Sustained in vivo cardiac protection by a rationally designed peptide that causes ɛ protein kinase C translocation

Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). ɛ Protein kinase C (ɛPKC) has been suggested to mediate preconditioning. Here, we describe an ɛPKC-selective agonist octapeptide, ψɛ receptor for activated C-kinase (ψɛRACK), derived from an ɛPKC sequence homologous to its anchoring protein, ɛRACK. Introduction of ψɛRACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased ɛPKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that ɛPKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this ɛPKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.

Essential role of POU–domain factor Brn-3c in auditory and vestibular hair cell development

The Brn-3 subfamily of POU–domain transcription factor genes consists of three highly homologous members—Brn-3a, Brn-3b, and Brn-3c—that are expressed in sensory neurons and in a small number of brainstem nuclei. This paper describes the role of Brn-3c in auditory and vestibular system development. In the inner ear, the Brn-3c protein is found only in auditory and vestibular hair cells, and the Brn-3a and Brn-3b proteins are found only in subsets of spiral and vestibular ganglion neurons. Mice carrying a targeted deletion of the Brn-3c gene are deaf and have impaired balance. These defects reflect a complete loss of auditory and vestibular hair cells during the late embryonic and early postnatal period and a secondary loss of spiral and vestibular ganglion neurons. Together with earlier work demonstrating a loss of trigeminal ganglion neurons and retinal ganglion cells in mice carrying targeted disruptions in the Brn-3a and Brn-3b genes, respectively, the Brn-3c phenotype reported here demonstrates that each of the Brn-3 genes plays distinctive roles in the somatosensory, visual, and auditory/vestibular systems.

Domain-specific gene disruption reveals critical regulation of neuregulin signaling by its cytoplasmic tail

Neuregulins are a multi-isoform family of growth factors that activate members of the erbB family of receptor tyrosine kinases. The membrane-anchored isoforms contain the receptor-activating ligand in their extracellular domain, a single membrane-spanning region, and a long cytoplasmic tail. To evaluate the potential biological role of the intracellular domain of the membrane-anchored neuregulin isoforms, we used a domain-specific gene disruption approach to produce a mouse line in which only the region of the neuregulin gene encoding almost the entire intracellular domain was disrupted. Consistent with previous reports in which all neuregulin isoforms were disrupted, the resulting homozygous neuregulin mutants died at E10.5 of circulatory failure and displayed defects in neural and cardiac development. To further understand these in vivo observations, we evaluated a similarly truncated neuregulin construct after transient expression in COS-7 cells. This cytoplasmic tail-deleted mutant, unlike wild-type neuregulin isoforms, was resistant to proteolytic release of its extracellular-domain ligand, a process required for erbB receptor activation. Thus, proteolytic processing of the membrane-bound neuregulin isoforms involved in cranial ganglia and heart embryogenesis is likely developmentally regulated and is critically controlled by their intracellular domain. This observation indicates that erbB receptor activation by membrane-bound neuregulins most likely involves a unique temporally and spatially regulated “inside-out” signaling process that is critical for processing and release of the extracellular-domain ligand.

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.

Disappearance of body fat in normal rats induced by adenovirus-mediated leptin gene therapy

Sustained hyperleptinemia of 8 ng/ml was induced for 28 days in normal Wistar rats by infusing a recombinant adenovirus containing the rat leptin cDNA (AdCMV-leptin). Hyperleptinemic rats exhibited a 30–50% reduction in food intake and gained only 22 g over the experimental period versus 115–132 g in control animals that received saline infusions or a recombinant virus containing the β-galactosidase gene (AdCMV-βGal). Body fat was absent in hyperleptinemic rats, whereas control rats pair-fed to the hyperleptinemic rats retained ≈50% body fat. Further, plasma triglycerides and insulin levels were significantly lower in hyperleptinemic versus pair-fed controls, while fatty acid and glucose levels were similar in the two groups, suggestive of enhanced insulin sensitivity in the hyperleptinemic animals. Thus, despite equivalent reductions in food intake and weight gain in hyperleptinemic and pair-fed animals, identifiable fat tissue was completely ablated only in the former group, raising the possibility of a specific lipoatrophic activity for leptin.

Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity

Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, γc and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.

The pattern of gene expression in human CD15+ myeloid progenitor cells

We performed a genome-wide analysis of gene expression in primary human CD15+ myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes.

Identification of 10 novel snoRNA gene clusters from Arabidopsis thaliana

Ten novel small nucleolar RNA (snoRNA) gene clusters, consisting of two or three snoRNA genes, respectively, were identified from Arabidopsis thaliana. Twelve of the 25 snoRNA genes in these clusters are homologous to those of yeast and mammals according to the conserved antisense sequences that guide 2′-O-ribose methylation of rRNA. The remaining 13 snoRNA genes, including two 5.8S rRNA methylation guides, are new genes identified from A.thaliana. Interestingly, seven methylated nucleotides, predicted by novel snoRNAs Z41a–Z46, are methylated neither in yeast nor in vertebrates. Using primer extension at low dNTP concentration the six methylation sites were determined as expected. These snoRNAs were recognized as specific guides for 2′-O-ribose methylation of plant rRNAs. Z42, however, did not guide the expected methylation of 25S rRNA in our assay. Thus, its function remains to be elucidated. The intergenic spacers of the gene clusters are rich in uridine (up to 40%) and most of them range in size from 35 to 100 nt. Lack of a conserved promoter element in each spacer and the determination of polycistronic transcription from a cluster by RT–PCR assay suggest that the snoRNAs encoded in the clusters are transcribed as a polycistron under an upstream promoter, and individual snoRNAs are released after processing of the precursor. Numerous snoRNA gene clusters identified from A.thaliana and other organisms suggest that the snoRNA gene cluster is an ancient gene organization existing abundantly in plants.

Actin Depolymerization Affects Stress-Induced Translational Activity of Potato Tuber Tissue1

Changes in polymerized actin during stress conditions were correlated with potato (Solanum tuberosum L.) tuber protein synthesis. Fluorescence microscopy and immunoblot analyses indicated that filamentous actin was nearly undetectable in mature, quiescent aerobic tubers. Mechanical wounding of postharvest tubers resulted in a localized increase of polymerized actin, and microfilament bundles were visible in cells of the wounded periderm within 12 h after wounding. During this same period translational activity increased 8-fold. By contrast, low-oxygen stress caused rapid reduction of polymerized actin coincident with acute inhibition of protein synthesis. Treatment of aerobic tubers with cytochalasin D, an agent that disrupts actin filaments, reduced wound-induced protein synthesis in vivo. This effect was not observed when colchicine, an agent that depolymerizes microtubules, was used. Neither of these drugs had a significant effect in vitro on run-off translation of isolated polysomes. However, cytochalasin D did reduce translational competence in vitro of a crude cellular fraction containing both polysomes and cytoskeletal elements. These results demonstrate the dependence of wound-induced protein synthesis on the integrity of microfilaments and suggest that the dynamics of the actin cytoskeleton may affect translational activity during stress conditions.