Chiral HPLC with carbohydrate carbamates: Influence of support structure on enantioselectivity

The effect of particle size and pore size of the aminopropylated silica support for cellulose tris(phenylcarbamate) and tris(3,5-dimethylphenylcarbamate) chiral HPLC phases was investigated. It was necessary to reduce phase loading below 20% w/w as pore size and particle size were reduced, but high efficiency columns could be prepared at a 15% w/w loading on 5 and 2.5 μm supports with 120-Å-diameter pores. The 2.5 μm phase permits the use of relatively high flow rates and very efficient enantioselective separations of a range of chiral compounds could be achieved in less than 3 min. © 1994 Wiley-Liss, Inc.     

Cloning of proline-specific endopeptidase gene from Flavobacterium meningosepticum: expression in Escherichia coli and purification of the heterologous protein

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing. According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F. meningosepticum genomic DNA, employing the polymerase chain reaction technique. This fragment served as a molecular probe to isolate the respective gene. DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids. The coding region was cloned in different expression vectors of Escherichia coli. Transformed E. coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space. Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E. coli cells. Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E. coli cells is described. Correspondence to: G. Reipen     

Origin and clonal relationship of common inhibitory motoneurons CI1 and CI3 in the locust CNS

In the primordial thoracic ganglia of locust embryos, the bromodeoxiuridine (BrdU) technique for labelling proliferating cells and their progeny was combined with intracellular dye injection to investigate the origin and the clonal relationship of common inhibitory motoneurons. Common inhibitors 1 (CI₁) and 3 (CI₃) were found to be siblings, that is, they are produced by the division of one ganglion mother cell. This ganglion mother cell results from the first division of neuroblast 5–5, at about 30% of embryonic development. A large portion, at least, of the ganglion mother cells produced by subsequent divisions of neuroblast 5–5 give rise to interneurons with contralaterally ascending or descending axons and GABA-like immunoreactivity. Thus, CI₁ and CI₃ are more closely related to putative inhibitory interneurons than they are to other, that is, excitatory, motoneurons. Consistent with this, the CI somata are associated with cell bodies of putative inhibitory interneurons rather than with clusters of excitatory motoneuron somata. These results elicit speculations regarding the evolutionary origin of inhibitory motoneurons. 1994 John Wiley & Sons, Inc.     

Slow cortical potential biofeedback and the startle reflex

The negativity of slow cortical potentials (SCP) of the surface EEG is a measure of brain excitability, correlating with motor and cognitive preparation. Selfcontrol of SCP positivity has been shown to reduce seizure activity. Following SCP biofeedback from a central EEG electrode position, subjects gained bidirectional control over their SCP. The current study used a modified feedback methodology, and found a positive relationship between negativity and magnitude of EMG startle response (a measure of cortical and subcortical arousal, particularly aversive response disposition). Greater success in SCP differentiation was associated with self-report of less relaxation during negativity training.This research was supported by the Deutsche Forschungsgemeinschaft under grant No. SFB 307.     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Effect of prostaglandin E1 on chemiluminescence response and adherence of human polymorphonuclear leukocytes to human cultured endothelial cells of prostaglandin E1 treated polytraumatized patients

We investigated the effect of prostaglandin E1 on human polymorphonuclear leukocytes, in vivo. Polymorphonuclear leukocytes of a prostaglandin E1 and placebo study group were harvested and their function, as production of oxygen-derived metabolites and adherence to human cultured endothelial cells, was compared. Additionally, data obtained from polymorphonuclear leukocytes of a prostaglandin E1 and placebo group were compared with data obtained from polymorphonuclear leukocytes from 28 blood donors, who served as a control group. Production of oxygen-derived metabolites by polymorphonuclear leukocytes during contact with endothelial cells was measured by chemiluminescence. Chemiluminescence was significantly (p < 0.01) increased in the placebo group in comparison to the control group decreasing to values of control group after 6 d (post-trauma). Chemiluminescence response was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. Adherence of polymorphonuclear leukocytes (placebo group) to endothelial cells was significantly increased (p < 0.01) within the first 6 d post-trauma Following day 6, values were in the same range as values for the control group. Adherence was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. In conclusion, prostaglandin E1 at a dose of 20 ng/kg bw/min does not influence production of oxygenderived metabolites and adherence in polytraumatized patients in comparison to a placebo group. Additionally, production of oxygen-derived metabolites by polymorphonuclear leukocytes in response to endothelial cells is shown and it is evident that endothelial cells might influence production of oxygen derived metabolites by polymorphonuclear leukocytes.     

The agony of choice: Impact of the host animal species on the enzyme-linked immunosorbent assay performance for host cell protein quantification

Host cell proteins (HCPs) are inevitable process-related impurities in biotherapeutics commonly monitored by enzyme-linked immunosorbent assays (ELISAs). Of particular importance for their reliable detection are the anti-HCP polyclonal antibodies (pAbs), supposed to detect a broad range of HCPs. The present study focuses on the identification of suitable host animal species for the development of high-performance CHO-HCP ELISAs, assuming the generation of pAbs with adequate coverage and specificity. Hence, antibodies derived from immunization of sheep, goats, donkeys, rabbits, and chickens were compared concerning their amount of HCP-specific antibodies, coverage, and performance in a sandwich ELISA. Immunization of sheep, goats, donkeys, and rabbits met all test criteria, whereas the antibodies from chickens cannot be recommended based on the results of this study. Additionally, a mixture of antibodies from the five host species was prepared to assess if coverage and ELISA performance can be improved by a multispecies approach. Comparable results were obtained for the single- and multispecies ELISAs in different in-process samples, indicating no substantial improvement for the latter in ELISA performance while raising ethical and financial concerns.     

Chromosomal mapping in the mouse of eight K-channel-genes representing the four Shaker-like subfamilies Shaker, Shab, Shaw, and Shal

The four Shaker-like subfamilies of Shaker-, Shab-,Shaw-, and Shal-related K⁺ channels in mammals have been defined on the basis of their sequence homologies to the corresponding Drosophila genes. Using interspecific backcrosses between Mus musculus and Mus spretus, we have chromosomally mapped in the mouse the Shaker-related K⁺-channel genes Kcna1, Kcna2, Kcna4, Kcna5, and Kcna6; the Shab-related gene Kcnb1; the Shaw-related gene Kcnc4; and the Shal-related gene Kcnd2. The following localizations were determined: Chr 2, cen-Acra-Kcna4-Pax-6-a-Pck-1-Kras-3-Kcnb1 (corresponding human Chrs 11p and 20q, respectively); Chr 3, cen-Hao-2-(Kcna2, Kcnc4)-Amy-1 (human Chr 1); and Chr 6, cen-Cola-2-Met-Kcnd2-Cpa-Tcrb-adr/Clc-1-Hox-1.1-Myk-103-Raf-1-(Tpi-1, Kcna1, Kcna5, Kcna6) (human Chrs 7q and 12p, respectively). Thus, there is a cluster of at least three Shaker-related K⁺-channel genes on distal mouse Chr 6 and a cluster on Chr 2 that at least consists of one Shaker-related and one Shaw-related gene. The three other K⁺-channel genes are not linked to each other. The map positions of the different types of K⁺-channel genes in the mouse are discussed in relation to those of their homologs in man and to hereditary diseases of mouse and man that might involve K⁺ channels.