Hank's Medium Research Articles

Short- and Long-Term Storage of Non-Domesticated European Mouflon (Ovis aries musimon) Cumulus-Oocyte Complexes Recovered in Field Conditions.

Reproductive biotechnologies can be used as a supporting tool, through gamete conservation and in vitro embryo production, in the preservation of invaluable and irreplaceable animal genetic resources. In the present study, immature mouflon cumulus-oocyte complexes (COCs) collected from ovariectomized female ovaries underwent short- or long-term conservation (24 h maintained in Earle's/Hank's (EH) medium or vitrification) under field conditions and afterwards transported to the laboratory where they were cultured for in vitro maturation (IVM) and assessed for oocyte meiotic competence and bioenergetic-oxidative status. Utilization of both storage techniques led to COC morphology preservation, as well as cumulus expansion and oocyte meiotic resumption after the IVM procedure. Quantitative bioenergetic-oxidative parameters were reduced in vitrified oocytes compared with EH ones. Immature COC storage needs to be optimized in both domesticated and non-domesticated sheep as a part of the strategy to avoid the loss of valuable genotypes of these animal species.

Selection of Molecules with Immunological Potential from Excretory and Secretory Products from the Nematode Haemonchus placei by Cell Proliferation and Gene Expression Assays.

The nematodeHaemonchus placeiis a pathogenic parasite, the most seriously affecting ruminant's health and being responsible for enormous economic losses all over the world. The present protocol describes different in vitro techniques to select potential candidate antigens with immune-protective activity from excretory and secretory products (ESP) fromH. placeitransitory infective larvae (xL3). ESP from xL3were obtained from the in vitro infective larvae (L3) maintained in Hank's medium at 37 °C with 5% CO2for 48 h. Then, the presence of ESP proteins was confirmed by SDS-PAGE to be used in an in vitro proliferation assay with bovine peripheral blood mononuclear cells (PBMCs). The ESP were exposed to the PBMCs during two different periods (24 and 48 h). Genes associated with immune response against the nematode were analyzed using relative gene expression and bioinformatic tools. These are simple, economic, and helpful tools to identify potential immune-protective molecules under in vitro conditions for confirming the efficacy of future in vivo assays. Graphical overview.

Comparison of SARS-CoV-2 detection from combined nasopharyngeal/oropharyngeal swab samples by a laboratory-developed real-time RT-PCR test and the Roche SARS-CoV-2 assay on a cobas 8800 instrument

ObjectiveAlthough several assays have been developed to detect SARS-CoV-2 RNA in clinical specimens, their relative performance is unknown.MethodsThe concordance between the cobas 8800 SARS-CoV-2 and a laboratory developed (LD) reverse transcriptase-polymerase chain reaction (RT-PCR) assay was assessed on 377 combined nasopharyngeal/oropharyngeal swabs in Hanks medium.ResultsThe positive and negative agreement between these assays were 99.3 % (95 % CI, 97.3–99.9) and 77.1 % (95 % CI, 67.7–84.4), respectively, for an overall agreement of 93.6 % (95 % CI, 90.7–95.7) beyond random chance (kappa of 0.82, 95 % CI, 0.75−0.85). Of the 22 samples positive by cobas SARS-CoV-2 only, 9 were positive only for ORF-1 gene and had Cycle thresholds (Ct) > 35.1, 8 were positive only for the E gene with Ct > 35.5 and 5 were positive for both targets with Ct > 33.9. Samples positive only with the cobas assay were more often positive with only one gene target (77.3 %) than samples positive in both assays (16.9 %, p < 0.0001). Ct values in the cobas SARS-CoV-2 assay were significantly higher in the 279 samples testing positive in both assays (32.9 %, 95 % CI 32.3–33.6) compared to the 22 samples with discordant results (36.6 %, 95 % CI 36.2–37.1; p = 0.0009). An excellent correlation (r2 = 0.98) was obtained between Ct values of the ORF-1 and E targets in the cobas assays and a good correlation was obtained between LD RT-PCR test and cobas SARS CoV-2 ORF-1 target (r2 = 0.82).ConclusionOur study demonstrated an excellent concordance between a LD RT-PCR and the cobas SARS-CoV-2 tests on the 8800 platform.

SARS-CoV-2 detection by direct rRT-PCR without RNA extraction.

Rapid and reliable screening of SARS-CoV-2 is fundamental to assess viral spread and limit the pandemic we are facing. In this study, we compared direct rRT-PCR method (without RNA extraction) using SeeGene AllplexTM 2019-nCoV rRT-PCR with the RealStar® SARS-CoV-2 rRT-PCR kit (Altona Diagnostics). Furthermore, we assessed the impact of swab storage media composition on PCR efficiency. We show that SeeGene and Altona’s assays provide similar efficiency. Importantly, we provide evidence that RNA extraction can be successfully bypassed when samples are stored in UTM medium or in molecular water but not when samples are stored in saline solution and in Hanks medium.

Calcium Channel Opening Rather than the Release of ATP Causes the Apoptosis of Osteoblasts Induced by Overloaded Mechanical Stimulation

Background: Stress fracture is one of the most common overuse injuries in athletes. Overloaded mechanical stimulation is an important factor affecting stress fractures, but the mechanism is unclear. Methods: MC3T3-E1 cells and a polycaprolactone (PCL) scaffold were co-cultured, and finite element analysis (FEA) was used to analyze the load-carrying capability. Cell proliferation was investigated with CCK-8 assays. An alkaline phosphatase (AKP) activity assay was used to evaluate cell differentiation. Cell apoptosis was analyzed using Hoechst/ PI double-labeling, Caspase-3 activity and lactate dehydrogenase (LDH) activity assays. Realtime PCR and Western blotting were used to examine the gene and protein expression, respectively, of Caspase-3 and Caspase-9. Assays of the intracellular calcium with fluorescent probe technique and extracellular ATP with fluorometric assay kit were used to analyze the changes in the intracellular calcium concentration induced by calcium channel opening and the release of ATP, respectively, at different operation times. Results: When the apparent strain reached 10000 µε, the strain scope of fber at levels greater than 4000 µε was 60%. Overloading for 4 days and operation times of 0.5 h and 2 h increased the cell number and AKP secretion. However, apoptosis genes were activated at the same time, and the operation time of 2 h had a significantly greater effect than 0.5 h. At 8 days, the cell numbers were greater for the operation time of 0.5 h than for 2 h, and the 2-h groups had the fastest apoptosis rate. Overloading for 1 day increased intracellular calcium levels and ATP release. The increase in intracellular calcium could be blocked by the addition of N-ethylmaleimide (NEM) or Hank's medium. Overloading for 8 days increased intracellular calcium levels but decreased extracellular ATP, and verapamil blocked the increase in intracellular calcium. Conclusion: We found that a simultaneous ‘double effect’ on osteoblasts was induced by overloading, which promoted cell proliferation, differentiation and apoptosis. Short-term overloading could open the cell membrane calcium channels and release calcium stores to elevate intracellular calcium levels, thereby promoting the proliferation and differentiation of cells to a greater extent than the effect of apoptosis. For long-term overloading, calcium channel opening in the membrane could lead to overloading of intracellular calcium levels, inducing an apoptosis effect that is greater than the effect on proliferation and differentiation.

Sexagem de espermatozoides bovinos por centrifugação em gradiente descontínuo de densidade de Percoll

O objetivo neste trabalho foi desenvolver um método de seleção do sexo de espermatozoides bovinos por centrifugação em gradiente de densidade de Percoll. Utilizou-se sêmen congelado de touros mantidos em regime de colheita de sêmen. A fração de espermatozoides X ou Y foi separada por centrifugação em treze diferentes gradientes de densidade de Percoll formados por 1 a 12 camadas com densidades que variaram de 1,004 g/mL a 1,123 g/mL. As soluções com diferentes densidades foram preparadas misturando-se, em proporções diferentes, meio de cultura Hank's e uma solução estoque composta de NaCl 1,5 M e Percoll (1:9, v/v). Sobre cada gradiente foi colocado um total de 50 × 10(6) espermatozoides descongelados em 0,7 mL de meio Hank's e centrifugados a 250 X g por 30 minutos, em rotor horizontal, a 25°C. Os espermatozoides das frações superior e inferior foram tratados com Quinacrina Mustarda e analisados (200 deles) quanto à presença do corpúsculo-F. Dos espermatozoides encontrados no sedimento de dois gradientes, compostos de 8 e 12 frações com densidades variando entre 1,050 a 1,120 g/mL e 1,044 a 1,123 g/mL, respectivamente, visualizaram-se 25% com corpúsculo-F e os 75% restantes prováveis portadores do cromossomo X. O aumento na porcentagem de espermatozoides X após a centrifugação em gradiente de densidade permitirá que esse método de sexagem seja usado em larga escala na produção comercial de carne e leite bem como no teste de progênie.

Characterization andIn VitroControl of MPF Activity in Zebrafish Eggs

We describe the characterization of maturation-promoting factor (MPF) in zebrafish eggs and used different defined conditions to maintain its activity in vitro. MPF activity levels are high in freshly ovulated mature eggs and decline rapidly within 5 min after either fertilization or parthenogenetic activation. The MPF activity of eggs matured in vitro declines faster when the eggs are incubated in Hank's culture medium supplemented with 0.5% BSA (H-BSA) than when incubated in Chinook salmon ovarian fluid (CSOF). MPF activity in nonactivated, aged eggs remains high in H-BSA supplemented with 75 microM MG132 or 10 mM caffeine, but neither MG132 nor caffeine can sustain high MPF activity in activated eggs. MG132-treated eggs showed delayed completion of metaphase and extrusion of the second polar body. Nuclear staining of the activated eggs confirmed the correlation between their cell cycle stage and MPF activity at each time point. An embryotoxic effect was found when matured eggs were held in 100 microM of MG132 or 20 mM caffeine for 1 h. Calcium-depleted medium and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid also showed detrimental effects on the embryos. Conversely, nonactivated, aged matured eggs maintained high MPF activity and developmental potential when CSOF was used as a holding medium.

Expression of bystin in reactive astrocytes induced by ischemia/reperfusion and chemical hypoxia in vitro

In this study, we investigated the effects of ischemia/reperfusion and chemical hypoxia on the morphology, cell viability and expression of bystin and glial fibrillary acidic protein (GFAP) in primary cultured astrocytes which were prepared by the subculture method. The astrocytes in Hank's medium without glucose and serum (oxygen–glucose deprivation, ischemic cells) were first exposed to 1% O2 and then to 21% O2 (normoxia), or treated with different concentrations of CoCl 2 or NaN 3 for different periods. Relevant observations and measurements were then conducted. The findings showed that treatment with 1% O 2 for 0.5 or 3 h could induce a characteristic ‘reactive’ morphology and a significant increase in cell viability and total protein amount. The western blot analysis showed that treatment with 1% O 2 for 0.5 or 3 h also induced a significant increase in the expression of bystin and that the response of bystin to mild ischemia was much more sensitive than that of GFAP. Similar results were also found in the cells treated with mild chemical hypoxia. The data demonstrated for the first time that mild ischemia and hypoxia could activate astrocytes and that bystin is a much more sensitive marker in activated astrocytes induced by ischemia and hypoxia as compared to GFAP. The significant up-regulation of bystin suggests that bystin may play an important role in the activation of astrocytes as well as in the neuroprotective role of hypoxic and ischemic preconditioning.

Absorption of anthocyanins through intestinal epithelial cells. Effect of ethanol.

Anthocyanins are phenolic compounds that are consumed from many dietary sources such as red fruit, vegetables and red wine. They present a wide range of biological activities including antioxidant, anti‐inflammatory and anticarcinogenic activities. Bioavailability is a major issue regarding the biological effects of these compounds in vivo and remains unclear, with few data available on this matter.This work aimed to evaluate the absorption of anthocyanins on intestine using Caco‐2 cells. Anthocyanin extract, rich in malvidin‐3‐glucoside, was obtained from grape skins. Anthocyanins were tested on Caco‐2 cells cultured on transwell inserts for 25 days. The transported anthocyanins were detected by HPLC.Anthocyanins (200 μg/ml) were tested in Hanks medium, in absence or presence of 1% ethanol, for different incubation times. Our results showed that this transport was significantly increased in the presence of ethanol especially after 60 min of incubation. In addition, cells that were pretreated for 4 days with anthocyanins (200 μg/ml) showed an increase in their own transport. Cells viability was assayed and was not altered in these conditions. This result could indicate an interference of these compounds with expression/activity of the transporters involved on their own transport.Supported by Fundação para a Ciência e Tecnologia (POCI, FEDER, Programa Comunitário de Apoio, SFRH/BD/28160/2006).

The effect of peritoneal rest in combination therapy of peritoneal dialysis and hemodialysis: using the cultured human peritoneal mesothelial cell model

The effects of peritoneal rest for 24 h during peritoneal dialysis and hemodialysis combination therapy were investigated using cultured human peritoneal mesothelial cell (HPMC) models. Cell activity was investigated by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenylterazolium bromide (MTT) assay after exposing HPMCs to peritoneal dialysis fluids (PDFs) with different pH levels. The following PDFs (50 microl/well) were used for exposure durations of 30 or 240 min: acidic heat-sterilized PDFs (L-H PDF, pH 5.5) and neutral heat-sterilized PDFs (N-H PDF, pH 6.7). Control wells were exposed to M-199 Hanks medium containing 20% fetal bovine serum (FBS) for 30 or 240 min. Supernatants were then aspirated from each well and M-199 culture medium containing 20% FBS (50 microl) was added to each well to rest HPMCs for 24 h before investigation of MTT activity. The activity of HPMCs exposed to L-H PDF for 240 min decreased to approximately 20% and 15% when compared with controls (glucose concentrations of 1.36% and 3.86%, respectively; P < 0.01 versus control, Tukey-Kramer test), and to approximately 60% and 40% after exposure to N-H PDF for 240 min (glucose: 1.36% and 3.86%; P < 0.01). The activity of HPMCs exposed to L-H PDF for 240 min followed by rest was approximately 20% and 4% when compared with controls (glucose: 1.36% and 3.86%; P < 0.01) and was 93% and 96% when compared with controls after exposure to N-H PDF for 240 min followed by rest (glucose: 1.36% and 3.86%). These findings suggest that rest for 24 h after exposure to N-H PDF improves the activity of HPMCs.

Increase in Tetrahydrobiopterin Release from PC 12 Cells under Hypotonic Culture Conditions is Inhibited by HgCl2

Abstract Tetrahydrobiopterin (BH4) was released from PC 12 cells to the extracellular fluid. We found that the BH4 outward flow from the cells placed in Hanks medium was increased when NaCl concentration of the medium was decreased. Increase in the BH4 out flow was not observed when NaCl in the Hanks medium was substituted with sodium glutamate (0.14 M), choline chloride (0.14 M) or sucrosc (0.25 M). HgCl2, an inhibitor of water channel, aquaporin, prevented the increase in BH4 out flow caused by the hypotonic medium. The results suggested the presence of a site in BH4 transport which was stimulated by osmotic pressure and/or water influx.

Modification of demineralized bone matrix by a chemical route

Demineralized bone matrix (DBM) is a well known osteoinductive biomaterial being used in bone related therapies and recently in biomolecular delivery. To widen its usage in therapeutic delivery, the present study was focused on chemical modification of DBM by a grafting technique using glycidylmethacrylate (GMA) as a monomer and ceric ammonium nitrate (CAN) as an initiating system. The chemical modification of DBM was proved by using Fourier transform infrared (FTIR) spectroscopy to analyze functional groups before and after grafting. The grafting results and grafting mechanism are described in detail. The chemically modified DBM was evaluated for in-vitro therapeutic delivery by coupling the gentamicin through epoxy functional groups introduced by GMA. The loading and release of gentamicin was evaluated in Hank's medium at pH 7.4 and 37 °C under physiological conditions and it was found to release the drug for a prolonged period as compared to ungrafted DBM. Based on the experimental results, the chemical modification paves the way to utilization of DBM macromolecules in therapeutic delivery.

Effects of interleukin-8 and Helicobacter pylori on histamine release from isolated canine gastric mucosal mast cells.

In recent studies, the involvement of mast cells in the pathogenesis of Helicobacter pylori infection was suggested. In the present study, using isolated canine gastric mucosal mast cells, we undertook to elucidate the effects of interleukin-8 (IL-8) and H. pylori on histamine release from these cells. Enriched canine gastric mucosal mast cells (50% target cells) were incubated in Hanks medium with IL-8, or water extract or sonicate of H. pylori for 15 min at 37 degrees C. The content of histamine in the supernatants and the cell pellets after centrifugation was assayed with a histamine radioimmunoassay (RIA) kit. IL-8 (50 ng/ml) and concanavalin A (20 microg/ml) significantly increased histamine release from enriched gastric mucosal mast cells. Dose-dependent stimulation of histamine release by IL-8 (5-50 ng/ml) was also seen. Water extract and sonicate of H. pylori (10(8) bacteria) increased histamine release from mast cells. A concentration-dependent stimulation of histamine release by water extract or sonicate was also seen. The maximal response of histamine release was seen at the highest concentration of the water extract or sonicate. The results indicated that IL-8 and H. pylori had stimulatory effects on histamine release from canine gastric mucosal mast cells. The results imply that IL-8 and soluble factors of H. pylori may accelerate inflammation of the gastric mucosa via histamine release from mast cells.

Determining Relative Estrogenicity by Quantifying Vitellogenin Induction in Rainbow Trout Liver Slices

Precision cut tissue slices, by modeling the entire organ, are a valuable tool for studying protein induction or inhibition by test chemicals. This manuscript describes parameters to quantify relative estrogenicity of chemicals in rainbow trout liver slices by measuring vitellogenin (Vg) induction, a well-characterized biomarker of estrogen receptor signal transduction. Hank's medium (phenol-red free) supplemented with Hepes, sodium bicarbonate, and 1% bovine serum albumin was utilized. The experimental parameters were optimized using 1000 nM 17β-estradiol, a potent estrogen in rainbow trout that induces Vg production in vivo. The addition of trout serum and retention of the media was essential, probably to allow for the accumulation of Vg in the slices and media. Histological examination and ATP analyses indicated no toxicity in control or 17β-estradiol-treated liver slices after 120 h. Induction was 4-fold greater with 25% serum containing media compared to media with 10% serum. We observed Vg induction as great as 500-fold over controls at 96 h in liver slices and media containing 25% serum and 1000 nM 17β-estradiol. Controls without 17β-estradiol, incubated in media with 10 or 25% serum, exhibited no detectable Vg production, indicating that the induction seen was not from the media or serum. We observed that 48 h was required for significant Vg induction in the media and liver slices. Maximum induction in slices occurred at 96 h, whereas media Vg levels continued to increase to 120 h, suggesting a time delay between Vg production and excretion by the liver. The feasibility of this model to detect weak environmental estrogens was determined with 0–250 μM o,p′DDE and bisphenol A. Both compounds induced Vg in this model with EC50 values of 104 and 2 × 105 higher than E2, respectively. Our results indicate the importance of media, serum, and time selection for optimal Vg induction. This model allows for the determination of relative estrogenicity of chemicals in a controlled in vitro system while utilizing the advantages of precision cut slice technology.

Morphometric and densitometric study of the biogenesis of electron-dense granules in Fonsecaea pedrosoi

Fonsecaea pedrosoi is a polymorphic pathogenic fungus, etiological agent of chromoblastomycosis, that synthesizes a melanin-like pigment. Although this pigment has been described as a component of the outer layers of the cell wall, electron-dense cytoplasmic bodies have also been visualized. In this work, we have correlated the appearance of intracellular electron-dense granules with the melanization process in F. pedrosoi. For this, conidial forms were grown under conditions where melanin was not synthesized. Afterwards, cells were incubated in Hank's medium supplemented with bovine fetal serum, at 37°C, to stimulate the pigment production. The genesis of cytoplasmic bodies, with different stages of electron density, was demonstrated by transmission electron microscopy. The appearance of fungal acidic compartments, visualized by confocal laser scanning microscopy in cells stained with acridine orange, was time coincident with the formation of electron-dense granules observed by transmission electron microscopy. The quantification of granule numbers as well as morphometric and densitometric studies were performed.

Morphometric and densitometric study of the biogenesis of electron-dense granules inFonsecaea pedrosoi

Fonsecaea pedrosoi is a polymorphic pathogenic fungus, etiological agent of chromoblastomycosis, that synthesizes a melanin-like pigment. Although this pigment has been described as a component of the outer layers of the cell wall, electron-dense cytoplasmic bodies have also been visualized. In this work, we have correlated the appearance of intracellular electron-dense granules with the melanization process in F. pedrosoi. For this, conidial forms were grown under conditions where melanin was not synthesized. Afterwards, cells were incubated in Hank's medium supplemented with bovine fetal serum, at 37 degrees C, to stimulate the pigment production. The genesis of cytoplasmic bodies, with different stages of electron density, was demonstrated by transmission electron microscopy. The appearance of fungal acidic compartments, visualized by confocal laser scanning microscopy in cells stained with acridine orange, was time coincident with the formation of electron-dense granules observed by transmission electron microscopy. The quantification of granule numbers as well as morphometric and densitometric studies were performed.

In Vivo Identification, Survival, and Functional Efficacy of Transplanted Hepatocytes in Acute Liver Failure Mice Model by FISH Using Y-Chromosome Probe

Hepatocyte transplantation has excited much interest in lending temporary metabolic support to a failing liver following acute liver injury. The exact site from which they act and the clinical, biochemical, and histological changes in the recipient body following hepatocyte transplantation is yet to be worked out. The present study is an attempt to delineate location and function of transplanted hepatocytes and also the overall survival of these cells with a fluorescent in situ hybridization (FISH) technique using a Y-chromosome–specific probe in a carbon tetrachloride (CCl 4)-induced mice model of fulminant hepatic failure. Fifty-five syngenic adult Swiss female mice of approximately the same age and body weight were divided into three groups. Group-1 ( n = 15), which received mineral oil, served as a negative control. Group-II ( n = 15) received CCl 4 (3 mL/kg) 40% vol/vol in mineral oil, by gavage served as positive control for hepatic failure. Group-III ( n = 25) received intrasplenic transplantation of syngenic single cell suspension of hepatocytes in Hanks medium, after 30 h of CCl 4 administration. Male Swiss adult mice ( n = 15) served as donors of hepatocytes. The overall survival of animals in groups I to III was 100, 0, and 70%, respectively, by 2 wk of the study period. Transplanted hepatocytes were identified by Periodic Acid Schiff (PAS) staining and confirmed with a FISH technique using the Y-chromosome probe. The majority of exogenously transplanted hepatocytes were found in the liver and spleen sections even after 1 wk of hepatocyte transplantation. Transplanted cells were mostly found to be translocated into the sinusoids of the liver. Transplanted hepatocytes were found to be beneficial as a temporary liver support in a failing liver, significantly improving the survival of the animals. In the present study, the FISH technique was used to unequivocally distinguish the transplanted cells from the host, and thus describes a model for studying the distribution and survival of the transplanted cells.

In vivo identification, survival, and functional efficacy of transplanted hepatocytes in acute liver failure mice model by FISH using Y-chromosome probe.

Hepatocyte transplantation has excited much interest in lending temporary metabolic support to a failing liver following acute liver injury. The exact site from which they act and the clinical, biochemical, and histological changes in the recipient body following hepatocyte transplantation is yet to be worked out. The present study is an attempt to delineate location and function of transplanted hepatocytes and also the overall survival of these cells with a fluorescent in situ hybridization (FISH) technique using a Y-chromosome-specific probe in a carbon tetrachloride (CCl4)-induced mice model of fulminant hepatic failure. Fifty-five syngenic adult Swiss female mice of approximately the same age and body weight were divided into three groups. Group-1 (n = 15), which received mineral oil, served as a negative control. Group-II (n = 15) received CCl4 (3 mL/kg) 40% vol/vol in mineral oil, by gavage served as positive control for hepatic failure. Group-III (n = 25) received intrasplenic transplantation of syngenic single cell suspension of hepatocytes in Hanks medium, after 30 h of CCl4 administration. Male Swiss adult mice (n = 15) served as donors of hepatocytes. The overall survival of animals in groups I to III was 100, 0, and 70%, respectively, by 2 wk of the study period. Transplanted hepatocytes were identified by Periodic Acid Schiff (PAS) staining and confirmed with a FISH technique using the Y-chromosome probe. The majority of exogenously transplanted hepatocytes were found in the liver and spleen sections even after 1 wk of hepatocyte transplantation. Transplanted cells were mostly found to be translocated into the sinusoids of the liver. Transplanted hepatocytes were found to be beneficial as a temporary liver support in a failing liver, significantly improving the survival of the animals. In the present study, the FISH technique was used to unequivocally distinguish the transplanted cells from the host, and thus describes a model for studying the distribution and survival of the transplanted cells.

Role of hydrogen peroxide in the cytotoxic effects of UVA/B radiation on mammalian cells.

Effects of selenium (Se) deficiency on the sensitivity of murine leukemia L1210 cells to broad band UVA/B radiation (310-400 nm) have been investigated. Cells rendered glutathione peroxidase (GPX) deficient by shortterm (2-3 week) growth in 1%, serum/RPMI medium without added Se [L.Se(-) cells] were found to be much less resistant to clonally assessed UVA/B lethality than Se-supplemented controls [L.Se(+) cells]. By contrast, long-term ( > 20 week) Se-deprived [L'.Se(-)] cells whose catalase (CAT) activity was elevated > 100-fold were far more resistant to UVA/B than L.Se(+) cells. Similar trends were observed for cells irradiated in 1% serum/RPMI or Hank's medium. Whereas the CAT inhibitor 3-amino-1,2,4-triazole had no effect on L.Se(+) photosensitivity, it produced a large increase in L'.Se(-) photosensitivity. These findings are consistent with H2O2 intermediacy in photokilling and suggest that L1210 cells depend mainly on GPX for protection against this species but switch to overexpressed CAT after chronic Se deprivation. In agreement with this, steady-state H2O2 levels measured by H2O2 electrode during UVA/B exposure were higher in L.Se(-) than L.Se(+) suspensions but much lower (barely detectable) in L'.Se(-) suspensions. Cytotoxic effects of UVA/B and variations thereof resulting from Se manipulation could be mimicked by treating cells with glucose oxidase in the presence of D-glucose, providing further support for H2O2 involvement. Whether UVA/B-generated H2O2 is directly cytotoxic or gives rise to a more damaging species such as hydroxyl radical (HO) is presently unknown.

A threshold decrease for electrically stimulated motor responses of isolated aging outer hair cells from the pigmented guinea pig.

When outer hair cells are isolated from guinea pig cochleas and are placed in normal Hank's medium, they exhibit aging as a slow tonic reduction in length and increase in diameter. During this time the lateral subsurface cisternae become progressively vesiculated and the optical density of the border seen under phase-contrast microscopy decreases. A study of 65 outer hair cells was carried out using video imaging of this process. The base of each cell bonded to the Petri dish and the motility of the cuticular plate was recorded in two ways. To quantify the slow contraction of each preparation, the dimensions of the cell were measured from video replay. Displacements of the cuticular plate in response to an alternating electric field in line with the cell axis were also monitored using a video tracking technique. The amplitude of a 1 Hz stimulus required to cause a visually detectable motor response above baseline noise decreased as the cell degraded. Typically, fresh cylindrical cells exhibiting high optical contrast showed relatively small movements for field strengths up to 2 kVm-1. However, as the cell depolarized, the rigidity initially decreased and the cell could respond to field strengths down to 0.1 kVm-1 before cell death ultimately occurred. Such a threshold phenomenon in the isolated OHC has not been demonstrated directly until now. This result explains the variability of electromotility in aging in vitro preparations from the cochlea.