Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry

To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.     

Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation

An experimental investigation was undertaken to measure the intrinsic elastic properties of several of the microstructural components of human vertebral trabecular bone and tibial cortical bone by the nanoindentation method. Specimens from two thoracic vertebrae (T-12) and two tibiae were obtained from frozen, unembalmed human male cadavers aged 57 and 61 years. After drying and mounting in epoxy resin nanoindentation tests were conducted to measure Young's modulus and the hardness of individual trabeculae in the vertebrae and single osteons, and interstitial lamellae in the tibiae. Measurements on the vertebral trabeculae were made in the transverse direction, and the average Young's modulus was found to be 13.5 +/- 2.0 GPa. The tibial specimens were tested in the longitudinal direction, yielding moduli of 22.5 +/- 1.3 GPa for the osteons and 25.8 +/- 0.7 GPa for the interstitial lamellae. Analysis of variance showed that the differences in the measured moduli are statistically significant. Hardness differences among the various microstructural components were also observed.     

Oligomeric state of human erythrocyte band 3 measured by fluorescence resonance energy homotransfer

The oligomeric state of the erythrocyte anion exchange protein, band 3, has been assayed by resonance energy homotransfer. Homotransfer between oligomeric subunits, labeled with eosin-5-maleimide at Lys430 in the transmembrane domain, has been demonstrated by steady-state and time-resolved fluorescence spectroscopy, and is readily observed by its depolarization of the eosin fluorescence. Polarized fluorescence measurements of HPLC-purified band 3 oligomers indicate that eosin homotransfer increases progressively with increasing species size. This shows that homotransfer also occurs between labeled band 3 dimers as well as within the dimers, making fluorescence anisotropy measurements sensitive to band 3 self-association. Treatment of ghost membranes with either Zn2+ or melittin, agents that cluster band 3, significantly decreases the anisotropy as a result of the increased homotransfer within the band 3 clusters. By comparison with the anisotropy of species of known oligomeric state, the anisotropy of erythrocyte ghost membranes at 37 degrees C is consistent with dimeric and/or tetrameric band 3, and does not require postulation of a fraction of large clusters. Proteolytic removal of the cytoplasmic domain of band 3, which significantly increases the rotational mobility of the transmembrane domain, does not affect its oligomeric state, as reported by eosin homotransfer. These results support a model in which interaction with the membrane skeleton restricts the mobility of band 3 without significantly altering its self-association state.     

Immunolocalisation of thrombospondin 1 in human, bovine and rabbit cornea

Light- and electron-microscopic immunohistochemical techniques were used to investigate the distribution of the matricellular protein thrombospondin 1 in normal human, bovine and rabbit cornea. Light-microscopic immunoreactivity for thrombospondin 1 was observed in the epithelial basement membrane, posterior Descemet's membrane and endothelium of human and bovine cornea. The bulk of the stroma, the stromal cells (keratocytes) and the anterior part of Descemet's membrane in human and bovine cornea were devoid of detectable thrombospondin 1 and the protein could not be demonstrated in any of the layers of the rabbit cornea. Electron-microscopic immunogold studies of human and bovine cornea revealed that thrombospondin 1 labelling of corneal endothelial (and basal epithelial) cells included focal deposits at cell membranes. It is postulated that thrombospondin 1 regulates interactions between cells and their basement membrane, and perhaps cell-to-cell interactions, in the normal human and bovine corneal endothelium and basal epithelium.     

A topographic study of ERPs elicited by visual feature discrimination

We report the cloning of a zebrafish paired-type homeobox gene, Alx, closely related to the murine Chx10 and the gold fish Vsx-I homeodomain proteins. Alx is first expressed at about 12 h post-fertilization (hpf) when optic vesicles appear. Its expression is restricted to the early retinal neuroepithelium, whereas no signal can be detected in the optic placode. Later, Alx expression follows the differentiation of the neural retina. Inhibition experiments with antisense oligonucleotides resulted in specific eye malformations which are reminiscent of the phenotype of ocular retardation (or) mice, caused by a spontaneous Chx10 mutation. The expression of other developmentally relevant genes such as pax(zf-a), pax(zf-b) and krx-20 was not affected in the antisense treated embryos.     

Perceptual-cognitive and personality development of Mexican and Anglo-American children as measured by human figure drawings.

Presents longitudinal and cross-sectional human figure drawing data for 394 8.7-17.7 yr old Mexican and American school children. The factors of sex, age, socioeconomic status, culture, and year of repeated testing were investigated in factorial complex analyses of variance for Goodenough-Harris scores in the 1st and 2nd figures drawn and for masculinity-femininity ratings on both sex figures drawn. Percentages of Ss drawing self-sex figures 1st are presented. The importance of cultural variables as determinants of the behaviors involved in performance on these measures was evident from the significant main effects and complex interactions obtained. Cultural differences in rearing practices linked with sex of S were reflected in the poorer degree of sexual differentiation in the drawings of American, as compared with Mexican, Ss. Important methodological and interpretative issues in cross-cultural research and clinical application are discussed (e.g., those involved in employing uniculturally developed instruments with culturally different populations). (17 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effect of photic stimulation on energy metabolism in the human brain measured by 31P-MR spectroscopy

Effect of photic stimulation (PS) on energy metabolism in the human occipital cortex was examined by using phosphorus-31 magnetic resonance spectroscopy in 9 normal subjects. Phosphocreatine (PCr)/total phosphorus signal peak area ratio significantly decreased from 12.3% to 10.9% during the 12 minutes of PS (P < 0.05). PCr once returned to a normal level after PS (11.9%) but significantly decreased again 12-18 minutes after PS (10.8%; P < 0.05). Intracellular pH increased from 7.08 to 7.16 during PS, although this increase was not significant. These results suggest that functional alteration of energy metabolism in the brain is different from that in muscles.     

The methodological bases of topographic neuropathomorphology

This paper describes the major features of topographic and anatomical studies of CNS structures, CSF circulation system, extracerebral structures and vertebral column. These methods include brain fixation, which is able to prevent post mortem brain deformation due to decalcification (even for block-preparations like "brain-skull base" or "posterior cerebral fossa-cervical spine"). The author also presents the following methods: spatial reconstruction of the brain and skull base subtracted from the series of slices of different thickness and various planes of transection; biopsy investigation of cerebral midline structures and CSF system in relation with skull base and convex structures; staged microdissection of tumors; filling of arterial and venous systems. All these modalities were used in examining the topographical features of craniopharyngiomas, hypophyseal adenomas, parasagittal and sagittal meningiomas, pineal tumors, VIII cranial nerve neurinomas, arachnoid cysts and in developing surgical approaches to the tumors of the third ventricle, skull base and foramen magna.     

Nitrogen narcosis measured by dual-task performance.

Two hyperbaric studies tested detrimental effects of 188-ft sea water gauge (fswg) air pressure. In each experiment, 8 males aged 22–32 yrs, qualified for hyperbaric exposures, executed single-task controls of a choice reaction time (RT) task and a pursuit tracking task, as well as their dual-task combinations. All tasks were tested 0, 10, and 188 fswg. Exp I was designed to measure the effects of nitrogen narcosis on 2 successive weekly dives. No improvement specific to the 188 fswg depth was found on the 2nd dive. It is concluded that the prior exposure did not result in measurable adaptation to narcosis. At 188 fswg, the rate of information transmission in the choice RT task was slowed and tracking error increased. Dual-task requirements resulted in poorer tracking but left choice RT performance unaffected. In Exp II, half of the Ss stopped at 19 fswg before proceeding to depth. They showed a decrement in performance at 188 fswg equal to, or greater than, that found for the remaining Ss, who used the standard procedure of descending to depth directly. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Hemodialysis access recirculation measured by ultrasound dilution

The most widely used clinical method for measuring recirculation in the access device is based on urea dilution. The three simultaneous blood samples required during hemodialysis interrupt the treatment, and results of chemical analysis are often delayed for several days. Alternatively, detecting recirculation by dilution of arterial blood caused by a bolus of normal saline injected into the venous blood line has several advantages. In this study, an ultrasound sensor clamped onto the arterial line entering the dialyzer was used to detect such dilution from a reduction in sound velocity observed in the saline diluted blood. Within the target range, the change in ultrasound velocity (ultrasound dilution) is linearly correlated with the dilution of whole blood by normal saline. The same sensor was also used to measure flow in the blood line using an established ultrasound transit-time method. During 34 hemodialyses in 28 patients, only 3 patients had detectable recirculation measured by ultrasound dilution. To further evaluate the sensitivity of the new method the dialysis lines were reversed during hemodialysis in the 25 patients with no recirculation. After this, all had detectable recirculation ranging from 10 to 60%. The mean error of duplicate measurements was 3.9 +/- 2.8%. Recirculation by ultrasound dilution correlated closely with recirculation measured by urea dilution (r = 0.9156, p < 001). The data suggest that the ultrasound dilution method is both sensitive and accurate. Ease of use and immediate availability of results added to the clinical usefulness of this method for evaluating the integrity of the hemodialysis access.     

Expression and release of tumor necrosis factor-alpha by explants of mouse cornea

PURPOSE: To elucidate a possible target of immunosuppressive agents widely used in the treatment of corneal disorders, the authors determined whether corneal cells are capable of expressing and releasing tumor necrosis factor-alpha (TNF alpha) on lipopolysaccharide (LPS) stimulation, and they investigated whether TNF alpha production can be modulated by pharmacologic agents. METHODS: Trephined central corneas from C57BL/6 mice were kept in culture for 3 days. Release of TNF alpha after a 24-hour stimulation with LPS (1 microgram/ml) into the culture medium was determined both by bioassay and by enzyme-linked immunosorbent assay. Expression of TNF alpha mRNA after 6-hour stimulation was examined by polymerase chain reaction. Immunofluorescent staining on cryostat sections of cultured corneas was performed to localize TNF alpha in the tissue. Corneal explants were pretreated with immunosuppressive agents (prednisolone, budesonide, cyclosporin A) for 48 hours, followed by 6-or 24-hour stimulation with LPS in the continuous presence of the agents. RESULTS: Lipopolysaccharide stimulated TNF alpha release into the culture medium. The addition of budesonide (10(-7) M) or prednisolone (10(-6) M) significantly inhibited LPS-induced TNF alpha release, whereas cyclosporin A (10(-7) - 10(-5) M) had no marked effect. Levels of TNF alpha mRNA in corneal explants increased fivefold after stimulation with LPS. Immunohistochemical staining revealed that TNF alpha was expressed in the epithelial cells. Budesonide markedly decreased mRNA expression and abolished immunostaining of TNF alpha stimulated by LPS. CONCLUSIONS: TNF alpha is produced and released by the epithelial cells of mouse central cornea in response to LPS. Contrary to cyclosporin A, corticosteroids such as prednisolone and budesonide potently inhibit TNF alpha production.     

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.     

Preoperative and intraoperative topographic diagnosis of insulinomas

Altogether 120 patients with organic hyperinsulinism underwent clinical examination and treatment (38 male, 82 female, mean age 44.2 +/- 4.6 years). The cause of hyperinsulinism was benign insulinomas in 96 (80.0%), malignant tumors in 9 (7.5%), and hyperplasia of beta cells in 6 (5.0%). In 9 (7.5%) patients the origin of hyperinsulinism was not diagnosed. The tumor was localized in the head, body, and tail of the pancreas in 31.8%, 36.4%, and 31.8% of cases, respectively. Intraoperative ultrasonography (IOUS) was undertaken in 37 patients, and in 83 cases only intraoperative palpation was done. Arterial stimulated venous sampling (ASVS) was performed in 17 patients (blood was sampled from the right hepatic vein for determination of the insulin level after arterial stimulation by calcium gluconate in different parts of the pancreas). The sensitivity of ultrasonography (US) was 29.5%, computed tomography (CT) 24.2%, angiography 55.9%, superselective angiography (branches of the celiac trunk) 72.2%, and intraoperative palpation 90.0%. ASVS showed an accuracy of 90.0%. Combining angiography with ASVS gave an exact diagnosis of hyperinsulinism in 100% of cases, and IOUS revealed tumors in 100% of cases. Hyperplasia of beta-cells was diagnosed only by means of ASVS. A total of 117 patients underwent surgery, including distal resection of pancreas (n = 39), enucleation of tumor (n = 70), and laparotomy (n = 8). The postoperative mortality associated with insulinomas was 7.7%. The frequency of postoperative complications was 43.6%. Benign insulinomas recurred at a rate of 5.4%. Patients with malignant insulinomas had a 5-year survival of 66.0%. The diagnosis of insulinomas was achieved by a combination of selective angiography, ASVS, and IOUS.