Search PubMed⌕ Search

Biomedical subjects

L H Pinto

Publications and source records attributed to L H Pinto.

At least 37 records · Page 2Linked to original sources
PubMed1995

Pharmacological and genetic approaches for the study of circadian rhythms in mammals.

Two different approaches have been utilized to study the controlling mechanisms that underlie the generation and entrainment of circadian rhythms in mammals. The use of specific drugs to alter the period and/or the phase of circadian rhythms has provided new insights into both the pathways by which environmental information reaches the mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) and the cellular and neurochemical events within the SCN itself which are involved in circadian rhythmicity. A second approach, which seeks to exploit genetic differences in the properties of the circadian system, holds the promise of eventually defining the cellular and molecular events that are part of the clock itself, the events that underlie the entrainment of the circadian clock by environmental factors, and the expression of overt rhythms driven by the clock. It is anticipated that the pharmacological and genetic approaches to the study of circadian rhythms will complement each other as the underlying physiological mechanisms of the circadian clock system become defined.

Afferent Pathways↗
PubMed1995

Activation of the M2 ion channel of influenza virus: a role for the transmembrane domain histidine residue.

To test the hypothesis that transmembrane domain histidine residue 37 of the M2 ion channel of influenza A virus mediates the low pH-induced activation of the channel, the residue was changed to glycine, glutamate, arginine, or lysine. The wild-type and altered M2 proteins were expressed in oocytes of Xenopus laevis and membrane currents were recorded. The mass of protein expressed in individual oocytes was measured using quantitative immunoblotting and correlated with membrane currents. Oocytes expressing the M2-H37G protein had a voltage-independent conductance with current-voltage relationship similar to that of the wild-type M2 channel. The conductance of the M2-H37G protein was reversibly inhibited by the M2 ion channel blocker amantadine and was only very slightly modulated by changes in pHout over the range pH 5.4 to pH 8.2. Oocytes expressing the M2-H37E protein also had a voltage-independent conductance with a current-voltage relationship similar to that of the wild-type M2 channel. The conductance of the M2-H37E protein was reversibly inhibited by amantadine and was also only very slightly modulated by changes in pHout over the range pH 5.4 to pH 8.2. These slight alterations in conductance of the mutant ion channels on changes in pHout are in striking contrast to the 50-fold change in conductance seen for the wild-type M2 channel over the range pH 4.5 to pH 8.2. The specific activity of the M2-H37G protein was 1.36 +/- 0.37 microA/ng and the specific activity of the M2-H37E protein was 30 +/- 3 microA/ng at pH 6.2. These values of specific activity greatly exceed that of the wild-type protein at the same pH (0.16 + 0.01 micro A/ng). Oocytes expressing the M2-H37K and M2-H37R mutant proteins could not be studied because the oocytes did not survive more than a few hours in culture. Oocytes expressing the M2-H37E mutant protein also had a voltage-activated Cl- conductance that was observed only for oocytes that expressed a mass of protein exceeding a large threshold value. These results are consistent with protonation of histidine residue 37 as an essential step in the activation of the wild-type M2 ion channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Viral and cellular small integral membrane proteins can modify ion channels endogenous to Xenopus oocytes.

A slowly activated, inward current could be evoked from Xenopus oocytes in response to application of a strong (approximately -190 mV) hyperpolarizing pulse. However, a much lesser hyperpolarization (approximately -130 mV) was able to evoke a similar current from oocytes that expressed the cellular proteins IsK and phospholemman, the synthetic protein SYN-C, and the NB protein of influenza B virus. All of these currents were carried principally by Cl-, and they had similar blocker profiles. The time course (the function of time that described the current increase during a hyperpolarizing voltage-clamp pulse, i.e., activation kinetics) varied from one batch of oocytes to another, but did not vary within each batch with the type of protein expressed. This slowly activated, inward current evoked by hyperpolarization to approximately -130 mV required the expression of a characteristic, minimum level of each of the proteins IsK, SYN-C, and NB. However, not every integral membrane protein expressed in oocytes allowed substantial inward currents to be generated at -130 mV. Oocytes that expressed large amounts of the M2 protein of influenza A virus, which is known to possess an intrinsic cation channel activity, did not display a Cl- current when hyperpolarized to -130 mV. These results suggest that expression of any of the four proteins-IsK, phospholemman, SYN-C, or NB- acts as an activator of an endogenous Cl- conductance.

Animals↗
PubMed1995

Analysis of the posttranslational modifications of the influenza virus M2 protein.

The sites of posttranslational modifications of the influenza A virus M2 protein were examined, and the effect of these modifications on the M2 protein ion channel activity was analyzed. Cysteine residues 17 and 19 in the M2 protein ectodomain form disulfide bonds. The cytoplasmic tail is posttranslationally modified by palmitoylation, and mutagenic studies support the view that cysteine residue 50 is the site for fatty acylation. In addition, the cytoplasmic tail of the M2 protein was found to be posttranslationally modified by the addition of phosphate to specific serine residues. Site-directed mutagenesis of serine residues in the M2 protein cytoplasmic tail, combined with phosphoamino acid analysis, indicated that serine residue 64 is the predominant site for phosphorylation but that serine residues 82, 89, and 93 were also phosphorylated but to much lesser extents. Disulfide-bond formation, palmitoylation, and phosphorylation occurred on M2 protein expressed in mammalian cells infected with influenza virus, in mammalian cells in which the M2 protein was expressed from DNA expression vectors, and when the M2 protein was expressed in oocytes of Xenopus laevis. The membrane currents of oocytes of Xenopus laevis expressing wild-type and site-specifically altered forms of the M2 protein, to ablate posttranslational modifications, indicated that none of the posttranslational modifications significantly affected the ion channel activity of the M2 protein in oocytes. Therefore, these data do not indicate a functional role for posttranslational modifications of the M2 protein in its ion channel activity.

Amino Acid Sequence↗
PubMed1995

The Shaker-like potassium channels of the mouse rod bipolar cell and their contributions to the membrane current.

RT PCR on mRNA from enzymatically dissociated, isolated bipolar cells showed that these neurons express the Shaker-like K+ channels Kv1.1, Kv1.2, and Kv1.3. Immunohistochemical localization showed each channel to have a unique subcellular distribution: Kv1.1 immunoreactivity was detected in the dendrites and axons terminal, whereas Kv1.2 and Kv1.3 subunits were localized to the axon and the postsynaptic membrane of the rod ribbon synapse, respectively. Whole-cell patch-clamp recordings indicated that the activation voltage of the delayed rectifier current of the isolated bipolar cell and the inhibitory constants for current blockade by TEA, 4-AP, and Ba2+ were similar to these same properties measured for Kv1.1 expressed in oocytes. However, the TEA and 4-AP inhibitory constants for the bipolar cell current differed from the inhibitory constants for Kv1.2 or Kv1.3. These results suggest that the current of the isolated rod bipolar cell is most similar to Kv1.1 but that all three channels may function in the intact retina to allow complex modulation of retinal synaptic signals.

Animals↗
PubMed1994

Direct measurement of the influenza A virus M2 protein ion channel activity in mammalian cells.

The influenza A virus M2 integral membrane protein has an ion channel activity which is thought to play an essential role in the uncoating process of influenza virus in infected cells and, for some strains of influenza virus, in maintaining the hemagglutinin in its pH neutral form during transport through the trans Golgi network. To demonstrate directly that the M2 protein forms an ion channel in mammalian cells, the M2 protein was expressed in CV-1 cells by using an SV40-M2 recombinant virus and the whole cell membrane currents were recorded. It was found that the whole cell current was activated by low pH and inhibited by the M2 ion channel-specific blocker, amantadine hydrochloride. Expression of an altered M2 protein that contains a deletion of four residues in the transmembrane domain (M2-del28-31) and that when found in influenza virus confers amantadine resistance, resulted in a current that was activated by hyperpolarization of the membrane, was pH insensitive, and was resistant to block by amantadine. The data obtained in mammalian cells for the wild-type M2 and M2-del28-31 protein ion channel activities were very similar to those obtained when using the heterologous oocyte expression system.

Amantadine↗
PubMed1994

Forward and reverse genetic approaches to behavior in the mouse.

Modern molecular genetic and genomic approaches are revolutionizing the study of behavior in the mouse. "Reverse genetics" (from gene to phenotype) with targeted gene transfer provides a powerful tool to dissect behavior and has been used successfully to study the effects of null mutations in genes implicated in the regulation of long-term potentiation and spatial learning in mice. In addition, "forward genetics" (from phenotype to gene) with high-efficiency mutagenesis in the mouse can uncover unknown genes and has been used to isolate a behavioral mutant of the circadian system. With the recent availability of high-density genetic maps and physical mapping resources, positional cloning of virtually any mutation is now feasible in the mouse. Together, these approaches permit a molecular analysis of both known and previously unknown genes regulating behavior.

Animals↗
PubMed1994

Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior.

In a search for genes that regulate circadian rhythms in mammals, the progeny of mice treated with N-ethyl-N-nitrosourea (ENU) were screened for circadian clock mutations. A semidominant mutation, Clock, that lengthens circadian period and abolishes persistence of rhythmicity was identified. Clock segregated as a single gene that mapped to the midportion of mouse chromosome 5, a region syntenic to human chromosome 4. The power of ENU mutagenesis combined with the ability to clone murine genes by map position provides a generally applicable approach to study complex behavior in mammals.

Animals↗
PubMed1994

Reconstitution of the influenza virus M2 ion channel in lipid bilayers.

M2, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M2 baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25-500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25-90 pS). (3) The channels were selective for cations; tNa = 0.75 when 150 mM NaCl bathed both sides of the membrane. (4) Amantadine reduced the probablity of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs approximately Na > Li. The pH activation, amantadine block and ion selectivity of the M2 protein ion channel in bilayers are consistent with those observed on expression of the M2 protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M2 protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.

Amantadine↗
PubMed1994

Influenza A virus M2 ion channel protein: a structure-function analysis.

A structure-function analysis of the influenza A virus M2 ion channel protein was performed. The M2 protein of human influenza virus A/Udorn/72 and mutants containing changes on one face of the putative alpha helix of the M2 transmembrane (TM) domain, several of which lead to amantadine resistance when found in virus, were expressed in oocytes of Xenopus laevis. The membrane currents of oocytes expressing mutant M2 ion channels were measured at both normal and low pH, and the amantadine-resistant mutant containing the change of alanine at residue 30 to threonine was found to have a significantly attenuated low pH activation response. The specific activity of the channel current of the amantadine-resistant mutants was investigated by measuring the membrane current of individual oocytes followed by quantification of the amount of M2 protein expressed in these single oocytes by immunoblotting analysis. The data indicate that changing residues on this face of the putative alpha helix of the M2 TM domain alters properties of the M2 ion channel. Some of the M2 proteins containing changes in the TM domain were found to be modified by addition of an N-linked carbohydrate chain at an asparagine residue that is membrane proximal and which is not modified in the wild-type M2 protein. These N-linked carbohydrate chains were further modified by addition of polylactosaminoglycan. A glycosylated M2 mutant protein (M2 + V, A30T) exhibited an ion channel activity with a voltage-activated, time-dependent kinetic component. Prevention of carbohydrate addition did not affect the altered channel activity. The ability of the M2 protein to tolerate deletions in the TM domain was examined by expressing three mutants (del29-31, del28-31, and del27-31) containing deletions of three, four, and five residues in the TM domain. No ion channel activity was detected from expression of M2 del29-31 and del27-31, whereas expression of M2 del28-31 resulted in an ion channel activity that was activated by hyperpolarization (and not low pH) and was resistant to amantadine block. Examination of the oligomeric form of M2 del28-31 indicated that the oligomer is different from wild-type M2, and the data were consistent with M2 del28-31 forming a pentamer.

Amantadine↗
PubMed1994

Glycine receptors in the retinas of normal and spastic mutant mice.

PURPOSE: Spastic mutant mice have abnormal gait and righting behavior, and the responses of their retinal ganglion cells have recently been shown to be abnormal. The former defects have been linked to a reduction of glycine-receptor density in the spinal cord of spastic mutants, but the cause of the retinal defects has not yet been determined. The authors thus tested for reduced glycine-receptor density in the mutant retina by comparing the levels of glycine receptors in the retinas of spastic mutant mice with those found in normal mice. METHODS: Indirect immunofluorescence histochemistry was employed, using monoclonal antibodies directed against the alpha- and beta-subunits of the receptor and against the 93-kd cytoplasmic receptor-associated protein, gephyrin. RESULTS: In normal mice, all glycine-receptor antibodies labeled two laminae of the inner plexiform layer (IPL): a broad band in the distal third of the IPL and a narrow band in the middle of the IPL. Lighter labeling was also seen in the outer plexiform layer with these antibodies. In spastic mutant mice, the glycine-receptor labeling of the IPL was reduced markedly. However, the overall structure of the spastic mutant retina was not disrupted because the distribution and intensity of both a presynaptic marker (synaptophysin) and a marker for the rod bipolar cell (protein kinase C) in the mutant retina were indistinguishable from those in normal retinas. CONCLUSIONS: The glycine-receptor distribution in normal mice was consistent with that previously reported for the rat and with the distribution of glycine responsiveness of dissociated rodent bipolar cells. The reduced levels of glycine receptors in spastic mice help explain the abnormal ganglion cell responses in the spastic mutant.

Animals↗
PubMed1993

Mus spretus-specific LINE-1 DNA probes applied to the cloning of the murine pearl locus.

LINE-1 is the major family of long, interspersed, repetitive DNA sequences found in mammalian genomes. The mouse species Mus spretus contains large LINE-1 subfamilies that are distinguishable from the LINE-1 elements of laboratory Mus domesticus strains by their content of particular nucleotide differences. Oligonucleotides containing these differences act as M. spretus-specific LINE-1 hybridization probes. We have used these probes as a novel genetic tool in conjunction with an interspecific hybrid congenic mouse, in which the M. spretus allele of the pearl gene has been transferred onto a M. domesticus background. From a lambda library prepared from this congenic mouse, four clones were isolated by hybridization to the M. spretus-specific probes. After derivation of genetic markers from these clones, two of them were found to be linked to the pearl gene. These markers are the first two of up to 75 that could be isolated to support cloning the pearl gene. Considering the interspersed nature of LINE-1, we propose that species-specific LINE-1 probes could also be used to isolate markers for many other target genes.

Animals↗
PubMed1993

Reduced light sensitivity of the circadian clock in a hypopigmented mouse mutant.

Pink-eyed dilution (p/p) is a recessive mutation in mice which results in reduced pigmentation of the retinal pigment epithelium, as well as alterations in visual pathways and function. We investigated whether this mutation also affects light information reaching the circadian clock. Entrainment to a 12 h light 12 h dark cycle and the free-running period in constant darkness were not affected by this mutation. Phase shifts in response to 1 h light pulses consisting of bright white light at either circadian time 16 or 24 also did not differ between mutant and wild-type C57BL/6J mice. However, when 5 min, 502 nm light pulses of 1.2 x 10(-1) microW/cm2 or 4 x 10(-2) microW/cm2 were given at circadian time 16, the mutant mice responded with significantly smaller phase shifts than the wild-type mice. When animals were transferred to constant light, the free-running period of wild-type mice was longer than that of mutant mice, a finding which is consistent with a sensitivity difference between mutant and wild-type mice. Horseradish peroxidase tracing of retinal innervation of the hypothalamic suprachiasmatic nuclei (SCN)--the location of a circadian pacemaker--revealed a reduced innervation of the SCN in mutant mice compared with wild-type mice. The total volume of the SCN, as determined by neutral red stain, was also reduced in mutant mice, although not to as great an extent as the retinal innervation. Taken together, these results indicate that while basic characteristics of circadian clock function are not altered by the pink-eyed dilution mutation, the sensitivity of the clock to light is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block.

The influenza A virus M2 integral membrane protein has ion channel activity which can be blocked by the antiviral drug amantadine. The M2 protein transmembrane domain is highly conserved in amino acid sequence for all the human, swine, equine, and avian strains of influenza A virus, and thus, known amino acid differences could lead to altered properties of the M2 ion channel. We have expressed in oocytes of Xenopus laevis the M2 protein of human influenza virus A/Udorn/72 and the avian virus A/chicken/Germany/34 (fowl plague virus, Rostock) and derivatives of the Rostock ion channel altered in the presumed pore region. The pH of activation of the M2 ion channels and amantadine block of the M2 ion channels were investigated. The channels were found to be activated by pH in a similar manner but differed in their apparent Kis for amantadine block.

Amantadine↗
PubMed1992

Influenza virus M2 protein has ion channel activity.

The influenza virus M2 protein was expressed in Xenopus laevis oocytes and shown to have an associated ion channel activity selective for monovalent ions. The anti-influenza virus drug amantadine hydrochloride significantly attenuated the inward current induced by hyperpolarization of oocyte membranes. Mutations in the M2 membrane-spanning domain that confer viral resistance to amantadine produced currents that were resistant to the drug. Analysis of the currents of these altered M2 proteins suggests that the channel pore is formed by the transmembrane domain of the M2 protein. The wild-type M2 channel was found to be regulated by pH. The wild-type M2 ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection.

Amantadine↗
PubMed1992

Receptive field organization of retinal ganglion cells in the spastic mutant mouse.

1. We examined the receptive field properties of retinal ganglion cells in the isolated, superfused retinae of spastic mutant mice (B6C3Fe-spa/spa) that did not have the retinal degeneration (rd) phenotype. Glycine receptor density in the spastic mutant is greatly reduced in all areas of the CNS that have been examined. Phenotypically normal litter-mates were used as controls. Radial sections from the retinae of both spastic and normal animals were examined with light and electron microscopy and no differences were observed. The planimetric density of the cell bodies in the inner nuclear layer did not differ between the normal and mutant animals, about 400 cm-2. The absolute dark-adapted sensitivity of spastic ganglion cells was greater (271 +/- 69.0 impulses quanta-1 rod-1) than that of normal ganglion cells (47.7 +/- 10.4 impulses quanta-1 rod-1; P < 0.01). 2. Extracellular recordings of retinal ganglion cell responses to circular and annular stimuli, centred on the receptive field, were used to construct peri-stimulus-time histograms. In normal retinae, an annular stimulus elicited a response that was characteristic of the surround response mechanism of receptive fields with antagonistic centre-surround organization. In the mutant retina, annular stimuli did not elicit a surround-type response; instead, a centre-type response was recorded. 3. Illumination of the receptive field periphery attenuated centre-type responses in ganglion cells from both spastic and normal retinae. Centred circular stimuli of various areas (14, 35, 78, 122, 235, 783 deg2) were presented to the receptive fields. For mutant and normal ganglion cells, the response to the largest stimulus was smaller than that to an intermediate-sized stimulus. 4. The effect of strychnine, a glycine receptor antagonist, on the response to circular stimuli was examined. Very low concentrations of strychnine attenuated the light response in mutant retinae (apparent inhibitory binding constant KI = 8.1 x 10(-13) M). In normal animals, the light response was also attenuated by strychnine, but the apparent KI was much higher (apparent KI = 1 x 10(-7) M). 5. In normal ganglion cells, the sustained component of the light response was much more attenuated by strychnine than was the transient component. Interestingly, ganglion cells from spastic retinae did not exhibit a sustained component, even at stimulus luminances that evoked responses near threshold.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Ocular↗
PubMed1991

A randomized trial comparing radiation therapy versus concomitant radiation therapy and chemotherapy in carcinoma of the thoracic esophagus.

From September 1982 to December 1985, 59 previously untreated patients with Stage II squamous cell carcinoma of the thoracic esophagus were randomly assigned to receive radiation therapy (RT) alone versus the concomitant use of RT and chemotherapy (CT) with 5-fluorouracil (5-FU), mitomycin C, and bleomycin (RT + CT). Thirty-one patients were randomized to the RT regimen and 28 to the RT + CT regimen. The complete local response rate was 58% for the RT group and 75% for the RT + CT group (P = 0.77). The median duration of response was 8 months for both groups. The overall 5-year survival rates were 6% and 16% (P = 0.16) for the RT and RT + CT groups, respectively. Acute toxicities were more pronounced in the RT + CT group. This clinical trial did not detect a difference in outcome with combined-technique therapy. This result must be interpreted with caution because of the small number of patients entered in this trial. Confirmation of the value or lack of value for combined therapy will require additional larger clinical trials.

Adult↗