To investigate whether the 60-kDa heat shock chaperonin protein (hsp60) is present on surface of mammalian cells, we used immunogold labeling intact cells and backscattered electron imaging to image gold particles. Chinese hamster ovary human leukemic CD4-positive T-cell line CEM-SS glass coverslips were labeled using affinity-purified monoclonal polyclonal antibodies specific for hsp60 30 nm markers. Cells imaged scanning mode conventional transmission microscope. Backscattered provided...

Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level individual microtubules double-label immunofluorescence. The haptenized tubulin localized an anti-fluorescein antibody a second conjugated fluorescein. Total identified anti-tubulin secondary rhodamine. Contrary to recent studies (Salmon,...

We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, adrenal gland. Immunogold labeling tissues embedded acrylic resin LR Gold strong mitochondria all However, anterior...

To study the interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules, we have developed a quadruple fluorescence labeling procedure to visualize all four structures in same cell. We applied this approach cellular organization control cells treated with microtubule drugs vinblastine or taxol. Endoplasmic reticulum was visualized by staining glutaraldehyde-fixed dye 3,3′-dihexyloxacarbocyanine iodide. After detergent permeabilization, triple...

ABSTRACT Giardia lamblia trophozoites contain a complex endomembrane system as demonstrated by fluorescence and cryoelectron microscopy. The was weakly detected in live cells using the fluorescent membrane dye 3,3′-dihexyloxacarbocyanine iodide. definitive identification of endoplasmic reticulum required development molecular label. We expressed Giardial Bip Escherichia coli raised polyclonal antibody to purified protein. In western blots, specific for did not react with human, monkey rodent...

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, first molar equivalent this ligand 93% bound to human serum albumin. With addition palmitate, free spin label concentration decreases greatly, by almost 80%, in presence fatty acid:albumin ratio 3:1 4:1. spectral characteristics are also affected. changes seen intensity and splitting between high...

Giardia lamblia trophozoites contain a 60-kDa protein recognized in immunoblots by antibody to mammalian hsp60, localized mitochondria eukaryotic cells. The cellular distribution of this is evaluated immunofluorescence microscopy using monoclonal human polyclonal rodent and 2 antibodies mycobacterial 65-kDa antigen, prokaryotic hsp60 homolog. All these antibodies, except 1, which specific for give punctate labeling pattern throughout the cytoplasm, indicating that concentrated at discrete...

Outgrowth of neurites in culture is used for assessing neurotrophic activity. Neurite measurements have been performed very slowly using manual methods or more efficiently with interactive image analysis systems. In contrast, medium-throughput and noninteractive neurite screens has not well described. The authors report the performance an automated acquisition system (IN Cell Analyzer 1000) assay. Neuro-2a (N2a) cells were plated 96-well plates exposed to 6 conditions retinoic acid....

Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as an anti-inflammatory donor. We determined whether small volume transfusion hyperoncotic is neuroprotective in a porcine model traumatic injury (TBI) without accompanying hemorrhagic shock (HS)....

ABSTRACT Giardia lamblia trophozoites contain acetylated α‐tubulin but lack detectable levels of tyrosinolated α‐tubulin, as demonstrated in immunoblots with monoclonal antibodies specific for these tubulin forms. By immunofluorescence microscopy, is localized axonemes, median bodies and the adhesive disk. Post‐embeddment immunogold labeling thin sections cells was used to evaluate acetylation at level individual microtubules by electron microscopy. Cells were fixed glutaraldehyde embedded...

Abstract We have examined the effect of ethacrynic acid on mitochondrial morphology and distribution as well cellular toxicity in cultured human fibroblasts, African Green Monkey B‐SC‐1 kidney cells, Chinese hamster ovary cells. Treatment above cells with 66 μM causes no reduction cell viability after 2 h but is cytotoxic upon prolonged (6–7 days) exposure. Ethacrynic treatment for up to found cause novel shape changes redistribution mitochondria, assessed by immunofluorescence electron...

Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as a donor. We determined whether small volume transfusion hyperoncotic is neuroprotective in polytrauma model traumatic injury (TBI) plus hemorrhagic shock. Guinea pigs were used because, like...

The binding isotherm and unique electron spin resonance spectral characteristics of a monoanionic label (1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) dianionic (1-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) are used to prove the steroid modulation serum albumin properties. Effects selected number steroids (progesterone, testosterone, estradiol, aldosterone, estriol, corticosterone, deoxycorticosterone,...

Of the deaths attributed to cancer, 90% are due metastasis. Treatments that prevent or cure metastasis remain elusive. Low expression of extracellular superoxide dismutase (EcSOD SOD3) has been associated with poor outcomes and increased metastatic potential in multiple types cancer. Here, we characterize antimetastatic therapeutic mechanisms a macromolecular SOD3-mimetic polynitroxyl albumin (PNA, also known as VACNO). PNA is human serum conjugated nitroxide groups acts an SOD-mimetic. Here...

We used quantitative immunogold electron microscopy to evaluate the subcellular distribution of cytochrome‐c in normal rat tissues, employing a wide variety monoclonal and polyclonal antibodies against mammalian cytochrome‐c. Immunogold labeling tissues embedded acrylic resin LR Gold shows highly specific mitochondria all examined, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen thyroid. In pancreatic acinar cells...