A microchamber for polarographic assay

A glass micro-chamber which allows polarographic assay in a volume of 180 microliter is described. The conical shape of this chamber allows efficient mixing with a Teflon magnetic flea. This chamber facilitates the study of the small quantities of mitochondria obtained from human tissue biopsies or animal sources. The polarographic assay of mouse liver mitochondria is described.     

A stirred microchamber for oxygen consumption rate measurements with pancreatic islets

Improvements in pancreatic islet transplantation for treatment of diabetes are hindered by the absence of meaningful islet quality assessment methods. Oxygen consumption rate (OCR) has previously been used to assess the quality of organs and primary tissue for transplantation. In this study, we describe and characterize a stirred microchamber for measuring OCR with small quantities of islets. The device has a titanium body with a chamber volume of about 200 microL and is magnetically stirred and water jacketed for temperature control. Oxygen partial pressure (pO(2)) is measured by fluorescence quenching with a fiber optic probe, and OCR is determined from the linear decrease of pO(2) with time. We demonstrate that measurements can be made rapidly and with high precision. Measurements with betaTC3 cells and islets show that OCR is directly proportional to the number of viable cells in mixtures of live and dead cells and correlate linearly with membrane integrity measurements made with cells that have been cultured for 24 h under various stressful conditions.     

PKC signaling at fertilization in mammalian eggs

Protein kinase C (PKC) has been proposed to regulate major egg activation events during mammalian fertilization. Most of the evidence supporting this assumption has first been obtained using pharmacological activation and inhibition of the kinase, while egg activation was assessed by checking for exocytosis of the cortical granules, extrusion of the second polar body and formation of pronuclei. However, results have been inconclusive and sometimes contradictory regarding the exact role of PKC in regulating egg activation events. The PKC family is composed of various isotypes, which differ in their modular structures and regulatory properties. Hence the need to re-examine the roles of egg PKCs more specifically. Mammalian eggs express many PKC isotypes, the roles of which have been investigated using immunodetection, isotype-specific inhibition and, more recently, live imaging of fluorescent chimaeras. Here, I review the recent development of PKC research in mammalian fertilization and the evidence for a specific role for certain PKC isotypes in fertilization-induced egg activation.     

Immunofluorescence microchamber technique for characterizing isolated organelles

We describe a rapid technique for the localization and quantitation of specific proteins on organelles bound to microscope chambers. Disposable chambers are constructed from glass slides and provide a platform for the binding of organelles and subsequent immunofluorescence and biochemical assays. Several studies are presented to demonstrate the utility of this technique. Kinesin was visualized in postnuclear supernatants. Golgi and endoplasmic reticulum bound quantitatively to chambers. Endocytic vesicles prepared from rat liver that had been injected in situ with Texas red-labeled asialoorsomucoid allowed for simultaneous detection of asialoorosomucoid, asialoglycoprotein receptor, caveolin 1, and microtubules. Asialoglycoprotein receptor colocalized with asialoorosomucoid-containing vesicles, whereas many of the caveolin 1 structures had no asialoorosomucoid or asialoglycoprotein receptor. The microchambers were also used to measure the binding to endocytic vesicles of exogenously added Rab5 and to monitor the ATP-dependent acidification of endocytic vesicles using the fluorescent dye acridine orange.     

Flow microchamber for the Linnic interference microscope

A flow microchamber for the Linnic microscope was constructed. The volume of the microchamber is 6-10 microliters. The microchamber may be used in two working regimes: (a) under conditions of periodical change of solutions during the analysis of the same object and (b) under conditions of continuous flow of solutions. The microchamber was used for studying the birefringence of skeletal muscle sarcomere at rigor and relaxed states.     

A role for microwave processing in the dry preservation of mammalian cells

Dry preservation involves removing water from samples so that degradative biochemical processes are slowed and extended storage is possible. Recently this approach has been explored as a method for preserving living mammalian cells. The current work explores the use of microwave processing to enhance evaporation rates and to improve drying uniformity, thereby overcoming some of the challenges in this field. Mouse macrophage cells (J774) were pre-incubated in full complement media containing 50 mM trehalose, for 18-h, to allow for endocytosis of trehalose. Droplets of experimental and control (no intracellular trehalose) cell suspensions were placed on coverslips in a microwave cavity. Water was evaporated using intermittent microwave heating (600 W, 30 s intervals). Samples were dried to various moisture levels, rehydrated, and then survival was assessed after a 45-min recovery period using Calcein-AM/PI fluorescence and Trypan Blue exclusion assays. The metabolic activity of dried cells (4.3 gH(2)O/gdw) was assessed after rehydration using a resazurin reduction assay. Apoptosis levels were also measured. Post- rehydration survival correlated with the final moisture content achieved, consistent with other drying methods. Intracellular trehalose provided protection against injury associated with moisture loss. Metabolic assays revealed normal growth in surviving cells, and these survival levels were consistent with results from apoptosis assays (P > 0.05). Brightfield and fluorescence images of microwave-dried samples revealed a uniform distribution of cells within the dried matrix and profilometry analysis demonstrated that solids were uniformly distributed throughout the sample. Microwave-processing successfully facilitated rapid and uniform dehydration of cell-based samples.     

A digital image processing system for quantitating dynamic morphology in cultured mammalian cells

An automated, video-driven system has been developed which can quantitate dynamic cell morphology in cultured mammalian cells. This system is based upon the Personal Image Analysis System and is assisted by a video-enhanced contrast microscopy with a computer-aided digital image processing unit and a time-lapse video technique. Various parameters for cell motility including locomotion (vectorial translation) and accompanying shape changes can be simultaneously analyzed. Here, we describe this system and demonstrate its application in Balb/c 3T3 cell culture. This system represents a new tool for exploring subtleties of mammalian cell behavior.     

The dynamics of calcium oscillations that activate mammalian eggs

It has been known for some time that mammalian eggs are activated by a series of intracellular calcium oscillations that occur shortly after sperm egg membrane fusion. Recent work has identified a novel sperm specific phospholipase C zeta as the likely agent that stimulates the calcium oscillations in eggs after sperm-egg membrane fusion. PLCzeta is stimulated by low intracellular calcium levels in a manner which suggests that there is a regenerative feedback of calcium release and PLCzeta induced inositol 1,4,5-trisphophate (InsP(3)) production in eggs. This implies calcium oscillations in fertilizing mammalian eggs are driven by underlying oscillations of InsP(3). This model of oscillations is supported by the response of mouse eggs to sudden increases in InsP(3). The cellular targets of calcium oscillations include calmodulin-dependent protein kinases, protein kinase C and mitochondria. There is evidence that eggs might be best activated by multiple calcium increases rather than a single calcium rise. As yet we do not fully understand how the target of calcium in a mammalian egg might decode the patterns of calcium changes that can occur during egg activation.     

A data processing pipeline for mammalian proteome dynamics studies using stable isotope metabolic labeling

In a recent study, in vivo metabolic labeling using (15)N traced the rate of label incorporation among more than 1700 proteins simultaneously and enabled the determination of individual protein turnover rate constants over a dynamic range of three orders of magnitude (Price, J. C., Guan, S., Burlingame, A., Prusiner, S. B., and Ghaemmaghami, S. (2010) Analysis of proteome dynamics in the mouse brain. Proc. Natl. Acad. Sci. U. S. A. 107, 14508-14513). These studies of protein dynamics provide a deeper understanding of healthy development and well-being of complex organisms, as well as the possible causes and progression of disease. In addition to a fully labeled food source and appropriate mass spectrometry platform, an essential and enabling component of such large scale investigations is a robust data processing and analysis pipeline, which is capable of the reduction of large sets of liquid chromatography tandem MS raw data files into the desired protein turnover rate constants. The data processing pipeline described in this contribution is comprised of a suite of software modules required for the workflow that fulfills such requirements. This software platform includes established software tools such as a mass spectrometry database search engine together with several additional, novel data processing modules specifically developed for (15)N metabolic labeling. These fulfill the following functions: (1) cross-extraction of (15)N-containing ion intensities from raw data files at varying biosynthetic incorporation times, (2) computation of peptide (15)N isotopic incorporation distributions, and (3) aggregation of relative isotope abundance curves for multiple peptides into single protein curves. In addition, processing parameter optimization and noise reduction procedures were found to be necessary in the processing modules in order to reduce propagation of errors in the long chain of the processing steps of the entire workflow.     

Information processing in mammalian olfactory system

In recent years, considerable progress has been made in understanding how the olfactory system uses neural space to encode sensory information. In this review, we focus on recent studies aimed at understanding the organizational strategies used by the mammalian olfactory system to encode information. The odorant receptor gene family is discussed in the context of its genomic organization as well as the specificity of olfactory sensory neurons. These data have important consequences for the mechanisms of odorant receptor gene choice by a given sensory neuron. Division of the olfactory epithelium into zones that express different sets of odorant receptors is the first level of input organization. The topographical relationship between periphery and olfactory bulb represents a further level of processing of information and results in the formation of a highly organized spatial map of information in the olfactory bulb. There, local circuitry refines the sensory input through various lateral interactions. Finally, the factors that may drive the development of such a spatial map are discussed. The onset of expression and the establishment of the zonal organization of odorant receptor genes in the epithelium are not dependent upon the presence of the olfactory bulb, suggesting that the functional identity of olfactory sensory neurons is determined independently of target selection. © 1996 John Wiley & Sons, Inc.     

Parallel processing in the mammalian retina

Our eyes send different 'images' of the outside world to the brain - an image of contours (line drawing), a colour image (watercolour painting) or an image of moving objects (movie). This is commonly referred to as parallel processing, and starts as early as the first synapse of the retina, the cone pedicle. Here, the molecular composition of the transmitter receptors of the postsynaptic neurons defines which images are transferred to the inner retina. Within the second synaptic layer - the inner plexiform layer - circuits that involve complex inhibitory and excitatory interactions represent filters that select 'what the eye tells the brain'.     

Anomalous RNA substrates for mammalian tRNA 3' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. The enzyme can also recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA. To investigate the interaction between 3' tRNase and substrates, we tested various anomalous pre-tRNA-like complexes for cleavage by pig 3' tRNase. We examined how base mismatches in the acceptor stem affect 3' tRNase cleavage of RNA complexes, and found that even one base mismatch in the acceptor stem drastically reduces the cleavage efficiency. Mammalian 3' tRNase was able to recognize complexes between target RNAs and 5'-half tDNAs, and cleave the target RNAs, although inefficiently, whereas the enzyme had no activity to cleave phosphodiester bonds of DNA. A relatively long RNA target, the Escherichia coli chloramphenicol acetyltransferase (CAT) mRNA, was cleaved by 3' tRNase in the presence of appropriate 5'-half tRNAs. We also demonstrated that an RNA complex of lin-4 and lin-14 from Caenorhabditis elegans can be recognized and cleaved by pig 3' tRNase.     

Meiotic drive of noncentromeric loci in mammalian meiosis II eggs

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Cytoskeletal sheets appear as universal components of mammalian eggs.

The eggs of two mammalian species have been shown to contain novel cytoskeletal elements, referred to as cytoskeletal sheets, which undergo stage-specific changes in spatial organization at three key developmental transitions, fertilization, compaction, and blastocyst formation. If cytoskeletal sheets have an integral role in these developmental transitions, the sheets should be present in the eggs of other mammals as well. We examined the eggs of four additional species to determine if sheets were present. Our results indicate that sheets were present and they can be categorized into two classes based on their surface appearance. Cytoskeletal sheets in eggs of hamsters and rats have a smooth surface appearance, while eggs from humans, cows, pigs, and mice have a fibrous surface appearance. In addition, we observed that species-specific variations exist in the width of the sheets and in the density of the sheets (i.e., number per micron 2) in the eggs. These species-specific variations may relate to the role of the sheets during early development.     

Pressure-driven perfusion culture microchamber array for a parallel drug cytotoxicity assay

This article reports a pressure-driven perfusion culture chip developed for parallel drug cytotoxicity assay. The device is composed of an 8 x 5 array of cell culture microchambers with independent perfusion microchannels. It is equipped with a simple interface for convenient access by a micropipette and connection to an external pressure source, which enables easy operation without special training. The unique microchamber structure was carefully designed with consideration of hydrodynamic parameters and was fabricated out of a polydimethylsiloxane by using multilayer photolithography and replica molding. The microchamber structure enables uniform cell loading and perfusion culture without cross-contamination between neighboring microchambers. A parallel cytotoxicity assay was successfully carried out in the 8 x 5 microchamber array to analyze the cytotoxic effects of seven anticancer drugs. The pressure-driven perfusion culture chip, with its simple interface and well-designed microfluidic network, will likely become an advantageous platform for future high-throughput drug screening by microchip.     

Specialized cytoskeletal elements in mammalian eggs: structural and biochemical evidence for their composition

Mammalian eggs and embryos contain an extensive detergent-resistant cytoskeletal network, including many elements which have been referred to as sheets in hamster eggs. In this study we examined the structure of the sheet-like components by using embedment-free sections and freeze-fracture electron microscopy and found that the sheets are composed of both filamentous and particulate components. In addition, exposure to a high salt extraction medium resulted in the disappearance of the sheets at the ultrastructural level. SDS-polyacrylamide gel electrophoresis of the cell fractions revealed four stainable proteins solubilized by the high salt extraction with one of the proteins being greatly enriched. Because these cytoskeletal sheets undergo an extensive reorganization coincident with key events during early development they serve as internal markers for the establishment of polarity and subsequent differentiation of the first embryonic epithelium, the trophectoderm.