Controlled delivery of lipophilic agents to cell cultures for in vitro toxicity and biocompatibility assays.

In this report, we present a novel method for delivering lipophilic compounds to cell cultures. The delivery system is based on a nanoemulsion stabilized by phospholipids. These nanoemulsions are well tolerated by cell cultures, such as TK6 lymphoblastoid cells and can be used to deliver defined amounts of encapsulated lipophilic compounds into cells. We measured the growth inhibition of TK6 lymphoblastoid cells caused by different oils, UV-filters and fragrances to determine the biocompatibility or the toxicity of these compounds in simple cell culture experiments. Our data show that the applied nanoemulsion technology is also very suitable to study biological effects of the UV-A-irradiated compounds in cell culture assays.

Improving skin function with CM-glucan, a biological response modifier from yeast.

Preparations from yeast have been used for a long time for cosmetic and pharmaceutical purposes. Studies have identified glucan from the cell wall of baker's yeast as an immunologically active agent. Glucan is a poly beta-( 1-3)-linked glucopyranose of high molecular weight and belongs to the class of compounds known as biological response modifiers. Glucan preparations are involved in the activation of the body's natural defence mechanisms and in the acceleration of the skin's wound healing processes. In the skin, Langerhans' cells and keratinocytes are the immunologically competent cells. Recent studies indicate that UV irradiation can deplete the number and viability of these cells (immunosuppression). The use of non-specific immune-stimulators, such as glucan, is a new approach for improving the function of stressed skin. We have developed a process to modify pure glucan from baker's yeast to carboxymethyl glucan (CM-glucan), a water soluble product suitable for topical formulations. The functional properties of this new compound have been investigated in vitro and in vivo. Cell culture experiments showed that CM-glucan protects skin cells against the depletion of antioxidant molecules upon UV-A irradiation and promotes the growth of keratinocytes. In placebo controlled studies with healthy volunteers, the pretreatment of skin with CM-glucan offered substantial protection against skin damage caused by a detergent challenge or UV-A irradiation. In addition, CM-glucan enhanced the renewal rate of the stratum corneum.

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, XI. Engineering thermostability and activity of lactate dehydrogenases from bacilli.

An extensive comparative structural analysis of lactate dehydrogenase (LDH) sequences from thermophilic, mesophilic and psychrophilic bacilli revealed characteristic primary structural differences. These specific amino-acid substitutions were found in the entire LDH molecule. However, in certain regions of the LDH an accumulation of these exchanges could be detected. These regions seem to be particularly important for the temperature adaptation of the enzyme. The influence of one of such regions at the N-terminus on stability and activity of LDHs was analysed by the construction of hybrid mutants between LDH sequences from thermophilic, mesophilic and psychrophilic bacilli and also by site-directed mutagenesis experiments at five different positions. The substitutions of Thr-29 or Ser-39 to Ala residues in the LDH from the mesophilic B. megaterium increased the thermostability of the enzyme drastically (15 degrees C). An increase of 20 degrees C could be observed when both amino-acid substitutions were introduced. These amino-acid substitutions resulted in an increase of Km for pyruvate and led to a three-fold reduction of the activity (kcat/Km) at 40 degrees C compared with the wild type enzyme. The influence of these amino-acid substitutions was also investigated in the LDHs from thermophilic and psychrophilic bacilli. The high heat resistance of the LDH from the thermophilic B. stearothermophilus was not altered by the Ala to Thr and Ser substitutions at positions 29 and 39, respectively. This indicates a cooperatively stabilized conformation of this LDH. However, in this mutant of the B. stearothermophilus LDH the activity (kcat/Km) was increased two-fold.

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, X. Analysis of structural elements responsible for the differences in thermostability and activation by fructose 1,6-bisphosphate in the lactate dehydrogenases from B. stearothermophilus and B. caldolyticus by protein engineering.

The amino-acid sequences of the lactate dehydrogenases (LDH) from B. stearothermophilus and B. caldolyticus differ at only 10 positions. The properties of these enzymes however show substantial differences. The LDH from B. stearothermophilus is activated by Fru-P2 and has a higher thermostability (10 degrees C) than the enzyme from B. caldolyticus which cannot be activated by Fru-P2. To correlate these functional differences to the structural properties, we have constructed a set of hybrid- and point-mutants of the two LDHs. The amino acids at positions 207, 209B, and 209C could be identified to confer the property of activation by Fru-P2 to the enzymes. This part of the enzyme is to a large extent also responsible for the different thermostabilities of these two proteins.

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, VI. Nucleotide sequences of lactate dehydrogenase genes from the thermophilic bacteria Bacillus stearothermophilus, B. caldolyticus and B. caldotenax.

Based on the previously determined amino-acid sequence of lactate dehydrogenase from B. stearothermophilus, an oligonucleotide probe was synthesized and used to clone the structural genes for lactate dehydrogenase from B. stearothermophilus, B. caldolyticus and B. caldotenax. The nucleotide sequences of the entire LDH genes from these three thermophilic bacilli were determined by the method of Maxam and Gilbert. The nucleotide sequence of the LDH gene from B. stearothermophilus is exactly identical to the one published recently; it agrees with the experimentally determined amino-acid sequence except at three positions. The amino-acid homologies among these thermophilic enzymes are 90% or more. The LDH genes are efficiently expressed in E. coli.

Purification, amino-acid sequence and some properties of the ferredoxin isolated from Bacillus acidocaldarius.

Ferredoxin was isolated from the aerobic, thermophilic and acidophilic bacterium Bacillus acidocaldarius and its sequence of 78 amino acids completely determined by automated Edman degradation of the protein and of peptides derived from chemical cleavage between aspartic acid and proline and from enzymatic digestions. The optical spectrum of the oxidized protein has a broad maximum around 400 nm. The ferredoxin is thermostable: its absorbance begins to decrease only at incubation over 71 degrees C. The number of iron and inorganic sulphur atoms per molecule was determined to be 5.3 and 5.0, respectively. The calculated molar extinction coefficient was 23 000 M-1 X cm-1, the molecular mass of the apoferredoxin 8 872 Da. Contrary to all expectations, the sequence of B. acidocaldarius ferredoxin shows very little homology to that of B. stearothermophilus but closely resembles that of Thermus thermophilus.