Quantitative analysis of activation and inactivation of asymmetry currents in biological membranes,based on a conformational transition model

Summary A basic voltage-dependent conformational transition mechanism is proposed. It comprises one relatively fast conversion between two individual states which are comparatively slowly coupled with a third state. Having introduced voltage as an additional parameter of state, standard methods of thermodynamics and rate theory are employed to describe the equilibrium and kinetic behavior of the system. In particular, a quantitative discussion is given regarding the asymmetrical displacement currents generated by switching on and off a voltage pulse. Effects of temperature pulse duration, and application of a conditioning prepulse are examined. The results provide a comprehensive basis for a quantitative analysis of pertinent experimental work. The so far presented measuring data can indeed be very well described along these lines.     

High precision immunoscanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold.

Ultra-small colloidal gold (less than 1 nm), bound to Fab fragments provides the shortest practical specific marker system to date and can be used in concert with field emission scanning electron microscopes to precisely locate antigenic sites. An "in-lens" FE-SEM equipped with a highly sensitive single crystal YAG-detector for backscattered electrons, as well as the use of advanced specimen preparation techniques based on cryofixation, are among the indispensible prerequisites. A T-even type Escherichia coli bacteriophage, Tu II*-46, was chosen to study properties of the immunogold labeling system. Distinct regions on the tail fibers of this phage were labeled with Fab fragments derived from antibodies against the related phage Tu II*-6. The tail fibers are composed of pairs of homologous proteins, thus offering two identical antigenic sites at the same locus on the tail fibers. Fab fragments can be visualized in the SEM at high accelerating voltage (30 kV) without any additional marker. This permits comparison of the labeling characteristics of unmarked and colloidal gold-marked Fab fragments. Unmarked Fab fragments often bind by pairs (two singlet Fab fragments bound opposed to each other along the axis of the tail fiber). The labeling efficiency of unmarked Fab fragments was greater than that of ultra-small gold-labeled Fab fragments. Binding by pairs was not seen after labeling with ultra-small gold-Fab fragments. The conjugates used in this study exhibited one colloidal gold per Fab fragment.     

A two-step enzymatic synthesis of dipeptides

A simple system is introduced to produce dipeptides continuously by enzyme catalyzed condensation of amino acid esters and amino acid amides. Synthesis of N-terminal free dipeptide-amides is achieved by means of carboxypeptidase Y. The peptide-amide is deamidated utilizing a newly isolated peptide-amide is deamidated utilizing a newly isolated peptide-amidase. Separation of substrates and products is accomplished by anion-exchange chromatography. Modeling of the reactions shows that the two reactions have to be carried out in a cascade of two reactors in order to prevent hydrolysis of the peptide by the carboxypeptidase. Continuous production of Kyotorphin (H-TyrArg-OH) with a space-time yield of 257 g/L . d shows the feasibility of this concept.     

A Cyanobacterial Gene Encoding Peptidyl-Prolyl cis-trans Isomerase

The rotA gene encoding peptidyl-prolyl cis-trans isomerase has been identified, sequenced, and shown to be transcribed in the cyanobacterium Synechococcus sp. PCC7942. Inactivation of the gene by replacement of a region containing the open reading frame with a gene conferring kanamycin resistance resulted in merodiploids containing both the wild type and the modified genomic region. We were not able to isolate a kanamycin-resistant mutant in which all the genomic wild-type copies were substituted, which suggests that such replacement could have been lethal.     

Inositol tetrakisphosphates as second messengers induce Ca(++)-dependent chloride currents in Xenopus laevis oocytes.

Microinjection of inositol 1,3,4,5-tetrakisphosphate or inositol 1,4,5-trisphosphate induced distinct chloride membrane currents in defolliculated Xenopus laevis oocytes. To decide whether these Cl(-)-currents were due to the injected compounds or their metabolic products, [3H]Ins(1,3,4,5)P4 or [3H]Ins(1,4,5)P3 were injected into oocytes and their metabolites were analyzed by HPLC. Our results indicate that Ins(1,3,4,5)P4 itself or its metabolite Ins(1,3,4,6)P4 is able to induce Cl(-)-membrane currents, most likely by increasing the cytosolic Ca(++)-concentration.