Minimal nutritional requirements for immobilized yeast

The effect of reduced nutritional levels (particularly nitrogen source) for immobilized K. fragilis type yeast were studied using a trickle flow, "differential" plug flow type reactor with cells immobilized by adsorption onto an absorbant packing matrix. Minimizing nutrient levels in a feed stream to an immobilized cell reactor (ICR) might have the benefits of reducing cell growth and clogging problems in the ICR, reducing feed preparation costs, as well as reducing effluent disposal costs. In this study step changes in test feed medium nutrient compositions were introduced to the ICR, followed by a return to a basal medium. Gas evolution rates were monitored and logged on a continuous basis, and effluent cell density was used as an indicator of cell growth rate of the immobilized cell mass. Startup of the reactor using a YEP medium showed a rapid buildup of cells in the reactor during the initial 110 h operation. The population density then stabilized at 1.6 x 10(11) cells/g sponge. A defined medium containing a complex mix of essential nutrients with an inorganic nitrogen source (ammonium sulfate) was able to maintain 90% of the productivity in the ICR as compared to the YEP medium, but proved unable to promote growth of the immobilized cell mass during startup. Experiments on reduced ammonium sulfate in the defined medium, and reduced yeast extract and peptone in YEP medium indicated that stable productivity could be maintained for extended periods (80 h) in the complete absence of any nutrients besides a few salts (potassium phosphate and magnesium sulfate). It was found that productivity rates dropped by 35-65% from maximal values as nitrogenous nutrients were eliminated from the test mediums, while growth rates (as determined by shed cell density from the reactor) dropped by 75-95%. Thus, nutritional deficiencies largely decoupled growth and productivity of the immobilized yeast which suggests productivity is both growth- and non-growth-associated for the immobilized cells. A yeast extract concentration of 0.375 g/L with or without 1 g/L ammonium sulfate was determined to be the minimum level which gave a sustained increase in productivity rates as compared to the nutritionally deficient salt medium. This represents a 94% reduction in complex nitrogenous nutrient levels compared to standard YEP batch medium (3 g/L YE and 3.5 g/L peptone).     

Enzyme-Linked Immunosorbent Assay of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.     

Pseudouridine residues in the 5′-terminus of uridine-rich nuclear RNA I (U1 RNA)

The primary nucleotide sequence was reported earlier for U1 RNA (Reddy et al, (1974) J. Biol. Chem. $\text{249}$, 6486–6494), an snRNA implicated in splicing of HnRNAs. In view of the presence of homologous pseudouridine (ψ) residues in 5′-ends of several highly conserved U-snRNAs and the recent report of modified bases in the U1 RNA structure (Branlant et al, (1980) Nucleic Acids Res. $\text{8}$, 4143–4154) a study was made for the presence of ψ and other modified nucleotides in the 5′-end of the U1 RNA. Identification of ψ residues at positions 6 and 7, shows the 5′-sequence of U1 RNA is: m₃², 2,7 GpppAm-Um-A-C-ψ-ψ-A-C-C-U-G-G-C-A-G-G-G-G-A-G-A-U-A-C. The ψ residues in place of U at positions 6 and 7 may affect the binding of U1 RNA at intron-exon splice junctions.     

The distribution of two highly repeated DNA sequences within Drosophila melanogaster chromosomes

In situ hybridization using ³H-RNA probes has been used to localize the sequences found in two satellites of density 1.705 g/cc and 1.672 g/ cc to specific sites within the chromosomal complement. A detailed analysis of the sites on the X chromosome was carried out using the scute series of inversions to relate the heterochromatic breakpoint relative to the location of the sequence on this chromosome. It has also been possible to establish the order of arrangement of 1.705 and 1.672 DNA at the heterochromaticeuchromatic junction on chromosome 3(R). A mitotic map is provided. The T_m of hybrids formed in situ showed that the hybrids were representative of the sequences being analyzed. The two satellites also were traced through a number of purification procedures to show that a covalent linkage may be likely between the 1.705 g/cc and 1.672 g/cc satellite as predicted from in situ hybridization analyses.     

Estimating thermal properties of simulated aquatic soils from their composition

Heat penetration and thermal lag in the submersed soil surrounding the roots of aquatic plants depends on two fundamental thermal properties of the substrate, volumetric heat capacity (C_V) and thermal conductivity (k). The relationship of these parameters to the fractions of organic and mineral matter, gas and water in natural and simulated aquatic soils was investigated. The gas fraction was found to be insignificant and it was possible to make good estimates of C_V and k from a knowledge of substrate water content alone.     

The mechanism of Cl− transport at the plasma membrane ofChara corallina I. Cotransport with H+

Summary Cl^– transport into cells ofChara corallina was studied in relation to that of other ions which have been proposed as cosubstrates for the Cl^– transport system. Although there appears to be a partial mutual dependence between K⁺ and Cl^– for transport in intact cells, this is not apparent in cells which have been perfused internally. Moreover, in intact cells, the fluxes of K⁺ and Cl^– show a large degree of independence in their responses to Cl^– starvation. Cl^– transport is electrogenic in a direction indicating the transport of excess positive charge into the cell. In the absence of any other likely counter ion, it is suggested that Cl^– is cotransported with H⁺. Response of Cl^– influx to internal and external pH in perfused cells is consistent with this suggestion. There appears, in addition, to be a role for ATP in transport as judged by fourfold stimulation of Cl^– influx in perfused cells when 1mm ATP is incorporated in the perfusion medium.     

The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer.

The measurement of the efficiency of F?rster long-range resonance energy transfer between donor (D) and acceptor (A) luminophores attached to the same macromolecular substrate can be used to estimate the D-A separation, R. If the D and A transition dipoles sample all orientations with respect to the substrate (the isotropic condition) in a time short compared with the transfer time (the dynamic averaging condition), the average orientation factor less than K2 greater than is 2/3. If the isotropic condition is not satisfied but the dynamic averaging condition is, upper and lower bounds for less than K2 greater than, and thus R, may be obtained from observed D and A depolarizations, and these limits may be further narrowed if the transfer depolarization is also known. This paper offers experimental protocols for obtaining this reorientational information and presents contour plots of less than K2 greater than min and less than K2 greater than max as functions of generally observable depolarizations. This permits an uncertainty to be assigned to the determined value of R. The details of the D and A reoreintational process need not be known, but the orientational distributions are assumed to have at least approximate axial symmetry with respect to a stationary substrate. Average depolarization factors are derived for various orientational distribution functions that demonstrate the effects of various mechanisms for reorientation of the luminophores. It is shown that in general the static averaging regime does not lend itself to determinations of R.     

The interaction of the polyene antibiotic lucensomycin with cholesterol in erythrocyte membranes and in model systems. III. Characterization of spectral parameters.

The variations of optical density and fluorescence of lucensomycin are good indices of the binding of this polyenic antibiotic to membranes. The former parameter reflects more generally the binding to any site present in the membrane, while the latter is more specific for binding to cholesterol. The chromophore of the lucensomycin-cholesterol complex has a relatively long lifetime, is almost immobile in the membrane, and is not accessible to water-soluble fluorescence-quenching agents. The stoichiometry, evaluated fluorometrically, corresponds to about two cholesterol molecules per polyene. In colloidal cholesterol suspensions, the extent of binding as a function of free polyene concentration is described by rectangular hyperbolae, the dissociation constant being, however, dependent on the sterol concentration. In erythrocyte membranes, on the other hand, and even more markedly in model systems containing appropriate solvents, the combination between lucensomycin and the sterol sites is described by sigmoid titration curves, indicative of cooperative effects, and probably due to solvation of cholesterol.