Temporal characterization of growth of fathead minnow (Pimephales promelas) larvae during sublethal hydrogen cyanide exposure

Growth of fathead minnow yolk sac larvae was characterized from changes in dry weight and total content and concentrations of RNA, DNA and protein in fish exposed to a sublethal level of HCN (58 micrograms/l) and in age matched controls. Cyanide toxicosis occurred within 24 hr of exposure as evidenced by significant reductions in protein and RNA content and RNA/DNA ratio of larvae. After 96 hr exposure to HCN, larvae exhibited the same growth rate and protein synthetic rate (RNA/DNA) as control fish. HCN toxicosis and recovery is rapid and at least partial tolerance to HCN develops within 96 hr of exposure in larval fathead minnows.     

Two color light scattering identifies physical differences between lymphocyte subpopulations

Forward angle light scattering of two different wavelengths by cells in a flow cytometer was used to investigate physical differences between lymphocytes of different lineage, functional subclass and developmental stage. Correlation of the ultraviolet (UV: 351 nm and 364 nm) and 488 nm light scattering signals produced by lymphoid cells demonstrated that the two signals were not equivalent and that they placed different emphasis on the physical parameters characterizing lymphocytes. Both small T and B lymphocytes from peripheral lymphoid tissues and mitogenically activated large T and B lymphocyte blasts were discriminated by both wavelengths. Differences between the Lyt-2 negative and Lyt-2 positive T lymphocyte subsets were also apparent. Two color light scattering could also discriminate between immature thymocytes and mature peripheral T cells and between small bone marrow cells and mature peripheral B cells. In bone marrow an increase in UV light scattering coincided with the appearance of cell surface immunoglobulin on small cells. These data establish that two color light scattering is a sensitive probe for distinguishing cells of apparently similar morphology and that it can be used to study the physical changes that occur during lymphoid cell differentiation.     

Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.     

Energy and biological evolution—I. The equilibrium states of biochemical processes

The Thom gradient model of morphogenesis poses the followinga posteriori problem: “From the observed morphology of a given natural process (effect) determine the dynamics of the process (cause)”. In this paper we consider the classicala priori problem: “Given the cause (dynamics) determine the effect (resultant morphology)”. We find that in biochemical processes the mechanisms for energy activation, energy-matter interaction and energy dissipation determine the dynamics. Furthermore there exists basic energy mechanisms which drive the equilibrium states through the elementary catastrophes of Thom. A comparison with current theories shows that our models describe open ecological food chains and their dynamical systems generalize the equations of organisation posed by M. Eigen. Work supported by a Research Associateship of the International Centre for Theoretical Physics, P.O.B. 586, Miramare, 34100 Trieste, Italy.     

Rapid electrocatalytic procedure for hydrogenase kinetic determination in the H2 evolution direction

The linear sweep voltammetric method is used as a new approach for kinetic determination with enzymes accepting reversible redox couples as cosubstrate. A monolayer of hydrogenase molecules is grafted onto a glassy carbon electrode which is both the support of the enzyme and the detector of the activity. Reduced viologen concentration in the enzyme microenvironment is controlled by the electrode potential. The catalytic current produced by the enzyme allows an easy kinetic constant determination without the classical constraints found in hydrogenase kinetic measurements.     

Membrane chloride transport measured using a chloride-sensitive fluorescent probe

Transport of chloride across cell membranes through exchange, cotransport, or conductive pathways is a subject of great biological importance. Current methods of measurement are restricted in their sensitivity, time resolution, and applicability. A new transport measurement technique has been developed on the basis of the fluorescence quenching by chloride of the dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). SPQ fluorescence quenching by chloride is rapid (less than 1 ms) and sensitive, with a greater than 50% decrease in fluorescence at 10 mM chloride. SPQ fluorescence is not altered by other physiological anions or by pH and can be used to measure both neutral and conductive transport processes. The high water solubility and membrane permeability properties of SPQ make it ideal for use in both membrane vesicles and cells. Chloride transport determined with SPQ was validated by measurement of erythrocyte chloride/anion exchange and membrane vesicle chloride conductance.     

Purification and characterization of a membrane-bound protein kinase from spinach thylakoids

A protein kinase was isolated from spinach thylakoid membranes by solubilization with octyl glucoside and cholate. The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, and sucrose density centrifugation, followed by affinity chromatography on either Affi-Gel blue (yielding denatured enzyme) or on histone cross-linked to Sepharose (yielding active enzyme). Electrophoresis on denaturing polyacrylamide gels, followed by staining with silver, revealed the kinase as a single band corresponding to an apparent molecular mass of 64 kDa. The active enzyme underwent autophosphorylation and could be detected by autoradiography following incubation with [gamma-32P]ATP and Mg2+ ion. The specific phosphotransferase activity of purified kinase was approximately 30 nmol of phosphate min-1 (mg protein)-1 with lysine-rich histone (III-S or V-S) as substrate; casein was phosphorylated at approximately 30% of this rate. The physiological substrate for the kinase is presumed to be light-harvesting chlorophyll a/b protein complex. In solubilized form, this was phosphorylated at approximately 10% of the rate observed with histone III-S as substrate, or 10-100 times slower than the estimated rate of phosphorylation of the light-harvesting complex in situ. Possible reasons for this shortfall are considered. The kinase is proposed as the principal effector of thylakoid protein phosphorylation and associated State transition phenomena.