RELATIONSHIP BETWEEN NUCLEOSIDE DIPHOSPHATE KINASE ACTIVITY AND LIGHT-LIMITED GROWTH RATE IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Light-limited cultures of the marine diatom Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (3H clone) were grown over a range of growth rates between 0.06 and 1.64 d^?1. Variations in cell volume, cell quotas of carbon, nitrogen, and protein, and maximal activity of the enzyme nucleoside diphosphate kinase (NDPK) were measured and examined as a function of growth rate. NDPK from T. pseudonana showed K_m values of 0.24 and 0.68 mM for thymidine 5′-diphosphate and adenosine 5′-triphosphate (ATP), respectively, which are similar to those found for NDPK from a variety of organisms, from bacteria to mammals. An apparent activation enthalpy of 3.52 kCal·mol^?1 was determined from Arrhenius plots. No thermodynamic transition points were noted over a temperature range from 10° to 25°C. NDPK activity was significantly correlated with growth rate but not with cell volume, carbon, nitrogen, or protein; for interspecific comparisons, normalization of enzyme activity to cell number may be most meaningful. NDPK activity per cell versus growth rate followed a U-shaped relationship, being relatively constant between 0.5 and 1.0 d^?1 and rising at higher and lower growth rates. Over this range, enzyme activity may be regulated by substrate concentration (ATP or other nucleoside triphosphates) or by adenylate energy charge. At higher growth rates where energy charge and substrate concentrations are probably high, changes in enzyme concentration appear to be required. The reasons for a rise in enzyme activity at low growth rate is unclear. Simultaneous measurement of nucleoside di- and triphosphate levels alongside NDPK measurements may help clarify the relationship, but these preliminary experiments indicate that NDPK is of limited usefulness as an index of in situ growth rate.     

Loss of antibody productivity is highly reproducible in multiple hybridoma subclones

An immunoglobulin G (IgG(2b)) producing hybridoma cell line (S3H5/gamma2bA2) was cloned and subcloned. Twenty subclones were grown in parallel while being adapted in a stepwise fashion to serum-free medium. Following adaptation to serum-free medium, it was found that 16 of the 20 subclones remained at a relatively constant proportion of nonproducing cells. Three of the remaining subclones transiently deviated from this balance but eventually returned toward this population composition. One subclone continued to lose productivity. A population balance was reached at approximately 8% of the population being nonproducers. The loss of antibody productivity was thus highly reproducible. (c) 1993 John Wiley & Sons, Inc.     

Temperature dependence of thermal inactivation rate constants of bacterial spores in a glassy state

Summary Differential scanning calorimetry data obtained from corn embryos is consistent with the hypothesis of their glassy state. This work extends that hypothesis to explain the speculation about the high heat resistance of bacterial spores. By considering the protoplast to be in a glassy solid-state, it can be assumed that the configurational rearrangements of the key life dependent polymer chain backbones (DNA, etc.) are extremely slow, thereby ceasing thermal motions. It is assumed that at the glass transition temperature, the spore protoplast undergoes a discontinuity in the thermal expansion coefficient, and above this critical temperature, the rate of thermal inactivation of spores is free volume dependent and can be described adequately by the William, Landel and Ferry (WLF) equation. Glass transition temperatures forBacillus stearothermophilus andClostridium botulinum spores, obtained by fitting the inactivation rate data to the WLF equation, indicate a decrease in the inactivation rates with increasing glass-transition temperatures.     

Acid-Catalyzed nucleophilic substitution and racemization of 3-methoxy-N-desmethyldiazepam enantiomers in methanol

pK_a1 values of 3-methoxy-N-desmethyldiazepam in acetonitrile and methanol containing various acid concentrations were determined by spectrophotometry to be 3.5 and 1.3, respectively. Temperature-dependent racemization of enantiomeric 3-methoxy-N-desmethyldiazepam in methanol containing 0.5 M H₂SO₄ was studied by circular dichroism spectropolorimetry and the racemization reactions were found to follow apparent first-order kinetics. Thermodynamic parameters of the racemization reaction were found to be: E_act = 18.8 kcal/mol, and at 25°C: ΔH^? = 18.3 kcal/mol, ΔS^? = ?14.8 entropy unit, and ΔG^? = 22.7 kcal/mol, respectively. The racemization had an isotope effect (k_H/k_D) of 1.6 at 42°C. Based on the results of this report and those of earlier reports by other investigators, a nucleophilically solvated C3 carbocation intermediate resulting from either a P (plus) or an M (minus) conformation is proposed to be an intermediate and responsible for the stereoselective nucleophilic substitution and the subsequent racemization of 3-methoxy-N-desmethyldiazepam enantiomers. © 1993 Wiley-Liss, Inc.     

Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Determination of the coupling relationships between the respiratory reactions and miscellaneous observations concerning rate-limiting steps

The linear sequence of steps involved in the oxidation of extramitochondrial succinate by O₂ in bovine heart mitochondria was examined by a steady-state kinetic method to determine whether or not freely diffusible intermediates occur between the various inhibitor-sensitive steps. The kinetic method is based on the facts (1) that if two inhibitor-sensitive steps within a sequence are linked by a freely diffusible intermediate, inhibition of one will make the other less rate limiting in the overall reaction and thus will increase the amount of inhibitor of the other step required for half-maximal inhibition of the overall reaction, and (2) that if the two steps are not linked in this manner, inhibition of one will make the other more rate limiting and thus will decrease the amount of inhibitor of the other required for half-maximal inhibition. These two types of coupling relationships between steps were designated as sequential and fixed, respectively. The results indicate the existence of freely diffusible intermediates (sequential coupling relationships) between the succinate transport and succinate dehydrogenase reactions, between the succinate dehydrogenase and cytochromebc ₁ reactions, and between the cytochromesbc ₁ andaa ₃ reactions. Uncoupling respiration from phosphorylation results in the coupling relationship between thebc ₁ andaa ₃ reactions becoming partially fixed. This change is accompanied by marked decreases in the degrees to which thebc ₁ andaa ₃ reactions limit the overall reaction and appears to account for the large uncoupler-induced releases of inhibition at the levels of thebc ₁ andaa ₃ reactions observed previously by others. It is suggested that cytochromec is the freely diffusible intermediate between thebc ₁ andaa ₃ reactions and that the uncoupler-induced changes occur as a result of formation of functional and highly efficient supercomplexes between cytochromec and the cytochromesbc ₁ andaa ₃ complexes.     

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture

Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm²) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to platic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium. Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared in Clin. Res. 30: 93A; 1982.     

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm.Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.     

Opsonizing effect of serum and albumin gland extracts on the elimination of human erythrocytes from the circulation of Helix pomatia

The removal of human erythrocytes of the A₁ and B types from the circulation of the gastropod Helix pomatia follows an exponential curve. The elimination of the nonself particles is apparently dependent on serum opsonins as preincubation of A₁ and B erythrocytes in Helix serum increases the rate of their clearance. This conclusion is supported by our finding that secondary doses of nonsensitized A₁ erythrocytes injected 12–19 hr after a similar primary dose are cleared very slowly; however, the clearance rate returns to normal if erythrocytes comprising the second dose are preincubated with Helix serum. Furthermore, the elimination of second-dose A₁ erythrocytes is strongly enhanced after their pretreatment with agglutinating extracts of the albumin glands from H. pomatia and Cepaea (Helix) nemoralis. On the other hand, no opsonizing effect was obtained by pre-incubating A₁ erythrocytes in the agglutinating extract of the sponge Aaptos papillata.     

Mechanism of insulin aggregation and stabilization in agitated aqueous solutions

Undesirable aggregation of aqueous insulin solutions remains a serious obstacle in the development of alternative methods of diabetes therapy. We investigated the fundamental nature of the aggregation mechanism and proposed stabilization strategies based on a mathematical model for the reaction scheme. Insulin aggregation kinetics in the presence of solid-liquid and air-liquid interfaces were monitored using UV spectroscopy and quasielastic light scattering (QELS). Experimental observations were consistent with our model of monomer denaturation at hydrophobic surfaces followed by the formation of stable intermediate species which facilitated subsequent macroaggregation. The model was used to predict qualitative trends in insulin aggregation behavior, to propose stabilization strategies, and to elucidate mechanisms of stabilization. In the absence of additives, insulin solutions aggregated completely (more than 95% of the soluble protein lost) within 24 h; with sugarbased nonionic detergents, no detectable loss occurred for more than 6 weeks. (c) 1992 John Wiley & Sons, Inc.