HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Transformation and rescue of a flightless Drosophila tropomyosin mutant.

In the Drosophila flightless mutant Ifm(3)3, a transposable element inserted into the alternatively spliced fourth exon of the tropomyosin I (TmI) gene prevents proper expression of Ifm-TmI, the tropomyosin isoform found in indirect flight muscle. We have rescued the flightless phenotype of Ifm(3)3 flies using P-element-mediated transformation with a segment of the Drosophila genome containing the wild-type TmI gene plus 2.5 kb of 5' flanking and 2 kb of 3' flanking DNA. The inserted TmI gene is expressed with the proper developmental and tissue specificity, although its level of expression varies among the five transformed lines examined. These conclusions are based on analyses of flight, myofibrillar morphology, and TmI RNA and protein levels. A minimum of two copies of the inserted TmI gene per cell is necessary to restore flight to most of the flies in each line. We also show that the Ifm-TmI isoform is expressed in the leg muscle of wild-type flies and is decreased in Ifm(3)3 leg muscle. Homozygous Ifm(3)3 mutants do not jump. The ability to jump can be restored with a single copy of the wild-type TmI gene per cell.     

Quantitative survey of sieve tube distribution in foliar terminal veins of ten dicot species

Quantitative data on sieve tubes in foliar terminal veins (vein endings) were added to the meager published information from only five dicot species. Correlations with other minor vein configurations were also explored. Leaf samples from ten species of dicots (Oxalis nelsonii, O. pes-capri, O. rubra, O. stricta [Oxalidaceae], Caesalpinia pulcherrima. Glycine max, Trifolium repens [Leguminosae], Ampelamus albidus [Asclepidaceae], Eupatorium rugosum [Asteraceae], and Polygonum convolvulus [Polygonaceae]) were selected for two quantitative procedures: 1) a survey of the arrangement of terminal veins and distribution of sieve tubes in terminal veins in 100 areoles per species using stained leaf clearings; and 2) a search for correlations of sieve tube distribution with number and branching patterns of terminal veins, and with sizes of areoles using image analysis. Two Oxalis species (O. pes-capri and O. stricta) had the smallest areoles and virtually no sieve tubes in any terminal vein. Polygonum convolvulus, at the other extreme, had sieve tubes extending to the tips of most terminal veins. The other species had various intermediate sieve tube configurations. The data indicate that species with few or no sieve tubes associated with their terminal veins, regardless of the number of terminal veins per areole, have smaller areoles. These results may have implications regarding the entry of leaf photosynthates into the vascular system.     

Ten-year stability of EEG biofeedback results for a hyperactive boy who failed fourth grade perceptually impaired class

Ten years ago, the first successful application of a clinical,private-practice based, EEG 14-Hz biofeedback training regimen for the treatment of learning disorders was performed by the author. After the 10-year-old boy, with presenting symptomology including a developmental reading disorder, hyperactivity, and an educational classification of perceptually impaired, continued symptom free for a period oftwo years, his case was submitted for publication. Ten years after his termination from successful treatment, his ongoingly normal social and academic functioning is noted and his EEG brainwave signature examined and compared with a population of 24 used-to-be learning disabled, one-half of which had a pretreatment state including the educational classification of perceptually impaired. This 10-year follow-up confirms the long-term stability of the results of this EEG 14-Hz biofeedback regimen. Current findings on recent medical research identifying a major cerebral locus of dysfunction for hyperkinesis and how it supports the electrode placements of this clinical office setting regimen is also discussed.     

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.     

Morphological and temporal variation in early embryogenesis contributes to species divergence in Malawi cichlid fishes

The cichlid fishes comprise the largest extant vertebrate family and are the quintessential example of rapid “explosive” adaptive radiations and phenotypic diversification. Despite low genetic divergence, East African cichlids harbor a spectacular intra- and interspecific morphological diversity, including the hyper-variable, neural crest (NC)-derived traits such as coloration and craniofacial skeleton. Although the genetic and developmental basis of these phenotypes has been investigated, understanding of when, and specifically how early, in ontogeny species-specific differences emerge, remains limited. Since adult traits often originate during embryonic development, the processes of embryogenesis could serve as a potential source of species-specific variation. Consequently, we designed a staging system by which we compare the features of embryogenesis between three Malawi cichlid species—Astatotilapia calliptera, Tropheops sp. ‘mauve’ and Rhamphochromis sp. “chilingali”—representing a wide spectrum of variation in pigmentation and craniofacial morphologies. Our results showed fundamental differences in multiple aspects of embryogenesis that could underlie interspecific divergence in adult adaptive traits. First, we identified variation in the somite number and signatures of temporal variation, or heterochrony, in the rates of somite formation. The heterochrony was also evident within and between species throughout ontogeny, up to the juvenile stages. Finally, the identified interspecific differences in the development of pigmentation and craniofacial cartilages, present at the earliest stages of their overt formation, provide compelling evidence that the species-specific trajectories begin divergence during early embryogenesis, potentially during somitogenesis and NC development. Altogether, our results expand our understanding of fundamental cichlid biology and provide new insights into the developmental origins of vertebrate morphological diversity.     

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white-matter regions of rat brain, and in Vibratome sections as well as paraffin sections.     

Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.