Effect of cholesterol on human erythrocyte membrane. A spin label study

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.     

Age- and Sex-Dependency of Laser Speckle Flowgraphy Measurements of Optic Nerve Vessel Microcirculation

Purpose

To investigate the relationship between various characteristics of a normal population and laser speckle flowgraphy (LSFG) measurements of mean blur rate (MBR) in the optic nerve head (ONH).

Methods

A total of 189 eyes of 189 normal subjects (93 male, 96 female, mean age 45 ± 14 years old, age range: 20–72) without any history of hypertension, hyperlipidemia or diabetes were enrolled. ONH microcirculation was measured with LSFG and overall MBR (MA), vessel-area MBR (MV), and tissue-area MBR (MT) were derived from these measurements. The statistical association of these measurements with characteristics such as sex, age, intraocular pressure (IOP) and systolic blood pressure (SBP) was then determined.

Results

There was a trend towards decreased IOP and MV and increased SBP with age (P = 0.002, P = 0.035, and P = 0.006, respectively). Furthermore, IOP, MV and SBP were correlated with age (r = -0.23, P = 0.011; r = -0.24, P < 0.001; and r = 0.30, P < 0.001, respectively). Separate multiple regression analyses of independent contributing factors revealed that sex and IOP contributed to MA (P < 0.001 and P = 0.002, respectively), sex, IOP, and age contributed to MV (P < 0.001, P = 0.003, and P = 0.024, respectively), while only IOP contributed to MT (P = 0.003).

Conclusion

In a normal population, MBR was affected by IOP in both the large vessel and capillary areas of the ONH, but not by SBP. MV was also affected by age and sex, while MT was stable independent of age or sex.     

Effects of exposure to a circularly polarized 50-Hz magnetic field on plasma and pineal melatonin levels in rats

We sought to determine whether a 6-week exposure to a 50-Hz rotating magnetic field influences melatonin synthesis by 11–18 week-old Wistar-King male rats. Rats were exposed continuously to a rotating magnetic field at 1, 5, 50, or 250 μT (spatial vector rms) for 6 weeks, except for twice-weekly breaks of about 2 h for cleaning of cages and feeding. The rats were housed in exposure and sham-exposure facilities, which were located in the same room, under a 12:12 light-dark photoperiod (lights on at 06:00 h). The room was constantly illuminated by 4 small, dim red lights (< 0.07 lux in dark period). Levels of plasma and pineal gland melatonin were determined by radioimmunoassay. A significant decrease of melatonin was observed between the control group and groups exposed to a magnetic field at a flux density in excess of l μT during the night time, but no statistical differences were found among the exposed groups. These results indicate that subchronic exposure of albino rats to a 50-Hz rotating magnetic field influences melatonin production and secretion by the pineal gland. © 1993 Wiley-Liss, Inc.     

Electron spin relaxation of synthetic melanin and melanin-containing human tissues as studied by electron spin echo and electron spin resonance

Electron spin lattice relaxation times (T1) and the phase memory times (Tm) were obtained for the synthetic melanin system from 3-hydroxytyrosine (dopa) by means of electron spin echo spectroscopy at 77 degrees K. Saturation behavior of the ESR spectra of melanins in melanin-containing tissue and of the synthetic melanin was also determined at the same temperature. The spin lattice relaxation time and the spectral diffusion time of the synthetic melanin are very long (4.3 ms and 101 microseconds, respectively, in the solid state), and the ESR signal saturates readily at low microwave powers. On the other hand, ESR spectra of natural melanins from the tissues chosen for this study, as well as those of synthetic melanins which contain Fe3+ of g = 4.3 and Mn2+ of g = 2, are relatively difficult to saturate compared with samples without such metal ions. These results show clearly that a large part of those two metal ions in sites responsible for the ESR spectral components with these particular g values are coordinated to melanin in melanin-containing tissue, and modify the magnetic relaxation behavior of the melanin. Accumulations of these metal ions in melanins are different from system to system, and they increase in the order: hair (black), retina and choroid (brown), malignant melanoma of eye and skin, and lentigo and nevus of skin.     

Cell age-dependent changes in deformability and calcium accumulation of human erythrocytes

The deformability of human erythrocytes was measured in a rheoscope, as a function of intracellular calcium content (varied with ionophore (A23187) and CaCl2) without complete ATP depletion and echinocytic transformation. Loading calcium into intact erythrocytes (calcium content: 16.8 mumol/1 packed cells = 1.48 amol per cell), the cell volume and energy charge gradually decreased. Further, the membrane fluidity of the lipid portion decreased without crosslinking of membrane proteins. A distinct transition from deformable to undeformable cells was observed by the rheoscope technique: i.e., 50% transition occurred at 40-50 mumol calcium/1 packed cells (= 3.5-4.0 amol per cell) and more than 90% above 100 mumol/1 packed cells (= 6.5 amol per cell) at a shear stress of 140 dyn/cm2. The deformable cells maintained their deformability to ellipsoidal disks independent of the average calcium content. The underformable cells, separated as high-density cells by density gradient centrifugation after calcium-loading, showed lower glucose-6-phosphate dehydrogenase activity than low-density-deformable cells; thus, the calcium-loaded, undeformable cells were presumably in vivo aged cells. The younger cells, fractionated as low-density cells from intact erythrocytes, were more deformable than aged cells. Upon calcium-loading, the younger cells restored their cell volume and deformability, while the aged cells, containing originally more calcium and less ATP, decreased their volume and became undeformable. Therefore, calcium accumulation by ionophore-CaCl2 takes place in preference to aged cells of lower energy metabolism, and leads to cellular dehydration and loss of deformability, due to condensed hemoglobin and altered membrane organization.     

Resonance Raman study on complexes of medium-chain acyl-CoA dehydrogenase.

Resonance Raman (RR) spectra of the complex of pig kidney medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA and of the purple complex formed upon the addition of octanoyl-CoA to the dehydrogenase were obtained. RR spectra were also measured for the complexes prepared by using isotopically labeled compounds, i.e., [3-13C]-, [1,3-13C]-, and [2,4-13C2]acetoacetyl-CoA; [1-13C]octanoyl-CoA; the dehydrogenase reconstituted with [4a-13C]- and [4,10a-13C2]FAD. Both bands of oxidized flavin and acetoacetyl-CoA were resonance-enhanced in the 632.8 nm excited spectra of the acetoacetyl-CoA complex; this confirms that the broad long-wavelength absorption band is a charge-transfer absorption band between oxidized flavin and acetoacetyl-CoA. The 1,622 cm-1 band was assigned to the C(3)=O stretching mode coupling with the C(2)-H bending mode of the enolate form of acetoacetyl-CoA and the bands at 1,483 and 1,119 cm-1 were assigned to bands associated with the C(2)=C(1)-O- moiety. Both bands of fully reduced flavin and the substrate were resonance-enhanced in the 632.8 nm excited spectra of the purple complex. As the enzyme is already reduced, the substrate must be oxidized to octenoyl-CoA; the complex is a charge-transfer complex between the reduced enzyme and octenoyl-CoA. The low frequency value of the 1,577 cm-1 band, which is associated with the C(2)-C(1)=O moiety of the octenoyl-CoA, suggests that the enzyme-bound octenoyl-CoA has an appreciable contribution of C(2)=C(1)-O-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line,LM217

Background

Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines.

A nuclear GFP/β-galactosidase fusion protein as a marker for morphogenesis in living Drosophila

A general, non-invasive method to trace morphogenesis in living Drosophila was developed. To label specific cells, green fluorescence protein (GFP) of jellyfish Aequorea victoria was expressed by the Ga14-UAS system. Green-fluorescence from GFP fused to the nuclear localization signal was detectable in polytene larval tissue, but not in diploid tissue. Further fusion to bacterial β-galactosidase produced GFPN-lacZ, which fluoresced brightly in several diploid larval and embryonic tissues. GFPN-lacZ was used to trace dynamic cell movement during the formation of the embryonic tracheal system. These results indicate that GFPN-lacZ can be used to mark specific cells to study cell movement and gene expression in living animals.     

DIDS increases K+ secretion through an I sKchannel in apical membrane of vestibular dark cell epithelium of gerbil

Vestibular dark cell epithelium secretes K⁺ via I _sKchannels in the apical membrane. The previous observation that disulfonic stilbenes increased the equivalent short circuit current (I _sc) suggested that these agents might be useful investigative tools in this tissue. The present experiments were conducted to determine if the increase in I _scwas associated with an increase in K⁺ flux and if the effect was directly on the I _sKchannel or indirectly via a cytosolic intermediary. Measurements of transepithelial K⁺ flux with the K⁺-selective vibrating probe and of changes in net cellular solute flux by measurements of epithelial cell height showed that 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) increased K⁺ flux by a factor of 1.96±0.71 and caused net solute efflux. The apical membrane was partitioned with a macropatch pipette and DIDS was applied either to the membrane outside the pipette, inside the pipette or to the entire apical membrane. DIDS inside the pipette increased the current across the patch, the membrane conductance, the slowly-inactivating (I _sK) component of the membrane current and shifted the reversal voltage toward the equilibrium potential for K⁺. DIDS outside the patch decreased the patch current and conductance, consistent with shunting of current away from the membrane patch. These findings strongly support the notion that DIDS increases K⁺ secretion through I _sKchannels in the apical membrane of vestibular dark cell epithelium by acting directly on the channels or on a tightly colocalized membrane component.We thank Dr. Peter J.S. Smith and Alan Shipley of the National Vibrating Probe Facility at the Marine Biological Laboratory at Woods Hole, MA for their support and assistance in the measurements of K⁺ flux. This work was supported by National Institutes of Health grants R01-DC00212, R29-DC1098 and P41-RR01395.