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Biomedical subjects

P Rath

Publications and source records attributed to P Rath.

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

Palama Settlement: 100 years of serving a neighborhood's needs.

The founding of Palama Settlement brought to those who might not be able to afford it public health nurses for maternal care and nutrition, well-baby clinics, tuberculosis clinics, medical and dental clinics, and eventually major support of medical needs during and after the attack on Pearl Harbor. Palama Settlement celebrates its centennial year with many of its early functions assumed by state and private organizations, but it is prepared to enter the next 100 years of service to the community. Palama was founded by James Arthur Rath with the purpose of serving the community; many people today remember their childhood and Palama Settlement.

Community Health Services↗
PubMed1994

A redirected proton pathway in the bacteriorhodopsin mutant Tyr-57-->Asp. Evidence for proton translocation without Schiff base deprotonation.

Light-driven proton pumping in bacteriorhodopsin involves deprotonation of the retinylidene Schiff base during M formation and reprotonation during N formation as key steps. This study reports on the spectroscopic characterization of the bacteriorhodopsin mutant Tyr-57-->Asp (Y57D). The results reveal that although formation of the M intermediate and Schiff base deprotonation is blocked, the mutant still exhibits a significant level of light-driven proton translocation. The photocycle of Y57D involves formation of K and L intermediates accompanied by the normal chromophore isomerization and changes in the hydrogen bonding of Asp-96 and Asp-115. However, an additional Asp residue deprotonates during formation of the L intermediate along with a transmembrane alpha-helical structural change that normally occurs upon N formation. We postulate that proton transport in Y57D occurs through a redirected pathway that does not involve the deprotonation of the Schiff base. Chromophore isomerization, which normally results in the transfer of a proton from the Schiff base to Asp-85, instead causes the deprotonation of Asp-57 in Y57D, most likely through an interaction involving Asp-212. This deprotonation of Asp-57 causes the release of a proton into the extracellular medium. Reprotonation of Asp-57 occurs through the Schiff base reprotonation pathway, which consists of a hydrogen-bonded network of residues spanning from Asp-96 to Asp-212. The results also indicate that the transmembrane alpha-helical structural changes observed during N formation (Rothschild, K.J., Marti, T., Sonar, S., He, Y.W., Rath, P., Fischer, W., Bousche, O., and Khorana, H. G. (1993) J. Biol. Chem. 268, 27046-27052) do not require deprotonation of Asp-96 or of the Schiff base.

Aspartic Acid↗
PubMed1994

The Schiff base counterion of bacteriorhodopsin is protonated in sensory rhodopsin I: spectroscopic and functional characterization of the mutated proteins D76N and D76A.

Both sensory rhodopsin I (SR-I), a phototaxis receptor, and bacteriorhodopsin (BR), a light-driven proton pump, share residues which have been identified as critical for BR functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions both as the Schiff base counterion and proton acceptor. We found that substituting an Asn for Asp76 (D76N) in SR-I has no effect on its visible absorption unlike the analogous mutation (D85N) in BR which shifts the absorption to longer wavelengths. The mutated proteins D76N and D76A are also fully functional as phototaxis receptors in contrast to BR, where the analogous substitutions block proton transport. D76N was also found to exhibit a spectrally normal SR587-->S373 transition. However, FTIR difference spectroscopy reveals that two bands in the SR587-->S373 difference spectrum at 1766/1749 cm-1 (negative/positive), assigned to the C=O stretch mode of a carboxylic acid, disappear in D76N, although no changes are observed in the carboxylate region. In addition, the kinetics and yield of this photoreaction are altered. On this basis, it is concluded that, unlike Asp85 in bacteriorhodopsin, Asp76 is protonated in SR-I and undergoes an increase in its hydrogen bonding during the SR587-->S373 transition. This model accounts for the difference in color of SR-I and BR and the finding that Asn can substitute for Asp76 without greatly altering the SR-I phenotype. Interestingly, parallels exist between this residue and Asp83 in the visual receptor rhodopsin which has recently been found to exist in a protonated form and to undergo an almost identical change in hydrogen bonding during rhodopsin activation.

Bacteriorhodopsins↗
PubMed1994

Photoactivation of rhodopsin involves alterations in cysteine side chains: detection of an S-H band in the Meta I-->Meta II FTIR difference spectrum.

FTIR difference spectroscopy has been used to study the role of cysteine residues in the photoactivation of rhodopsin. A positive band near 2550 cm-1 with a low frequency shoulder is detected during rhodopsin photobleaching, which is assigned on the basis of its frequency and isotope shift to the S-H stretching mode of one or more cysteine residues. Time-resolved studies at low temperature show that the intensity of this band correlates with the formation and decay kinetics of the Meta II intermediate. Modification of rhodopsin with the reagent NEM, which selectively reacts with the SH groups of Cys-140 and Cys-316 on the cytoplasmic surface of rhodopsin, has no effect on the appearance of this band. Four other cysteine residues are also unlikely to contribute to this band because they are either thio-palmitylated (Cys-322 and Cys-323) or form a disulfide bond (Cys-110 and Cys-187). On this basis, it is likely that at least one of the four remaining cysteine residues in rhodopsin is structurally active during rhodopsin photoactivation. The possibility is also considered that this band arises from a transient cleavage of the disulfide bond between cysteine residues 110 and 187.

Amino Acid Sequence↗
PubMed1993

Asp96 deprotonation and transmembrane alpha-helical structural changes in bacteriorhodopsin.

The M-->N transition in the photocycle of bacteriorhodopsin involves the transfer of a proton from Asp96 to the retinylidene Schiff base, possibly through a network of hydrogen-bonded amino acid residues and water molecules (Rothschild, K. J., He, Y. W., Sonar, S., Marti, T., and Khorana, H. G. (1992) J. Biol. Chem. 267, 1615-1622). A conformational change of the protein backbone is also observed during this transition. In this work, we have investigated the effects of replacing the residue Thr46, which might be part of this chain, with an aspartic acid. Both Fourier transform infrared and resonance Raman spectroscopy show that the chromophore structure of this mutant (T46D) is normal. However, N formation is accelerated and N decay is significantly slowed compared to wild-type bacteriorhodopsin. This effect causes the N intermediate to accumulate under steady-state illumination thereby facilitating spectroscopic studies under normal pH conditions. Fourier transform infrared difference spectroscopy reveals that like native bacteriorhodopsin, N formation in T46D involves deprotonation of Asp96, reprotonation of the Schiff base, and a change in the backbone secondary structure. However, in contrast to bacteriorhodopsin, bands assigned to the C = O stretch mode of the carboxylic acid group of Asp96 are upshifted by 10 cm-1 reflecting a change in the Asp96 environment and a drop in its effective pKa throughout the photocycle. This change in the pKa can directly account for changes in the photocycle kinetics and indicates that Asp96 deprotonation/protonation are the rate limiting steps in the formation and decay of the N intermediate. By studying the effects of H/D exchange, evidence is found that the backbone structural changes involve transmembrane alpha-helices. It is proposed that these structural changes serve to modulate the local environment and protonation state of Asp96 during the photocycle and are also essential for formation of the proton conducting hydrogen bonded network which functions during Schiff base reprotonation.

Aspartic Acid↗
PubMed1993

Fourier transform infrared difference spectroscopy of rhodopsin mutants: light activation of rhodopsin causes hydrogen-bonding change in residue aspartic acid-83 during meta II formation.

Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis have been used to investigate structural changes which occur during rhodopsin photoactivation at the level of individual amino acid residues. The rhodopsin-->bathorhodopsin FTIR difference spectra of the mutants Asp-83-->Asn (D83N) and Glu-134-->Asp (E134D) incorporated into membranes are similar to that of native rhodopsin in the photoreceptor membrane, demonstrating that the retinal chromophores of these mutants undergo a normal 11-cis to all-trans photoisomerization. Two bands assigned to the C = O stretching mode of Asp and/or Glu carboxylic acid groups are absent in the D83N rhodopsin-->metarhodopsin II FTIR difference spectrum. Corresponding changes are not observed in the carboxylate C = O stretching region. The most straightforward explanation is that the carboxylic acid group of Asp-83 remains protonated in rhodopsin and its bleaching intermediates but undergoes an increase in its hydrogen bonding during the metarhodopsin I-->metarhodopsin II transition. The mutant E134D produced a normal rhodopsin-->bathorhodopsin and rhodopsin-->metarhodopsin II difference spectrum, but a fraction of misfolded protein was observed, supporting earlier evidence that Glu-134 plays a role in proper protein insertion and/or folding in the membrane.

Animals↗
PubMed1993

Hydrogen bonding interactions with the Schiff base of bacteriorhodopsin. Resonance Raman spectroscopy of the mutants D85N and D85A.

The bacteriorhodopsin (bR) mutants Asp-85-->Asn (D85N) and Asp-85-->Ala (D85A) have a red-shifted chromophore absorption and exhibit no proton pumping (Otto, H., Marti, T., Holz, M., Mogi, T., Stern, L., Engel, F., Khorana, H. G., and Heyn, M. P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1018-1022) consistent with the hypothesis that Asp-85 functions as a counterion and proton acceptor for the retinal Schiff base (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520). Resonance Raman spectroscopy reveals that these mutants contain a mixture of all-trans and 13-cis/C = N syn chromophores, similar to dark-adapted purple membrane and acid-induced or deionized blue membrane. At high NaCl concentrations, both mutants adopt a predominantly all-trans chromophore structure similar to acid purple membrane. A comparison of the Schiff base C = NH+ stretch frequency (vC = N) and deuterium isotope shift for D85N, D85A as well as various forms of bR, including light-adapted bR, blue membrane, and acid purple membrane, provides information about hydrogen bonding interactions to the Schiff base. D85N has as strong a hydrogen bond as light-adapted bR despite the loss of the negative charge at residue 85. In contrast, D85A has a weaker hydrogen bond. These results can be explained if a direct interaction exists between the Schiff base and Asn-85 in D85N and between the Schiff base and a substituted water molecule in D85A. Many of the properties of wild type bR, D85N, D85A, blue membrane, and acid purple membrane can be explained on the basis of changes in the local hydrogen bonding near the Schiff base.

Alanine↗
PubMed1993

Fourier transform Raman spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: formation of a stable O-like species during light adaptation and detection of its transient N-like photoproduct.

Near-infrared FT-Raman spectroscopy can be used to measure the vibrations of the bacteriorhodopsin (bR) chromophore without the disadvantage of conventional visible resonance Raman spectroscopy, where the visible excitation drives the bR photoreactions. We utilized this technique to investigate the light-dark adaptation of bacteriorhodopsin and the mutant Tyr-185-->Phe (Y185F) at room temperature in solution. Compared to wild-type bR, both the FT-Raman and resonance Raman spectra of the light-adapted Y185F displayed new features characteristic of the vibrations of the O intermediate. Light adaptation of Y185F was found to involve a 13-cis, C=N syn-->all-trans isomerization of the retinal chromophore which produces a species similar to bR570 and a second O-like species. Dark adaptation, which was much slower in Y185F compared to wild-type bR, involved a parallel decay of the bR570 and O-like species and resulted in a decreased all-trans:13-cis ratio compared to wild type. Further evidence for the existence of an O-like species in Y185F comes from pump-probe Raman difference spectroscopy, where a red pump beam is found to produce a species very similar to the N intermediate in the photocycle. This species is shown by stroboscopic Raman measurements to exist transiently even at high pH. We postulate that when the Y185F chromophore has an all-trans structure the effective pKa of Asp-85 and Asp-212 is elevated in Y185F due to the disruption of the Asp-212/Tyr-185 hydrogen bond, thereby accounting for the increased protonation of these residues in the O-like species.

Bacteriorhodopsins↗
PubMed1993

Balloon mitral valvotomy by using the Twin-AT catheter: immediate results and complications in 110 patients.

Balloon mitral valvotomy, using a new Twin AT catheter (two balloons attached side by side over one shaft), was performed in 110 consecutive cases. The age of the patients ranged from 19-78 yr (mean 46 +/- 15). From a total of 94 females and 16 males, 23 of the patients (22%) had mitral valve calcification, 47 patients (46%) had atrial fibrillation, and 39 patients (37%) had mitral regurgitation (< +2). Twenty patients (18%) presented with restenosis following surgical commissurotomy. Total catheterization time was 101 +/- 26 min and the duration of the valvotomy procedure was 37 +/- 21 min in these cases. For the entire population, there was a significant reduction in mitral valve gradient (15 +/- 6 to 4.8 +/- 2.6 mmHg, p < .001), an increase in mitral valve area (MVA) (1.1 +/- 0.3 to 2.35 +/- 0.7 cm2, p < .001), and a decrease in mean pulmonary arterial pressure (31 +/- 12 to 26 +/- 11, p < .002) after the balloon mitral valvotomy. Sixteen patients (14%) developed significant left to right shunt, and in 22 patients (20%) mitral regurgitation increased moderately but without resulting in emergency valve replacement. There was one incidence of embolic episode and one pericardial tamponade. Adequate hemodynamic results (MVA > 1.5 cm2 and % increase in MVA > or = 50%) without major complications were obtained in 99 cases. In 9 patients with severely diseased valve (2 previous commissurotomy, one restenosis after balloon valvotomy), or small left ventricular cavity, insufficient results were obtained by the Twin-AT catheter.(ABSTRACT TRUNCATED AT 250 WORDS)

Balloon Occlusion↗
PubMed1992

Improved myocardial ischemic response and enhanced collateral circulation with long repetitive coronary occlusion during angioplasty: a prospective study.

OBJECTIVES: The goal of the study was to evaluate the progressive increase in ischemic threshold with multiple sequential transient coronary occlusions and to assess the role of the collateral circulation in adaptation to ischemia. BACKGROUND: It has been observed that the duration of balloon inflations during coronary angioplasty can be gradually prolonged during subsequent dilations with a reduction in patient symptoms and diminished ischemic electrocardiographic (ECG) changes. Although the mechanism has not been fully explained, recruitment of coronary collateral circulation induced by repeated coronary occlusion has been reported. The stimuli for recruitment and the natural history of coronary collateral circulation are not understood. METHODS: Seventeen patients with isolated stenosis of the left anterior descending coronary artery and a normal left ventricle were enrolled. Angioplasty consisted of five successive prolonged inflations. Sequential changes in clinical, intracoronary ECG and left ventricular indexes of myocardial ischemia were examined. Coronary collateral channels were evaluated during balloon inflations by ipsilateral and contralateral injections of contrast medium and hemodynamically by occlusion pressure. RESULTS: An improved tolerance to myocardial ischemia with repetitive coronary occlusions was demonstrated by a significant reduction of angina, ST segment deviation, left ventricular filling pressure and less impairment of ejection fraction. Left ventricular wall motion abnormalities remained unchanged. Collateral angiographic grade did not change in 7 patients and increased in 10. CONCLUSIONS: This study confirms a progressive adaptation of myocardial ischemia to repetitive coronary occlusions and supports the concept that sequential episodes of myocardial ischemia are a stimulating factor for the recruitment of collateral channels in humans. These results also suggest that enhancement of recruitable collateral circulation might be an underlying mechanism of myocardial ischemic preconditioning.

Adult↗
PubMed1991

Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization.

We report that the replacement of Leu-93 in bacteriorhodopsin by Ala (L93A) or Thr (L93T) slows down the photocycle by approximately 100-fold relative to wild-type bacteriorhodopsin. Time-resolved visible absorption spectroscopy and resonance Raman experiments, respectively, show the presence of long-lived O-like and N-like intermediates in the photocycles of the above mutants. We infer the existence of an equilibrium between the N and O intermediates in the photocycles of these mutants. The L93A and L93T mutants exhibit normal proton pumping under continuous illumination, suggesting that the decay of the N and/or O intermediate, and consequently, proton translocation, can be accelerated by the absorption of a second photon. Since the 13-cis----all-trans reisomerization of retinal is completed during the decay of the N and O intermediates, we conclude that the interaction of Leu-93 with retinal is important in this phase of the photocycle. This conclusion is supported by a recent structural model of bacteriorhodopsin that suggests that Leu-93 is near the C-13 methyl group of retinal.

Alanine↗
PubMed1991

Resonance Raman studies of the HOOP modes in octopus bathorhodopsin with deuterium-labeled retinal chromophores.

Resonance Raman spectra of the hydrogen out-of-plane (HOOP) vibrational modes in the retinal chromophore of octopus bathorhodopsin with deuterium label(s) along the polyene chain have been obtained. In clear contrast with bovine bathorhodopsin's HOOP modes, there are only two major HOOP bands at 887 and 940 cm-1 for octopus bathorhodopsin. On the basis of their isotopic shifts upon deuterium labeling, we have assigned the band at 887 cm-1 to C10H and C14H HOOP modes, and the band at 940 cm-1 to C11H = C12H Au-like HOOP mode. Except for a 26 cm-1 downward shift, the C11H = C12H Au-like wag appears to be little disturbed in octopus bathorhodopsin from the chromophore in solution since its changes upon deuterium labeling are close to those found in solution model-compound studies. We found also that the C10H and C14H HOOP wags are also similar to those in the model-compound studies. However, we have found that the interaction between the C7H and C8H HOOP internal coordinates of the chromophore in octopus bathorhodopsin is different from that of the chromophore in solution. The intensity of the C11H = C12H and the other HOOP modes suggests that the chromophore of octopus bathorhodopsin is somewhat torsionally distorted from a planar trans geometry. Importantly, a twist about C11 = C12 double bond is inferred. Such a twist breaks the local symmetry, resulting in the observation of the normally Raman-forbidden C11H = C12H Au-like HOOP mode. The twisted nature of the chromophore, semiquantitatively discussed here, likely affects the lambda max of the chromophore and its enthalpy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.

Biophysical Phenomena↗
PubMed1991

A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores.

The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.

Animals↗
PubMed1987

Resonance raman spectroscopy of an ultraviolet-sensitive insect rhodopsin.

We present the first visual pigment resonance Raman spectra from the UV-sensitive eyes of an insect, Ascalaphus macaronius (owlfly). This pigment contains 11-cis-retinal as the chromophore. Raman data have been obtained for the acid metarhodopsin at 10 degrees C in both H2O and D2O. The C = N stretching mode at 1660 cm-1 in H2O shifts to 1631 cm-1 upon deuteriation of the sample, clearly showing a protonated Schiff base linkage between the chromophore and the protein. The structure-sensitive fingerprint region shows similarities to the all-trans-protonated Schiff base of model retinal chromophores, as well as to the octopus acid metarhodopsin and bovine metarhodopsin I. Although spectra measured at -100 degrees C with 406.7-nm excitation, to enhance scattering from rhodopsin (lambda max 345 nm), contain a significant contribution from a small amount of contaminants [cytochrome(s) and/or accessory pigment] in the sample, the C = N stretch at 1664 cm-1 suggests a protonated Schiff base linkage between the chromophore and the protein in rhodopsin as well. For comparison, this mode also appears at approximately 1660 cm-1 in both the vertebrate (bovine) and the invertebrate (octopus) rhodopsins. These data are particularly interesting since the absorption maximum of 345 nm for rhodopsin might be expected to originate from an unprotonated Schiff base linkage. That the Schiff base linkage in the owlfly rhodopsin, like in bovine and in octopus, is protonated suggests that a charged chromophore is essential to visual transduction.

Animals↗
PubMed1986

Prophylactic oophorectomy in premenopausal women with stage II breast carcinoma.

Prophylactic oophorectomy, as an additional treatment for stage II breast cancer, is controversial. In a retrospective study, a group of 37 premenopausal women with stage II infiltrating duct carcinoma and one to three positive axillary lymph node involvement after modified radical mastectomy and bilateral oophorectomy were compared to a matched group of 34 women treated by modified radical mastectomy only. Prophylactic oophorectomy prolonged the disease free interval significantly as compared to the control group. However, it did not prolong survival. This raises the question whether the prolongation of survival achieved by late oophorectomy in women with advanced breast cancer is preferable to an improvement in quality of life resulting from longer disease free intervals.

Actuarial Analysis↗
PubMed1983

Survival data of Israeli Jewish patients with squamous cell carcinoma of the cervix treated by irradiation.

During a 9-year period 61 Israeli Jewish Patients with histologically confirmed invasive squamous cell carcinoma of the uterine cervix (SUC) were treated by irradiation alone. A large proportion of the patients (44.2%) were more than 60 years old and only 27.9% were diagnosed as having stage IB disease. This is attributed to low awareness and infrequent cytologic screening. There is a trend for a large proportion of stage IB patients among those younger than 59 compared to those older than 60 years. The overall 5-year survival rate was 52.2% and the survival of patients with stage IB (72.6%) was significantly higher than in more advanced stages. In addition to clinical stage, age at diagnosis and method of irradiation also influenced survival rates.

Adult↗