Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitis B virus

A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3. Lac+ colonies of the Escherichia coli M182 (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-ß-galactosidase fusion protein. This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to >90% homogeneity by affinity chromatography on p-aminophenyl-ß-D-thiogalactoside-Sepharose. It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw [pre-S(120-145)] or with antibodies to hepatitis B virus. pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-ß-galactosidase fusion protein. These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles. Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-ß-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B

EF hand-mediated Ca2+- and cGMP-signaling in photoreceptor synaptic terminals

Photoreceptors, the light-sensitive receptor neurons of the retina, receive and transmit a plethora of visual informations from the surrounding world. Photoreceptors capture light and convert this energy into electrical signals that are conveyed to the inner retina. For synaptic communication with the inner retina, photoreceptors make large active zones that are marked by synaptic ribbons. These unique synapses support continuous vesicle exocytosis that is modulated by light-induced, graded changes of membrane potential. Synaptic transmission can be adjusted in an activity-dependent manner, and at the synaptic ribbons, Ca2+- and cGMP-dependent processes appear to play a central role. EF-hand-containing proteins mediate many of these Ca2+- and cGMP-dependent functions. Since continuous signaling of photoreceptors appears to be prone to malfunction, disturbances of Ca2+- and cGMP-mediated signaling in photoreceptors can lead to visual defects, retinal degeneration (rd), and even blindness. This review summarizes aspects of signal transmission at the photoreceptor presynaptic terminals that involve EF-hand-containing Ca2+-binding proteins

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4–6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4–6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons

The Calcineurin-Binding, Activity-Dependent Splice Variant Dynamin1xb Is Highly Enriched in Synapses in Various Regions of the Central Nervous System

In the present study, we generated and characterized a splice site-specific monoclonal antibody that selectively detects the calcineurin-binding dynamin1 splice variant dynamin1xb. Calcineurin is a Ca2+-regulated phosphatase that enhances dynamin1 activity and is an important Ca2+-sensing mediator of homeostatic synaptic plasticity in neurons. Using this dynamin1xb-specific antibody, we found dynamin1xb highly enriched in synapses of all analyzed brain regions. In photoreceptor ribbon synapses, dynamin1xb was enriched in close vicinity to the synaptic ribbon in a manner indicative of a peri-active zone immunolabeling. Interestingly, in dark-adapted mice we observed an enhanced and selective enrichment of dynamin1xb in both synaptic layers of the retina in comparison to light-adapted mice. This could be due to an illumination-dependent recruitment of dynamin1xb to retinal synapses and/or due to a darkness-induced increase of dynamin1xb biosynthesis. These latter findings indicate that dynamin1xb is part of a versatile and highly adjustable, activity-regulated endocytic synaptic machinery