Biomedical subjects

S A Sundberg

Publications and source records attributed to S A Sundberg.

7 recordsLinked to original sources

PubMed2000

High-throughput and ultra-high-throughput screening: solution- and cell-based approaches.

The trend towards assay miniaturization for high-throughput and ultra-high-throughput screening continues to spur development of homogeneous, fluorescence-based assays in higher density, smaller volume microplate formats. Recently, first-generation microfluidic devices have been designed for performing continuous-flow biochemical and cell-based assays. These devices provide orders-of-magnitude reduction in reagent consumption, and offer the potential for implementing high-throughput screening in formats that integrate up-front compound handling with unique assay functionality.

Animals↗

PubMed1990

Distribution of charge on photoreceptor disc membranes and implications for charged lipid asymmetry.

A novel spin labeling technique is used to determine both the inner and outer surface potentials of isolated rod outer segment disc membranes and of reconstituted membranes containing rhodopsin with defined lipid compositions. It is shown that these potentials can be accounted for in a consistent manner by the accepted model of rhodopsin, the known lipid composition, and the Gouy-Chapman theory, provided the charged lipid is asymmetric in the membrane, with approximately 75% on the external surface.

Amino Acid Sequence↗

PubMed1990

Characterization of Halobacterium halobium mutants defective in taxis.

Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.

Bacterial Proteins↗

PubMed1986

Properties of a second sensory receptor protein in Halobacterium halobium phototaxis.

A second slow-cycling retinylidene protein, in addition to slow-cycling (sensory) rhodopsin (SR), can be bleached with hydroxylamine and regenerated with all-trans retinal in photosensory signaling Halobacterium halobium membranes. Flash photolysis shows this protein undergoes a photochemical reaction cycle characterized by photoconversion of its ground state (lambda max 480 nm) to a species with lambda max less than or equal to 360 nm, which thermally regenerates the 480-nm species with a t1/2 of 260 msec at 25 degrees C, under conditions in which SR photocycles at 650 msec in the same membranes. Mutants characterized with respect to their phototaxis behavior are identified which contain SR and the 480-nm pigment, the latter ranging from undetectable to a concentration equal to that of SR. Receptor mutants lacking all phototaxis sensitivity lack both of the photochemically reactive proteins. The mutant properties contribute to an accumulation of behavioral and spectroscopic evidence that the 480-nm pigment is a second sensory photoreceptor in H. halobium. NaDodSO4-polyacrylamide gel electrophoresis of [3H]retinal-labeled membrane proteins from the mutants indicates SR and the 480-nm pigment contain distinct chromophoric polypeptides differing in their migration rates. The data implicate polypeptides of 25,000 Mr and 23,000 Mr as retinal-binding polypeptides of SR and the 480-nm protein, respectively.

Bacterial Proteins↗

PubMed1986

Excitation signal processing times in Halobacterium halobium phototaxis.

Phototaxis responses of Halobacterium halobium were monitored with a computerized cell-tracking system coupled to an electronic shutter controlling delivery of photostimuli. Automated analysis of rates of change in direction and linear speeds provided detection of swimming reversals with 67 ms resolution, permitting measurement of distinct phases of the responses to attractant and repellent stimuli. After stimulation, there was a latency period in which the population reversal frequency was unchanged, followed by an excitation phase in which reversal frequency increased, and a slower adaptation phase in which reversal frequency returned to its prestimulus value. A step-decrease in illumination of the attractant receptor slow-cycling or sensory rhodopsin (SR) (lambda max, 587 nm) was interpreted by the cells as an unfavorable stimulus and, after a minimum latency of 0.70 +/- 0.14 s, induced swimming reversals with the peak response occurring 1.34 +/- 0.07 s after onset of the stimulus. Two distinct repellent responses in the near UV/blue were observed. One was a reversal response to 400 nm light, which was dependent on orange-red background illumination as expected for the photointermediate repellent form of SR (lambda max, 373 nm). The minimum latency of this response was approximately the same as that of the SR attractant system. The second was a reversal response with shorter minimum latency (0.40 +/- 0.07 s) to light of longer wavelength (450 nm) than absorbed by the known SR repellent form. This result confirms recent findings of an additional repellent photosystem in this spectral range. Further, the longer wavelength repellent response is independent of orange-red background illumination, indicating that the photoreceptor mediating this response is not a photointermediate of SR.

Bacteriorhodopsins↗

PubMed1986

Investigation of surface potential asymmetry in phospholipid vesicles by a spin label relaxation method.

In earlier work, Castle and Hubbell (1976) demonstrated the use of a spin-labeled amphiphile as a probe for the electrostatic potential at the outer surface of charged phospholipid vesicles. In recent experiments, we have shown that the hydrophobic anion tetraphenylboron (TPB) promotes transbilayer migration of the probe molecule. Relaxation data recorded following the rapid mixing of the probe with TPB-containing vesicle samples provides information about the electrostatic potentials at both the outer and inner vesicle surfaces. The measured potentials for both surfaces of asymmetrically screened vesicles were found to be in good agreement with theoretical values calculated using their known surface charge density. The method is also sensitive to transmembrane potentials as indicated by the response of the label to potentials created with the use of potassium concentration gradients and valinomycin.

Cyclic N-Oxides↗

PubMed1985

Selection and properties of phototaxis-deficient mutants of Halobacterium halobium.

A method for isolating phototaxis-deficient (Pho-) mutants of Halobacterium halobium was developed. The procedure makes use of a flashing repellent light to induce frequent reversals of swimming direction by responsive cells, thereby impeding their migration along a small capillary and resulting in a spatial separation of the parent population and a population enriched for Pho- cells. Two classes of Pho- mutants were obtained by this selection scheme: those which have lost the chemotactic response (Che-) as well as phototaxis sensitivity (general taxis mutants), and those which are defective in steps specific to phototaxis (photosignaling mutants). In the latter class, several retinal synthesis mutants were isolated, as well as a strain which fit the expected properties of a mutant lacking a functional photoreceptor protein. On the basis of spectroscopic and swimming behavior studies, the retinal-containing protein, slow-cycling or sensory rhodopsin (SR), was previously proposed to be a dual-function sensory receptor mediating both attractant and repellent photosensing. The receptor mutant Pho81 fulfills two predictions which provide direct genetic evidence for this proposal. The mutant has lost SR photoactivity as determined by spectroscopic measurements, and it has simultaneously lost both attractant and repellent phototaxis sensitivity. Comparison of [3H]retinal-labeled membrane proteins from the mutant and its SR-containing parent implicated a 25,000 Mr polypeptide as the chromophoric polypeptide of SR.

Chemotaxis↗