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A Virtanen

Publications and source records attributed to A Virtanen.

At least 19 recordsLinked to original sources
PubMed1997

The murine IgM secretory poly(A) site contains dual upstream and downstream elements which affect polyadenylation.

Regulation of polyadenylation efficiency at the secretory poly(A) site plays an essential role in gene expression at the immunoglobulin (IgM) locus. At this poly(A) site the consensus AAUAAA hexanucleotide sequence is embedded in an extended AU-rich region and there are two downstream GU-rich regions which are suboptimally placed. As these sequences are involved in formation of the polyadenylation pre-initiation complex, we examined their function in vivo and in vitro . We show that the upstream AU-rich region can function in the absence of the consensus hexanucleotide sequence both in vivo and in vitro and that both GU-rich regions are necessary for full polyadenylation activity in vivo and for formation of polyadenylation-specific complexes in vitro . Sequence comparisons reveal that: (i) the dual structure is distinct for the IgM secretory poly(A) site compared with other immunoglobulin isotype secretory poly(A) sites; (ii) the presence of an AU-rich region close to the consensus hexanucleotide is evolutionarily conserved for IgM secretory poly(A) sites. We propose that the dual structure of the IgM secretory poly(A) site provides a flexibility to accommodate changes in polyadenylation complex components during regulation of polyadenylation efficiency.

Alternative Splicing↗
PubMed1996

Inducible nuclear factors binding the IgM heavy chain pre-mRNA secretory poly(A) site.

Two alternative forms of IgM heavy-chain mRNA are produced from a common precursor mRNA as a result of competition between cleavage/poly(A) addition at the upstream (secretory) poly(A) site and cleavage/poly(A) addition at the downstream (membrane) poly(A) site coupled with splicing. The efficiency of cleavage at the secretory poly(A) site is thought to play a crucial role in this alternative processing. We therefore examined RNA binding factors recognizing the secretory poly(A) site, in the absence of the splicing option, to look for transacting factors that may play a role in cleavage/polyadenylation efficiency at this site. Purified primary B cells produce the secretory form of mu mRNA when stimulated with lipopolysaccharide (LPS) and the membrane form of mu mRNA when their antigen receptors are ligated by anti-mu antibodies. We compared RNA binding factors in nuclear extracts from cells produced by these different stimulatory conditions and show that induction of the secretory form of mu mRNA by LPS correlates with the induction of a 28-32-kDa secretory poly(A) site-specific polypeptide which is also present in the plasmacytoma cell line J558L. Visualization of the 28-32-kDa polypeptide in UV cross-linking assays depends on a GU-rich element downstream of the secretory poly(A) site. We show that this GU-rich region enhances polyadenylation efficiency in vivo by transfection of luciferase reporter constructs into the plasmacytoma J558L. We also examined nuclear extracts from B cells doubly stimulated with LPS and anti-mu antibodies in which expression of the secretory form of mu mRNA is selectively inhibited. This inhibition may be due to a down-regulation of polyadenylation at the secretory poly(A) site or an up-regulation of the competitive splicing process. This form of stimulation does not lead to the disappearance of the 28-32-kDa polypeptide, but to an enhanced binding of a 50-55-kDa factor which binds both the secretory and membrane poly(A) site. We report the first detection of changes in RNA binding factors taking place at the secretory poly(A) site which correlate with the expression of different forms of mu mRNA produced by primary B cells under different stimulation conditions.

Animals↗
PubMed1995

Estimation of reference change limits using patient data.

Two approaches for deriving reference change limits from patient data are described. In the direct method, hospital database information is used for the selection of appropriate reference groups. If database information is not sufficient or reliable enough, but still most of the source data can be considered as health-related, an indirect method can be applied in the calculation of rough estimates for reference change limits. A computer program developed by us, GraphROC for Windows, includes both methods for the estimation of change limits from patient data. Time between specimen collections should be included as one classifying factor in the selection of source data. When only one previous result is available for comparison, change limits based on the reference sample group form the only available guide for clinical interpretation. However, when several previous results are available and the within-subject variances for the considered analyte are known to be heterogeneous between individuals, the clinical interpretation should rather be based on application of time series analysis.

Data Interpretation, Statistical↗
PubMed1995

Reliability and adequacy of discharge diagnosis databases in the production of reference values.

Discharge diagnoses provide a possibility to select patients individually and then to establish reference values for both "pathological" and control groups. Currently, the available diagnostic information is still at its infancy and should be carefully evaluated before the reference values based on those groups are utilized. It is anticipated that electronic storage of diagnostic and therapeutic information will be applied more commonly in the future as the development of computers makes it easier. The advanced utilization of laboratory data challenges physicians both in the clinical and laboratory side to participate in this development in order to make the information systems serve their actual needs more closely.

Clinical Laboratory Information Systems↗
PubMed1995

Effect of platelet count on serum and plasma potassium: evaluation using database information from two hospitals.

The availability of retrospective data from potassium (K+) analyses from two hospitals, one using serum and the other plasma for electrolyte measurements, offered us the possibility to investigate the effect of blood platelet count on serum and plasma K+ concentrations. A weak correlation between plasma K+ and platelet count was observed. The in vitro increase of serum K+ in proportion to the platelet count has clinical significance in conditions, where it may impede the detection of an underlying true K+ disorder. Nomograms and correction factors, based on the correlation between platelet count and serum K+, have been suggested also in some recent reports. In the present study unselected routine patient data was used as source data. The effect of platelet count on the concentration of K+ in serum was lower than reported in previous studies, as indicated by the regression analysis. An increase of 1000 x 10(9)/l in the blood platelet count would cause an increase of about 0.7 mmol/l in the serum K+ concentration (p < 0.0001, r = 0.155). The weak correlation between platelet count and serum K+ does not support the application of platelet-count-based correction of serum K+ level in thrombocytosis. The laboratory should notify the clinician of the significance of the in vitro increase of K+ caused by increased platelet count. K+ should be measured from plasma in such cases.

Artifacts↗
PubMed1995

Poly (A) polymerases in the nucleus and cytoplasm of frog oocytes: dynamic changes during oocyte maturation and early development.

Poly(A) can be added to mRNAs both in the nucleus and in the cytoplasm. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs provides a common mechanism of translational control. In this report, to begin to understand the regulation of polyadenylation activities during early development, we analyze poly (A) polymerases (PAPs) in oocytes and early embryos of the frog, Xenopus laevis. We have cloned and sequenced a PAP cDNA that corresponds to a maternal mRNA present in frog oocytes. This PAP is similar in size and sequence to mammalian nuclear PAPs. By immunoblotting using monoclonal antibodies raised against human PAP, we demonstrate that oocytes contain multiple forms of PAP that display different electrophoretic mobilities. The oocyte nucleus contains primarily the slower migrating forms of PAP, whereas the cytoplasm contains primarily the faster migrating species. The nuclear forms of PAP are phosphorylated, accounting for their retarded mobility. During oocyte maturation and early postfertilization development, preexisting PAPs undergo regulated phosphorylation and dephosphorylation events. Using the cloned PAP cDNA, we demonstrate that the complex changes in PAP forms seen during oocyte maturation may be due to modifications of a single polypeptide. These results demonstrate that the oocyte contains a cytoplasmic polymerase closely related to the nuclear enzyme and suggest models for how its activity may be regulated during early development.

Amino Acid Sequence↗
PubMed1994

Multiple forms of poly(A) polymerases in human cells.

We have cloned human poly(A) polymerase (PAP) mRNA as cDNA in Escherichia coli. The primary structure of the mRNA was determined and compared to the bovine PAP mRNA sequence. The two sequences were 97% identical at the nucleotide level, which translated into 99% similarity at the amino acid level. Polypeptides representing recombinant PAP were expressed in E. coli, purified, and used as antigens to generate monoclonal antibodies. Western blot analysis using these monoclonal antibodies as probes revealed three PAPs, having estimated molecular masses of 90, 100, and 106 kDa in HeLa cell extracts. Fractionation of HeLa cells showed that the 90-kDa polypeptide was nuclear while the 100- and 106-kDa species were present in both nuclear and cytoplasmic fractions. The 106-kDa PAP was most likely a phosphorylated derivative of the 100-kDa species. PAP activity was recovered in vitro by using purified recombinant human PAP. Subsequent mutational analysis revealed that both the N- and C-terminal regions of PAP were important for activity and suggested that cleavage and polyadenylylation specificity factor (CPSF) interacted with the C-terminal region of PAP. Interestingly, tentative phosphorylation sites have been identified in this region, suggesting that phosphorylation/dephosphorylation may regulate the interaction between the two polyadenylylation factors PAP and CPSF.

Animals↗
PubMed1994

Reference intervals developed from data for hospitalized patients: computerized method based on combination of laboratory and diagnostic data.

We utilized the databases of a hospital information system to select for determination of reference values various individual hospitalized patients on the basis of their diagnoses at discharge. The nonparametric 2.5-97.5% "health-related" reference intervals were calculated for hemoglobin concentration, mean corpuscular volume (MCV), and erythrocyte count for both sexes. After excluding patients with diseases possibly affecting erythrocyte variables, we obtained a final group of 1786 women and 1450 men, ages 20-65 years, who were studied in age groups of 20-30, 30-45, 45-55, and 55-65 years. The upper reference limits of the MCV results obtained from hospitalized patients were higher than those produced conventionally from healthy individuals, as would be intuitively suggested by clinical experience. This method, based on selection by diagnosis, could be applicable to various analytes measured in hospital laboratories, provided sufficient data are available as databases.

Adult↗
PubMed1993

Method for determining reference changes from patients' serial data: example of cardiac enzymes.

Changes between serial laboratory test results can be significant, even if none of the individual results exceeds the reference interval. We developed a statistical method for the calculation of reference change limits from routine patients' data for situations in which the majority of the patients can be considered suitable reference subjects. The method was applied to cardiac enzyme data [creatine kinase (CK; EC 2.7.3.2), creatine kinase isoenzyme MB (CK-2), lactate dehydrogenase (LD; EC 1.1.1.28), and lactate dehydrogenase isoenzyme 1 (LD-1)] from 2029 consecutive patients. We used hospital discharge diagnoses to exclude patients with the diagnosis of myocardial infarction or myocarditis, but we also studied the characteristics of the method on unselected patients' data. The reference change limits derived from the diagnosis-selected patient group were as follows (U/L, activity measurements in serum at 37 degrees C according to Scandinavian recommendations): CK from -39 to 27, CK-2 from -8 to 7, LD from -86 to 85, and LD-1 from -19 to 15. Similar limits were obtained by conventional statistical methods from a group of 29 hospitalized patients with no myocardial symptoms. Our results suggest that it is possible to produce clinically applicable reference change limits from routine data.

Adolescent↗
PubMed1992

Properties of a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mammalian mRNA.

A HeLa cell 3'-exonuclease with properties of a mammalian mRNA poly(A) tail-removing enzyme has been characterized. The exonuclease shows high specificity for the poly(A) tail, and it is single strand-specific and requires a 3'-hydroxyl group for its activity. During degradation 5'-AMP is liberated as a product, and a 3'-OH group is left on the last adenosine residue of the remaining poly(A) tail. The activity is inhibited by 5'-AMP and can be competed by poly(A)-containing mRNA or poly(A). Based on these findings we propose a reaction pathway for poly(A) tail removal catalyzed by the HeLa cell poly(A) tail-specific 3' exonuclease.

Base Sequence↗
PubMed1992

Quality control of multichannel hematology analyzers. Bivariate on-line comparison of MCV and MCH values for the detection of random errors.

Volume and hemoglobin content of red blood cells are physiologically interdependent variables but are measured independently in multichannel hematologic analyzers. Therefore correlation of these results to each other can be used as an additional step in internal quality control. To facilitate precise correlation, data were collected from 44,873 consecutive routine determinations of red blood cell parameters. Gaussian distributions were fitted to the original distributions and bivariate elliptical acceptance limits were calculated for simultaneous evaluation of mean corpuscular volume and mean corpuscular hemoglobin results. With the proposed method, rare combinations of the three directly measured red blood cell parameters (hemoglobin, red blood cell count, and mean corpuscular volume) can be identified for the detection of random analytical or preanalytical errors. This mean corpuscular volume-mean corpuscular hemoglobin rule could be added to internal quality-control programs of hematologic analyzers to improve detection of unusual results and possible random errors.

Diagnosis, Computer-Assisted↗
PubMed1992

Role of intradental A- and C-type nerve fibres in dental pain mechanisms.

The arousal of the two components of pain (the first rapid or sharp pain and the second dull pain) are considered to be related to activation of A delta- and C-type nociceptive primary afferents, respectively. The same dichotomy of pain sensations may also exist in teeth, although due to the short distance between the site of stimulation and the brain the two sensations might not be as clearly separated as in stimulation of, for example, the extremities. The sensations evoked by stimulation of human teeth vary according to the type of the stimuli applied. Low-intensity electrical stimulation is able to induce non-painful (prepain) sensations. At high current intensities pain is evoked. Drilling, probing and air-drying of exposed dentin induce only pain. Most studies also indicate that thermal stimulation only induces painful sensations. The quality of dental pain can vary. Typically, dentinal stimulation of teeth with healthy pulps induces sharp pain. On the other hand intense heat stimulation can result in dull pain which radiates to a wider area of the face and jaws. This component of the stimulus-induced pain seems to share some characteristics of toothache associated with painful pulpitis. Single fibre recordings of intradental nerve activity in experimental animals have shown that in addition to A-fibres a considerable number of C-type primary afferents innervate the dental pulp. This is in accordance with the results of neuroanatomical studies, which indicate that 70-80% of pulpal axons in human, monkey, dog, and cat teeth are unmyelinated. Intradental A- and C-fibre groups seem to be functionally different and can be activated separately by certain external stimuli. Comparison of the response characteristics of the pulp nerve fibres and the sensations induced from human teeth indicate that: 1) A-fibres are responsible for the sensitivity of dentine and thus for the mediation of the sharp pain induced by dentinal stimulation, 2) Prepain sensations induced by electrical stimulation result from activation of the lowest threshold A-fibres some of which can be classified as A beta-fibres according to their conduction velocities. Comparison of the responses of the A beta- and A delta-fibres indicate that they belong to the same functional group, 3) Intradental C-fibres are activated only if the external stimuli reach the pulp proper. Their activation may contribute to the dull pain induced by intense thermal stimulation of the tooth and to that associated with pulpal inflammation.

Animals↗
PubMed1992

Reference intervals for 24-h urinary normetanephrine, metanephrine, and 3-methoxy-4-hydroxymandelic acid in hypertensive patients.

The urinary excretion of normetanephrine, metanephrine, and 3-methoxy-4-hydroxymandelic acid was quantified by HPLC in hypertensive patients who were being routinely investigated for the exclusion of pheochromocytoma. The data were used to calculate reference intervals for these analytes. Age- and sex-specific 95% reference intervals for 24-h urinary excretion were as follows (mumol/24 h): normetanephrine in men ages 16-35 years (n = 17) 0.7-3.4, in men older than 35 years (n = 121) 0.8-5.1, in women ages 16-35 years (n = 41) 0.5-2.1, and in women older than 35 years (n = 144) 0.6-3.3 mumol/24 h; metanephrine in men (n = 138) 0.3-2.0, and in women (n = 185) 0.2-1.3; and 3-methoxy-4-hydroxymandelic acid in men (n = 142) 10-54, and in women (n = 184) 9-38. Normetanephrine or metanephrine excretion rates or both exceeded the proposed reference intervals in seven of the eight patients with pheochromocytoma. Determination of 3-methoxy-4-hydroxymandelic acid excretion did not yield any additional diagnostic information.

Adolescent↗
PubMed1991

A simple procedure for isolation of eukaryotic mRNA polyadenylation factors.

We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeable and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.

Cell Nucleus↗
PubMed1991

In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease.

We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.

Base Sequence↗
PubMed1991

Adenovirus E4-dependent activation of the early E2 promoter is insufficient to promote the early-to-late-phase transition.

The adenovirus E4 ORF6/7 protein has been shown to activate the cellular transcription factor E2F. E2F activation leads to activation of the adenovirus early E2 promoter which controls the production of viral DNA replication proteins. In the present study an adenovirus type 5 cDNA mutant, H5ilE4L, was constructed. This mutant is capable of making the ORF6/7 polypeptide but lacks the coding sequences for all other E4 products. H5ilE4L trans activates the early E2 promoter to wild-type levels, but still it is defective for viral DNA replication. A mutant expressing ORF6 in addition to ORF6/7, H5ilE4I, is normal for viral DNA replication. This indicates that activation of the early E2 promoter is insufficient to promote efficient viral DNA replication and that another E4-encoded function is necessary. The ORF6 protein seems to provide this function. We suggest that ORF6/7-induced activation of E2F is not necessary for adenovirus growth in HeLa cells. Rather, this activation might be of importance in the normal, growth-arrested host cell, since E2F has been shown to bind to the promoter regions of a number of immediate-early genes involved in regulation of cell proliferation (M. Mudryj, S. W. Hiebert, and J. R. Nevins, EMBO J. 9:2179-2184, 1990).

Adenovirus Early Proteins↗
PubMed1990

An adenovirus E4 gene product trans-activates E2 transcription and stimulates stable E2F binding through a direct association with E2F.

The adenovirus E4 gene encodes a trans-activating function that can stimulate the E2 promoter. E2 promoter sequences required for E4 trans-activation are identical to those required for E1A trans-activation, and these principally are the E2 promoter binding factor (E2F) binding sites. Furthermore, full activation of E2F DNA binding activity requires both E1A and E4 action. Analysis of a series of mutant E4 viruses identifies open reading frame (orf) 6/7 of the E4 transcription unit as that required for activation of E2F binding activity. In addition, the assay of various E4 cDNAs demonstrates that the E4 orf 6/7 also is responsible for the trans-activation of E2 transcription. Translation of the E4 orf 6/7 mRNA, but not a control mRNA, in a reticulocyte extract generates an activity that can stimulate cooperative binding of E2F in vitro, consistent with recent in vivo assays that demonstrate a role for the E4 gene in E2F stable complex formation. This stimulation is due to a direct interaction of the E4 protein with E2F since an antibody that recognizes the E4 orf 6/7 polypeptide detects this E4 protein in the E2F-DNA complex. We conclude that the E4 orf 6/7 product interacts with the E2F factor altering binding to allow formation of a stable complex that results in a stimulation of transcription.

Adenovirus Early Proteins↗