Spindle Dynamics and the Role of γ-Tubulin in Early Caenorhabditis elegans Embryos

gamma-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that gamma-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::gamma-tubulin or GFP::beta-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of gamma-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of gamma-tubulin. gamma-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that gamma-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.     

Effects of DNA charge and length on the electrophoretic mobility of intercalated DNA

DNA molecules ranging in size from 1 to 630 kilobase pair and intercalated with either ethidium bromide (EtBr) or propidium iodide (PI) were electrophoresed in 1% agarose at four different electric field strengths. The extent of intercalation of EtBr under the conditions of our electrophoresis experiments was determined by a spectroscopic technique, whereas the extent of intercalation of PI was inferred from previous studies. The effects of the increase in DNA contour length and the concomitant decrease of linear charge density were separated based on our analysis of the mobility data. We conclude that the main factor responsible for the reduced electrophoretic mobility of intercalated DNA is the diminished linear charge density and not the increased contour length. © 1994 John Wiley & Sons, Inc.     

μOrgano: A Lego?-Like Plug & Play System for Modular Multi-Organ-Chips

Human organ-on-a-chip systems for drug screening have evolved as feasible alternatives to animal models, which are unreliable, expensive, and at times erroneous. While chips featuring single organs can be of great use for both pharmaceutical testing and basic organ-level studies, the huge potential of the organ-on-a-chip technology is revealed by connecting multiple organs on one chip to create a single integrated system for sophisticated fundamental biological studies and devising therapies for disease. Furthermore, since most organ-on-a-chip systems require special protocols with organ-specific media for the differentiation and maturation of the tissues, multi-organ systems will need to be temporally customizable and flexible in terms of the time point of connection of the individual organ units. We present a customizable Lego^®-like plug & play system, μOrgano, which enables initial individual culture of single organ-on-a-chip systems and subsequent connection to create integrated multi-organ microphysiological systems. As a proof of concept, the μOrgano system was used to connect multiple heart chips in series with excellent cell viability and spontaneously physiological beat rates.     

Nitrogen Cycling of Active Bacteria within Oligotrophic Sediment of the Mid-Atlantic Ridge Flank

Microbial ecology within oligotrophic marine sediment is poorly understood, yet is critical for understanding geochemical cycles. Here, 16S rRNA sequences from RNA and DNA inform the structure of active and total microbial communities in oligotrophic sediment on the western flank of the Mid-Atlantic Ridge. Sequences identified as Bacillariophyta chloroplast were detected within DNA, but undetectable within RNA, suggesting preservation in 5.6-million-year-old sediment. Statistical analysis revealed that RNA-based microbial populations correlated significantly with nitrogen concentrations, whereas DNA-based populations did not correspond to measured geochemical analytes. Bioenergetic calculations determined which metabolisms could yield energy in situ, and found that denitrification, nitrification, and nitrogen fixation were all favorable. A metagenome was produced from one sample, and included genes mediating nitrogen redox processes. Nitrogen respiration by active bacteria is an important metabolic strategy in North Pond sediments, and could be widespread in the oligotrophic sedimentary biosphere.     

Microbial models of soil metabolism: biotransformations of danofloxacin

Danofloxacin is a new synthetic fluoroquinolone antibacterial agent under development for exclusive use in veterinary medicine. Such use could lead to deposition of low levels of danofloxacin residues in the environment in manure from treated livestock. This study was conducted to evaluate the potential for indigenous soil microorganisms to metabolize danofloxacin. Cultures of 72 soil microorganisms representing a diverse panel of bacteria, fungi and yeast were incubated with danofloxacin mesylate substrate and samples analyzed periodically by high performance liquid chromatography for loss of danofloxacin and formation of metabolites. Some samples were further analyzed by liquid chromatography-mass spectrometry and mass spectrometry to confirm metabolite identification. Twelve organisms, representing eight different genera, biotransformed danofloxacin to metabolites detectable by the chromatographic methods employed. Two Mycobacterium species, two Pseudomonas species, and isolates of Nocardia sp, Rhizopus arrhizus and Streptomyces griseus all formed N-desmethyldanofloxacin. The formation of the 7-amino danofloxacin derivative, 1-cyclopropyl-6-fluoro-7-amino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid by cultures of Candida lipopytica, Pseudomonas fluorescens, two Mycobacterium species and three Penicillium species demonstrates the propensities of these cultures to completely degrade the piperazine ring. At least two additional and unidentified metabolite peaks were observed in chromatograms of Aspergillus nidulans and Penicillium sp cultures. Radiolabled [2-¹⁴C]danofloxacin added to cultures of the fungus Curvularia lunata was apparently mineralized, with approximately 31% of the radiolabel recovered as volatile metabolites after 24 h of incubation, indicating the susceptibility of the quinolone ring to microbial metabolic degradation. Received 09 December 1996/ Accepted in revised form 09 April 1997     

The physiology of hibernation in common map turtles (Graptemys geographica).

Map turtles from Wisconsin were submerged at 3 degrees C in normoxic and anoxic water to simulate extremes of potential respiratory microenvironments while hibernating under ice. In predive turtles, and in turtles submerged for up to 150 days, plasma PO2, PCO2) pH, [Cl-], [Na+], [K+], total Mg, total Ca, lactate, glucose, and osmolality were measured; hematocrit and body mass were determined, and plasma [HCO3-] was calculated. Turtles in anoxic water developed a severe metabolic acidosis, accumulating lactate from a predive value of 1.7 to 116 mmol/l at 50 days, associated with a fall in pH from 8.010 to 7.128. To buffer lactate increase, total calcium and magnesium rose from 3.5 and 2.0 to 25.7 and 7.6 mmol/l, respectively. Plasma [HCO3-] was titrated from 44.7 to 4.3 mmol/l in turtles in anoxic water. Turtles in normoxic water had only minor disturbances of their acid-base status and ionic statuses; there was a marked increase in hematocrit from 31.1 to 51.9%. This study and field studies suggest that map turtles have an obligatory requirement for a hibernaculum that provides well-oxygenated water (e.g. rivers and large lakes rather than small ponds and swamps) and that this requirement is a major factor in determining their microdistribution.     

Responses of cultured rat trigeminal ganglion neurons to bitter tastants

The initial steps in taste and olfaction result from the activation by chemical stimuli of taste receptor cells (TRCs) and olfactory receptor neurons (ORNs). In parallel with these two pathways is the chemosensitive trigeminal pathway whose neurons terminate in the oral and nasal cavities and which are activated by many of the same chemical stimuli that activate TRCs and ORNs. In a recent single unit study we investigated the responses of rat chorda tympani and glossopharnygeal neurons to a variety of bitter-tasting alkaloids, including nicotine, yohimbine, quinine, strychnine and caffeine, as well as capsaicin, the pungent ingredient in hot pepper. Here we apply many of these same compounds to cultured rat trigeminal ganglion (TG) neurons and measure changes in intracellular calcium [Ca2+]i to determine whether TG neurons will respond to these same compounds. Of the 89 neurons tested, 34% responded to 1 mM nicotine, 7% to 1 mM caffeine, 5% to 1 mM denatonium benzoate, 22% to 1 mM quinine hydrochloride, 18% to 1 mM strychnine and 55% to 1 microM capsaicin. These data suggest that neurons from the TG respond to the same bitter-tasting chemical stimuli as do TRCs and are likely to contribute information sent to the higher CNS regarding the perception of bitter/irritating chemical stimuli.