A Ca-Dependent Early Functional Heterogeneity in Amoebae ofDictyostelium discoideum,Revealed by Flow Cytometry

When freshly starved amoebae ofDictyostelium discoideumare loaded with the Ca²⁺-specific dye indo-1/AM and analyzed in a fluorescence-activated cell sorter, they exhibit a quasi-bimodal distribution of fluorescence. This permits a separation of the population into two classes: H, or “high Ca²⁺-indo-1 fluorescence,” and L, or “low Ca²⁺-indo-1 fluorescence.” Simultaneous monitoring of Ca²⁺-indo-1 and Ca²⁺-chlortetracycline fluorescence shows that by and large the same cells tend to have high (or low) levels of both cytoplasmic and sequestered Ca²⁺. Next we label H cells with tetramethylrhodamine isothiocyanate (TRITC) and mix them in a 1:4 ratio with L cells. In the slugs that result, TRITC fluorescence is confined mainly to the anterior prestalk region. This implies that amoebae with relatively high Ca²⁺at the vegetative stage tend to develop into prestalk cells and those with low Ca²⁺into prespores.Polysphondylium violaceum,a cellular slime mold that does not possess prestalk and prespore cells, also does not display a Ca²⁺-dependent heterogeneity at the vegetative stage or in slugs. Finally, confirming earlier findings with the fluorophore fura-2 (Azharet al., Curr. Sci.68, 337–342 (1995)), a prestalk–prespore difference in cellular Ca²⁺is present in the cells of the slugin vivo.These findings are discussed in light of the possible roles of Ca²⁺for cell differentiation inD. discoideum.     

Molecular assembly of proteins and conjugated polymers: Toward development of biosensors

A molecular assembly in which a conjugated polymer is interfaced with a photodynamic protein is described. The conjugated polymer, functionalized with biotion, is designed such that it can be physisorbed on or chemically grown off a glass surface. The streptavidin-derivatized protein is immobilized on the biotinylated polymer matrix through the strong biotin-streptavidin interactions. The assembly, built on the surface of an optical fiber or on the inside walls of a glass capillary, forms an integral part of a biosensor for the detection of environmental pollutants such as organophosphorus-based insecticides. The Protein in the system can be replaced by any biological macromolecule of interest. We study one specific case, the enzyme alkaline phosphatase. The enzyme catalyzes a reaction producing an intermediate compound that chemiluminesces, and the chemiluminescence singnal is monitored to detect and quantify insecticides such as paraoxon and methyl parathion. Preliminary results indicate ppb level detection with response time less than 1 minute. (c) 1995 John Wiley & Sons, Inc.     

Variation of mitochondrial size during the cell cycle: A multiparameter flow cytometric and microscopic study.

BACKGROUND: Changes in mitochondrial structure and size are observed in response to alterations in cell physiology. Flow cytometry provides a useful tool to study these changes in intact cells. We have used flow cytometry and digital fluorescence microscopy to analyze the variations in mitochondrial size in relation to specific phases of the cell cycle. METHODS: Supravital staining of rat fibroblasts was done with Hoechst 33342 and rhodamine 123, and cells were analyzed in a dual-laser flow cytometer. Synchronized cells at various stages of the cell cycle were analyzed for changes in mitochondrial size. These cells were also examined by electron microscopy, digital fluorescence microscopy and computerized image analysis to compare the lengths of the mitochondria. RESULTS: By using fluorescence pulse width analysis, we observed two populations of mitochondria in intact cells. The percentage of cells with small and large mitochondria at specific stages of the cell cycle indicated that mitochondrial size increases during the cell cycle; early G1 phase cells had the smallest mitochondria and the mitotic phase cells had the largest mitochondria. These results were confirmed by microscopic analysis of cells. CONCLUSIONS: Flow cytometry can distinguish the relative mitochondrial size in intact cells, and in combination with digital microscopy it can be used to study mitochondrial variation during the cell cycle.     

Signalling mucin Msb2 Regulates adaptation to thermal stress in Candida albicans

Temperature is a potent inducer of fungal dimorphism. Multiple signalling pathways control the response to growth at high temperature, but the sensors that regulate these pathways are poorly defined. We show here that the signalling mucin Msb2 is a global regulator of temperature stress in the fungal pathogen Candida albicans. Msb2 was required for survival and hyphae formation at 42°C. The cytoplasmic signalling domain of Msb2 regulated temperature‐dependent activation of the CEK mitogen activated proteins kinase (MAPK) pathway. The extracellular glycosylated domain of Msb2 (100‐900 amino acid residues) had a new and unexpected role in regulating the protein kinase C (PKC) pathway. Msb2 also regulated temperature‐dependent induction of genes encoding regulators and targets of the unfolded protein response (UPR), which is a protein quality control (QC) pathway in the endoplasmic reticulum that controls protein folding/degradation in response to high temperature and other stresses. The heat shock protein and cell wall component Ssa1 was also required for hyphae formation and survival at 42°C and regulated the CEK and PKC pathways.     

Use of the fish enzyme system in monitoring water quality: effects of mercury on tissue enzymes

1. Rosy barb (Puntius conchonius) were exposed to 181 micrograms/l mercuric chloride for 48 h and the activity of acid and alkaline phosphatases (AcP and AIP), aspartate aminotransferase (AAT), alanine aminotransferase (AIAT), lactic dehydrogenase (LDH), and acetylcholinesterase (AchE) were measured in vivo in several organs. 2. The AcP activity was inhibited in the liver, gills, kidneys, and gut but stimulated in the gonads. With the exception of kidney, the AIP activity showed an increase in all the organs examined. The AAT and AIAT were generally inhibited in different organs. An increase in LDH activity occurred in the cardiac and skeletal muscles while the AchE activity was considerably lowered in the brain, gills, and liver. 3. In vitro exposure to mercury at concentrations ranging between 10(-10) and 10(-4) M, inhibited the AIP, AAT, AIAT, LDH, and AchE activities in the tissues examined. The AcP activity was also depressed in all the tissues except in the testes, in which a marginal increase was noted. 4. The in vivo and in vitro effects of Hg were not of similar quality implying sequestration of toxic cations in the intact animals.     

On the mechanism of hydrogen-deuterium exchange in bacteriorhodopsin.

Continuous-flow resonance Raman experiments carried out in bacteriorhodopsin show that the exchange of a deuteron on the Schiff base with a proton takes place in times shorter than 3 ms. Exchange mechanisms based on a base-catalyzed deprotonation followed by reprotonation of the Schiff base are excluded. A mechanism is suggested in which a water molecule interacts directly with the Schiff base deuteron in a concerted exchange mechanism. It appears that in the dark, the binding site is more accessible to neutral water molecules than to charged protons.     

Restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from mapped loci of rice (Oryza sativa L.) genomic DNA

Summary Thirty mapped Indica rice genomic (RG) clones were partially sequenced from each end. From such sequence data, pairs of oligonucleotides were synthesized to act as primers for polymerase chain reaction (PCR) amplification of the corresponding loci in crude total DNA preparations. The PCR products from DNA of Indica varieties were of the sizes expected from the sizes of the corresponding RG clones. However, size polymorphisms were seen between PCR products from Indica and Japonica varieties, and among wildOryza species. Restriction fragment length polymorphism (RFLP) was observed between PCR products of Indica varieties simply by electrophoretic analysis of restricted products, without the need for Southern hybridization or radiolabelling. The RFLPs noted between varieties ARC6650 and Phalguna were inherited in recombinant inbred lines derived from a cross between them. The RFLPs were detectable in PCR products amplified from DNA extracted by a simple procedure from single seedlings or leaves, and revealed genetic heterogeneity in cultivated lines. An approach is described that is relevant to the acceleration of classical plant breeding through molecular techniques.     

Resonance Raman spectroscopy of octopus rhodopsin and its photoproducts

We report here the resonance Raman spectra of octopus rhodopsin and its photoproducts, bathorhodopsin and acid metarhodopsin. These studies were undertaken in order to make comparisons with the well-studied bovine pigments, so as to understand the similarities and the differences in pigment structure and photochemical processes between vertebrates and invertebrates. The flow method was used to obtain the Raman spectrum of rhodopsin at 13 degrees C. The bathorhodopsin spectrum was obtained by computer subtraction of the spectra containing different photostationary mixtures of rhodopsin, isorhodopsin, hypsorhodopsin, and bathorhodopsin, obtained at 12 K using the pump-probe technique and from measurements at 80 K. Like their bovine counterparts, the Schiff base vibrational mode appears at approximately 1660 cm-1 in octopus rhodopsin and the photoproducts, bathorhodopsin and acid metarhodopsin, suggesting a protonated Schiff base linkage between the chromophore and the protein. Differences between the Raman spectra of octopus rhodopsin and bathorhodopsin indicate that the formation of bathorhodopsin is associated with chromophore isomerization. This inference is substantiated by the chromophore chemical extraction data which show that, like the bovine system, octopus rhodopsin is an 11-cis pigment, while the photoproducts contain an all-trans pigment, in agreement with previous work. The octopus rhodopsin and bathorhodopsin spectra show marked differences from their bovine counterparts in other respects, however. The differences are most dramatic in the structure-sensitive fingerprint and the HOOP regions. Thus, it appears that although the two species differ in the specific nature of the chromophore-protein interactions, the general process of visual transduction is the same.