A kinetic analysis of myogenesis in vitro.

Conditions which yielded reproducible growth kinetics with extensive, relatively synchronous differentiation are described for chick muscle cultures. The effects of cell density and medium changes on the timing of cell fusion were examined. Low-density cultures which received a change of medium at 24 hr after plating show the highest rate of cell fusion, increasing from 15 to 80% fused cells in a 10 hr period. These optimal culture conditions were employed to reexamine two questions from the earlier literature on muscle culture: (a) can cells which normally would fuse at the end of one cell cycle be forced to go through another cell cycle before fusion; and (b) how soon after its final S period can a cell complete fusion? In answer to the first question, it was found that if the medium is changed, many cells which would otherwise fuse can be made to undergo another cell cycle before fusion. In the second case, radioautographs were made from cultures incubated with tritiated thymidine for various times at the beginning of the fusion period. These show labeled nuclei in myotubes as early as 3 hr after the beginning of the incubation period. This indicates that cells can fuse as early as the beginning of the G(1) period, and suggests that there is not an obligatory exit from the cell cycle or a prolonged G(1) period before cell fusion and differentiation during myogenesis.

Repair DNA synthesis in differentiated embryonic muscle cells.

The differentiation of embryonic skeletal muscle cells is closely coupled with the cessation of normal DNA replication. Once these cells begin to differentiate, they normally never undergo semiconservative replication of DNA during the entire life time of the muscle cell. Cessation of DNA synthesis has been shown to be accompanied by a loss of 80-90% of the replicative DNA polymerase activity of these cells. Despite this loss the studies reported here demonstrate that muscle cells retain the ability to synthesize DNA of a repair type after UV irradiation. These results suggest that the control exercised over semiconservative DNA synthesis during differentiation of these cells does not extend to repair synthesis after UV irradiation.

A genomic analysis of adult T-cell leukemia.

Adult T-cell leukemia (ATL) is an intractable malignancy of CD4+ T cells that is etiologically associated with infection by human T-cell leukemia virus-type I. Most individuals in the chronic stage of ATL eventually undergo progression to a highly aggressive acute stage. To clarify the mechanism responsible for this stage progression, we isolated CD4+ cells from individuals in the chronic (n=19) or acute (n=22) stages of ATL and subjected them to profiling of gene expression with DNA microarrays containing >44,000 probe sets. Changes in chromosome copy number were also examined for 24 cell specimens with the use of microarrays harboring approximately 50,000 probe sets. Stage-dependent changes in gene expression profile and chromosome copy number were apparent. Furthermore, expression of the gene for MET, a receptor tyrosine kinase for hepatocyte growth factor (HGF), was shown to be specific to the acute stage of ATL, and the plasma concentration of HGF was increased in individuals in either the acute or chronic stage. HGF induced proliferation of a MET-positive ATL cell line, and this effect was blocked by antibodies to HGF. The HGF-MET signaling pathway is thus a potential therapeutic target for ATL.

Consensus methods for finding and ranking DNA binding sites. Application to Escherichia coli promoters.

There have been many different approaches employed to define the "consensus" sequence of various DNA binding sites and to use the definition obtained to locate and rank members of a given sequence family. The analysis presented here enlists two of these approaches, each in modified form, to develop a highly efficient search protocol for Escherichia coli promoters and to provide a relative ranking of these sites showing good agreement with in vitro measurements of promoter strength. Schneider et al. have applied Shannon's index of information content to evaluate the significance of each position within the consensus of a family of aligned sequences. In a formal sense, this index is only applicable to a group of sequences, providing at each position a negative entropy value between zero (random) and two bits (total conservation of a single base) for sequences in which all bases are equally represented. A method for evaluating how well an individual sequence conforms to the information content pattern of the consensus is described. A function is derived, by analogy to the information content of the sequence family, for application to individual sequences. Since this function is a measure of conformity, it can be used in a search protocol to identify new members of the family represented by the consensus. A protocol for locating E. coli promoters is presented. The Berg-von Hippel statistical-mechanical function is also tested in a similar application. While the information content function provides a superior search protocol, the Berg-von Hippel function, when scaled at each position by the information content, does well at ranking promoters according to their strength as measured in vitro.

A back-propagation neural network predicts absorption maxima of chimeric human red/green visual pigments.

The absorption spectra of human red and green visual pigments have peak wavelengths, lambda max, that differ by 31 nm, yet the opsins differ in only 15 amino acids. Mutagenesis studies have demonstrated that seven of the 15 amino acids determine the spectral shift. We trained neural networks to predict the lambda max of any red/green chimeric protein. Seven mutants were excluded from the original training set. The trained networks were able to predict the lambda max for the excluded mutants. As an additional test, five new chimeric pigments were constructed and lambda max determined. The neural networks correctly predicted the lambda max of all five mutants. The use of neural networks is a novel approach to the problem of wavelength modulation in visual pigments.

A Canadian multicenter, double-blind study of paroxetine and fluoxetine in major depressive disorder.

BACKGROUND: Recent studies have suggested clinical differences among selective serotonin reuptake inhibitors. In a 12-week randomized, multicenter, double-blind trial, the antidepressant and anxiolytic efficacy of the selective serotonin reuptake inhibitors paroxetine and fluoxetine was compared in patients with moderate to severe depression. METHODS: A total of 203 patients were randomized to fixed doses (20 mg/day) of paroxetine or fluoxetine for the first six weeks of therapy. From week 7-12, dosing could be adjusted biweekly, as required (paroxetine 20-50 mg/day, and fluoxetine 20-80 mg/day). The mean prescribed doses were paroxetine 25.5 mg/day (range 20.0-40.2 mg/day), and fluoxetine 27.5 mg/day (range 20.0-59.5 mg/day). Emergence of motor nervousness or restlessness was assessed using the ESRS scale for akathisia. RESULTS: Both active treatments demonstrated comparable antidepressant efficacy (HAM-D, CGI). Anxiolytic activity of the two drugs (COVI, STAI, HAM-D) was also comparable. However, paroxetine was found to be superior to fluoxetine on two subscore measures at week 1 of therapy (HAM-D Agitation item, p < 0.05; Psychic Anxiety item, p < 0.05), with no differences detected after week 2. The overall incidence of adverse effects was comparable in the two treatment groups. Constipation, dyspepsia, tremor, sweating and abnormal ejaculation were more common in paroxetine-treated subjects, whereas nausea and nervousness were more frequent in fluoxetine-treated patients. Weight loss was more common in the fluoxetine versus paroxetine group (11.88% versus 2.94%, respectively). ESRS scores for akathisia were low throughout the study and showed little change. LIMITATIONS: Differences observed between the two drugs in antianxiety effects were limited to two measures of anxiety among several others. DISCUSSION: The data indicate that paroxetine and fluoxetine have comparable antidepressant and anxiolytic efficacy. Paroxetine appears to produce an earlier improvement in agitation and psychic anxiety symptoms compared with fluoxetine.

A Diabetes Outcome Progression Trial (ADOPT): baseline characteristics of Type 2 diabetic patients in North America and Europe.

AIMS: To examine baseline characteristics of patients recruited into ADOPT, a multinational trial comparing three oral glucose-lowering monotherapies. METHODS: Between April 2000 and June 2002, 4360 patients aged 30-75 years with Type 2 diabetes diagnosed for < 3 years and remaining on diet therapy alone with fasting plasma glucose levels (FPG) between 7.0 and 10.0 mmol/l were enrolled by 488 North American and European centres. Medical histories, anthropometric data and laboratory measurements were determined using common methodologies. RESULTS: The mean (SD) age of the patients was 57 (10) years, body mass index 32.2 (6.4) kg/m(2), HbA(1c) 7.4 (0.9)%; 58% were male, 88% Caucasian and 15% smoked. North American Caucasians (NAC) were younger, more obese, and more insulin resistant than European Caucasians (EUC), but had better pancreatic B-cell function. NAC had lower total, low-density lipoprotein- and high-density liporpotein-cholesterol concentrations with higher triglyceride concentrations and were more often on lipid-lowering treatment. They had lower blood pressure levels but were equally likely to be on antihypertensive treatment. Metabolic syndrome was more frequent and microalbuminuria less frequent in NAC. Within North America, NAC had lower HbA(1c) concentrations than Blacks, Hispanics and Asians despite similar or higher FPG and 30-min postchallenge glucose concentrations. CONCLUSIONS: Caucasian North American and European ADOPT patients differ with respect to adiposity, insulin resistance and metabolic syndrome prevalence. North American Blacks, Hispanics and Asians had lower HbA(1c) concentrations than NAC despite similar or higher glucose concentrations. These phenotypic differences may influence the progression of Type 2 diabetes and the response to initial oral glucose-lowering monotherapy.

Training back-propagation neural networks to define and detect DNA-binding sites.

A three layered back-propagation neural network was trained to recognize E. coli promoters of the 17 base spacing class. To this end, the network was presented with 39 promoter sequences and derivatives of those sequences as positive inputs; 60% A + T random sequences and sequences containing 2 promoter-down point mutations were used as negative inputs. The entire promoter sequence of 58 bases, approximately -50 to +8, was entered as input. The network was asked to associate an output of 1.0 with promoter sequence input and 0.0 with non-promoter input. Generally, after 100,000 input cycles, the network was virtually perfect in classifying the training set. A trained network was about 80% effective in recognizing 'new' promoters which were not in the training set, with a false positive rate below 0.1%. Network searches on pBR322 and on the lambda genome were also performed. Overall the results were somewhat better than the best rule-based procedures. The trained network can be analyzed both for its choice of base and relative weighting, positive and negative, at each position of the sequence. This method, which requires only appropriate input/output training pairs, can be used to define and search for any DNA regulatory sequence for which there are sufficient exemplars.

Growth and differentiation during myogenesis in the chick embryo.

A general method for describing the complex dynamics of cell populations over an extended period of growth and differentiation in a developing tissue is presented. The measurements required to produce a unique description are defined. Skeletal muscle development in the thigh and breast of the chick embryo is analyzed, using this method, during the period of embryonic development between Day 7 and Day 17. A unique quantitation of growth and differentiation for the period from Day 11 to Day 17 is developed. The pectoralis major is compared with the averaged behavior of the thigh musculature. In each case, a single partitioning rule holds for more than 10 generations during the main myogenic period. In the pectoralis, approximately 51% of the cells entering G1 in each generation continue in the cell cycle; in the thigh, which experiences substantially greater overall growth, approximately 58% of the cells entering G1 in each generation continue in the cell cycle. No significant cell death is detected. Thus, in each case, the absolute number of myoblasts is increasing while the fractional value of myoblasts in the population is decreasing over a fivefold range. These results are discussed in terms of several quantitative models for the possible basis of the observed population dynamics.

Escherichia coli promoters: neural networks develop distinct descriptions in learning to search for promoters of different spacing classes.

Back-propagation neural networks were trained to recognize promoter sequences of each of the three major spacing classes found in E. coli. These networks were trained with the object of maximizing their ability to generalize while maintaining the level of false positive identifications at a fraction of 1 percent. These objectives were generally met. Networks for the 16 base spacing class captured between 78 and 100% of previously unseen promoters in different tests; networks for the 17 base class identified 97% of the test promoters; networks for the 18 base class identified 79% of the test promoters. A tandem poll of networks for all three spacing classes produced a cumulative false positive level of less than 0.5%. In each case, the weight matrices used by the networks in their classification were analyzed to determine the relative weight assigned to the occurrence of a given base at a given position within the promoter. In this fashion, an approximate description of the network's definition of the promoter can be obtained.

A general procedure for locating and analyzing protein-binding sequence motifs in nucleic acids.

In the last decade, two tools, one drawn from information theory and the other from artificial neural networks, have proven particularly useful in many different areas of sequence analysis. The work presented herein indicates that these two approaches can be joined in a general fashion to produce a very powerful search engine that is capable of locating members of a given nucleic acid sequence family in either local or global sequence searches. This program can, in turn, be queried for its definition of the motif under investigation, ranking each base in context for its contribution to membership in the motif family. In principle, the method used can be applied to any binding motif, including both DNA and RNA sequence families, given sufficient family size.

A general method for modeling cell populations undergoing G1----G0 transitions during development.

The transition from the dividing state to a non-dividing, terminally differentiated state is common to the history of most populations of cells during development. Quantifying such transitions and events related to them is often difficult, even in those cases for which there is a good tissue culture model, because the process is asynchronous and occurs against a background of continued extensive growth. A general model for analyzing these complex population changes is presented here. In the absence of definitive data, the model provides projections of the possible range, under a given set of boundary values, for the rate of terminal differentiation, the overall growth rate, and the degree of cell death. On the other hand, given data on the rate of DNA accumulation, dividing cell fraction, and generation time, the model provides the effective partitioning coefficient between the dividing and non-dividing states averaged over the population, at a given time. These data also allow for an assessment of the degree of actual cell death against a background in which significant numbers of cells are withdrawing from the cell cycle. The types of data required with respect to the model's ability to resolve the nature of a G0 transition "window" within the cell cycle are also discussed.

Symmetry, homology, and phrasing in the recognition of helical regulatory sequences in DNA.

Regulatory regions in DNA which have been sequenced have generally been found to contain one or more axes of two-fold rotational symmetry. If this symmetry is to be maintained in the helical sequence, the axis of rotation must be aligned with one of the two dyad axes of the helix. This is equivalent to saying that the rotational symmetry of the sequence can only be seen from certain viewing points in a circuit about the helix. More surprising is the fact that new symmetrical sequence arrangements can be seen at +/- 36 degrees, +/- 72 degrees, +/- 108 degrees, and +/- 144 degrees relative to the point at which the rotational symmetry is seen. This "amplification" of symmetry suggests a three-dimensional approach to sequence analysis. A specific reading frame, suggested by the geometry of the helix, is examined with regard to its elucidation of intra- and inter-sequence homologies. Two sequences are thus identified as being recurrent in a number of different regulatory sequences.

Escherichia coli promoters. I. Consensus as it relates to spacing class, specificity, repeat substructure, and three-dimensional organization.

Fifty-two of the best characterized Escherichia coli promoters in the Hawley and McClure [1983) Nucleic Acids Res. 8, 2237-2255) listing were used to determine the distribution of information content in promoters and to describe the basic features underlying the existence of several different promoter spacing classes, which are defined by the number of bases separating the -35 and -10 regions. The contact regions at -35 and -10 do not, on the average, contain sufficient information to specify a promoter. The search for additional specifying bases led to two conclusions: 1) the consensus nucleotide sequence in the noncontact regions of a promoter appears to be distinct for each of the major promoter spacing classes; 2) promoters appear to contain a 15-20 base subset of the 40-50 additional optimal noncontact bases. This improved view of the extended consensus sequence allows the detection of a 10-base degenerate palindrome which may be the basic unit of promoter structure. Contiguous direct repeats of this sequence produce a sequence closely related to the consensus for the 18-base pair spacing class. This underlying structure is also evidenced in the 17- and 16-base pair spacing classes; however, the start points of the fourth and subsequent repetitions of the sequence element are moved one and two bases upstream, respectively, relative to their location in the 18-base pair spacing class. These consensus sequences, when viewed in a helical format, all present the opportunity for two alternative sets of a dyad repeat. The -35 region is common to both sets and is paired with an extended -10 region in one set and with a pseudo-10 region in the other. Possible implications of these arrangements are discussed.

Escherichia coli promoters. II. A spacing class-dependent promoter search protocol.

A computer search protocol for finding Escherichia coli bacterial and phage promoters is presented. This protocol relies heavily on the description of promoter sites developed in the preceding paper (O'Neill, M. C. (1989) J. Biol. Chem. 264, 5522-5530), with particular emphasis on the existence of a distinct consensus sequence for each of the three major spacing classes. The input sequence is tested independently for promoters with 16, 17, or 18 bases separating the -35 and -10 regions. Within a given spacing group, a series of six tests is employed to define possible promoters. These tests were developed empirically to identify members of the known promoter database with high efficiency while producing a minimal level of false positives. The degree to which this aim is met is discussed in the context of searches of random sequence, of pBR322, and of lambda.

Molecuar model of the DNA interaction site for the cyclic AMP receptor protein.

A topological model of the DNA binding site for the cyclic AMP receptor protein (CRP) is presented. A consensus sequence drawn from the known CRP binding sites has several symmetrical subregions that are spatially resolved onto different faces of the DNA helix. Consideration of available biochemical and genetic data suggest one particular choice among the possible symmetrical arrangements. In this case, the sequence of its helical form presents nearly the same pattern of exposed base pairs on two faces of the helix. These two faces are separated by a helix angle of 72 degrees; the similar sequences that are exposed in the grooves occur in opposite orientations on the two faces. We propose that this inverted symmetry arrangement provides each of the identical subunits of the CRP with a similar recognition region within the overall site. In gal and ara, the site appears to accommodate a single molecule of the CRP; in lac, the site repeats the symmetrical arrangement and should accommodate two molecules of the CRP.

The Zymomonas mobilis glf, zwf, edd, and glk genes form an operon: localization of the promoter and identification of a conserved sequence in the regulatory region.

The Zymomonas mobilis genes that encode the glucose-facilitated diffusion transporter (glf), glucose-6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), and glucokinase (glk) are clustered on the genome. The data presented here firmly establish that the glf, zwf, edd, and glk genes form an operon, in that order. The four genes of the operon are cotranscribed on a 6.14-kb mRNA. The site of transcriptional initiation for the polycistronic message was mapped by primer extension and nuclease S1 protection analysis. The glf operon promoter region showed significant homology to other highly expressed Z. mobilis promoters, but not to consensus promoters from other bacteria. The highly expressed Z. mobilis promoter set contains two independent, overlapping, conserved sequences that extend from approximately bp -100 to +15 with respect to the transcriptional start sites. Expression of the glf operon was shown to be subject to carbon source-dependent regulation. The mRNA level was threefold higher in cells grown on fructose than in cells grown on glucose. This increase was not the result of differential mRNA processing when cells were grown on the different carbon sources, nor was it the result of differential transcript stability. Degradation of the 6.14-kb glf operon mRNA was biphasic, with initial half-lives of 11.5 min in fructose-grown cells and 12.0 min in glucose-grown cells. Thus, the higher level of glf operon mRNA in fructose-grown cells is the result of an increased rate of transcription. The importance of increasing glf expression in cells growing on fructose is discussed.

Stimulation of spermatogonial DNA synthesis in slug gonad by a factor released from cerebral ganglia under the influence of long days.

Incorporation of [3H]thymidine into gonadal DNA was shown to increase 1 week after implantation into an immature slug (Limax maximus) of a "brain" (circumesophageal ring of ganglia) from a male-phase donor. Light microscope autoradiography revealed that in stimulated gonads labeling was localized primarily in the nuclei of spermatagonia. Implant-stimulated spermatogonial DNA synthesis was found to depend upon implantation of supraesophageal (cerebral) ganglia. Neither subesophageal ganglia implants nor immature supraesophageal implants had an effect. Thymidine incorporation could also be stimulated by exposure of slugs to long-day lightcycles (LD 16:8) for 3 to 4 weeks. Similar duration of long-day treatment was also adequate to trigger male-phase development even after animals were returned to short days (LD 8:16). The results are consistent with the view that 3 to 4 weeks of long-day lightcycles are required to promote irreversibly the release from slug cerebral ganglia of a male-phase gonadotropic factor which directly or indirectly promotes spermatogonial proliferation.

Male gonadotrophic factor in brain and blood of photoperiodically stimulated slugs.

A pulmonate male gonadotrophic factor (MGF) has been described that is released from cerebral ganglia of male-phase slugs (Limax maximus). This factor produces, directly or indirectly, an increase in spermatogonial proliferation as determined by in vivo incorporation of [3H]thymidine into gonadal DNA. In the present investigation MGF activity was demonstrated in saline homogenates of male-phase cerebral ganglia by injecting homogenates into immature slugs for 5 consecutive days and assaying gonadal [3H]thymidine incorporation on Day 7. Dose-response data indicate that daily administration of as little as 0.1 brain equivalent can produce a significant stimulation in incorporation. Comparison of brain homogenates from immature (short-day) and male-phase (long-day) animals has shown that male-phase cerebral ganglia contain substantially more MGF activity than immature ganglia. Similar injection experiments using slug blood plasma showed that activity is present in male-phase blood but not in the blood of short-day immatures. MGF activity in long-day brain homogenates and blood plasma was found to be associated with a molecular weight fraction of 50 to 100 kDa obtained by ultrafiltration. Activity could be reduced or destroyed by treatment with trypsin or by heating. The present findings suggest that MGF is a proteinaceous factor of substantial size. It appears that both the synthesis and the secretion of MFG are stimulated in slugs that are in their male developmental phase as a result of prior exposure to long-day photoperiods.

Replication of animal viruses in differentiating muscle cells: influenza virus A.

Cells were cultured from the breast muscle of 11- to 12-day-old chick embryos and were grown under conditions optimal for the development of the cells into terminally differentiated, fused myotubes. Myotubes were infected with influenza virus A/Ann Arbor/6/60(H2N2) at high multiplicity, and synthesis of virus-specific proteins and RNAs was detected by haemadsorption, fluorescence microscopy and/or isotope labelling and electrophoresis techniques. Provided that myotubes were maintained at temperatures below 39 degrees C after infection, production of virus components and yield of infectious virus in these cells was similar to those observed in infected chick kidney cells. However, if cells were maintained at temperatures of 39 degrees to 40 degrees C after infection, virus nucleoprotein was prominent in the nuclei, and synthesis of virus-specific polypeptides and of plus-strand RNA was reduced about fourfold to 20-fold compared to that detected at lower temperatures. Moreover, infectious virus was not produced when temperatures of 39 to 40 degrees C were used during virus replication. The results demonstrate that under suitable conditions avian myotubes formed in culture resemble epithelioid cells in their ability to support the productive replication of influenza virus.