Molecular species delimitation methods recover most song-delimited cicada species in the European Cicadetta montana complex.

Molecular species delimitation is increasingly being used to discover and illuminate species level diversity, and a number of methods have been developed. Here, we compare the ability of two molecular species delimitation methods to recover song-delimited species in the Cicadetta montana cryptic species complex throughout Europe. Recent bioacoustics studies of male calling songs (premating reproductive barriers) have revealed cryptic species diversity in this complex. Maximum likelihood and Bayesian phylogenetic analyses were used to analyse the mitochondrial genes COI and COII and the nuclear genes EF1α and period for thirteen European Cicadetta species as well as the closely related monotypic genus Euboeana. Two molecular species delimitation methods, general mixed Yule-coalescent (GMYC) and Bayesian phylogenetics and phylogeography, identified the majority of song-delimited species and were largely congruent with each other. None of the molecular delimitation methods were able to fully recover a recent radiation of four Greek species.

Design, synthesis, and biological activity of methoctramine-related polyamines as putative G(i) protein activators.

The universal template approach provided a prospect of modifying methoctramine (2) structure. Thus, polyamines 3-7 were designed in which the flexibility of the diaminohexane spacer of 2 was replaced by a bipiperidinyl moiety. In electrically stimulated guinea pig left atria, these novel polyamines, unlike prototype 2, displayed a potent intrinsic activity, which was in contrast with the muscarinic antagonism shown in binding studies by some of them (3 and 4) and was inhibited by benzalkonium chloride, an inhibitor of G(i) proteins.

A modified technique for securing oro-nasal tubes.

Secure positioning of orally or nasally placed endotracheal and gastric tubes is a necessity for the patient with burns. This article describes a technique for securing these tubes with a simple modification to avoid compression on the ear that may lead to subsequent necrosis and infection.

Attention deficit hyperactivity disorder among adults with severe and profound mental retardation.

The present study investigated the incidence of attention deficit hyperactivity disorder in a sample of 86 adults with severe to profound mental retardation. Participants were evaluated by supervisory staff using the diagnostic criteria from the Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, 1994) and Conners' (1990) Hyperactivity Index. Using the most conservative measure, 15% of the sample met the diagnostic criteria. This measure was not influenced by the subjects' chronological age, adaptive age, IQ, or gender. Implications of this finding for continued research and practice were discussed.

The human cytomegalovirus origin of DNA replication (oriLyt) is the critical cis-acting sequence regulating replication-dependent late induction of the viral 1.2-kilobase RNA promoter.

Plasmid constructs containing the 1.2-kb RNA promoter from the long terminal repeat region of human cytomegalovirus (HCMV) display the early-phase regulation of this promoter but lack the characteristic late induction (E. J. Wade, K. M. Klucher, and D. H. Spector, J. Virol. 66:2407-2417, 1992). To determine if the HCMV origin of replication (oriLyt) was necessary and sufficient for the late induction of the 1.2-kb RNA promoter, we cloned a 9.6-kbp segment of the origin of replication onto the p456 OCAT plasmid containing the 1.2-kb RNA promoter. This plasmid was designated ori456 OCAT. A control construct, which contains all of the same sequences as the ori456 OCAT construct except that a 2.4-kbp segment derived from HCMV EcoRI segment U is inverted in orientation to disrupt the origin function, was designated inv456 OCAT. After electroporation into human fibroblast cells and infection with HCMV 24 h later, ori456 OCAT replicated and showed the same early and late transcription pattern as the authentic viral 1.2-kb RNA. Under similar conditions, the inv456 OCAT neither replicated nor showed late induction. Experiments using plasmids synthesized in bacteria lacking methylation activity demonstrated that the late induction was not dependent on the change in methylation state of the plasmids. Ganciclovir, an inhibitor of the HCMV DNA polymerase, was used to demonstrate the replication dependence of the expression of the virally encoded 1.2-kb RNA, while the nearby early 2.7-kb RNA was unaffected. Ganciclovir also inhibited the late induction of the chloramphenicol acetyltransferase gene from ori456 OCAT, while expression from inv456 OCAT increased. Site-specific mutations in two previously identified important regulatory elements of the 1.2-kb RNA promoter, the AP1-binding site and the CATA site, indicated that these sites continue to contribute to promoter activity at late times but that the replication-dependent late induction acts independently of these sites. Possible mechanisms underlying the late induction are discussed.

An AP-1 binding site is the predominant cis-acting regulatory element in the 1.2-kilobase early RNA promoter of human cytomegalovirus.

To extend our analysis of the regulation of human cytomegalovirus (HCMV) early gene expression, we examined a transcription unit located in the terminal repeats of the long segment of the viral genome. This region encodes a major 1.2-kb RNA which is induced at early times in infection but undergoes its largest increase in abundance after the onset of viral DNA replication. To identify the important cis-acting regulatory elements for this gene, two constructs were prepared for use in transient expression assays. One contained 413 bp of the upstream sequence and 43 bp of the leader sequence fused to the gene for chloramphenicol acetyltransferase (CAT). The second construct included 1,722 bp upstream of the start site of the 1.2-kb RNA, the entire transcribed region with an additional 166-bp insert derived from the CAT gene as an assayable marker, and 2,393 bp downstream of the polyadenylation signal. Both constructs were individually transfected into human fibroblast cells, and the cells were infected with HCMV. RNA specified by the hybrid construct was initiated at the correct position and accumulated with the same kinetics as the authentic viral transcript at early times in the infection but did not undergo the increase in abundance at late at late times. By 5'-end-deletion analysis, we determined that the promoter for the 1.2-kb RNA contains a number of cis-acting elements, the most significant of which are the TATA-like sequence CATAAA at -30 and a sequence corresponding to the binding site for the transcription factor AP-1 at -75. Using extracts prepared from HeLa cells as well as from infected and uninfected fibroblasts in gel retardation assays, we obtained evidence for the specific interaction of a cellular factor(s) with the AP-1 binding site. The pattern of binding differed in the HeLa and fibroblast cells but did not change as a function of the HCMV infection. However, the functional importance of the AP-1 binding site and its key role in the regulation of the 1.2-kb RNA was supported by analysis of constructs containing specific point mutations at this site in gel retardation and transient expression assays. Site-specific mutations in the AP-1 consensus sequence, which resulted in the complete loss of binding to cellular factors, eliminated the basal activity and reduced the inducible promoter activity by eightfold.