Biomedical subjects

A Portela

Publications and source records attributed to A Portela.

At least 55 records · Page 3Linked to original sources

PubMed1990

Location of a highly conserved neutralizing epitope in the F glycoprotein of human respiratory syncytial virus.

Trypsin digestion of the purified F protein from human respiratory syncytial virus (Long strain) generated a set of fragments in the amino-terminal third of the F1 subunit which contained the epitope 47F involved in neutralization. Sequencing of five escape mutant viruses selected with monoclonal antibody 47F allowed us to map precisely two amino acid residues (262 and 268) of the F1 subunit which are essential for the integrity of this important epitope. The results are discussed in terms of the mechanisms involved in virus neutralization and the design of potential synthetic vaccines.

Amino Acid Sequence↗

PubMed1990

Frame shift mutations as a novel mechanism for the generation of neutralization resistant mutants of human respiratory syncytial virus.

The genetic characterization of four previously reported mutants of human respiratory syncytial (RS) virus resistant to monoclonal antibody 63G is described. Sequences of the G protein genes were obtained from: (i) mRNA derived cDNA recombinants, (ii) direct mRNA sequencing and (iii) amplified vRNA derived cDNAs. The results obtained indicate that the original escape mutants, recovered from individual plaques, contained heterogeneous viral populations. This heterogeneity affected the number of adenosine residues present after nucleotides 588 or 623 of the G protein gene. Mutant viruses recovered after a second plaque purification step generated homogeneous sequences but contained single adenosine insertions or deletions at those two sites compared with the Long sequence. These genetic alterations introduced frameshift changes which are reflected in both the antigenic and structural properties of the mutant G proteins. The origin and importance of frameshift mutations in the RS virus G protein gene are discussed.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of the fusion and phosphoprotein genes of human respiratory syncytial (RS) virus Long strain: evidence of subtype genetic heterogeneity.

The nucleotide and deduced amino acid sequences of the fusion (F) and phosphoprotein (P) genes of the Long strain of human respiratory syncytial (RS) virus have been determined from cDNA copies cloned into pBSV9 shuttle vector. Comparison of these sequences with their counterparts of other strains reveals genetic heterogeneity within the same subtype. The percentage of nucleotide and amino acid changes occurring in both proteins is similar. Thus, the Long F and P proteins share 97.9% and 98.3% amino acid identity, respectively, with their homologs of the A2 strain. Nevertheless the F2 subunit of the fusion protein accumulates 3.1 times more amino acid changes than the F1 subunit. In addition, the percentage of nucleotide changes in the 3' extracistronic sequences is 6 times higher in the P than in the F gene. These results are discussed in terms of selective pressures operating in the evolution of RS virus in nature.

Amino Acid Sequence↗

PubMed1987

Permanent cell lines established from ts-COS cells that regulate by temperature the amplification and expression of cloned genes.

Temperature-sensitive COS cells have been transformed at restrictive temperature with SV40 replicons containing the neo or pac markers. Puromycin-resistant cell clones maintained at the restrictive temperature contain the pac gene integrated into the cell DNA. However, when the cells are shifted to the permissive temperature the pac gene is amplified in episomal forms up to 2-4 X 10(4) copies per cell. Concomitant with this, an induction of 35-300 fold in the levels of puromycin acetyl transferase activity is observed, leading to the accumulation of the enzyme up to 10-60 mU/mg of total cell protein. A band of apparent molecular weight 26,500 daltons is observed by polyacrylamide gel electrophoresis of induced culture extracts, that accounts for approximately 3% of the newly synthesized protein. The expression of non-selectable genes can also be regulated, as shown by the induction of influenza virus nucleoprotein synthesis in transformed cells. These results indicate that the ts-COS cells can be used as a highly efficient, regulable mammalian expression system.

Acetyltransferases↗

PubMed1986

Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance.

The gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger has been cloned next to the SV40 early promoter in a mammalian cells-Escherichia coli shuttle vector. When this construction was introduced into VERO cells it expressed the relevant enzymic activity. Moreover, the puromycin N-acetyl transferase gene has been used as a dominant marker for the selection of transformed mammalian cells able to grow in the presence of the antibiotic.

Acetyltransferases↗

PubMed1986

Construction of cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes.

Monkey cell lines have been transformed with a mixture of plasmids pSV2neo and pSLVa232N, a derivative of plasmid pSLVa232 (Portela et al., 1985b). Plasmid pSLVa232N contained the influenza virus genes encoding non-structural proteins under the control of the SV40 late promoter in pSLts1 vector that includes the SV40 ori and the tsA209 T-antigen gene. At restrictive temperature, plasmid sequences remained stably integrated in the cell genome, but upon temperature shift-down, defined circular DNA molecules were generated and amplified up to 2000-5000 copies/cell. Restriction analysis, Southern blot hybridization and partial sequencing indicate that one such episome, pC5, was derived from the integrated plasmid sequences by a homologous recombination event that led to deletion of the pBR322 sequences included in pSLVa232N. Concomitant with gene amplification, an induction of 20-65-fold in the expression of NS1 and NS2 proteins was observed after temperature shift-down. Thus, gene cloning into vector pSLts1 and transformation at restrictive temperature of cells permissive for SV40 DNA replication, appears to be a useful strategy for the controlled amplification and expression of cloned genes.

Animals↗

PubMed1985

Regulation of gene amplification and expression in cells that constitutively express a temperature sensitive SV40 T-antigen.

Simian cells have been transformed with SV40 origin-defective recombinant plasmids containing the tsA209 T-antigen gene. These plasmids contain deletions of either 5 or 52 nucleotides that include the BglI site at the SV40 ori, are defective for replication in COS-1 cells but retain a functional SV40 early promoter. Two cell lines transformed with these plasmids, U4 and S7, and their respective clonal derivatives E5 and F11, contain the tsA209 T-antigen gene integrated into the cell DNA and express T-antigen as detected by immunoprecipitation and immunofluorescence. These cells behave as ts-COS cells, since they complement in a temperature dependent manner the replication of an SV40 derived recombinant plasmid. When transfected with recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) gene cloned into SV40 replicons, ts-COS cells were able to regulate the induction of the CAT activity by temperature. The ratios of CAT activity observed at permissive versus restrictive temperature were in the range of 20-400. Thus, these ts-COS cells are useful systems for the regulated expression of cloned genes in simian cells.

Acetyltransferases↗

PubMed1985

A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression.

Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.

Animals↗

PubMed1985

Oriented synthesis and cloning of influenza virus nucleoprotein cDNA that leads to its expression in mammalian cells.

The influenza virus nucleoprotein gene has been cloned by a procedure that involves direct cDNA synthesis onto the primer-vector pBSV9, a pBR322-SV40 recombinant plasmid. dT-tailed pBSV9 was used to prime the synthesis of cDNA on a template of in vitro synthesized viral mRNA. The synthesis of ds-cDNA was initiated by a specific oligodeoxynucleotide and the resulting recombinant was circularized by intramolecular ligation. Recombinant pSVa963 contained the viral nucleoprotein gene directly fused to the SV40 early promoter region included in pBSV9 and followed by a dA:dT tail and the SV40 polyadenylation signal. When pSVa963 was used to transfect COS-1 cells, the presence of three NP-specific mRNAs of 1600, 1900 and 2500 nucleotides in length could be detected. Pulse labelling experiments of COS-1 transfected cells and immunobinding to a nucleoprotein monoclonal antibody indicated the synthesis of nucleoprotein. This nucleoprotein accumulated in the nucleus of transfected cells at a level similar to that found in infected cells. The vector and method described may be useful for the specific cloning and expression of any mRNA for which a 5'-terminal sequence is known.

Animals↗

PubMed1985

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review.

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.

Base Sequence↗

PubMed1983

Evolution of the influenza virus neuraminidase gene during drift of the N2 subtype.

The complete genetic information for the neuraminidase (NA) gene of influenza virus A/Bangkok/1/79 has been cloned by in vitro synthesis of dsDNA, insertion into pBR322 plasmid, and transformation of Escherichia coli. The nucleotide sequence of the NA gene has been determined by the Maxam and Gilbert method. It is 1466 nucleotides long and contains a single open reading frame with a coding capacity for 469 amino acids. When compared to the NA genes of the N2 strains A/Victoria/3/75, A/Udorn/72, A/NT/60/68, and A/RI/5-/57, 90% of the nucleotide positions and 87% of the amino acid positions remained invariant. Forty-two nucleotide changes and 14 amino acid changes accumulated in the period 1975-1979, but the general structure of the protein appeared to remain constant.

Amino Acid Sequence↗

PubMed1979

Temperature dependence on the passive and dynamic electrical parameters of muscle cells.

Measurements of the temperature dependence in the range from 10 C to 30 C on the passive and dynamic electrical properties of single frog muscle cell following Arrhenius relation have been made. The propagated responses (V-t) and conduction velocity theta were analyzed following the H-H propagated cable equation. The ionic current-membrane potential relationship (I-t) was calculated from the phase-plane trajectory analysis (V-V) of the action potential curve (V-t). All the rate and time constants of the excitation and propagation processes kr, kNa, kK, tau Na, tau K, the negative conductance (-gNa) and the ionic conductances gNa, gK, influencing the evolution of the curves (V-t), (V-V) and (I-V) are correlated. The magnitudes of the resting (Vr) and action potential amplitude (Vs), the excitation potential (V*), the negative after potential (Vn), and the sodium equilibrium potential (VNa); the magnitudes of the maximum rate of rise and fall of the spike (V+) and (V-) and those corresponding to the inward INa and outward IK ionic currents, were analyzed. Two general classes of findings were obtained. One group of action potential parameters theta, V+, V-, Vn, kr, kNa, kK, tau Na, tau K, -gNa, gNa, gK, INa and IK is strongly temperature dependent with Q 10 S approximately 2 and energy of activation E approximately 10 kcal/mole. The other group of parameters, Gm (passive conductance), Cm (capacitance), tau, Vr, V*, Vs, and VNa are slightly temperature dependent, with Q10's lower than 1.4. This study contributes deeply to the analysis of temperature effects on the electrical cell responses to adequate stimuli. This temperature dependence analysis was designed to detect possible "masked" actions of microwave radiation on cell membrane functions.

Action Potentials↗

PubMed1979

Quantitation of chronic microwave radiation effects on muscle cell electrical excitable properties: a temperature dependence analysis of the H-H cable and membrane current parameters of irradiated cells.

Frogs Rana pipiens maintained under constant laboratory environmental conditions were subjected daily to chronic microwave exposure with pulsed microwave (2.88 GHZ) at a power density of 10 mW/cm2, during 0.1 hour, for periods up to 100 days. The whole body Specific Absorption Rate (SAR) was of 1.5 mW/g per 1 mW/cm2. The passive and dynamic electrical parameters of sartorius muscle cells from control and irradiated frogs as a function of temperature in the range from 10 C to 30 C, have been analyzed. This temperature dependence analysis, presented in another work, was planned to be used in this paper for detecting those possible "masked" chronic microwave effects in complex membrane mechanisms highly temperature sensitive, and tightly related to the excitation and propagation of bioelectrical responses. The temperature dependence of the passive (Vr, Gm, Cm, and tau) and the active cell electrical parameters (theta, V*, Vs, VNa, Vn, V+, V-, kr, kNa, kK, tau Na, tau K, -gNa, gNa, gK, INa, and IK) was not altered by chronic microwave exposure. The striking observation reported in another work, about the presence of two groups of cell electrical parameters, characterized by their dependence with temperature, was reproduced on the irradiated muscle cells. Results have indicated that there was not muscle cell cumulative bioeffects resulting from microwave exposure to 10 mW/cm2, over a 0.1 hour period.

Action Potentials↗

PubMed1978

Digital interface for bioelectrical data acquisition system.

The action potential digitalized and stored by means of a fast solid state electronic waveform recording system (Digitalizer) can be directly read out on a printer or in a computer for further processing. It has been designed a digital interface allowing the automatization of these bioelectrical data acquisition and printing process.

Action Potentials↗

PubMed1978

Digitalizer system and memory for electric transients.

It was designed a fast solid state electronic waveform recording system (Digitalizer), which can record, store and display action potentials or any type of event, either single pulse or repetitive, as a function of time. Above all, its capacity to record fast transients and to give the output in analog and digital form, makes this electronic device more convenient that conventional recorders or single trace oscilloscope systems.

Action Potentials↗

PubMed1978

Temperature effects on the properties of the Hodgkin-Huxley propagated action potential model determined by computed solutions and phase-plane analysis.

The equation for membrane potential (V) of the squid giant axon or some muscle cells, which is the heart of the Hodgkin-Huxley model for the action potential, can be written in three ways: first, as a partial differential equation in time and space; second, as an ordinary differential equation in time, assuming uniform wave propagation for the action potential, and third, as an even simpler ordinary differential equation for the potential at a point, so-called "space clamp" conditions. Solutions were computed for the first two of these equations, at three different temperatures, and the results compared. Temperature dependence of the appropriate parameters was calculated from a simple exponential relationship. Significant changes in the quantitative predictions of the model were found as the temperature was changed from 6.3 C to 18.5 C. Phase-plane (V v V, I v V) analysis has been used to examine the nature of these differences.

Action Potentials↗

PubMed1977

X-radiation actions on the neuromuscular transmission.

The effects of high doses of X-radiation (100 kilorads) on the neuromuscular transmission of isolated sciatic nerve-sartorius muscle preparations of the frog, as evaluated by bioelectrical characteristics, were explored. Intracellular microelectrode recordings after X-irradiation showed that the resting, excitation and action potentials of nerve fibers approaching the synaptic terminal region of the motor end-plate became lessened, and also presented a slower velocity of impulse propagation, earlier than that observed in muscle cells. After forty minutes following the irradiation period, the neuromuscular transmission became blocked, although muscle fibers still responded to direct electrical stimulation. Records taken at the motor end-plate region of muscle cells, demonstrated the presence of postsynaptic miniature end-plate potentials (m.e.p.p.'s), the sequence of which fits closely into a random Poisson distribution. X-irradiation elicited an increase of the rate of m.e.p.p.'s and induced membrane changes over fine terminal nerve branches, leading into a failure to initiate and propagate action potentials. Only as time progressed, this nerve bioelectrical impairment was accompanied by a similar one in muscle cells, associated to the inability to develop contractile tension. The increase of m.e.p.p.'s frequency due to depolarization by a high K+ concentration, of presynaptic nerve membranes of control and irradiated preparations, was reversed by a high Mg+--Ca2+--free media. However, a concentration of Mg2+, which normally reduced quantal release of acetylcholine (ACh), without altering presynaptic nerve membrane potentials, failed to modify the spontaneous basal frequency of m.e.p.p.'s, both in irradiated and control preparations. The findings of the present study suggest that the presynaptic ACh synthesis, storage, and availability for ACh liberation are not early affected by X-rays, i.e. at a time when transmission from nerve to muscle had already failed. On the contrary, the most precocius membrane changes following X-irradiation, seem related to the mechanisms underlying nerve resting, excitation, and action potentials. The smallest presynaptic nerve terminal fibers close to the motor end-plate, are the most radiosensitive portion of the myoneural junction. Indeed, their bioelectric characteristics are early affected by X-rays, resulting in a failure to generate and propagate nerve impulses which lead to a blockade of neuromuscular transmission.

Action Potentials↗

PubMed1976

Programmed stimulator for threshold of action potential.

A programmed digital stimulator is described, in which the pulse generator can be regulated in frequency and duration. The output pulses amplitude increases automatically by increments under 5% of the previous step, sequence which allows a much better approximation to the threshold value of action potential. A photographic camera is synchronized for recording. The power supplies, pulse duration generator, binary counter, converter, pulse control, and detector circuits are given in detail.

Action Potentials↗