Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles

Background: This follow-up study aims to determine the physical parameters which govern the differential radiosensitization capacity of two tumor cell lines and one immortalized normal cell line to 1.9 nm gold nanoparticles. In addition to comparing the uptake potential, localization, and cytotoxicity of 1.9 nm gold nanoparticles, the current study also draws on comparisons between nanoparticle size and total nanoparticle uptake based on previously published data.

Methods: We quantified gold nanoparticle uptake using atomic emission spectroscopy and imaged intracellular localization by transmission electron microscopy. Cell growth delay and clonogenic assays were used to determine cytotoxicity and radiosensitization potential, respectively. Mechanistic data were obtained by Western blot, flow cytometry, and assays for reactive oxygen species.

Results: Gold nanoparticle uptake was preferentially observed in tumor cells, resulting in an increased expression of cleaved caspase proteins and an accumulation of cells in sub G1 phase. Despite this, gold nanoparticle cytotoxicity remained low, with immortalized normal cells exhibiting an LD50 concentration approximately 14 times higher than tumor cells. The surviving fraction for gold nanoparticle-treated cells at 3 Gy compared with that of untreated control cells indicated a strong dependence on cell type in respect to radiosensitization potential.

Conclusion: Gold nanoparticles were most avidly endocytosed and localized within cytoplasmic vesicles during the first 6 hours of exposure. The lack of significant cytotoxicity in the absence of radiation, and the generation of gold nanoparticle-induced reactive oxygen species provide a potential mechanism for previously reported radiosensitization at megavoltage energies.

ISOLATION, LOCALIZATION, AND CARDIOVASCULAR ACTIVITY OF TACHYKININS FROM THE STOMACH OF THE BOWFIN AMIA-CALVA

The bowfin is an extant representative of an ancient group of ray-finned fish with evolutionary connections to modern teleosts. A peptide with substance P-like immunoreactivity was isolated from an extract of bowfin stomach and its primary structure was established as Ser-Lys-Ser-His-Gln-Phe-Tyr-Gly-Leu-Met-NH2. This amino acid sequence resembles mammalian substance P only in the COOH-terminal region of the peptide. A second tachykinin with neurokinin A-like immunoreactivity isolated from the extract comprises 23 amino acid residues and shows limited structural similarity to mammalian neuropeptide-gamma. A randomly distributed population of cells in the gastric glands of the bowfin were immunostained with an antiserum raised against substance P, but no immunopositive structures were identified in the surface epithelium, lamina propria, or the nerve plexuses of the submucosa. Bolus injections of synthetic bowfin substance P (0.1-10 nmol/kg) into the bulbus arteriosus of unanesthetized bowfin resulted in a significant and dose-dependent rise in vascular resistance and arterial blood pressure (P < 0.01) and a fall in cardiac output (P < 0.05) without change in heart rate. After 5-10 min, arterial pressure and vascular resistance returned to preinjection levels, but cardiac output significantly (P < 0.05) increased over baseline values. The response to the peptide was unaffected by pretreatment of the animals with phentolamine. The study has shown that the stomach of the bowfin synthesizes tachykinins with novel structural features that display cardiovascular activity in this species.

Urotensin II in the central nervous system of the frog Rana ridibunda: Immunohistochemical localization and biochemical characterization

Urotensin II (UII) is traditionally regarded as a product of the neurosecretory cells in the caudal portion of the spinal cord of jawed fishes. A peptide related to UII has been recently isolated from the frog brain, thereby providing the first evidence that UII is also present in the central nervous system of a tetrapod. In the present study, we have investigated the distribution of UII-immunoreactive elements in the brain and spinal cord of the frog Rana ridibunda by immunofluorescence using an antiserum directed against the conserved cyclic region of the peptide. Two distinct populations of UII-immunoreactive perikarya were visualized. The first group of positive neurons was found in the nucleus hypoglossus of the medulla oblongata, which controls two striated muscles of the tongue. The second population of immunoreactive cell bodies was represented by a subset of motoneurons that were particularly abundant in the caudal region of the cord (34% of the motoneuron population). The telencephalon, diencephalon, mesencephalon, and metencephalon were totally devoid of UII-containing cell bodies but displayed dense networks of UII-immunoreactive fibers, notably in the thalamus, the tectum, the tegmentum, and the granular layer of the cerebellum. In addition, a dense bundle of long varicose processes projecting rostrocaudally was observed coursing along the ventral surface of the brain from the midtelencephalon to the medulla oblongata. Reversed-phase high-performance liquid chromatography analysis of frog brain, medulla oblongata, and spinal cord extracts revealed that, in all three regions, UII-immunoreactive material eluted as a single peak which exhibited the same retention time as synthetic frog UII. Taken together, these data indicate that UII, in addition to its neuroendocrine functions in fish, is a potential regulatory peptide in the central nervous system of amphibians. (C) 1996 Wiley-Liss, Inc.

Localization of erythropoietin gene expression in proximal renal tubular cells detected by digoxigenin-labelled oligonucleotide probes

Erythropoietin (EPO) is the main humoral stimulus of erythropoiesis. In adult mammals, the kidney releases EPO in response to hypoxic stress. Conflicting data have suggested either renal tubular or peritubular cell origins of EPO synthesis in vivo. In situ hybridization studies were performed to define further the kidney cell type(s) capable of increasing EPO gene expression during hypoxic stimulation. EPO gene expression was stimulated in mice exposed to acute hypobaric hypoxia. Kidneys from hypoxic and control normoxic mice were obtained. Six digoxigenin-labelled oligonucleotide probes complementary to EPO exon sequences were utilized for in situ hybridization for EPO messenger RNA. Positive hybridization signals were identified in some proximal tubular cells, confined to the inner third of the renal cortex of hypoxic mouse kidney.

Speciation and distribution of arsenic and localization of nutrients in rice grains

Arsenic (As) contamination of rice grains and the generally low concentration of micronutrients in rice have been recognized as a major concern for human health. Here, we investigated the speciation and localization of As and the distribution of (micro)nutrients in rice grains because these are key factors controlling bioavailability of nutrients and contaminants. Bulk total and speciation analyses using high-pressure liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICP-MS) and X-ray absorption near-edge spectroscopy (XANES) was complemented by spatially resolved microspectroscopic techniques (micro-XANES, micro-X-ray fluorescence (micro-XRF) and particle induced X-ray emission (PIXE)) to investigate both speciation and distribution of As and localization of nutrients in situ. The distribution of As and micronutrients varied between the various parts of the grains (husk, bran and endosperm) and was characterized by element-specific distribution patterns. The speciation of As in bran and endosperm was dominated by As(III)-thiol complexes. The results indicate that the translocation from the maternal to filial tissues may be a bottleneck for As accumulation in the grain. Strong similarities between the distribution of iron (Fe), manganese (Mn) and phosphorus (P) and between zinc (Zn) and sulphur (S) may be indicative of complexation mechanisms in rice grains.

Speciation and localization of arsenic in white and brown rice grains

Synchrotron-based X-ray fluorescence (S-XRF) was utilized to locate arsenic (As) in polished (white) and unpolished (brown) rice grains from the United States, China, and Bangladesh. In white rice As was generally dispersed throughout the grain, the bulk of which constitutes the endosperm. In brown rice As was found to be preferentially localized at the surface, in the region corresponding to the pericarp and aleurone layer. Copper, iron, manganese, and zinc localization followed that of arsenic in brown rice, while the location for cadmium and nickel was distinctly different, showing relatively even distribution throughout the endosperm. The localization of As in the outer grain of brown rice was confirmed by laser ablation ICP-MS. Arsenic speciation of all grains using spatially resolved X-ray absorption near edge structure (micro-XANES) and bulk extraction followed by anion exchange HPLC-ICP-MS revealed the presence of mainly inorganic As and dimethylarsinic acid (DMA). However, the two techniques indicated different proportions of inorganic:organic As species. A wider survey of whole grain speciation of white (n=39) and brown (n=45) rice samples from numerous sources (field collected, supermarket survey, and pot trials) showed that brown rice had a higher proportion of inorganic arsenic present than white rice. Furthermore, the percentage of DMA present in the grain increased along with total grain arsenic.

Language and crossed finger localization in patients with schizophrenia

Language deficits are frequently reported in studies of patients with schizophrenia. The present study sought to test the hypothesis that such deficits are related to callosal function in this group. The FAS test of verbal fluency and Perin's Spoonerisms test of phonological processing were the tests of language. Callosal function was assessed using a Crossed Finger Localisation Test (CFLT), which is a measure of the interhemispheric transfer of somatosensory information. Patients with schizophrenia performed less well than controls on measures of language function. as well as on the CFLT. Significant positive correlations between CFLT performance and language function were present in the patient group, but not the control group. These findings extend on previous studies that report functional abnormalities of the corpus callosum in schizophrenia and are consistent with the hypothesis that language deficits in schizophrenia are related to impaired callosal functioning in this group. However, other explanations cannot be ruled Out.

Metamaterial as a controllable template for nanoscale field localization

We report that subwavelength localization of light in the near-field of a double-periodic photonic metamaterial may be efficiently controlled by the polarization and wavelength of the incident radiation. A dramatic variation in the periodic near-field landscapes, including a transition from a pattern of isolated subwavelength plasmon hot-spots to a blurred, low contrast pattern, accompanied by a change in the pattern's symmetry has been observed in the proximity of an aluminum nanowire "fish-scale" nanostructure. Hot-spots as small as 0.23 lambda have been achieved and their position has been controlled by tuning the wavelength of incident light across the dipole absorption resonance of the metamaterial. A simple switch of the polarization state can lead to a spatial period doubling in the landscape pattern.

Retinal and choroidal responses to panretinal photocoagulation: an ultrastructural perspective

BACKGROUND: There have been few histological or ultrastructural studies of the outer retina and choriocapillaris following panretinal photocoagulation therapy. This investigation examines the long-term morphological effects of panretinal photocoagulation in two patients with type II diabetes who had received laser treatment more than 6 months prior to death.

METHODS: Regions of retina and choroid from each patient were fixed in 2.5% glutaraldehyde, dissected out and examined using light microscopy and scanning and transmission electron microscopy.

RESULTS: After removing the neural retina, scanning electron microscopy of non-photocoagulated areas of the eye cups revealed normal cobblestone-like retinal pigment epithelial (RPE) cells. Regions with laser scars showed little RPE infiltration into the scar area, although large rounded cells often appeared in isolation within these areas. Sections of the retina and choroid in burn regions showed a complete absence of the outer nuclear layer and photoreceptor cells, with the inner retinal layers lying in close apposition to Bruch's membrane. Non-photocoagulated regions of the retina and choroid appeared normal in terms of both cell number and cell distribution. The RPE layer was absent within burn scars but many RPE-like cells appeared markedly hypertrophic at the edges of these regions. Bruch's membrane always remained intact, although the underlying choriocapillaris was clearly disrupted at the point of photocoagulation burns, appearing largely fibrosed and non-perfused. Occasional choroidal capillaries occurring in this region were typically small in profile and had plump non-fenestrated endothelium.

CONCLUSIONS: This study outlines retinal and choroidal cell responses to panretinal photocoagulation in diabetic patients and demonstrates an apparent reduction in the capacity of these tissues to repair laser damage.

The combined effects of diabetes and ionising radiation on the rat retina: an ultrastructural study

The combined effect of STZ-diabetes and ionising radiation on the rat retina was investigated. Wistar rats, which had been diabetic for 6 months, were irradiated with a single dose of x-rays (1500 cGy) and the ultrastructural effects evaluated at 4-10 mths post-irradiation. At 4 months post-irradiation, the outer nuclear layer of the retina was greatly reduced in thickness and the photoreceptor outer segments were disorganised and reduced in length. In addition, the nerve fibre layer contained many cytoid bodies and there were many redundant basement membrane tubes throughout the inner retina. By 6 months post-irradiation, the photoreceptor cells were virtually absent, bringing the external limiting membrane into close apposition to the RPE. Throughout large areas of the outer retina, RPE cells were hypertrophic and some had proliferated into the inner retina. In many regions, proliferating retinal capillaries were observed within the RPE layer, and at 8 months post-irradiation, some vessels extended into the inner retina accompanied by RPE cells. At 10 months post-irradiation, the RPE was atrophic and degenerative with retinal glial cells coming into contact with Bruch's membrane. In some areas, the glia which had breached Bruch's membrane had invaded the underlying choroid. Where glial cells contacted the choriocapillaries, the vessels assumed the appearance of retinal vessels with plump endothelia and no fenestrations. This study has described a progressive inner retinal ischemia, with cytoid bodies, capillary non-perfusion and general atrophy of the inner retina intensifying markedly with increasing post-irradiation time.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural findings in solar retinopathy

This study documents the ultrastructural findings in a case of solar retinopathy, 6 days after sungazing. A malignant melanoma of the choroid was diagnosed in a 65-year-old man. On fundoscopy, the macula was normal. The patient agreed to stare at the sun prior to enucleation. A typical solar retinopathy developed, characterised by a small, reddish, sharply circumscribed depression in the foveal area. Structural examination of the fovea and parafovea revealed a spectrum of cone and rod outer segment changes including vesiculation and fragmentation of the photoreceptor lamellae and the presence of discrete 100-120 nm whorls within the disc membranes. Many photoreceptor cells, particularly the parafoveal rods, also demonstrated mitochondrial swelling and nuclear pyknosis. Scattered retinal pigment epithelial cells in the fovea and parafovea showed a degeneration characterised by loss of plasma membrane specialisations, swelling of the smooth endoplasmic reticulum and changes in the fine structure of the lipofuscin granules. The good visual prognosis in solar retinopathy was attributed to the resistance of the foveal cone cells to photochemical damage.