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PubMed2006

Increased resistance of lipofuscin-loaded prematurely senescent fibroblasts to starvation-induced programmed cell death.

Alterations of cellular structures often found in ageing cells is mainly the result of production of reactive oxygen species and a consequence of aerobic life. Both oxidative stress and decreased degradative capacity of lysosomal system cause accumulation of intralysosomal age-related pigment called lipofuscin. To investigate the influence of lipofuscin on cell function, we compared survival of lipofuscin-loaded and control human fibroblasts following complete starvation induced by exposure to phosphate-buffered saline (PBS). Starving of control fibroblasts resulted in lysosomal alkalinisation, relocation of cathepsin D to the cytosol, caspase-3 activation and, finally, cell death, which became evident 72 h after the start of exposure to PBS. Increase of lysosomal pH was significantly less prominent in lipofuscin-loaded cells than in controls and was accompanied neither by leakage of cathepsin D nor by caspase-3 activation even 96 h after the initiation of starvation. Suppression of autophagy by 3-methyladenine (3-MA) accelerated cell death, while inhibition of cathepsin D delayed it, implying an important role of autophagy in cell survival during starvation and showing the involvement of lysosomes in starvation-induced cell death. Disturbed apoptotic response found in lipofuscin-loaded cells can be interpreted as an example of hormesis--an adaptation to low doses of otherwise harmful agents, in this case of lipofuscin, which has a protective effect at moderate amounts but becomes toxic at large quantities.

Adenine↗

PubMed2003

Precision and safety of the pulsed electron avalanche knife in vitreoretinal surgery.

BACKGROUND: We have developed a new surgical instrument, called the pulsed electron avalanche knife (PEAK; Carl Zeiss Meditec, Jena, Germany), for precise, "cold," and tractionless dissection of tissue in liquid media. OBJECTIVE: To evaluate the 3-dimensional damage zone induced by the PEAK compared with 2 other standard intraocular surgical instruments, diathermy and retinal scissors. METHODS: Damage zone and minimum safe distance were measured in vitro on chick chorioallantoic membrane and in vivo on rabbit retina with the use of propidium iodide staining. RESULTS: The PEAK produced a paracentral zone of cellular structure disruption surrounding a crater and a peripheral zone of structurally intact but abnormally permeable cells. The instrument induced a damage radius that varied from 55 to 300 micro m for the range of voltages and pulses typically used during surgery. For comparison, damage radius for microsurgical scissors was 50 micro m, and for diathermy, 400 to 850 micro m. The PEAK also damaged tissue up to 1.4 mm away by the creation of water flow that formed at the tip of convex probes during collapse of a cavitation bubble. Concave probes, which prevent formation of the water jet, eliminated this effect. CONCLUSIONS: The PEAK operated well within accept-able safety limits and may greatly facilitate both posterior segment surgeries (eg, membrane dissection and sheathotomy) and anterior segment procedures (eg, capsulotomy, nonpenetrating trabeculectomy, and iridectomy).

Allantois↗

PubMed1982

Neuropathology of schizophrenia.

Histologic sections were made from selected regions of the brains of 25 schizophrenic patients aged 21 to 54 years (mean, 43.5 years) who died during their hospitalization at St Elizabeths Hospital, Washington, DC. These sections were compared with similarly prepared sections from the same brain regions of 28 nonschizophrenic patients of similar age, who were hospitalized and died at the same hospital during the same period, and with sections from 20 age-matched, nonpsychiatric patients who died in a general hospital. Although few differences were apparent in material from schizophrenic and nonpsychiatric cases in sections stained for cellular structures, myelin, or axis cylinders, Holzer's stain for glial fibrils demonstrated increased fibrillary gliosis that affected principally the periventricular structures of the diencephalon, the periaqueductal region of the mesencephalon, or the basal forebrain in three fourths of the brains from schizophrenic subjects. Hypothalamus, midbrain tegmentum, and substantia innominata were most often affected. The nature and distribution of the findings suggest previous or low-grade inflammation. Review of the clinical history and course of each case allows some correlations to be made between clinical signs and histopathologic data.

Adult↗

PubMed1999

Measurement of thermal effects on the optical properties of prostate tissue at wavelengths of 1,064 and 633 nm.

BACKGROUND AND OBJECTIVE: The extent of thermal injury during laser prostatectomy is dependent on the light distribution in laser-irradiated tissue. As tissue is irradiated, the optical properties change as a function of temperature due to an alteration of molecular and cellular structure. The purpose of the present study was to determine how the exposure of both fresh and previously frozen canine prostate tissue to elevated temperatures affects the optical properties. STUDY DESIGN/MATERIALS AND METHODS: Optical properties were measured by using a double integrating sphere spectrophotometer with an inverse adding-doubling algorithm. Measurements were made at two wavelengths (1,064 nm and 633 nm) on samples heated in a waterbath in 5 degree-10 degree increments for 10 min through a 50 degrees C temperature range. RESULTS: Upon coagulation, the absorption coefficient of fresh tissue decreased from the baseline measurement for both wavelengths (0.027 +/- 0.003 to 0.019 +/- 0.002 for lambda = 1,064 nm; 0.073 +/- 0.007 to 0.061 +/- 0.006 for lambda = 633 nm). However, the scattering coefficient increased sharply from the baseline measurement following coagulation (3.06 +/- 0.26 to 6.05 +/- 0.29 for lambda = 1,064 nm; 4.89 +/- 0.23 to 7.22 +/- 0.30 for lambda = 633 nm). Thermal coagulation occurred during exposure to temperatures between 60 degrees C and 70 degrees C. CONCLUSION: Data obtained in this study indicate that thermal coagulation of tissue alters the optical properties. The extent to which these changes occur was found to be dependent on wavelength and freshness of tissue. These results are significant because they suggest how thermally induced changes in the optical properties may limit the depth of light penetration in tissue thus compromising treatment.

Algorithms↗

PubMed1999

R-cadherin expression in the developing and adult zebrafish visual system.

Cell adhesion molecules in the cadherin family have been implicated in histogenesis and maintenance of cellular structure and function in several organs. Zebrafish have emerged as an important new developmental model, but only three zebrafish cadherin molecules have been identified to date (N-cadherin, paraxial protocadherin, and VN-cadherin). We began a systematic study to identify other zebrafish cadherins by screening zebrafish cDNA libraries using an antibody raised to the cytoplasmic domain of mouse E-cadherin. Here, we report a partial cDNA with extensive sequence homology to R-cadherin. Spatial and temporal expression of this putative zebrafish R-cadherin was examined in embryos and adults by Northern analysis, RNase protection, and in situ hybridization. R-cadherin message increased during embryogenesis up to 80 hours postfertilization (hpf) and persisted in adults. In the embryonic brain, R-cadherin was first expressed in groups of cells in the diencephalon and pretectum. In adult zebrafish brain, R-cadherin continued to be expressed in several specific regions including primary visual targets. In the retina, R-cadherin was first detected at about 33 hours postfertilization in the retinal ganglion cell layer and the inner part of the inner nuclear layer. Expression levels were highest during periods of axon outgrowth and synaptogenesis. Retrograde labeling of the optic nerve with 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) followed by in situ hybridization confirmed that a subset of retinal ganglion cells in the embryo expressed R-cadherin message. In the adult, R-cadherin expression continued in a subpopulation of retinal ganglion cells. These results suggest that R-cadherin-mediated adhesion plays a role in development and maintenance of neuronal connections in zebrafish visual system.

Age Factors↗

PubMed1996

Mammalian myosin I alpha is concentrated near the plasma membrane in nerve growth cones.

To determine if unconventional myosins play a role in nerve outgrowth, antibodies specific for rat brain derived mammalian myosin I alpha (MMI alpha) were used to label cultured rat superior cervical ganglion nerve cells. Observations were made at both the light and electron microscopic level of resolution using preparative procedures designed to enhance the ability to precisely determine the relationship between antibody label and cellular structures in order to map the distribution and structural association of this myosin. Immunofluorescence showed that MMI alpha has a punctate distribution throughout the nerve cell body, neurites, and growth cones. In growth cones, MMI alpha staining is sometimes elevated in thin peripheral regions of high actin content at the leading edge. Immunoelectron microscopy using colloidal gold conjugated antibodies showed that in growth cones MMI alpha is absent from membranous organelles and is concentrated primarily in the cell cortex adjacent to the cell membrane. The cortical label is equally distributed between upper and lower membranes. The plasma membrane association of the MMI alpha label persists under conditions in which the actin cytoskeleton is perturbed or removed, suggesting a direct association between a fraction of MMI alpha and the plasma membrane. MMI alpha label is also associated with the non-cortical actin cytoskeleton. Partial disruption of the actin cytoskeleton using cytochalasin B causes redistribution of only a subset of MMI alpha label. These data suggest a complex relationship between MMI alpha, the actin cytoskeleton, and the plasma membrane in the growth cone. In contrast to its localization in the growth cone, in neuronal cell bodies MMI alpha is also associated with tubulovesicular structures. This suggests that at this location MMI alpha may either act as an organelle motor or is passively transported to the plasma membrane on vesicles.

Actins↗

PubMed1996

Site-directed mutagenesis enabled preparation of a functional fluorescent analog of profilin: biochemical characterization and localization in living cells.

The preparation of fluorescent profilin analogs for binding and spectroscopic studies, in vitro and in vivo, has been hampered by the poor chemical reactivity of this protein in its native form. We have addressed this problem by labeling a mutant, chemically reactive form of profilin. Site-directed mutagenesis was first used to replace a serine residue in a non-essential domain with a reactive cysteine residue. The mutant protein was expressed in Escherichia coli and reacted with tetramethylrhodamine iodoacetamide. In vitro assays indicated that the fluorescent profilin maintained its ability to bind actin, polyproline, and PIP2, to inhibit actin polymerization, and to stimulate actin nucleotide exchange. Fluorescence spectroscopy showed that neither the excitation nor the emission of the analog was sensitive to the interaction with actin or polyproline. However, binding of PIP2 caused a 75% quenching of the fluorescent signal, suggesting a dramatic change in the immediate environment of the probe. When the fluorescent profilin was microinjected into living NRK cells, it became localized at cell-cell junctions and discrete sites near the anterior end, where it colocalized with aggregates of unpolymerized actin. Different engineered forms of profilin with fluorophores located at defined sites should greatly facilitate the study of its interactions with various ligands and cellular structures.

Animals↗

PubMed1997

Characterization and localization of profilin in pollen grains and tubes of Lilium longiflorum.

Pollen tubes show a rapid and dramatically polarized growth in which the actin cytoskeleton appears to play a central role. In order to understand the regulation of actin we characterized its associated protein, profilin, in pollen tubes of Lilium longiflorum. By using purified polyclonal antibodies prepared against bean root profilin [Vidali et al., 1995: Plant Physiol. 108:115-123] we detected in pollen grains and tubes two profilin polypeptides with molecular masses of 14.4 and 13.4 KDa, and an identical isoelectric point of 5.05. Profilin comprises approximately 0.47% of the total grain protein, with actin being approximately 1.4%. We were unable to detect a statistically significant profilin increase after germination, while the actin increased approximately 68%. We also spatially localized the distribution of profilin using immunocytochemistry of fixed cells at both the light and electron microscope level, and by fluorescent analog cytochemistry on live cells. The results show that profilin is evenly distributed throughout the cytoplasm and does not specifically associate with any cellular structure.

Actins↗

PubMed1999

Comparison of cholinergic and histaminergic axons in the lateral geniculate complex of the macaque monkey.

The cholinergic and histaminergic projections have important neuromodulatory functions in the ascending visual pathways, so we compared the pattern and mode of innervation of the two projections in the lateral geniculate complex (dorsal lateral geniculate nucleus and pregeniculate nucleus) of the macaque monkey. Brain tissue from macaques was immunoreacted by means of antibodies to choline acetyltransferase (ChAT) or to histamine and processed for light and electron microscopy. A dense plexus of thin, highly branched ChAT-immunoreactive axons laden with varicosities was found in all layers of the dLGN including the koniocellular laminae and in the pregeniculate nucleus. ChAT label was more dense in magnocellular layers 1 and 2 than in parvocellular layers 3-6 and relatively sparse in the interlaminar zones. Varicosities associated with the cholinergic axons had an average of three conventional asymmetric synapses per varicosity, and these appeared to contact dendrites of both thalamocortical cells and interneurons. Histamine-immunoreactive axons were distributed homogeneously throughout all laminar and interlaminar zones of the dLGN, but were denser in the pregeniculate nucleus than in the dLGN. Histaminergic axons branched infrequently and were typically larger in caliber than cholinergic axons. The overwhelming majority of varicosities were found en passant and rarely displayed conventional synapses, despite the abundance of synaptic vesicles, and were not associated preferentially with specific cellular structures. The innervation of the macaque dLGN complex by cholinergic and histaminergic systems is consistent with their proposed role in state dependent modulation of thalamic activity. The dense and highly synaptic innervation by cholinergic axons supports the proposal of additional involvement of these axons in functions related to eye movements.

Animals↗

PubMed1998

APE/Ref-1 responses to oxidative stress in aged rats.

Chronic oxidative stress has been hypothesized to be a major contributor to the aging process. The continued exposure to reactive oxygen species (ROS) generated by oxidative metabolism or environmental sources can damage critical cellular structures and be responsible for some age-related pathology. The exposure of rodents to 100% oxygen, isobaric hyperoxia, increases ambient ROS levels and significantly increases apoptosis in brain. The deleterious effects of ROS also include increased lipid peroxidation, protein oxidation, and DNA damage. Although differences in the relative amounts of oxidative stress in young and old brains have been observed, the mechanisms responsible for impaired aging-associated DNA repair processes have not been characterized. We measured DNA levels of the DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/Ref-1) protein by Western blot analysis in the brains of young (3-month) and old (30-month) male rats exposed to isobaric hyperoxia. Given that APE/Ref-1 is the rate-limiting enzyme in the repair pathway of apurinic/apyrimidinic sites generated in DNA by oxidative damage, we assumed that APE/Ref-1 protein levels were a good reflection of ongoing DNA base excision repair. Isobaric hyperoxia stimulated APE/Ref-1 expression in the hippocampus and basal forebrain of young rats experiencing 100% oxygen for 6 hr, while aged rats showed no significant changes in APE/Ref-1 protein levels in all brain areas at any time tested (0-48 hr) after hyperoxia. Differences in the stress-induced levels of expression of DNA repair enzymes may contribute to apoptotic increases and pathology associated with the aging process.

Aging↗

PubMed1996

Long-term cryopreserved allogeneic nervous and vascular tissue transfers in monkeys.

Four adult Japanese monkeys were used. Donor tissues were harvested from the radial artery, saphenous vein and median nerve, 10 cm in length, and cryopreserved at -196 degrees C in a stepwise manner for long-term preservation for more than 6 months. After rapid thawing of the tissue, the allogeneic tissue transfer was performed of the radial artery to the radial artery, the saphenous vein to the ulnar artery and the median nerve to the median nerve between male and female monkeys. After one year follow-up, angiography was performed and the transferred tissue was harvested for scanning electron microscopic examination and histological examination. As a result, the allogeneic artery in the radial artery was completely patent with no stenosis, whereas there was obstruction of the allogeneic vein in the ulnar artery. Good axonal recovery of the median nerve was observed on histological examination. The cellular structure of neurovascular tissue can be maintained even after long-term cryopreservation. The antigenicity of tissue may be reduced significantly by being frozen, and cryopreserved allogeneic artery can be transferred successfully without the use of immunosuppressive agents. In this report, clinical aspects and the great potential of allogeneic neurovascular tissue transfer, especially in combination with arterial transfer, using cryopreservation are discussed.

Animals↗

PubMed2001

Transient electric fields induced by mechanically assisted corrosion of Ti-6Al-4V.

Samples of Ti-6Al-4V were immersed in physiological solution and abraded via an electrochemical scratch method to observe the development of transient electric fields a finite distance from the scratch event. Transient electric fields were detected near both potentiostatically held and freely corroding samples. Transient currents measured by a potentiostatically held PtIr microelectric probe near a potentiostatically held sample were opposite in sign to those of the mechanically induced sample currents and were found to change character with sample potential, probe potential, and distance from the scratch event. Transient probe currents measured near a freely corroding sample were of the opposite sign as the sample transient near the primary site of oxidation, but were of the same sign near the primary site of reduction. The measured transients are a direct result of the electrochemical processes ongoing during oxide fracture and repassivation and can be sensed several millimeters remote from the abrasion site. A model for the generation of these fields is presented. Possible effects that these potentials may have on cellular structures surrounding an implant are proposed.

Alloys↗

PubMed2000

Force Spectroscopy of Molecular Systems-Single Molecule Spectroscopy of Polymers and Biomolecules.

How do molecules interact with each other? What happens if a neurotransmitter binds to a ligand-operated ion channel? How do antibodies recognize their antigens? Molecular recognition events play a pivotal role in nature: in enzymatic catalysis and during the replication and transcription of the genome; it is also important for the cohesion of cellular structures and in numerous metabolic reactions that molecules interact with each other in a specific manner. Conventional methods such as calorimetry provide very precise values of binding enthalpies; these are, however, average values obtained from a large ensemble of molecules without knowledge of the dynamics of the molecular recognition event. Which forces occur when a single molecular couple meets and forms a bond? Since the development of the scanning force microscope and force spectroscopy a couple of years ago, tools have now become available for measuring the forces between interfaces with high precision-starting from colloidal forces to the interaction of single molecules. The manipulation of individual molecules using force spectroscopy is also possible. In this way, the mechanical properties on a molecular scale are measurable. The study of single molecules is not an exclusive domain of force spectroscopy; it can also be performed with a surface force apparatus, laser tweezers, or the micropipette technique. Regardless of these techniques, force spectroscopy has been proven as an extraordinary versatile tool. The intention of this review article is to present a critical evaluation of the actual development of static force spectroscopy. The article mainly focuses on experiments dealing with inter- and intramolecular forces-starting with "simple" electrostatic forces, then ligand-receptor systems, and finally the stretching of individual molecules.

Journal Article↗

PubMed2026

Ultrasound-Actuated Gene Editing in Human Kidney Organoids.

Efficient delivery of gene editing ribonucleoproteins (RNPs) into the interior of solid tissues remains a key hurdle to the clinical translation of non-viral CRISPR-Cas9 technologies. Here, we report acoustically-actuated peptide nanoemulsions (NPeps) that can be spatiotemporally guided and activated by ultrasound to ballistically deliver RNPs into cells within the bulk of dense 3D cellular structures. Using human kidney organoids as a model, we demonstrate NPep vectors improve the spatial profile of gene editing in the organoid mass relative to commercial lipofection reagents, without disruption of tissue structure or qualitative viability features. This technologic paradigm is poised to advance imaging-guided, deep tissue RNP delivery modalities to expand the clinical diagnostic and therapeutic potential of CRISPR-Cas9 editing strategies.

Humans↗

PubMedDate not supplied

Genetics of signal transduction and the effect of maternal smoking on sex ratio of offspring.

We investigated the possible effects of ACP1 on the relationship between maternal smoking and sex ratio of offspring. ACP1 is a highly polymorphic enzyme involved in signal transduction of growth factors, glucose metabolism, flavoenzyme activity, and T-cell activation. A sample of 357 consecutive newborns from the population of Rome and a sample of 362 consecutive newborns from the population of Penne were studied. In both populations, among female newborns from smoking mothers there was a higher proportion of the A phenotype (associated with the lowest enzymatic activity) and a lower proportion of carriers of the *C allele (associated with the highest enzymatic activity) compared to female infants from nonsmoking mothers. In newborns from smoking mothers there was a positive correlation between sex ratio and ACP1 activity. The data suggest intrauterine selection induced by smoke that is dependent on sex and the ACP1 genotype of the zygote. It has been shown that low ACP1 activity is associated with high activity of the flavoenzyme glutathione reductase, an enzyme that has an important role in the protection of cellular structures from oxidative damage. Thus, the A phenotype may have a protective effect against oxidative damage by cigarette smoking through an enhancement of glutathione reductase activity.

Birth Weight↗

PubMed1982

Ultrastructural evidence for steroid-hormone production in the adrenal of the marsupial Isoodon macrourus, at birth.

To determine whether the adrenal gland of the bandicoot is actively producing steroid hormones at birth, the cellular structure was examined by electron microscopy, and the concentration of cortisol in the gland was determined by radioimmunoassay. Two distinct cell populations were seen in the adrenal gland. One group of cells possessed dark staining granules, normally observed in catecholamine-secreting cells, and the second group contained large amounts of smooth endoplasmic reticulum and mitochondria with tubulo-vesicular cristae. Both features are characteristic of steroid-secreting cells. A concentration of 0.094 ng of cortisol per adrenal was determined by pooling glands from nine individual newborn bandicoots. This evidence of cortisol secretion from the bandicoot adrenal at birth indicates that, as in many eutherians, the marsupial adrenal may play an important role in determining the length of gestation.

Adrenal Glands↗

PubMed1986

Adipocyte development in primary rat cell cultures: a scanning electron microscopy study.

This study utilized scanning electron microscopy (SEM) techniques to observe primary cultures of stromal-vascular (SV) cells derived from postnatal rat inguinal adipose tissue. Cells were grown on collagen-coated, fibronectin-coated, or uncoated glass coverslips. Coverslips were normally fixed in glutaraldehyde, osmium tetroxide, dehydrated, and critical-point-dried. Other coverslips were frozen in isopentane (cooled in LN2) and dried or fixed in Baker's formalin for demonstration of inosine diphosphatase (IDPase) by X-ray microprobe analysis (XRMA). Adipocyte morphologies were similar on all substrates. At 2 days of culture, actin cables were detected extending from developing adipocytes. No difference in actin cable structure, cellular shape, or lipid accumulation was observed among the different substrates. Some stromal cells did not accumulate lipid but proliferated into a multilayer by 9 days in culture. Inosine diphosphatase was detected in the Golgi apparatus of developing adipocytes utilizing the technique of XRMA. This study demonstrates the potential for using SEM and XRMA techniques to define morphological features and cytochemical markers of adipocytes in vitro and the response of primary cultured rat SV cells to other attachment substrates.

Adipose Tissue↗

PubMed1988

Presence and distribution of vimentin in cynomolgus monkey trabecular cells.

The role of the trabecular meshwork in the ocular outflow tract has made it the object of considerable study. Recent work has examined the presence and function of microfilaments and microtubules in the cytoskeleton of cultured cynomolgus monkey trabecular cells. In this study, we used an indirect immunofluorescence technique to investigate the presence and distribution of the intermediate filament vimentin in cultured cynomolgus monkey trabecular cells. The cytoskeletal active agents cytochalasin B, colchicine, nocodozole, and taxol were also employed to investigate the role of vimentin in these cells. Vimentin formed a network of filaments that radiated throughout the cytoplasm from the nucleus to the cellular projections and cell membrane. The extensiveness of the vimentin network, and the cell shape, were observed to vary according to the degree of cell confluence, the degree of cell spread, and the degree of cell/cell contact. Cells in the less-confluent periphery had extensive vimentin networks and greater cell spreading and were polygonal in shape. Cells in the more confluent areas had a less-extensive vimentin network, underwent less cell spreading, and were primarily fusiform in shape. The change in cellular morphology induced by colchicine, nocodozole and taxol was proportional to the extensiveness and the degree of change of the vimentin network. Our observations have identified a proportional association between the extensiveness of the vimentin network, changes in the vimentin organization, and alterations in cellular morphology that is suggestive of a role for vimentin in determining cellular structure and shape.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗