Fickean and Non-Fickean Water Desorption During Vacuum Drying of <Emphasis Type="Italic">L. bulgaricus</Emphasis>

The recovery of Lactobacillus bulgaricus was studied in correlation to the kinetics of cell drying. When bacteria were dehydrated at 30 °C, either in the presence or the absence of sucrose, the drying kinetics corresponds to a Fickean diffusion in correspondence with a short lag time. In contrast, when the bacteria were dehydrated at 70 °C in the absence of sugar, the kinetics corresponds to an anomalous diffusion, and the lag time is four to five times higher than that at 30 °C. However, when drying at 70 °C was carried out in the presence of sucrose, drying kinetics turned into a Fickean process parallel to a substantial decrease in the lag time. The pattern of water desorption was correlated with the critical water activity. When the drying kinetics corresponds to a Fickean diffusion, the lag time started to increase at 0.7 water activity, but when the cells were dried at 70 °C, the damage started at 0.5 water activity. This observation indicates that the drying rate affects the pattern of water desorption, and it can change the value of critical water activity. These results put into relevance that the cell recovery is due to the drying history and that the recovery increase produced by sucrose can be related to the maintenance of kinetic barriers for water desorption.     

Effect of glucose feeding on net transport of plasma free fatty acids

The effect of a single glucose feeding upon the net inflow and outflow transport of plasma free fatty acids (FFA) has been studied in 75 unanesthetized rats. The animals were fasted for 22 +/- 2 hr; then 50 rats were refed 2 ml of 50% glucose by gastric intubation. At 0, 10-15, and 30-35 min after glucose refeeding, the rats were injected with palmitate-1-(14)C complexed to rat serum. The tracer dose included (131)I-labeled albumin. Plasma FFA concentration, (131)I concentration, and FFA-(14)C were measured at five time intervals after injection of the tracer dose. From these data the irreversible disposal rate, or net outflow transport, and the net inflow transport of plasma FFA were calculated. Estimations were based upon a special case of a general solution for measuring net inflow and outflow transport of a circulating metabolite. The general solution is independent of the number of compartments, how they are interconnected, the number of nonradioactive inflows, and where the inflows enter the system. Net inflow = net outflow transport = 7.6 micro eq/min in the fasted state and 3.5 micro eq/min in the new steady state that is reached 30-40 min after glucose refeeding. A very slight imbalance between the rates of net inflow and outflow transport could account for the rapid fall in plasma FFA concentration that results from a single glucose feeding. Theoretical and practical problems associated with studying inflow and outflow transport by means of the technique using a single injection of racer are discussed.     

Amino-Terminal Charge Affects the Periplasmic Accumulation of Recombinant Heregulin/EGF Hybrids Exported Using theEscherichia coliAlkaline Phosphatase Signal Sequence

AnEscherichia coliexpression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA–hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated byin vitroreplacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein inE. coliperiplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed inE. coliusing the PhoA signal sequence for protein export.     

Studies on anaerobic bacteria

Summary A new chromogenic anaerobe, Clostridium roseum nov. spec., has been found. It is characterized by: red-orange pigment, turning purplish on oxidation; gelatin liquefaction and other evidence of proteolysis; nitrate reduction; fermentation of various carbohydrates including pectin; close resemblance to Cl. acetobutylicum in corn mash fermentation, with the same neutral products, acetone, ethyl alcohol and butyl alcohol, in nearly the same ratios; agglutinative specificity and separation from Cl. acetobutylicum and Cl. felsineum, as well as several less nearly physiologically related butyric anaerobes.