Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis.

The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H. , Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170-1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an unusual 3/10 helix. Since beta-sheet residues play a role in a number of protein-RNA interactions, one or both of the beta-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both beta-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of beta-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an alpha-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.     

Immunohistochemical study of N-epsilon-carboxymethyl lysine (CML) in human brain: relation to vascular dementia

Background  

Advanced glycation end-products (AGEs) and their receptor (RAGE) occur in dementia of the Alzheimer's type and diabetic microvascular disease. Accumulation of AGEs relates to risk factors for vascular dementia with ageing, including hypertension and diabetes. Cognitive dysfunction in vascular dementia may relate to microvascular disease resembling that in diabetes. We tested if, among people with cerebrovascular disease, (1) those with dementia have higher levels of neuronal and vascular AGEs and (2) if cognitive dysfunction depends on neuronal and/or vascular AGE levels.     

Fish as Bioreactors: Transgene Expression of Human Coagulation Factor VII in Fish Embryos

A plasmid containing human coagulation factor VII (hFVII) complementary DNA regulated by a cytomegalovirus promoter was microinjected into fertilized eggs of zebrafish, African catfish, and tilapia. The active form of hFVll was detected in the fish embryos by various assays. This positive expression of human therapeutic protein in fish embryos demonstrates the possibility of exploitation of transgenic fish as bioreactors.     

Genetic structure of the LXS panel of recombinant inbred mouse strains: a powerful resource for complex trait analysis

The set of LXS recombinant inbred (RI) strains is a new and exceptionally large mapping panel that is suitable for the analysis of complex traits with comparatively high power. This panel consists of 77 strains—more than twice the size of other RI sets— and will typically provide sufficient statistical power (=0.8) to map quantitative trait loci (QTLs) that account for 25% of genetic variance with a genomewide p < 0.05. To characterize the genetic architecture of this new set of RI strains, we genotyped 330 MIT microsatellite markers distributed on all autosomes and the X Chromosome and assembled error-checked meiotic recombination maps that have an average F₂-adjusted marker spacing of 4 cM. The LXS panel has a genetic structure consistent with random segregation and subsequent fixation of alleles, the expected 3–4 × map expansion, a low level of nonsyntenic association among loci, and complete independence among all 77 strains. Although the parental inbred strains—Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS)—were derived originally by selection from an 8-way heterogeneous stock selected for differential sensitivity to sedative effects of ethanol, the LXS panel is also segregating for many other traits. Thus, the LXS panel provides a powerful new resource for mapping complex traits across many systems and disciplines and should prove to be of great utility in modeling the genetics of complex diseases in human populations.(Robert W. Williams and Beth Bennett)These authors contributed equally to this work.     

HLS5, a novel RBCC (ring finger, B box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1alpha, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.     

Putting the heat on sex determination

Sex determination and differentiation are inherently fascinating to both layperson and geneticist. Major advances have accelerated interest in the molecular genetic events mediating these processes in nematodes, flies, mice and humans. Far less attention has been paid to those organisms, particularly reptiles, where sex is determined by environmental cues. However, recent experimental evidence suggests that the two modes of sex determination may not only share common genetic elements, but may also be regulated by similar mechanisms. We argue that the ability to manipulate sex by temperature provides a particularly suitable model for exploring the molecular basis of this fundamental biological process.     

Overexpression or ablation of JNK in skeletal muscle has no effect on glycogen synthase activity

c-Jun NH₂-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle. electroporation; gene delivery; muscle contraction; exercise     

Prostate-specific antigen testing in Ontario: reasons for testing patients without diagnosed prostate cancer

BACKGROUND: The use of the prostate-specific antigen (PSA) test has been increasing rapidly in Canada since its introduction in 1988. The reasons for using the PSA test in patients without known prostate cancer are unclear. This paper reports on the first study in Canada to use physician records to assess the use of PSA testing. METHODS: A questionnaire was mailed to physicians attending 475 patients without diagnosed prostate cancer. The patients were randomly selected from 2 laboratory databases of PSA test records in the greater Toronto area during 1995. The physicians were asked to consult their patient records to avoid recall bias. Information obtained included physician''s specialty, patient''s age at time of PSA test and reason(s) for the test. RESULTS: There were 264 responses (56%), of which 240 (91%) were usable. Of these 240, 63% (95% confidence interval [Cl] 58%-70%) indicated that the test was conducted to screen for prostate cancer, 40% (95% Cl 34%-47%) said it was to investigate urinary symptoms, and 33% (95% Cl 27%-40%) responded that it was a follow-up to a medical procedure or drug therapy. More than one reason was permitted. Of 151 responses indicating screening as one reason for testing, 64% (95% Cl 56%-72%) stated that it was initiated by the patient, and 73% (95% Cl 65%-80%) stated that it was part of a routine examination. For 19%, both investigation of symptoms and screening asymptomatic patients were given as reasons for testing, and for another 19% both follow-up of a medical procedure and screening were given as reasons. Screening was recorded as a reason for testing far more commonly for patients seen by family physicians and general practitioners than for patients seen by urologists (67% v. 29%, p < 0.001). In contrast, the use of PSA testing to diagnose urinary symptoms was more common for patients seen by urologists than for those seen by family physicians and general practitioners (52% v. 37%, p = 0.044). No significant difference was found between physician groups in the use of PSA testing as a follow-up of a medical procedure (42% for urologists and 31% for family physicians and general practitioners). About 24% of the PSA test records were for patients younger than 50 and older than 70 years. PSA testing initiated by patients was more common in the practices of family physicians and general practitioners than in the practices of urologists (44% v. 13%, p < 0.001). INTERPRETATION: Screening for prostate cancer was the most common reason for PSA testing in our study group; it occurred most commonly in the family and general practice setting and was usually initiated by the patient. Differences in reasons for testing were identified by practice specialty. Although PSA screening for prostate cancer is sometimes recommended for men between 50 and 70 years of age, it is being conducted in men outside this age group.     

Honey bees can disseminate a microbial control agent to more than one inflorescence pest of oilseed rape

Pollen beetles (Meligethes aeneus) and cabbage seed weevils (Ceutorhynchus assimilis) are major pests of oilseed rape (Brassica napus) throughout Europe. In field cage experiments in both winter and spring rape, honey bees (Apis mellifera) effectively transported the entomopathogenic fungus Metarhizium anisopliae to the flowers, causing infection and mortality of both adult and larval pollen beetles, as well as of adult seed weevils. External conidiation was observed on many of the dead pest insects. Although some external conidiation also occurred on dead honey bees, reduction in honey bee colony size during the experiments appeared unrelated to the fungus. The potential of this technique for integration into pest management strategies for the crop, particularly in association with a trap crop, is discussed.