Mutants of purine metabolism in cultured mouse and hamster cells

The regulation of purine metabolism in mammalian cells is poorly understood presently. A variety of mutants in hamster and mouse is now available for these studies. It is clear that selective methods provide the potential for isolation of a greater variety and number of such mutants. It is predicted that the methods are now available for extensive study of the purine biosynthetic pathway with respect to both its genetic organization and cellular regulation. These mouse and hamster mutants will undoubtedly facilitate our understanding of this multistep pathway which until recently has been limited to the study of rare human mutants.     

The E3 Ubiquitin Ligase WWP1 Selectively Targets HER4 and Its Proteolytically Derived Signaling Isoforms for Degradation

In general, epidermal growth factor receptor family members stimulate cell proliferation. In contrast, at least one HER4 isoform, JM-a/Cyt1, inhibits cell growth after undergoing a two-step proteolytic cleavage that first produces a membrane-anchored 80-kDa fragment (m80^HER4) and subsequently liberates a soluble 80-kDa fragment, s80^HER4. Here we report that s80^HER4 Cyt1 action increased the expression of WWP1 (for WW domain-containing protein 1), an E3 ubiquitin ligase, but not other members of the Nedd4 E3 ligase family. The HER4 Cyt1 isoform contains three proline-rich tyrosine (PY) WW binding motifs, while Cyt2 has only two. WWP1 binds to all three Cyt1 PY motifs; the interaction with PY2 found exclusively in Cyt1 was strongest. WWP1 ubiquitinated and caused the degradation of HER4 but not of EGFR, HER2, or HER3. The HER4-WWP1 interaction also accelerated WWP1 degradation. Membrane HER4 (full length and m80^HER4, the product of the first proteolytic cleavage) were the preferred targets of WWP1, correlating with the membrane localization of WWP1. Conversely s80^HER4, a poorer WWP1 substrate, was found in the cell nucleus, while WWP1 was not. Deletion of the C2 membrane association domain of WWP1 allowed more efficient s80^HER4 degradation, suggesting that WWP1 is normally part of a membrane complex that regulates HER4 membrane species levels, with a predilection for the growth-inhibitory Cyt1 isoform. Finally, WWP1 expression diminished HER4 biologic activity in MCF-7 cells. We previously showed that nuclear s80^HER4 is ubiquitinated and degraded by the anaphase-promoting complex, suggesting that HER4 ubiquitination within specific cellular compartments helps regulate the unique HER4 signaling capabilities.     

Photosynthetic Oxygen Production Facilitates Translocation of 11C-Labelled Photoassimilates from Tendrils and Leaflets of Pisum sativum L

The translocation profiles of ¹¹C-photoassimilates from eithertendrils or leaflets of the compound leaf of Pisum sativum weresimilar in shape, speed and susceptibility to blockage by chillingand heat girdling. When the feed leaf component was exposedto an anaerobic gas stream consisting of N₂ gas supplementedwith 40 Pa CO₂, the export of previously-fixed ¹¹C-photoassimilatesfrom both leaflets and tendrils continued in the light, butstopped in the dark. However, in the light, translocation of¹¹C-assimilates from the leaflet was rapidly blocked by a flowof pure N₂ (i.e. anoxia). Movement of ¹¹C-assimilates from theleaf of another C₃ plant, sunflower, was similar to that fromthe pea leaflet. In contrast to both laminar leaf components,export from the tendrils was stopped under pure N₂ only in thedark. Taken together the data suggest that photosynthetic O₂production facilitated the movement of ¹¹C-assimilates in theabsence of exogenous O₂. The differences observed between thetendrils and the leaflets exposed to pure N₂ could be attributedto the greater capacity of tendrils to produce and recycle CO₂to support photosynthetic O₂ production in the light. Key words: Pea, ¹¹C-translocation, anoxia, tendril, leaflet     

Evaluation of 13 short tandem repeat loci for use in personal identification applications.

Personal identification by using DNA typing methodologies has been an issue in the popular and scientific press for several years. We present a PCR-based DNA-typing method using 13 unlinked short tandem repeat (STR) loci. Validation of the loci and methodology has been performed to meet standards set by the forensic community and the accrediting organization for parentage testing. Extensive statistical analysis has addressed the issues surrounding the presentation of "match" statistics. We have found STR loci to provide a rapid, sensitive, and reliable method of DNA typing for parentage testing, forensic identification, and medical diagnostics. Valid statistical analysis is generally simpler than similar analysis of RFLP-VNTR results and provides powerful statistical evidence of the low frequency of random multilocus genotype matching.     

Two independent mutational events in the loss of urate oxidase during hominoid evolution

Summary Urate oxidase was lost in hominoids during primate evolution. The mechanism and biological reason for this loss remain unknown. In an attempt to address these questions, we analyzed the sequence of urate oxidase genes from four species of hominoids: human (Homo sapiens), chimpanzee (Pan troglodytes), orangutan (Pongo pygmaeus), and gibbon (Hylobates). Two nonsense mutations at codon positions 33 and 187 and an aberrant splice site were found in the human gene. These three deleterious mutations were also identified in the chimpanzee. The nonsense mutation at codon 33 was observed in the orangutan urate oxidase gene. None of the three mutations was present in the gibbon; in contrast, a 13-bp deletion was identified that disrupted the gibbon urate oxidase reading frame. These results suggest that the loss of urate oxidase during the evolution of hominoids could be caused by two independent events after the divergence of the gibbon lineage; the nonsense mutation at codon position 33 resulted in the loss of urate oxidase activity in the human, chimpanzee, and orangutan, whereas the 13-bp deletion was responsible for the urate oxidase deficiency in the gibbon. Because the disruption of a functional gene by independent events in two different evolutionary lineages is unlikely to occur on a chance basis, our data favor the hypothesis that the loss of urate oxidase may have evolutionary advantages. Offprint requests to: C.T. Caskey     

Her4 mediates ligand-dependent antiproliferative and differentiation responses in human breast cancer cells

The function of the epidermal growth factor receptor (EGFR) family member HER4 remains unclear because its activating ligand, heregulin, results in either proliferation or differentiation. This variable response may stem from the range of signals generated by HER4 homodimers versus heterodimeric complexes with other EGFR family members. The ratio of homo- and heterodimeric complexes may be influenced both by a cell's EGFR family member expression profile and by the ligand or even ligand isoform used. To define the role of HER4 in mediating antiproliferative and differentiation responses, human breast cancer cell lines were screened for responses to heregulin. Only cells that expressed HER4 exhibited heregulin-dependent antiproliferative responses. In-depth studies of one line, SUM44, demonstrated that the antiproliferative and differentiation responses correlated with HER4 activation and were abolished by stable expression of a kinase-inactive HER4. HB-EGF, a HER4-specific ligand in this EGFR-negative cell line, also induced an antiproliferative response. Moreover, introduction and stable expression of HER4 in HER4-negative SUM102 cells resulted in the acquisition of a heregulin-dependent antiproliferative response, associated with increases in markers of differentiation. The role of HER2 in these heregulin-dependent responses was examined through elimination of cell surface HER2 signaling by stable expression of a single-chain anti-HER2 antibody that sequestered HER2 in the endoplasmic reticulum. In the cell lines with either endogenously (SUM44) or exogenously (SUM102) expressed HER4, elimination of HER2 did not alter HER4-dependent decreases in cell growth. These results suggest that HER4 is both necessary and sufficient to trigger an antiproliferative response in human breast cancer cells.     

Fixed Dystonia in Complex Regional Pain Syndrome: a Descriptive and Computational Modeling Approach

Background  

Complex regional pain syndrome (CRPS) may occur after trauma, usually to one limb, and is characterized by pain and disturbed blood flow, temperature regulation and motor control. Approximately 25% of cases develop fixed dystonia. Involvement of dysfunctional GABAergic interneurons has been suggested, however the mechanisms that underpin fixed dystonia are still unknown. We hypothesized that dystonia could be the result of aberrant proprioceptive reflex strengths of position, velocity or force feedback.