response have been reported previously. To determine whether the ADNFLE mutations have any common effects on the ACh response, we constructed two rat homologues (S252F and +L264) of the human ADNFLE mutations á4(S248F) and á4(777ins3), co-expressed them with rat â2 subunits in Xenopus oocytes, and studied the properties of the expressed receptors. We also constructed the rat double mutation V247I:S252F, which combined the S252F mutation with a second V247I mutation that converted the only rat/human residue substitution in the á4 M2 region to the corresponding human residue. All three Journal of Physiology (1998) 1. We constructed rat homologues (S252F and +L264) of two human á4 nicotinic mutations -á4(S248F) and á4(777ins3) -that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co-expressed them with wild-type rat â2 subunits in Xenopus oocytes. 2. The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use-dependent potentiation of the response during a train of brief 100 nÒ ACh pulses. Second, they delayed the rise times of the 5-15 nÒ (+L264) and 30 nÒ (S252F) ACh responses. Third, they reduced extracellular Ca¥-induced increases in the 30 ìÒ ACh response. 3. Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage-jump relaxations, enhanced steady-state desensitization and reduced the single-channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single-channel conductance. Neither mutation affected the number of surface receptors measured b...

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1 We studied the pharmacological properties of native rat brain and heterologously expressed rat a4b2 nicotinic receptors immunoprecipitated onto a ®xed substrate with the anti-a4 antibody mAb 299. 2 Immunodepletion with the anti-b2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained b2.

~~Anaplasma marginale is a rickettsia1 parasite of bovine erythrocytes causing world-wide economic losses in livestock production. Despite its importance, little is known about this rickettsia at a molecular level because it has not been cultured in zlitro, and there is no small-animal model. Although several genes have been cloned and sequenced, the gross genome structure of the organism has not yet been well characterized. We separated intact bovine erythrocytes from leucocytes, and determined the genome size of A. marginale by use of restriction endonuclease cleavage and pulsed-field gel electrophoresis (PFGE). A value of 56 mol% G + C was obtained for this genome by spectral analysis. Undigested A. marginale DNA failed to migrate under several different electrophoretic conditions, indicating a circular genome. Digestions of intact A. marginale DNA were performed using restriction endonucleases NotI, SfiI and Pad. Complete digestion with SfiI resulted in 12 distinct bands ranging in size from 14 to 170 kbp. Total size determined by addition of Sfl-digested fragments was approximately 1200 kbp. P a d cleaved the A. marginale genome from three different isolates into just three fragments, of 598, 557 and 97 kbp. Incomplete digestion produced a band measuring 1250 kbp. These results indicate that A. marginale has a circular genome between 1200 and 1260 kbp, with a G + C content of 56 mol%.

In this report, the genome of the Thai avian influenza virus A (H5N1); A/Chicken/Nakorn-Pathom/Thailand/CU-K2/04, isolated from the Thai avian influenza A (AI) epidemic during the early of 2004 was sequenced. Phylogenetic analyses were performed in comparison to AI viruses from Hong Kong 1997 outbreaks and other AI (H5N1) isolates reported during 2001-2004. Molecular characterization of the Thai AI (H5N1) HA gene revealed a common characteristic of a highly pathogenic AI (HPAI), a 20-codon deletion in the neuraminidase gene, a 5-codon deletion in the NS gene and polymorphisms of the M2 and PB2 genes. Moreover, the HA and NA genes of the Thai AI displayed high similarity to those of the AI viruses isolated from human cases during the same epidemic. Finally, our results demonstrated that the Thai AI emerged as a member of 2000's AI lineage with most of the genetic sequences closely related to the Influenza A/Duck/China/E319.2/03 (H5N1).

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