The repetitive calcium waves in the fertilized ascidian egg are initiated near the vegetal pole by a cortical pacemaker.

Ascidian eggs respond to fertilization with a series of repetitive calcium waves that originate mostly from the vegetal/contraction pole region (J. E. Speksnijder, C. Sardet, and L. F. Jaffe, 1990, Dev. Biol. 142, 246-249), where the myoplasm is concentrated during the first phase of ooplasmic segregation. This suggests that the myoplasm may be involved in initiating these calcium waves. To test this possibility, the starting position of the calcium waves was determined in eggs that had the subcortical, mitochondria-rich part of the myoplasm displaced by centrifugation. Such centrifuged eggs display four cytoplasmic layers: a large centrifugal yolk zone, a narrow clear zone, a mitochondria-rich layer, and a small clear zone at the centripetal pole. Imaging of the cytosolic calcium in centrifuged eggs that were injected with the calcium-specific photoprotein aequorin reveals a series of repetitive calcium waves after fertilization. About 70% of these waves start in the vegetal/contraction pole area, which is similar to the number of waves previously found to start in this area in uncentrifuged eggs. In contrast, only about 25% of the waves start close to the displaced mitochondria-rich layer. From this result it is concluded that the main wave initiation site is not displaced by the centrifugal forces that displace the subcortical, mitochondria-rich part of the myoplasm. Moreover, the observation that the animal-vegetal polarity of cortical components such as actin filaments and the endoplasmic reticulum has been retained after centrifugation further suggests that a cortical component located in the vegetal hemisphere--most likely the endoplasmic reticulum network in the cortical region of the myoplasm--is involved in initiating the repetitive calcium waves in the fertilized ascidian egg.     

E.E.G. and Personality Factors in Baby Batterers

Out of 35 parents who battered their children eight had an abnormal E.E.G. All of these were found to be psychopathic, of low intelligence, and to be persistent batterers. The presence of an abnormal E.E.G. strongly suggests that some baby batterers are more closely related to those who commit acts of violence and that taken as a whole they are not a homogenous group about whom it is safe to generalize. The possibility of a separate subgroup among baby batterers, therefore, needs close attention.     

Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.     

Electrocardiographic abnormalities associated with raised intracranial pressure.

Serial electrocardiographic (E.C.G.) recordings were taken in seven patients suffering from intracranial conditions, for which their intracranial pressure was directly and continuously monitored with a Konigsberg extradural transducer. The E.C.G. changes observed in patients with raised intracranial pressure were prominent U waves, ST-T segment changes, notched T waves, and shortening and prolongation of Q-T intervals. Two patients with normal intracranial pressure showed no E.C.G. abnormalities but also establish a relationship between E.C.G. abnormalities and changing intracranial pressure.     

Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.     

The snRNP E protein multigene family contains five pseudogenes with common mutations.

Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.     

A health centre E.C.G. services: its use and abuse.

An average of 10-5 E.C.G.s were recorded weekly in a health centre used by 32 general practitioners serving a population of almost 65,000. The main indication for an E.C.G. was chest pain (73%). 47% of the E.C.G.s were abnormal. A change in clinical diagnosis occurred in 28% of cases and in patient management in 16%. A significant number of these changes were unwarranted, however. It is recommended that the E.C.G.s should be recorded by suitably trained nurses and reported by a specially trained general practitioner. Further education of general practitioners in the clinical use of the E.C.G. is required.     

Characterization of an endoplasmic reticulum retention signal in the rubella virus E1 glycoprotein.

Rubella virus contains three structural proteins, capsid, E2, and E1. E2 and E1 are type I membrane glycoproteins that form a heterodimer in the endoplasmic reticulum (ER) before they are transported to and retained in the Golgi complex, where virus assembly occurs. The bulk of unassembled E2 and E1 subunits are not transported to the Golgi complex. We have recently shown that E2 contains a Golgi-targeting signal that mediates retention of the E2-E1 complex (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7-20, 1995). The focus of this study was to determine if E1 glycoprotein also contains intracellular targeting information. We constructed a series of chimeric reporter proteins by fusing domains from E1 to the ectodomains of two other type I membrane proteins which are normally transported to the cell surface, vesicular stomatitis virus G protein (G) and CD8. Fusion of the E1 transmembrane and cytoplasmic regions, but not analogous domains from two control membrane proteins, to the ectodomains of G and CD8 proteins caused the resulting chimeras to be retained in the ER. Association of the ER-retained chimeras with known ER chaperone proteins was not detected. ER localization required both the transmembrane and cytoplasmic regions of E1, since neither of these domains alone was sufficient to retain the reporter proteins. Increasing the length of the E1 cytoplasmic domain by 10 amino acids completely abrogated ER retention. This finding also indicated that the chimeras were not retained as a result of misfolding. In summary, we have identified a new type of ER retention signal that may function to prevent unassembled E1 subunits and/or immature E2-E1 dimers from reaching the Golgi complex, where they could interfere with viral assembly. Accordingly, assembly of E2 and E1 would mask the signal, thereby allowing transport of the heterodimer from the ER.     

Ultra-high expression of a thermally responsive recombinant fusion protein in E. coli

Elastin-like polypeptides (ELPs) are recombinant peptide-based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fraction of these amino acids in ELPs as compared to other cellular proteins, we hypothesized that intracellular pools of these amino acids can be selectively depleted and limit protein yields during expression. In this study, we examined how culture conditions and individual medium components affect protein yields by monitoring cell growth and protein expression kinetics of E. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining the underlying principles of superior fusion protein yields generated by the hyperexpression protocol, we further improved protein yields through the addition of glycerol and certain amino acids such as proline and alanine and found that amino acid concentrations and the type of basal medium used strongly influenced this beneficial effect. Surprisingly, amino acids other than those that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamic acid, also enhanced protein yields even in a nutrient-rich medium. Compared to commonly used Luria-Bertani medium, the protein yield was improved by 36-fold to the remarkable level of 1.6 g/L in shaker flask cultures with a modified medium and optimized culture conditions, which also led to a 8-fold reduction in the cost of the fusion protein. To our knowledge, this is the highest yield of an ELP-fusion protein purified from E. coli cultured in shaker flasks. This study also suggests a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides.     

Periodic calcium waves cross ascidian eggs after fertilization

Ascidian eggs respond to fertilization with one to two dozen periodic calcium pulses (J.E. Speksnijder, D.W. Corson, C. Sardet, and L.F. Jaffe, 1989a, Dev. Biol. 135, 182-190). We examined the spatial pattern of these pulses and found that they are initiated in discrete regions from which they propagate as waves. The first few pulses start in the animal hemisphere, whereas the later ones are mostly initiated near the vegetal pole. Such vegetal waves are often followed by a contraction of the egg surface. Since these waves are attenuated as they spread, they repeatedly expose the vegetal pole region to more calcium. The mechanism of these repetitive calcium waves and their possible role in establishing pattern or completing meiosis is discussed.     

Modification of hepatic vitamin E stores in vivo. II. Alterations in plasma and liver vitamin E content by 1,2-dibromoethane

Previous studies with methyl ethyl ketone peroxide (MEKP), a radical generator, showed depletion of plasma vitamin E and liver glutathione (GSH) levels prior to a decrease of liver vitamin E levels. Since hepatic pools of this vitamin may serve to maintain circulating levels of vitamin E under conditions of oxidative challenge, we have evaluated the similarity of response after treatment with 1,2-dibromoethane (DBE), a compound that is not known to generate oxyradicals or to induce lipid peroxidation in vivo. Treatment of normal rats with DBE caused a depletion in hepatic vitamin E levels 1 day after treatment; however, in contrast to our prior findings with MEKP this depletion after DBE treatment was observed in tandem with elevations in the plasma content of vitamin E. Liver vitamin E depletion was neither dependent upon a sustained liver GSH depletion nor upon hepatocellular death. Mobilization and export of hepatic vitamin E did not result in an immediate whole body redistribution of this vitamin in that pulmonary and renal levels of vitamin E remained normal under conditions of liver vitamin E depletion. Moreover, the stimulus that resulted in exportation of liver vitamin E was maintained by daily treatments with DBE. DBE caused a substantial elevation above control values in liver GSH content and these elevations were also maintained by daily DBE treatments. In experiments to assess the influence of prandial replacement of vitamin E on the extent of depletion in response to DBE treatment, rats were fed a vitamin E-deficient diet for 2 days prior to treatment. This short pulse of a vitamin E-deficient diet delayed (to 2 days) both the elevation in liver GSH content and the depletion of liver vitamin E and hastened (to 1 day) the elevation in plasma vitamin E concentration. These observations suggest the presence of at least two pools of liver vitamin E and that one of these pools, which comprises at least 30% of the total hepatic vitamin E content, is able to be mobilized and exported in response to chemical challenge. The stimulus that resulted in liver vitamin E exportation in response to DBE treatment seems to result from wholly intrahepatic processes and may not be a direct response to lipid peroxidation. Moreover, the similarity between the time-course and the extent of hepatic vitamin E depletion observed after treatment with either MEKP or DBE suggests a similarity in physiochemical processes that function to mobilize hepatic vitamin E stores.     

Rescue of cyclin D1 deficiency by knockin cyclin E.

D-type cyclins and cyclin E represent two very distinct classes of mammalian G1 cyclins. We have generated a mouse strain in which the coding sequences of the cyclin D1 gene (Ccnd1) have been deleted and replaced by those of human cyclin E (CCNE). In the tissues and cells of these mice, the expression pattern of human cyclin E faithfully reproduces that normally associated with mouse cyclin D1. The replacement of cyclin D1 with cyclin E rescues all phenotypic manifestations of cyclin D1 deficiency and restores normal development in cyclin D1-dependent tissues. Thus, cyclin E can functionally replace cyclin D1. Our analyses suggest that cyclin E is the major downstream target of cyclin D1.     

Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A.

We have examined E2F binding activity in extracts of synchronized NIH 3T3 cells. During the G0 to G1 transition, there is a marked increase in the level of active E2F. Subsequently, there are changes in the nature of E2F-containing complexes. A G1-specific complex increases in abundance, disappears, and is then replaced by another complex as S phase begins. Analysis of extracts of thymidine-blocked cells confirms that the complexes are cell cycle regulated. We also show that the cyclin A protein is a component of the S phase complex. Each complex can be dissociated by the adenovirus E1A 12S product, releasing free E2F. The release of E2F from the cyclin A complex coincides with the stimulation of an E2F-dependent promoter. We suggest that these interactions control the activity of E2F and that disruption of the complexes by E1A contributes to a loss of cellular proliferation control.     

北京地区宫颈癌HPV16上游调控序列、E6、E7癌基因序列初步分析

为检测HPV16上游调控序列(Upstream regulatory region, URR)、E6、E7癌基因变异在北京地区宫颈癌患者癌组织中的分布特征, 探讨该地区宫颈癌发生同HPV16变异株间的相关性, 文章以提取的31例HPV16检测阳性宫颈癌组织DNA为模板, 设计针对性引物扩增URR、E6、E7 3个目的片段, PCR产物直接测序并通过GenBank对比分析变异和分支鉴定情况。在所分析的宫颈癌组织中, URR是突变频率最高的片段, 其次为E7, 最保守的序列为E6。共发现热突变位点8个, 分别为URR序列上G7521A(100%)、C7435G(96.77%)、C24T(45.16%)、A7729C(45.16%)、G7839A(45.16%); E6序列上T178G(41.94%); E7序列上A647G(45.16%)、T846C(45.16%)。HPV16分支分布频率最广的是As型(54.84%), 其次为E型(45.16%)。研究结果提示, HPV16URR序列上G7521A、A7729C、G7839A, E6序列上T178G、T350G, E7序列上A647G、G658A等位点的变异可能与病毒致癌潜能及宫颈癌的发生相关。北京地区宫颈癌患者中As和E型可能是两种最主要的HPV16分支, 这有可能会为HPV疫苗的研制和感染治疗提供有价值的信息。As型和E型病毒在不同年龄组和不同肿瘤分期组的患者中分布频率有差异, 这可能会为揭示宫颈癌年轻化趋势提供新的线索。