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Continuous measurement of cerebral blood volume in rats with the photoelectric technique: effect of morphine and naloxone.

The validity of a photoelectric method for continuous cerebral blood volume (CBV) measurement was tested and modified for the rat's brain. A new way of introducing a miniature light source between the two hemispheres and fixing a light sensitive silicone blue cell to the outer surface of the parietal bone was developed. Light extinction factor of the rat's blood was determined experimentally (Eb rat = 1.38 +/- 0.15) in order to calculate absolute CBV value in this species, resulting in a 4.77 +/- 0.13 vol % absolute CBV value. Data obtained in anesthetized, artificially ventilated rats by simultaneous recording of CBV and local cerebral blood flow (H2-gas clearance technique) show that local hypothalamic blood flow decreased significantly after morphine (1.0 mg/kg s.c.), while total CBV remained unchanged. Opiate receptor blockade with naloxone (1.0 mg/kg s.c.) on the contrary, as well as naloxone and morphine administration, caused no change in local hypothalamic blood flow, but resulted in a significant increase of total cerebral blood volume.

Animals↗

Light-driven proton or chloride pumping by halorhodopsin.

Halorhodopsin from Halobacterium halobium was purified and reconstituted with lipids from purple membranes. The resulting protein-containing membrane sheets were adsorbed to a planar lipid membrane and photoelectric properties were analyzed. Depending on light conditions, halorhodopsin acted either as a light-driven chloride pump or as a proton pump: green light caused chloride transport and additional blue light induced proton pumping. In the living cell, both to these vectorial processes would be directed toward the cytoplasm and, compared to ion transport by bacteriorhodopsin, this is an inversed proton flow. Azide, a catalyst for reversible deprotonation of halorhodopsin, enhanced proton transport, and the deprotonated Schiff base in the 13-cis configuration (H410) was identified as the key intermediate of this alternative catalytic cycle in halorhodopsin. While chloride transport in halorhodopsin is mediated by a one-photon process, proton transport requires the absorption of two photons: one photon for formation of H410 and release of a proton, and one photon for photoisomerization of H410 and re-formation of H578 with concomitant uptake of a proton by the Schiff base.

Azides↗

Correlation between ciliary beat frequency and the structure of ciliated epithelia in pathologic human nasal mucosa.

The structure and function of ciliated epithelium were studied in 44 human nasal mucosa samples using a photoelectric method and scanning electron microscopy (SEM). The ciliary beat frequency (CBF) of cases with recurrent or chronic sinusitis was 9.1 +/- 5.4 Hz. In eight of the samples (18.2%) no ciliary activity was detected. The amount of ciliated cells, the orientation of cilia, epithelial metaplasia, and secretion were found to be explanatory factors accounting for the decreased ciliary activity. Ciliary disorientation and a lack of ciliated cells in SEM correlated with low ciliary activity. In cases where sinusitis secretion was not seen, the CBF was slower than in cases with mucus or mucopurulent secretion. Sinusitis with disoriented cilia, a loss of ciliated cells, and a lack of mucosal secretion is associated with a decreased CBF. This may lead to impaired mucociliary clearance and increase the risk of recurrent and chronic sinusitis.

Adolescent↗

Vasopressin stimulates ciliary motility of rabbit tracheal epithelium: role of V1b receptor-mediated Ca2+ mobilization.

Arginine vasopressin (AVP) has recently been shown to exist in and to be released from airway epithelial cells, but the physiologic role of this hormone in airway epithelial function is unknown. To determine whether AVP affects ciliary motility, and if so, to elucidate the mechanism of action and the subtype of AVP receptors involved, we measured ciliary beat frequency (CBF) and the intracellular Ca2+ concentration ([Ca2+]i) of cultured rabbit tracheal epithelium with a photoelectric method and the fura-2 fluorescence method, respectively. Addition of AVP caused a rapid increase in CBF, followed by a decline and a subsequent sustained response. The ciliary stimulatory action was dose dependent, the maximal peak increase from the baseline CBF being 20.6 +/- 4.7% (mean +/- SE, P < 0.001), and this effect was reduced to 5.9 +/- 2. 0% by the V1 receptor antagonist OPC-21268 (P < 0.01), but not by the V2 receptor antagonist OPC-31260. The AVP-induced increase in CBF was not altered by the protein kinase A (PKA) inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate triethylamine (Rp-cAMPS) or by Ca2+-free solution containing ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), but was abolished by pretreatment with thapsigargin. Exposure of cells to AVP elicited a transient increase in [Ca2+]i, an effect that was likewise abolished by thapsigargin. The rank-order potency of AVP analogues to increase [Ca2+]i was AVP = [deamino1, D-3-(pyridyl) Ala2-Arg8] vasopressin (DP-VP), a specific V1b receptor agonist > [Phe2, Ile3, Orn8] vasopressin (PO-VT), a V1a agonist > 1-desamino-8-D-arginine vasopressin (dDAVP), a V2 agonist. Moreover, OPC-21268 greatly attenuated the action of AVP, whereas OPC-31260 was without effect. These results suggest that AVP stimulates ciliary motility of rabbit tracheal epithelium through mobilization of Ca2+ from thapsigargin-sensitive stores, and that this effect may be mediated by V1b receptors.

Animals↗

VIP potentiates cholinergic effects on the mucociliary system in the maxillary sinus.

The neuropeptide vasoactive intestinal polypeptide (VIP), which is found in a population of cholinergic parasympathetic neurons in the airways, has no effects per se on mucociliary activity. In order to test the hypothesis that VIP may modulate cholinergic regulation of the mucociliary system, VIP was infused intraarterially (8.4 pmol/kg/min), and the response to challenges with methacholine in the maxillary sinus of rabbits were recorded with a photoelectric technique. Occurrence of VIP-like immunoreactivity in the rabbit maxillary sinus, maxillary nerve, and sphenopalatine ganglion was investigated. Immunoreactivity against VIP was found in nerve fibers in the subepithelial layer of the maxillary sinus and in numerous nerve cell bodies in the sphenopalatine ganglion. Infusion of VIP potentiated the mucociliary increase induced by methacholine. The mucociliary wave frequency change increased from 6.1% +/- 1.7% to 13.3% +/- 3.9% (0.01 micrograms/kg methacholine), from 11.6% +/- 3.6% to 18.8% +/- 2.2% (0.05 micrograms/kg) and from 17.0% +/- 3.0% to 27.4% +/- 3.6% (0.1 micrograms/kg). Both peak responses and response durations increased during infusions. In contrast, the vasodilating agent papaverine sulphate did not influence the mucociliary response to methacholine. The modulating effect of VIP on the mucociliary system, taken together with the morphologic observations, suggest that VIP may have a physiologic role in the regulation of the mucociliary system in the maxillary sinus.

Animals↗

The effect of antiallergic intranasal formulations on ciliary beat frequency of human nasal epithelium in vitro.

The nasal mucociliary clearance is an important defense mechanism of the upper respiratory tract. It is known to be influenced by many pharmacologic substances. We investigated the effects of three topical intranasal antiallergic formulations containing disodium cromoglycate (DNCG), dimethindene maleate (DMM), and azelastine-HCL (AZL) on nasal ciliary beat frequency (CBF) in vitro. Nasal ciliated cells were harvested from 16 healthy volunteers. Cells were diluted 1:10 in culture medium and incubated with a placebo formulation (PLAC), containing 0.022% benzalkonium chloride, which was part of all formulations, a registered 2% formulation of DNCG, a 0.1% formulation of DMM, and a registered 0.1% formulation of AZL. After an incubation period of 20 min, CBF was registered by a photoelectric measurement device. Under control conditions (CONTROL), CBF was 11.4 +/- 1.3 Hz. PLAC reduced CBF to 9.7 +/- 2.3 Hz (NS). DNCG reduced CBF to 9.7 +/- 2 Hz (NS). DMM reduced CBF to 7.2 +/- 1.7 Hz (P < or = 0.05 vs CONTROL, NS vs PLAC), and AZL reduced CBF to 0.9 +/- 1.8 Hz (P < or = 0.001 vs CONTROL, P < or = 0.001 vs DNCG, P < or = 0.001 vs PLAC). In conclusion, a possible influence of antiallergic intranasal formulations on nasal ciliary function has to be considered in clinical application.

Anti-Allergic Agents↗

Cure of Fisher rats bearing radioresistant F98 glioma treated with cis-platinum and irradiated with monochromatic synchrotron X-rays.

High-grade gliomas are usually of poor prognosis, and conventional radiotherapy, even combined with chemotherapy, still fails to improve the survival of patients. Here, we propose an innovative therapeutic approach combining synchrotron radiation with cis-diamminedichloroplatinum (II) (CDDP). As suggested previously, monochromatic synchrotron irradiation of CDDP at 78.8 keV, just above the 78.4 keV platinum absorption K-edge, leads to an enhanced photoelectric effect and an increased local toxicity. To select a particular radiation energy that could provide supra-additive effect, we used pulsed-field gel electrophoresis to assess yields of DNA double-strand breaks induced in rat F98 glioma cells after CDDP treatment combined with synchrotron X-rays. Thereafter, intracerebral CDDP injection combined with synchrotron X-rays was applied to Fisher rats bearing F98 glioma. CDDP concentrations were mapped by synchrotron X-ray microfluorescence. An extra number of more slowly repaired double strand breaks were observed when irradiating CDDP-treated F98 cells at 78.8 keV. In vivo treatments were then performed with different radiation doses and CDDP concentrations. All cell inoculations in rat brain resulted in tumor development, and tumor presence was controlled by computed tomography. Among all of the conditions tested, the combination of 3 micro g of CDDP with 15 Gy resulted in the largest median survival time (206 days). After 1 year, about 34% of treated rats were still alive. This preclinical finding, validated by molecular analysis, represents the most protracted survival reported with this radioresistant glioma model and demonstrates the interest in powerful monochromatic X-ray sources as new tools for cancer treatments.

Animals↗

[Leukocyte migration inhibition test in drug-induced pneumonitis].

The leukocyte migration inhibition test (LMIT) was performed in 8 cases of presumed drug-induced pneumonitis. The drugs involved were amiodarone in 3 cases, methotrexate in 2 cases, and fenofibrate, nadolol, and gold salt, each in one case. The agarose microdroplet technique for photoelectric readings of leukocyte migration inhibition was applied in the presence of a wide range of drug concentrations. LMIT was found to be positive in 6 of the 8 cases (75%). The presence of a positive LMIT indicates the elaboration of leukocyte migration inhibitory factor. These results suggest that cell-mediated immunity may play a role in the pathogenesis of drug-induced pneumonitis and that LMIT may be useful for the detection of causative drugs in patients with this condition.

Aged↗

Carboxyphenyl metalloporphyrins as photosensitizers of semiconductor film electrodes. A study of the effect of different central metals.

Free-base (P), Zn(II) (P(Zn)), Cu(II) (P(Cu)), Pd(II) (P(Pd)), Ni(II) (P(Ni)), and Co(II) (P(Co)) 5-(4-carboxyphenyl)-10,15,20-tris(4-methylphenyl) porphyrins were designed and synthesized to be employed as spectral senzitizers in photoelectrochemical cells. The dyes were studied adsorbed on SnO(2) nanocrystalline semiconductor and also in Langmuir-Blodgett film ITO electrodes in order to disclose the effect of molecular packing on the studied properties. Electron injection yields were obtained by fluorescence quenching analysis comparing with the dyes adsorbed on a SiO(2) nanocrystalline insulator. Back electron-transfer kinetics were measured by using laser flash photolysis. The unmetallized and metallized molecules have different singlet state energies, fluorescence quantum yields, and redox properties. The quantum yields of sensitized photocurrent generation are shown to be highly dependent on the identity of the central metal. It is shown that P(Ni) and P(Co) do not present a photoelectric effect. The other porhyrins present reproducible photocurrent, P(Pd) being the one that gives the highest quantum yield even in closely packet ITO/LB films. Photocurrent quantum yields increase as the dye ground-state oxidation potential becomes more anodic, which is in agreement with the observation, obtained by laser flash photolysis, that back electron-transfer kinetics decrease with the increase in the driving force for the recombination process. This effect could be exploited as a design element in the development of new and better sensitizers for high-efficiency solar cells involving porphyrins and related dyes.

Journal Article↗

Still looking for the Ivory Tower.

Following graduate training, which was disrupted by my changing schools and serving in the Navy in World War II, I arrived in Berkeley in 1948 as an instructor in the Biochemistry Department. Despite numerous academic reorganizations and a host of struggles over the University-imposed Loyalty Oath, dismissal of a faculty member because of political affiliations, free speech for students, and my resistance to mandatory retirement, I survived with the help of great graduate students, postdoctoral fellows, undergraduates, superb research assistants, and a supportive wife. Studies on structure of tobacco mosaic virus led to our investigating an ultracentrifuge anomaly and the construction of a synthetic boundary cell. In turn, this resulted in about 15 years of research on the ultracentrifuge and its application to the study of biological macromolecules. Among the latter, the discovery of large ribonucleoprotein complexes, now known as ribosomes, and chromatophores in photosynthetic microorganisms attracted the most attention. But it was the development of the photoelectric absorption optical system and the incorporation of the Rayleigh interferometer onto the ultracentrifuge that had the greatest impact on our further research. These tools, when applied to our initial research on E. coli aspartate transcarbamoylase (ATCase), led to the discovery of distinct subunits for catalysis and regulation and the global conformational change in the enzyme associated with its role in regulation. For almost 35 years we have been using the techniques of protein chemistry and molecular biology in studies of structural and conformational changes in the enzyme, the genes encoding the different polypeptides, subunit interactions, and assembly of the enzyme from six catalytic and six regulatory chains. Hybrids constructed from inactive mutants were used to demonstrate shared active sites requiring the joint participation of amino acid residues from adjoining polypeptide chains. ATCase is still being studied as a model for understanding allostery as a regulatory mechanism. Circularly permuted polypeptide chains are being used to study the folding and assembly pathways, and the recently determined crystal structure of the active nonallosteric catalytic subunit has led to new questions regarding the activated form of ATCase.

Aspartate Carbamoyltransferase↗

Meta-analysis of increases in micronuclei in peripheral blood lymphocytes after angiography or excretory urography.

Norman, A., Cochran, S. T. and Sayre, J. W. Meta-analysis of Increases in Micronuclei in Peripheral Blood Lymphocytes after Angiography or Excretory Urography. Radiat. Res. 155, 740-743 (2001). Meta-analysis of 10 studies confirms a significant increase in the frequency of micronuclei in peripheral blood lymphocytes after angiography or excretory urography; the weighted average increase is 4.2 (95% confidence interval 2.8-5.6) per 1000 binucleate lymphocytes, about the same increase in micronuclei as that produced in vitro by a diagnostic X-ray dose of 4 cGy. The analysis failed to reveal a significant effect of the specific contrast medium used in the X-ray examinations on the increased frequency of micronuclei. These results are consistent with the hypothesis that the effect of the contrast media is limited to the enhancement, by the photoelectric effect, of the X-ray dose absorbed by the lymphocytes irradiated while suspended in the contrast medium. Therefore, an estimate of increased cancer risk based on elevated frequencies of micronuclei or chromosome aberrations in peripheral blood lymphocytes may be greatly exaggerated whenever the radiation damage is largely confined to the cells circulating in the blood, as it is in people who have recently had X-ray examinations that use intravenous injections of contrast medium. Such examinations include angiography, excretory urography and CT scans, which are received annually by millions of people.

Angiography↗

Electron and photon spectra for three gadolinium-based cancer therapy approaches.

Some recent neutron capture therapy research has focused on using compounds containing the element gadolinium, which produces internal conversion and Auger cascade electrons. The low-energy, short-range Auger electrons are absorbed locally and increase cell killing dramatically as the gadolinium compounds are introduced into the cell nucleus and bind to the DNA. Detailed electron and photon spectra are needed for biophysical modeling and Monte Carlo calculations of damage to DNA. This paper presents calculated electron and photon spectra for three cases: thermal neutron absorption by (157)Gd, the beta-particle decay of (159)Gd, and the K-shell photoelectric event in gadolinium. The Monte Carlo sampling of atomic and nuclear transitions for each of the three cases was used to calculate a large number of representative decays. The sampled decays were used to determine average emissions and energy deposited in small spheres of tissue. The kinetic energy nuclear recoil from gamma-ray and electron emissions was calculated and found to be more than 10 eV for 26% of all (157)Gd neutron capture reactions.

Adult↗

Non-radiative deactivation pathways of subphthalocyanine and subnaphthalocyanine dyes and of their mixture.

Subphthalocyanine and subnaphthalocyanine dyes and their mixture were investigated by means of the spectroscopic and photoelectric methods. Absorption, fluorescence, steady-state and time-resolved photothermal measurements for the dyes and their mixture were done in order to get information about the radiative and non-radiative deactivation processes as competetive processes to charge separation. It was shown that energy transfer between the dyes improved the photocurrent generation in photoelectrochemical cells (PEC) based on In(2)O(3) and SnO(2) as an electrode. The possible participation of the dye triplet states in non-radiative processes was discussed.

Carbocyanines↗

Purinoceptors mediate renal vasodilation by nitric oxide dependent and independent mechanisms.

BACKGROUND: Adenosine triphosphate (ATP) and its metabolites including adenosine modulate renal vascular tone under physiological and pathophysiological conditions. Their effects are brought about by activation of membrane bound P1- and P2-purinoceptors located on smooth muscle and endothelial cells. In this study we analyzed the purinoceptor mediated dilation of rabbit and human renal arteries, and evaluated the possible involvement of endothelium-derived relaxing factors. METHODS: Segments of rabbit and human renal arteries were incubated and perfused with medium containing indomethacin. After preconstriction, drug induced changes in the vessel diameters were measured by a photoelectric device. RESULTS: ATP (EC50 = 1 mumol/liter), added intraluminally, caused maximal vasodilation of 80 to 100% of the preconstriction response in both species. This effect was inhibited by the P1-purinoceptor antagonist 8-p-(sulphophenyl)theophylline (100 mumol/liter), suggesting that it was in part due to breakdown of ATP to adenosine. The nature of purinoceptor mediated renal vasodilation was studied further in rabbit renal arteries. Adenosine (EC50 = 1 mumol/liter) as well as the P2Y-receptor agonists ADP beta S (EC50 = 0.4 mumol/liter) and 2-MeSATP (EC50 = 0.2 mumol/liter) dilated the arteries by 80 to 100%. The effects of 2-MeSATP, which were to a much lesser extent that of ADP beta S but not that of adenosine, were attenuated by the P2Y-antagonist reactive blue 2 (3 mumol/liter). Removal of the endothelium almost abolished the vasodilation induced by adenosine and ATP. In contrast, these dilator response were only slightly attenuated by the nitric oxide synthase blockers NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine (300 mumol/liter each), whereas acetylcholine and 2-MeSATP induced dilation was markedly reduced by NG-nitro-L-arginine methyl ester. CONCLUSIONS: P1-purinoceptors activated by adenosine dilate rabbit renal arteries by an endothelium-derived relaxing factor that appears to be distinct from nitric oxide. In contrast, P2Y-purinoceptor induced renal dilation is mediated by nitric oxide. ATP, the physiological activator of P2Y-purinoceptors, is rapidly broken down to adenosine in rabbit and human renal arteries. Therefore, in rabbit and human renal arteries the vasodilatory effect of exogenous ATP mainly results from P1-purinoceptor activation probably through its breakdown product, adenosine.

Adenosine↗

[The inhibitory effects of platelet activating factor (PAF) on ciliary activity of human paranasal sinus mucosa].

The effect of platelet activating factor (PAF) on human paranasal ciliated cells was investigated in vitro. Normal human paranasal sinus mucosa was obtained by surgical procedure and incubated with Eagle's MEM containing 10% FCS in the form of tissue culture. Ciliary activity was viewed at 37 degrees C under an inverted microscope equipped with a thermoregulator and a humidified CO2 chamber, recorded on video tapes and photoelectrically measured. Ciliary inhibition was observed by the treatment with PAF, in a dose dependent manner, at concentrations from 10(-10) M to 10(-6) M. The inhibitory effect of 10(-8) M PAF on ciliary activity was completely blocked when the mucosa was treated with 10(-6) M CV-3988 or 10(-6) M CV-6209 (specific PAF receptor antagonists). By the radioimmunoassay, the concentration of PAF in tissue culture was reduced by half within 12.5 min, and within 60 min it was only 5% of the initial concentration. There was no significant difference in ciliary inhibition between irrigation after a 60 min incubation with 10(-8) M PAF and non-irrigation. These results indicate that PAF inhibited ciliary activity directly and specifically, and induced irreversible damage primarily within the first 60 min after the challenge.

Cilia↗

Monte Carlo simulations and measurement of DNA damage from x-ray-triggered auger cascades in iododeoxyuridine (IUdR).

We investigated the DNA damage from Auger electrons emitted from incorporated stable iodine (127I), following photoelectric absorption of external x-rays. The effectiveness of the Auger electrons in producing DNA double-strand breaks (DSB) was determined theoretically, using Monte Carlo simulations of the radiation physics and chemistry, and was shown to be in reasonable agreement with DNA damage measured using the comet assay. The DSB yields were measured in CHO cells for 60Co (as a non-Auger-promoting radiation) and for tungsten-filtered 100 kVp x-rays capable of producing Auger electron emission. The theoretical study showed that on average, 2.5 Auger electrons were emitted for N-shell orbital vacancies and up to 10 Auger electrons were emitted from L1-shell vacancies. These Auger bursts produced approximately 0.03 DSB per N-shell vacancy and 0.3 DSB per K-shell or L-shell vacancy. The calculated yield of DSB from Auger cascades per unit dose (1 Gy) in water was approximately 1.7 for tungsten-filtered 100 kVp x-rays, assuming 20% IUdR substitution of thymidine. The comet assay yielded an experimental value of 3.6+/-1.6 per 1 Gy for the same conditions. The Monte Carlo simulations also demonstrated a high complexity of DSB produced by Auger cascades with virtually all DSB from inner shell orbitals (i.e. K, L shells) accompanied by compounded strand breakage and base damage, indicating a difficult lesion to repair. This finding agrees well with comet assay results of DNA repair, where an increase in the DSB yield in IUdR-sensitized cells was shown to persist after a time of 24 h. We conclude that Auger cascades in iodine produce a modest increase in the number of initial strand breaks of the order of 10% but the complex nature of these DSB makes them very difficult to repair or potentially prone to misrepair. The accentuated DNA damage may have major consequences for cell survival and may be exploitable in kilovoltage photon activation therapy (PAT) of tumors sensitized with iodine.

Animals↗

Fabrication and photoelectric response of poly(allylamine hydrochloride)/PM thin films by layer-by-layer deposition technique.

Thin films of poly(allylamine hydrochloride) (PAH) and bacteriorhodopsin (bR) embedded in purple membrane (PM) have been prepared by layer-by-layer (LBL) self-assembly technique. The results obtained by UV-Vis spectroscopy and atomic force microscopy (AFM) analysis showed that the biological activity of bR was preserved and PM fragments could be well oriented onto the ITO substrate. A photo-electrochemical cell with the structure of ITO/(PAH/PM)(n)/electrolyte (0.5M KCl)/Pt was fabricated and studied. The photocurrent peaks of (PAH/PM)(6) corresponding to light-on and light-off were about 200 and 100 nA/cm(2), respectively, with the former enhanced 30% higher than that of the reference films made of (PDAC/PM)(6).

Bacteriorhodopsins↗

Strand breaks in plasmid DNA, natural and brominated, by low-energy X-rays.

Single- and double-strand breaks were measured in Col E1 plasmid DNA, natural and partially brominated, irradiated with monoenergetic X-rays (from a synchrotron radiation source) on both sides of the K-absorption edge for Br. The fraction of the undamaged supercoiled form decreased exponentially with the photon dose; its yield in the brominated DNA did not exhibit any energy-dependence. This result is consistent with the calculated relative contributions of photoelectric interactions with individual component atoms which show that an electron flux originating in light atoms outweighs that from bromine. However, X-rays of energy above the Br K-edge appear slightly more efficient in producing double-strand breaks. This result, as well as those reported in the literature on the Auger enhancement of different biological endpoints in cells containing brominated DNA, seem to suggest that the positive charge and submolecular effects associated with the photo-absorption in bromine play some role in damaging processes, besides the initial distribution of deposited electron energy.

Bromine↗