Synthesis and transdermal permeation-enhancing activity of ketone, amide, and alkane analogs of Transkarbam 12

Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium-5-(dodecyloxycarbonyl)pentylcarbamate, T12) is a highly active transdermal permeation enhancer. In this study, ketone, amide, and alkane analogs of T12 have been synthesized and evaluated for their permeation-enhancing activity using porcine skin and theophylline as a model drug. Replacement of ester by methylene and ketone, respectively, led to a significant decrease of activity. Amide analogs displayed lower activity in 60% propylene glycol and were comparable to T12 in isopropyl myristate. An intramolecular H-bond between ester and ammonium-carbamate group was suggested to be important for the permeation-enhancing activity of T12.     

Isosteric and non-isosteric modification of carboxyl groups of papain

Guanidinated mercuri-papain (Gu-papain) was reacted with N-ethylbenzisoxazolium tetrafluoroborate at pH 4.2, 0 degree C, to yield highly reactive N-ethylsalicylamide esters. On varying the amount of reagent applied 2.5-10 carboxyl groups were modified. Appropriate plotting of the data indicated that all 12 groups exposed in the X-ray structure were modified to an extent of 80% in the final preparation, concomitant with a similar loss of activity towards N alpha-benzoyl-L-arginine ethyl ester. The preparations regained complete activity on saponification of the ester groups and removal of some oligomeric material by gel filtration. Considerable activity was recovered when the ester groups were completely replaced by amide groups by subjecting the esters to ammonolysis in 2 M ammonium acetate/ammonia (pH 9.2). The final preparation, after gel filtration, exhibited Km = 57 +/- 1 mM and kcat = 26 +/- 0.2 s-1 towards BAEE (native papain Km = 18 mM and kcat = 26 s-1). It may be concluded that replacement of a bulky modifying group by an isosteric one may cause considerable recovery of activity, emphasizing the importance of isostericity in suppressing the ionizing ability of ionizable groups; furthermore, that a large shift in overall charge, caused by amidation of all accessible carboxyl groups, does not affect the catalytic steps. The absence of effect of side-chain charges on the ion pair in the active site is briefly discussed.     

An opportunistic promoter sharing regulatory sequences with either a muscle-specific or a ubiquitous promoter in the human aldolase A gene.

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.     

Synthesis and transdermal permeation-enhancing activity of carbonate and carbamate analogs of Transkarbam 12

Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium-5-(dodecyloxycarbonyl)pentylcarbamate, T12) is a highly effective skin permeation enhancer. In this study, ester groups in the molecule of T12 were replaced by carbonate and carbamate ones, respectively. The in vitro permeation-enhancing activities were evaluated using porcine skin and compared with those of T12 and previously prepared series of amide, ketone, and alkyl analogs. According to the activities and behavior of the compounds in donor samples, ester group is essential for the activity of T12; its replacement not only decreases the enhancing potency, but is likely to change the mechanism of action.     

Characterization of a cardiac-specific enhancer, which directs {alpha}-cardiac actin gene transcription in the mouse adult heart

Expression of the mouse alpha-cardiac actin gene in skeletal and cardiac muscle is regulated by enhancers lying 5' to the proximal promoter. Here we report the characterization of a cardiac-specific enhancer located within -2.354/-1.36 kbp of the gene, which is active in cardiocytes but not in C2 skeletal muscle cells. In vivo it directs reporter gene expression to the adult heart, where the proximal promoter alone is inactive. An 85-bp region within the enhancer is highly conserved between human and mouse and contains a central AT-rich site, which is essential for enhancer activity. This site binds myocyte enhancer factor (MEF)2 factors, principally MEF2D and MEF2A in cardiocyte nuclear extracts. These results are discussed in the context of MEF2 activity and of the regulation of the alpha-cardiac actin locus.     

A conserved enhancer element differentially regulates developmental expression of CD5 in B and T cells

We previously identified an enhancer element upstream of the mouse cd5 gene that was required in reporter assays for the induction of cd5 promoter activity by BCR cross-linking. This element is highly conserved in placental mammals. To determine its physiological role, we have now generated mice with a targeted deletion of the enhancer. The result is the loss of CD5 expression in peritoneal and splenic B-1a cells of adult mice and an inability to induce CD5 by cross-linking of the BCR on splenic B-2 cells. Surprisingly, CD5 expression on B-1a cells of neonatal mice was only minimally compromised. Cd5 enhancer deletion also had only a modest effect on CD5 expression in the T lineage. Thus, this enhancer provides age- and tissue-specific regulation of CD5 expression and is an example of the utilization of different modes of regulation of expression in T and B cells.