bluuebunny/crossref_metadata_2025 huggingface dataset:
Predictors of HIV Serodiscordance among Couples in Southwestern Ethiopia
Authors: Habte Ermias , Yami Alemeshet , Alemseged Fissahye , Abdissa Yishak , Deribe Kebede , Memiah Peter , Biadgilign Sibhatu
Published: May 2013

Background:

With transmission of HIV occurring mainly through heterosexual contact, it is paramount to identify serodiscordant couples and implement preventive strategies that will protect the negative partner. The burden of serodiscordance and its predictors in Ethiopia is not clearly understood due to the dearth of data.

Objective:

To assess the prevalence and predictors of HIV serodiscordance among couples tested in Jimma University Specialized Hospital (JUSH) Voluntary Counseling and Testing (VCT) center.

Methods:

The study employed a case–control study design conducted at VCT center of JUSH in all registered serodiscordant couples and seroconcordant couples that were selected from the registered clients in the period from 2003 to 2010. A pretested structured questionnaire was used for data collection using medical chart abstraction. Data were entered, cleaned, and coded using Statistical Package of Social Sciences (SPSS) version 16.

Results:

The prevalence of serodiscordance in the study population was found to be 8.4%. Male and female discordants accounted for 5.8% (137) and 2.6% (62), respectively. Rare use of condom (adjusted odds ratio [AOR] = 7.2; 95% confidence interval [CI] = 1.59-32.54) and active tuberculosis (TB) at enrollment (AOR= 17.7; 95% CI = 2.3-139.2) were significantly found to be the predictors of serodiscordance.

Conclusion:

The prevalence of serodiscordance in the study area was found to be low, but it contributes to a clinically significant population that mandates implementation of preventive strategy. Sero-positive individuals who use condoms rarely should be encouraged to have their partners tested, and the association between active TB and serodiscordance underscores the need for further study.
Retention in Medical Care and Antiretroviral Treatment according to Skin Color in Southern Brazil
Authors: Zubaran Carlos , Michelim Lessandra , Foresti Katia , Medeiros Gregory , May William , Madi José Mauro
Published: May 2013

The aim of this study was to compare the retention in medical care and antiretroviral (ARV) treatment of individuals living with HIV and AIDS to antiretroviral therapy in southern Brazil according to their “race” or skin color. This study is part of a 225-day prospective trial, comprising 7 interviews, in which an intervention designed to improve adherence to treatment was tested. A convenience sample of 73 individuals living with HIV and/or AIDS enrolled in this follow-up procedure. The mean length of continuance in treatment was 161.5 (standard deviation [SD] = 18.6; 95% confidence interval [CI] = 125-198) and 138.4 (SD = 14.1; 95% C.I. = 111-166) days in the “nonwhite” and “white” categories, respectively. There was no significant difference between the 2 categories, χ2(1, n = 72) = 0.76, P = .38, which include similar levels of retention in medical care and treatment with ARV medications between groups of individuals categorized as white and nonwhite in this sample.
Oxidized low-density lipoproteins induce rapid platelet activation and shape change through tyrosine kinase and Rho kinase–signaling pathways
Authors: Wraith Katie S. , Magwenzi Simbarashe , Aburima Ahmed , Wen Yichuan , Leake David , Naseem Khalid M.
Published: May 2013

Key Points

Oxidized LDL stimulates rapid change in platelet shape through ligation of CD36. Ligation of CD36 by oxidized LDL simultaneously activates tyrosine and Rho kinase–dependent signaling pathways.
Characterization of the microbial community structure in Candidatus Liberibacter asiaticus-infected citrus plants treated with antibiotics in the field
Authors: Zhang Muqing , Powell Charles A , Guo Ying , Benyon Lesley , Duan Yongping
Published: May 2013

Background

Huanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. Previously, we reported a combination of Penicillin G and Streptomycin was effective in eliminating or suppressing the associated bacterium, ‘CandidatusLiberibacter asiaticus’ (Las).

Results

Here we report the bacterial composition and community structure in HLB-affected citrus plants during a growing season and while being treated with antibiotic combinations PS (Penicillin G and Streptomycin) and KO (Kasugamycin and Oxytetracycline) using the Phylochip™ G3 array. Both antibiotic treatments resulted in significantly lower Las bacterial titers (Pr<0.05) and hybridization scores. Of the 50,000+ available operational taxonomic units (OTUs) on PhyloChip™ G3, 7,028 known OTUs were present in citrus leaf midribs. These OTUs were from 58 phyla, of which five contained 100 or more OTUs,Proteobacteria(44.1%), Firmicutes(23.5%),Actinobacteria(12.4%),Bacteroidetes(6.6%) andCyanobacteria(3.2%). In the antibiotic treated samples, the number of OTUs decreased to a total of 5,599. The over-all bacterial diversity decreased with the antibiotic treatments, as did the abundance of 11 OTUs withinProteobacteria,Firmicutes,BacteroidetesandPlanctomycetes. Within theProteobacteria, ten OTUs representing the classγ-proteobacteria increased in abundance after four months of treatment, when the Las bacterium was at its lowest level in the HLB-affected citrus field plants.

Conclusions

Our data revealed thatProteobacteriawas constantly the dominant bacterial phylum recovered from citrus leaf midribs, with the α-proteobacterial and the γ-proteobacterial classes vying for prevalence. In addition, the level of bacterial diversity found in the leaf midribs of field citrus was greater than previously described. Bacterial cells in close proximity may be able to modify their microenvironment, making the composition of the microbial community an important factor in the ability of Las to cause HLB progression. A low Las level was seen as an annual fluctuation, part of the bacterial population dynamics, and as a response to the antibiotic treatments.
Intracardiac metastasis from known cervical cancer: a case report and literature review
Authors: Byun Seung Won , Park Sung Taek , Ki Eun Young , Song Hyun , Hong Suk Hee , Park Jong Sup
Published: May 2013

Cardiac metastasis from known cervical cancer is rare. Even through a routine check-up, this type of metastasis can present as pulmonary emboli. Suspicion of this diagnosis in an oncology patient with complicating pulmonary emboli but no evidence of deep vein thrombosis is important, especially in cervical cancer patients with extensive pelvic lymph node metastasis and vascular invasion of a primary tumor. Early recognition may aid in improving the prognosis. We present a case of intracardiac metastasis arising from a squamous carcinoma of the cervix in a patient with pulmonary tumor emboli and review other cases from the literature.
Monoubiquitination of EEA1 regulates endosome fusion and trafficking
Authors: Ramanathan Harish N , Zhang Guofeng , Ye Yihong
Published: May 2013

Background

Early endosomal autoantigen 1 (EEA1) is a membrane tethering factor required for the fusion and maturation of early endosomes in endocytosis. How the activity of EEA1 is regulated in cells is unclear.

Results

Here we show that endogenous EEA1 is prone to monoubiquitination at multiple sites, owing to an intrinsic affinity to ubiquitin conjugating enzymes (E2). The E2 interactions enable a ubiquitin ligase (E3) independent mechanism that decorate EEA1 with multiple mono-ubiquitin moieties. Expression of an ubiquitin-EEA1 chimera that mimics native mono-ubiquitinated EEA1 generates giant endosomes abutting the nucleus. Several lines of evidence suggest that this phenotype is due to increased endosome fusion and a simultaneous blockade on an endosome recycling pathway. The latter is likely caused by diminished endosome fission in cells expressing ubiquitin-EEA1.

Conclusion

Our results demonstrate that ubiquitination may dramatically affect the activity of an endosome fusion factor to alter endosome morphology and trafficking pattern, and thereby implicating an unexpected role of ubiquitin signaling in endocytosis.
“Marvellous Intellectual Feasts”: Arthur Lewis at the London School of Economics, 1933–48
Authors: Ingham Barbara , Mosley Paul
Published: May 2013

This article is concerned with the decade and a half spent by the development economist Arthur Lewis at the London School of Economics between 1933 and 1948. It discusses the intellectual traditions of the institution that Lewis joined and the various influences on the young economist. His research and teaching roles in London and Cambridge are covered, together with his work for the Fabian Society and his links with the anti-imperialist movements centered in London in the 1930s and 1940s. The aim of the essay is to shed light on this highly significant but little-known period in the career of the foremost development economist.
Ribosome profiling reveals resemblance between long non-coding RNAs and 5′ leaders of coding RNAs
Authors: Chew Guo-Liang , Pauli Andrea , Rinn John L. , Regev Aviv , Schier Alexander F. , Valen Eivind
Published: May 2013

Large-scale genomics and computational approaches have identified thousands of putative long non-coding RNAs (lncRNAs). It has been controversial, however, as to what fraction of these RNAs is truly non-coding. Here, we combine ribosome profiling with a machine-learning approach to validate lncRNAs during zebrafish development in a high throughput manner. We find that dozens of proposed lncRNAs are protein-coding contaminants and that many lncRNAs have ribosome profiles that resemble the 5′ leaders of coding RNAs. Analysis of ribosome profiling data from embryonic stem cells reveals similar properties for mammalian lncRNAs. These results clarify the annotation of developmental lncRNAs and suggest a potential role for translation in lncRNA regulation. In addition, our computational pipeline and ribosome profiling data provide a powerful resource for the identification of translated open reading frames during zebrafish development.
Genetic Evidence for Nucleotide Excision Repair of O6-Alkylguanine in Mammalian Cells
Authors: Boyle J. M. , Durrant L. G. , Wild C. P. , Saffhill R. , Margison G. P.
Published: May 2013

Human cells that lack O1-alkylguanine DNA alkyltransferase (AT) activity can remove O1-butylguanine (O1-wBuG) produced in cellular DNA by exposure to N-n-butyl-N-nitrosourea as determined by radioimmunoassay of enzyme digests of DNA. Fibroblasts from xeroderma pigmentosum (XP) complementation groups A and G that show <5 % unscheduled DNA synthesis following exposure to UVC failed to remove O1-nBuG. Hence it appears that O1- alkylguanine is repaired in cells that lack AT by a process that is defective in XP cells, presumably nucleotide excision repair. Neither V79 nor V79/79 Chinese hamster cell lines have AT activity and both are able to remove O1-nBuG from DNA. However, only V79/79 is able to remove O1MeG, suggesting some substrate specificity of the excision repair process. Comparison of relative levels of O1-alkylation by N-mcthyl-, N-ethyl-, N-propyl- and N-n-butyl-nitrosourea indicate that approximately equal levels of O1-alkylation are produced by equitoxic doses of these agents.
DNA Repair in Specific Sequences in Mammalian Cells
Authors: Smith Charles A.
Published: May 2013

To investigate the influence of function or activity of a DNA sequence on its repair, we have studied excision repair of a number of adducts in the non-transcribed, heterochromatic alpha DNA of monkey cells (by physically isolating the DNA) and also the removal of pyrimidine dimers in a number of genes in rodent and human cells (by an indirect assay using a dimer-specific endonuclease). In confluent cells, psoralen and aflatoxin B] (AFB1) adducts are produced in similar frequencies in alpha and in the rest of the DNA, but removal from alpha is severely deficient. Adducts of A’-acetoxy acetyl aminofluorene (NA-AAF) are formed in slightly higher frequencies in alpha, and removal is slightly deficient. The removal of thymine glycols from alpha DNA in gamma-irradiated cells is proficient, as is repair synthesis elicited by exposure to methyl methane sulphonate, dimethyl sulphate, or 254nm ultraviolet light (u.v.). Removal of AFB1 and NA-AAF adducts from alpha is enhanced by small doses of u.v. but not by X-rays or DMS. The quantum efficiency of conversion of psoralen monoadducts to crosslinks is much lower in alpha DNA. Taken together, these results suggest that the highly condensed chromatin structure of alpha hinders access of the repair system that acts on bulky adducts but not of systems for repair of specific base damage, u.v. damage may alter this chromatin structure directly or facilitate the action of some system that changes accessibility of chromatin to repair. The repair deficiencies are not observed in actively growing cells, in which chromatin structure may be less condensed due to DNA replication.

We have also demonstrated preferential excision repair of pyrimidine dimers in active genes. Dimers are efficiently removed from the essential dihydrofolate reductase (DHFR) and hydroxy- methylglutaryl CoA reductase genes in Chinese hamster ovary (CHO) cells and from the transcribed c-ahl proto-oncogene in the mouse cells. Both cell types remove few dimers from their overall genomes or from sequences distal to the DHFR gene; dimers are also removed poorly from the non-transcribed mouse c-mos gene. In human cells, dimers are removed more rapidly from the DHFR gene than from the genome as a whole. However, repair is as deficient in this gene in XP-C cells as it is in the entire genome.

These results suggest that resistance to DNA damage correlates better with repair of vital or active sequences than with overall repair levels and that mutagenic efficiency may vary according to the activity of the gene under study.
DNA Repair Mutants in Higher Eukaryotes
Authors: Collins A. , Johnson R. T.
Published: May 2013

Over the past ten years or so, we have seen a proliferation of reports of new cell lines of various vertebrate species, showing hypersensitivity to killing by DNA damaging agents. Regrettably, but predictably, there is no standard terminology to describe the mutants, and as a result the literature is liberally scattered with fragments of individualistic nomenclature. There is no way of imposing order at this stage, but it may be helpful to bring together in this chapter as much information as possible on the mutants now available. As well as being an aid for reference, this should serve as a pointer towards further investigation - either in characterizing the mutants we have, or in developing new ones to fill gaps in our knowledge.
Functional Expression of the Escherichia Coli Alkyltransferase Gene in Mammalian Cells
Authors: Margison G. P. , Brennand J.
Published: May 2013

Alkylating agents can produce a variety of biological effects in mammalian cells and organisms including toxicity, mutagenicity and malignant transformation. These agents react with oxygen and nitrogen atoms in DNA resulting in 12 products some of which are known to be eliminated from DNA by repair systems. One method of assessing the relative importance of a specific product in any of the biological effects of DNA alkylation would be to convert a cell line that is deficient in a particular repair function into a repair-proficient cell line and to determine whether this influences the magnitude of the effect. The cloning and expression in mammalian cells of the Escherichia coli DNA repair gene coding for the O1-alkylguanine-alkylphosphotriester dual alkyltransferase will be described. The E. coli gene product acts on damage produced in host cell DNA by treatment with methylnitrosourea, and reduces the toxicity and mutagenicity of this agent. The effects on the toxicity of a variety of other mono and bifunctional alkylating agents have also been assessed.
Recent Progress with the DNA Repair Mutants of Chinese Hamster Ovary Cells
Authors: Thompson L. H. , Salazar E. P. , Brookman K. W. , Collins C. C. , Stewart S. A. , Busch D. B. , Weber C. A.
Published: May 2013

Repair-deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant, which is noted for its very high sister-chromatid exchange frequencies. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to chlorodeoxyuridine (CldUrd). After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25-30 kilobases (kb) was present. Since the DNA molecules used to produce these transformants were sheared to <50 kb in size, the correcting gene should be small enough to clone in a cosmid vector.

Using drug-resistance markers to select for hybrids after fusion, we have done complementation experiments with ultraviolet light (u.v.)-sensitive mutants and have identified a sixth complementation group, line UV61. Additionally, CHO mutants UV27-1 and MMC-2, isolated in other laboratories, were found to belong to UV group 3, which is represented by line UV24.

To study the behaviour of transfected DNA molecules in repair-deficient cells, we treated plasmid pSN2gpt with either u.v. radiation or cA-diamminedichloroplatinum(II) (cA-DDP) and introduced the damaged DNA into normal CHO cells (AA8) and mutants UV4 and UV5. Unrepaired damage to the plasmid was indicated by loss of colony-forming ability of the transfected cells in selective medium containing mycophenolic acid. With u.v. damage, the differential survival of the cell lines was similar to that seen when whole cells are treated with u.v. However, with cw-DDP damage, mutant UV4 did not exhibit the extreme hypersensitivity (50-fold) that occurs when cells are treated. This result suggests that UV4 cells may be able to repair cross-links in transfected DNA.
Surface components involved in bacterial pathogen-plant host recognition
Authors: Sequeira Luis
Published: May 2013

During their initial association with plant hosts, pathogenic bacteria interact with plant cell walls. The results of this interaction appear to determine whether bacterial multiplication will take place. With one group of bacterial plant pathogens (e.g. Agrobacterium tumefaciens), attachment to the host surface appears essential for pathogenesis. With another group (e.g. Pseudomonas solanacearum), only those strains that do not attach to the host cell wall are able to multiply in the intercellular spaces. Attachment of many incompatible strains to tobacco mesophyll cell walls leads to a rapid hypersensitive response (HR) and a drastic reduction in bacterial multiplication. Our working hypothesis is that these differences in host response to strains of P. solanacearum are the result of a recognition response in which surface components of both host and pathogen play important roles.

Our approach is based on the use of spontaneous or transposon (Tn5)-generated mutants of strains K60 (virulent) and Bl (avirulent) that differ in surface components and in their ability to attach to host cells and to induce the HR. A study of the surface components of bacterial and tobacco cell walls has led to the tentative conclusion that bacterial lipopolysaccharide (LPS) and plant hydroxyproline-rich glycoproteins mediate initial attachment, apparently as a result of charge-charge interaction. This initial attachment is reversed by high salt concentrations during the first 15 min, but not thereafter. Firm attachment appears to depend on hydrophobic interactions mediated by bacterial pili. At the normal ionic strength of intercellular fluids, extracellular polysaccharide (EPS) appears to inhibit only the pili-mediated attachment. Several HR− mutants of strain Bl have been obtained by Tn5 insertion, but they remain avirulent on tobacco. We are examining the EPS, LPS and pili production, and the attachment characteristics of these strains.
Rhizobium attachment to clover roots
Authors: Mills Kim K. , Bauer Wolfgang D.
Published: May 2013

The adhesion of rhizobia to surfaces of clover roots was examined by an indirect plate-counting assay and phase-contrast microscopy. The number oí Rhizobium trifolii cells attached to clover root segments increased in approximately linear fashion during the first hour of incubation, but did not change appreciably thereafter. The addition of 30 mM-2-deoxy-D-glucose, which effectively inhibits binding of clover root lectin, did not promote the release of previously attached bacteria nor inhibit subsequent attachment to either root segments or root hairs. Rhizobia of several heterologous species attached to clover roots in numbers comparable to those of strains of R. trifolii, the homologous species. These results indicate that rhizobia have effective mechanisms of adhesion to non-host roots and that clover lectin contributed little or nothing to attachment under the conditions examined.
Cell wall glycoproteins: structure and function
Authors: Roberts K. , Grief C. , Hills G. J. , Shaw P. J.
Published: May 2013

Hydroxyproline-rich glycoproteins form an important, but little understood, structural component of most cell walls. Their occurrence, chemistry, synthesis, secretion, cross-linking and functions in higher plant cell walls will be briefly reviewed. Similar molecules also occur in other groups of plants; in particular, in the algae. In many of these they form highly ordered cell surface arrays, and we have studied these by high-resolution electron microscopy and computer image reconstruction. Some resulting three-dimensional models of these are presented. One particular glycoprotein, the major structural component of the cell wall of Chlamydonwnas reinhardii, has been investigated in some detail. The chemistry and structure of this glycoprotein, which we have called volvin, has. been studied and a family of monoclonal antibodies has been raised against it. Some of these antibodies appear to be specific to oligosaccharide side-chains and allow the localization of these substituents and their sites of synthesis. Immunofluorescence studies have shown that the expression of some of these antigenic determinants is developmentally regulated or cell-cycle-dependent. Immunogold labelling of thin sections has enabled the sites of synthesis and the method of secretion to be determined. These results will be discussed in the context of other cell wall glycoproteins, their relation to other glycoproteins, such as the mating agglutinin, and to their possible functions.
Differences and similarities in the neurotrophic growth factor requirements of sensory neurons derived from neural crest and neural placode
Authors: Lindsay Ronald M. , Barde Yves-Alain , Davies Alun M. , Rohrer Hermann
Published: May 2013

This article reviews recent studies that have examined differences and similarities in the neurotrophic growth factor requirements of neural crest-and neural placode-derived sensory neurons of the developing chick embryo. From in vitro experiments using both explant and dissociated, neuron-enriched cultures of spinal and cranial nerve sensory neurons, it has been established that only sensory neurons of neural crest origin are responsive, at least in terms of survival and neurite outgrowth, to mouse submandibular gland nerve growth factor (NGF). Sensory neurons derived from neural placodes (neurons of the ventrolateral portion of the trigeminal ganglion and the entire neuronal population of the vestibular, geniculate, petrosal and nodose ganglia) are largely unresponsive to NGF throughout embryonic development, but do respond to neurotrophic activity present in extracts of brain and various peripheral ‘end-organs’, such as heart or liver. By incubation of neuron-enriched cultures with radiolabelled [125I]NGF, followed by autoradiographic exposure, it has been demonstrated that placode-derived neurons, in marked contrast to those of neural crest origin, are completely devoid of specific cell surface receptors for NGF. In contrast to differences in their requirement and responsiveness to NGF, both placode-and crest-derived sensory neurons are responsive to the survival and neurite-promoting activity of a recently purified brain-derived neurotrophic factor (BDNF). It is postulated that all primary sensory neurons have a dual growth factor requirement during development; their survival being dependent on a supply of both a peripheral and a central ‘target’-derived neurotrophic factor. It appears that BDNF may act as common ‘central target-derived’ neurotrophic factor for both placode-and crest-derived sensory neurons, but that within peripheral tissues there are specific neurotrophic factors for each of these two classes of primary sensory neurons.
Analysis Of Pseudopodial Structure And Assembly With Viral Projections
Authors: A. Mortara Renato , L. E. Koch Gordon
Published: May 2013

The mechanisms by which cells extend motile pseudopodial projections are still poorly understood. Several fundamental mechanisms have been proposed on the basis of hydrostatic pressure, membrane addition and microfilament reorganization. A common focus of all such mechanisms is the growing tip of a pseudopodium. Yet some basic questions about the nature of the tip in natural pseudopodia remain obscure. However, one class of structure, the virus-tipped projections, often contains a well-defined particle, both morphologically and biochemically, and therefore provides a useful model system for the examination of the tips of cellular projections.

In P815 cells the virus-tipped projections are long, thin structures closely resembling filopodia in other cells. The apical virus particle is a retrovirus particle produced by the chronic infection existing in this cell line. In demembranated filopodia, the virus particle retains a tight association with a single actin microfilament. Biochemical analyses indicate that the major retroviral structural polypeptide Pr65 is an actin-binding protein that could provide the anchorage site for the actin filament.

The existence of a solid virus particle tethered by an actin filament to the cytoskeleton makes it very unlikely that these projections grow by membrane addition at the tip. The major positive implication is that the apex of a projection does not relinquish’ its interaction with the submembranous cytoskeleton during growth. Such an arrangement would be compatible with either a hydrostatic-pressure-driven or a cytoskeleton-driven mechanism of filopodial growth.
Dna transposition between plant organellar genomes
Authors: Stern David B.
Published: May 2013

Higher plant mitochondrial and chloroplast DNAs are known to share extensive sequence homologies. The present work addresses issues raised by these initial observations: (1) what is the distributive pattern of ctDNA sequences among different mitochondrial genomes, (2) what is the frequency of DNA transposition between the two organelles, (3) are the transposed ctDNA sequences transcribed? The results to be presented demonstrate that many ctDNA sequences, including identified genes, are widespread in mitochondrial genomes and in some cases are highly conserved. However, the distribution of any one particular sequence is sporadic, even within a plant family. Preliminary data, obtained in studies of watermelon, raise the possibility that some mtDNA transcripts share homology with ctDNA sequences.
Analysis of brome mosaic virus replication and aminoacylation functions by sitespecific mutagenesis
Authors: Hall Timothy C. , Marsh Loren E. , Dreher Theo W.
Published: May 2013

Brome Mosaic Virus (BMV) has a tripartite RNA genome; each RNA and the subgenomic RNA encoding the viral coat protein share a highly homologous region of about 200 nucleotides at the 3’ end, for which a tRNA-like structure has been proposed. Several sequences encoding functions, including replicase binding, initiation of (−) strand synthesis and tyrosine esterification are known to be nested within this region. Elongation factor EF-la binds to aminoacylated viral RNAs, but not to the uncharged forms. An additional function of the tRNA-like structure is to serve as a substrate for nucleotidyl transferase, which adds the terminal adenosine residue to the (+) sense virion RNAs. A template-dependent and template-specific replicase preparation from BMV-infected barley leaves has been characterized and extensively used for replication studies in vitro that complement studies in vivo using protoplasts. The replicase has been shown to initiate de novo both (−) strand synthesis on supplied (+) strand RNAs, and (+) strand subgenomic RNA synthesis on supplied (—) sense RNA3 templates. RNA transcripts obtained by transcription in vitro of cDNA clones containing desired base substitutions and deletions, have been supplied as templates for replication, aminoacylation and other assays. Use of such mutant RNAs has allowed the promoters for both (−) strand synthesis and for synthesis of the subgenomic (+) strand RNA to be characterized and defined. The same approach has also been used to reveal regions of the tRNA-like structure involved in the tyrosylation of the BMV RNAs. These experiments showed that, although regions important in aminoacylation and replication functions overlap, they are not identical. Some of the mutations tested in vitro have also been tested for infectivity in vivo using both barley plants and protoplasts. Mutants retaining replicase and nucleotidyl transferase template activity, but having lost aminoacylation capability are of special interest in that they should reveal the role of aminoacylation in the infection process.
The requirements for a high level of transposition of bacteriophage mu
Authors: Groenen Martien A. M. , Putte Pieter Van De
Published: May 2013

To analyse the functional and structural requirements of Mu transposition a special mini-Mu transposon was constructed. This mini-Mu, situated on a derivative of pBR322, has an easily selectable marker gene conferring chloramphenicol resistance (CamR) cloned between the ends of the mini-Mu. The genes required for transposition are cloned outside the ends and are under control of the PL promoter of bacteriophage λ.

To obtain a high frequency of transposition the A and B products are required. Under B−conditions both cointegrate formation and simple insertions are strongly reduced. The role of B in the transposition process is unknown and is discussed.

Deletion analysis of the ends of the mini-Mu has revealed the existence of multiple sites required for transposition. The same sites were found to be strong A-binding sites. In addition two weak A-binding sites were found. Point mutations introduced into these last sites show that they are at least as important for transposition as the strong binding sites. The organization of the binding sites is asymmetric in Mu. At the left end two binding sites are situated more than 100 bp away from the end. These affect specifically the frequency of transposition. The distance between these two sites and the site close to the left end is extremely critical. At the right end also three A-binding sites are found, but only two seem to be important for transposition.

Transposition with low frequency can be obtained with a mini-Mu with only one end. Secondary att sites are selected which all show some homology with an A-binding site. An absolute requirement for a 5’T at the artificial end is observed. Based on these results a simple scheme for the evolution of a transposon is discussed.
Structure and replication of geminivirus genomes
Authors: Davies J. W. , Stanley J. , Donson J. , Mullineaux P. M. , Boulton M. I.
Published: May 2013

The geminiviruses are a group of plant viruses containing single-stranded (ss) DNA in particles comprising two quasi-icosahedral units. Some are transmitted by whiteflies, others by leafhoppers. Comparisons were made of the genome organization and expression of cassava latent virus (CLV) and maize streak virus (MSV) and beet curly top virus (BCTV), each with distinct host range and insect vector species characteristics. From these studies, several indications as to the replication mechanism(s) are suggested.
Cell Behaviour During Active Cell Rearrangement: Evidence and Speculations
Authors: Keller Ray , Hardin Jeff
Published: May 2013

The cell behaviour and motility underlying cell rearrangement during gastrulation in amphibian and sea-urchin embryos are discussed. In particular, the cell behaviour of deep (non-epithelial) and epithelial cell populations that undergo cell rearrangement is compared and contrasted. Deep cell rearrangement in Xenopus laevis involves both convergence of cells towards the future dorsal midline and simultaneous axial extension of the mesodermal cell mass. Time-lapse cinemicrography and scanning electron microscopy suggest that asynchronous, repetitive motions of individual deep cells, involving local extensions and retractions of their margins, may provide the motive force for rearrangement. Such protrusive activity may be guided by local differences in cell-cell contacts in the marginal zone. Epithelial cell rearrangement in the sea-urchin embryo both elongates the archenteron and simultaneously closes the blastopore. Cell rearrangement is accompanied by stage-specific changes in protrusive activity and cell shape of the basal surfaces of cells in the wall of the gut rudiment, in contrast to the apical surfaces, which show little activity. These basal protrusions may be involved in the rearrangement process.
Stem cells: a problem in asymmetry
Authors: Wolpert Lewis
Published: May 2013

The special property of stem cells is that their development is asymmetric. They give rise both to cells that are identical to themselves and to cells that are different. The mechanism that provides this asymmetry may be intrinsic or extrinsic. Such mechanisms are considered within the context of other systems where asymmetric development occurs. The specification of mating types in yeast provides a clear example of a stem cell system generated intrinsically. In fission yeast it appears that the asymmetry is due to chromosomal differences: this is the only known mechanism for intrinsic asymmetry. While there is good evidence for intrinsic asymmetry in both plants and invertebrates - particularly the nematode - the mechanism is not known. In insects and vertebrates there is no well established example of intrinsic asymmetry if one excludes asymmetric cytoplasmic localization during cleavage of the egg. Asymmetry is thus due to environmental influences. Stem cell systems are usually well structured and the cell’s behaviour seems to be position-dependent. This is well established for the stem cells of hydra. By contrast it is claimed that the mammalian haemopoietic system is generated by an intrinsic, asymmetric, probabilistic mechanism - the validity of this view is questioned.
Marrow stromal stem cells
Authors: Owen Maureen
Published: May 2013

Evidence for the hypothesis that there are stromal stem cells present in the soft connective tissues associated with marrow and bone surfaces that are able to give rise to a number of different cell lines is reviewed. The lines are currently designated fibroblastic, reticular, adipocytic and osteogenic. Fibroblastic colonies, each derived from a single colony-forming unit fibroblastic (CFU-F), are formed when marrow cells are cultured in vitro. In vivo assays of tissue formed by CFU-F in open transplant or in diffusion chambers, have demonstrated that some CFU-F have a high ability for self renewal and multipotentiality whereas some have more limited potential. Preliminary investigations in vitro also support the hypothesis and have shown that CFU-F are a heterogeneous population of stem and progenitor cells and that their differentiation in vitro can be modified at the colony level. The stromal cells which survive and proliferate in vitro are highly dependent on culture conditions. The number and hierarchy of cell lines belonging to the stromal fibroblastic system are not yet fully elucidated and more specific markers and better assays for the different phenotypes are required before a greater understanding can be achieved. The possibility that the marrow stromal system is part of a wider stromal cell system of the body is proposed.
Differentiation of a bipotential glial progenitor cell: what controls the timing and the choice of developmental pathway?
Authors: Raff Martin C. , Lillien Laura E.
Published: May 2013

In the rat central nervous system (CNS) oligodendrocytes and type-2 astrocytes are thought to develop from a common precursor - the O-2A progenitor cell. Oligodendrocytes develop first and make myelin; type-2 astrocytes develop later and extend processes to nodes of Ranvier. The timing of differentiation of O-2A progenitor cells seems to depend on chemical signals secreted by another type of glial cell - the type-1 astrocyte. Type-1 astrocytes secrete platelet-derived growth factor (PDGF), which stimulates O-2A progenitor cell proliferation and drives the clock that controls the onset of oligodendrocyte differentiation, which is the constitutive pathway of progenitor cell development. Later, type-1 astrocytes are thought to secrete a CNTF-like protein that initiates type-2 astrocyte differentiation.
Epidermal stem cells in culture
Authors: Watt Fiona M.
Published: May 2013

Cultures of human epidermal keratinocytes retain many of the characteristics of the tissue from which they are derived, and are therefore useful as an experimental model for studying stem cell properties. The cultures provide evidence that the mechanisms regulating keratinocyte proliferation are complex. The dividing cell compartment is heterogeneous, containing some cells with stem cell characteristics and others that may correspond to transit amplifying cells. The overall growth rate of the cultures is influenced by a wide range of growth stimulatory and inhibitory molecules, but it is not clear whether responsiveness to these factors differs between the different subpopulations of dividing cells. Furthermore, keratinocytes are able to express molecules that regulate their own growth. The cultures are able to achieve at least a partial homeostatic balance between proliferation and terminal differentiation and can regenerate the suprabasal cell layers following injury. Finally, a range of new experimental approaches hold promise for future studies of how keratinocyte proliferation and terminal differentiation are controlled.
Targeting of oncoproteins to membranes by fatty acylation
Authors: Magee Anthony I. , Gutierrez Lourdes , Marshall Christopher J. , Hancock John F.
Published: May 2013

Post-translational modification of proteins with hydrophobic lipid-derived substituents is increasingly becoming recognized as a major route for targeting proteins to membranes. Glycosylphosphatidylinositol (GPI) anchors are found at the C terminus of a wide range of cell surface proteins, and may endow the cell with the ability to release them in a controlled fashion via specific phospholipases. We have concentrated on the direct attachment (acylation) of long-chain fatty acids (myristate, C14:0 and palmitate, C16:0) to proteins associated with the cytoplasmic face of cellular membranes. Two such proteins, the products of the src and ras oncogenes, require acylation respectively with myristate and palmitate for their membrane association and biological activity, including transformation. N-terminal myristoylation of p60ras seems to be a co- translational stable modification. However, our recent results show that post-translational modification of p21ras is a complex cascade of events involving proteolysis, methylation and thioesterification of palmitate. This last acylation event is dynamic in vivo and may regulate ras function. Enzymological studies of these modification events are in progress. A better understanding of acylation may provide targets for future pharmacological intervention.
Protein sorting in yeast: the role of the vacuolar protontranslocating ATPase
Authors: Kane Patricia M. , Yamashiro Carl T. , Rothman Joel H. , Stevens Tom H.
Published: May 2013

We are investigating the physiological roles of organelle acidification in yeast by two different approaches. First, we have identified two mutants which are defective in acidification of the yeast lysosome-like vacuole from among a collection of mutants which mis-sort soluble vacuolar proteins to the cell surface. These mutants have been helpful in identifying other cellular functions linked to acidification, such as the activation of vacuolar zymogens. We have complemented this classical genetic approach to acidification with direct biochemical and reverse genetic studies on the yeast vacuolar proton-translocating ATPase (H+-ATPase), the enzyme responsible for vacuolar network acidification. Our biochemical characterization of this enzyme indicates that it is a multisubunit complex with many structural similarities to other vacuolar H+-ATPases. Like the other vacuolar H+-ATPases characterized, it also shares some structural features with the FiF0-type ATPases of mitochondria, chloroplasts, and Escherichia coli. We are currently cloning the genes for the subunits of the yeast vacuolar H+-ATPase. Mutagenesis of the cloned genes will allow us to determine the phenotype of yeast cells expressing a vacuolar H+-ATPase altered in well controlled ways. We are also beginning to investigate how the subunits of the vacuolar H+-ATPase are assembled into the enzyme complex and targeted to their proper cellular location.
Towards an in vitro system for studying clathrin-coated pit function
Authors: Moore Mary Shannon , Anderson Richard G. W.
Published: May 2013

To elucidate the molecular basis of coated pit function, we are developing a method for reconstituting the endocytic cycle in a cell-free system. Using purified membranes attached to a solid substratum, we have successfully reconstituted coated pit assembly. We now show that the endogenous coated pits on these membranes spontaneously round up and leave the membrane at 37 °C. As measured by radioimmunoassay, clathrin loss is both pH- and temperature-dependent. The loss is also inhibited by pretreating the membranes with apyrase at 37 °C, which suggests that nucleotide triphosphates are required.
Import of proteins into the various submitochondrial compartments
Authors: Hartl Franz-Ulrich , Neupert Walter
Published: May 2013

Import of proteins into mitochondria can be subdivided into several distinct steps. (1) Mitochondrial proteins are synthesized on free ribosomes and are released into cytosolic pools. Nucleoside triphosphates are required to keep precursors in a conformation competent for import.

(2) Precursors are directed to mitochondria by specific targeting signals (in most cases contained in N-terminal presequences) and by binding to receptors on the surface of the outer membrane.

(3) Precursors interact with a component in the outer membrane which is believed to facilitate membrane insertion (‘general insertion protein’). (4) Outer membrane proteins are then directly routed to their final location. Proteins of all other submitochondrial compartments are directed into translocation contact sites between outer and inner membranes. Transfer into contact sites is dependent on the membrane potential (ΔΨ) across the inner membrane. (5) Presequences of precursors are cleaved in the matrix by the mitochondrial processing peptidase in cooperation with the processing enhancing protein. (6) Precursors of the intermembrane space or the outer surface of the inner membrane have to be re-translocated back across the inner membrane (‘conservative sorting’).

Cytochrome c is an exception to this general import pathway. The precursor, apocytochrome c, is directly translocated across the outer membrane into the intermembrane space in a process independent of ΔΨ.
Cellular and viral control of the initiation of DNA replication
Authors: Roberts James M. , D’urso Gennaro
Published: May 2013

Cell-free replication of SV40 DNA in extracts prepared from S phase cells is at least 20-fold more efficient than in extracts from G1 cells. The increased activity of S phase extracts correlates with the presence of an S phase-specific cellular factor that enhances DNA unwinding at the replication origin. This change in origin-DNA structure during the initiation of SV40 replication proceeds through at least three discrete steps which can be distinguished by their extent of topologic unwinding (linking differences of —1, —2 and —5). Specific DNA elements flanking the core origin enhance replication in vivo and facilitate the formation of the pre-initiation complexes, indicating that formation of these underwound conformations may be the limiting step in the initiation of DNA synthesis. In addition, the factor that activates DNA replication in extracts from S phase cells also enhances the formation of the most highly underwound —5 pre-initiation complex. These observations suggest that during SV40 replication, formation of the rate-limiting pre-initiation complex is the focus of at least three regulatory elements. Two of these are DNA sequences flanking the replication origin and the third is a cellular factor specific to the S phase cell.
DNA replication and cell cycle control in Xenopus egg extracts
Authors: Hutchison C. J. , Brill D. , Cox R. , Gilbert J. , Kill I. , Ford C. C.
Published: May 2013

Aspects of the regulation of DNA replication and mitosis have been studied using a cell-free extract of Xenopus eggs. The extract is characterized by repeated cycles of DNA replication and mitosis, which are accompanied by periodic synthesis and degradation of cyclins as well as fluctuations in the level of Histone Hl kinase activity. DNA replication in this system is dependent upon the formation of a nucleus. However, while nuclear structures are clearly required for initiation, a complete nuclear membrane does not appear to be necessary. Indirect immunofluorescence and DIC microscopy indicate that nuclear reformation from chromosomes occurs asynchronously around individual chromatids. Lamin polymerization, biotin-11-dUTP incorporation and association of polymerases with chromatin occur before membrane formation is complete. S phase nuclei are typified by the co-distribution of both anti-DNA polymerase a and anti-PCNA antibodies as discrete spots of fluorescence which align the chromatin. However, as DNA replication is terminated, PCNA fluorescence fades and DNA polymerase " dissociates from the chromatin and is redistributed throughout the nucleoplasm. By inhibiting DNA replication with aphidicolin, both DNA polymerase a and PCNA remain associated with the chromatin throughout prolonged incubation. Under these conditions mitosis is delayed by up to 70 min, although both the general rate of protein synthesis and more importantly the rate of cyclin synthesis and histone kinase activation are unaffected. Upon nuclear envelope breakdown and lamin dispersal, cyclins degrade; however, no chromosomes are formed, and both PCNA and DNA polymerase a remain associated with the chromatin. Also, histone kinase activity is maintained at elevated levels.
NSP1, a yeast protein located at the nuclear periphery, is required for the cell cycle of Saccharomyces cerevisiae
Authors: Hurt E. C.
Published: May 2013

Proteins located at the nuclear periphery and required for nuclear and cell division are not known in yeast. We recently identified a novel nucleoskeletal-like protein in Saccharomyces cerevisiae, which is in close proximity to the spindle pole body and the nuclear membrane. NSP1 has an essential function, since haploid yeast cells with a defective NSPJ gene cannot grow. In this report, we demonstrate that yeast cells which carry the NSP1 gene under the regulatory GALIO promoter exhibit abnormal cell morphology both under conditions where the level of NSP1 expression is greatly increased (the GALIO promoter is induced) or limited (the GALIO promoter is not induced). This result suggests that NSP1 is required for the coordinated cell cycle in S. cerevisiae.