Apinun Suprasert

Demonstration of substances of innate immunity in the integument of the Malayan pangolin ( Manis javanica )

Using immunohistochemistry, the study demonstrates substances of the innate immunity in the skin of the Malayan pangolin (Manis javanica), referring mainly to the epidermis. The results obtained showed clear reaction differences between the dorsolateral body region with its strong cover of hard horny scales and the abdominal body part with a thick soft stratum corneum and a dense cover of fine hairs. Regarding pathogen recognition receptors, positive reactions for Toll-like receptors were generally weak for TLR2, in contrast to TLR4, that exhibited strong reactions in the epidermis of both body regions, with increasing staining intensities towards the stratum corneum; ß-glucan receptors showed positive reactions only for l-ficolin and mannose-binding lectin, but not for dectin-1, and only at the abdomen. A generally positive staining for both body regions was obtained for all cationic antimicrobial peptides tested, whereby cathelicidin exhibited strong reaction intensities in all epidermal layers, ß-defensin 2 staining was often limited to the stratum basale and the stratum spinosum, and positive reactions for ß-defensin 3 appeared distinctly only in the epidermis of the abdomen; for these peptides, positive reaction staining could also be found in the outer epithelial root sheath of hair follicles, their glandular annexes, and free cells of the dermis. Lysozyme was found in the vital epidermis of both body regions studied, with strong staining limited to the dorsum; strong reactions were also visible in the hair follicles.

Immunohistochemical demonstration of keratins in the epidermal layers of the Malayan pangolin (Manis javanica), with remarks on the evolution of the integumental scale armour

Using immunohistochemistry, the study demonstrates the distribution of keratins (pankeratin with CK1-8, 10, 14-16, 19; keratins CK1, 5, 6, 9, 10; hair keratins AE13, AE14) in the epidermis of the Malayan pangolin (Manis javanica). A varying reaction spectrum was observed for pan-keratin, with body region-dependent negative to very strong reaction intensities. The dorsolateral epidermis exhibited positive reactions only in its vital layers, whereas the abdominal epidermis showed strong positive reactions in the soft two outer strata. The single acidic and basic-to-neutral (cyto)keratins produced clear variations compared to the pan-keratin tinging. For example, CK1 appeared in all epidermal layers of both body regions, except for the ventral stratum corneum, whereas CK5, 6, 9, 10 were restricted to the soft ventral epidermis. Here, distinctly positive reactions were confined to the stratum granulosum, except for CK6 that appeared in the soft stratum corneum. A different staining pattern was obvious for the hair keratins, i.e., positive reactions of AE13 concentrated only in the granular layer of the dorsal epidermis. In the abdominal epidermis, remarkable tinging for AE14 was visible in the stratum basale, decreasing toward the corneal layer, but was also found in the outer root sheath cells of the hair follicles in the ventral body part. Our findings are discussed related to the evolution of the horny dorsal scales of the pangolin, which may have started from the tail root, projecting forward to the head.

Characterization of Salmonella Typhimurium DNA gyrase as a target of quinolones

Quinolones exhibit good antibacterial activity against Salmonella spp. isolates and are often the choice of treatment for life-threatening salmonellosis due to multi-drug resistant strains. To assess the properties of quinolones, we performed an in vitro assay to study the antibacterial activities of quinolones against recombinant DNA gyrase. We expressed the S. Typhimurium DNA gyrase A (GyrA) and B (GyrB) subunits in Escherichia coli. GyrA and GyrB were obtained at high purity (>95%) by nickel-nitrilotriacetic acid agarose resin column chromatography as His-tagged 97-kDa and 89-kDa proteins, respectively. Both subunits were shown to reconstitute an ATP-dependent DNA supercoiling activity. Drug concentrations that suppressed DNA supercoiling by 50% (IC50 s) or generated DNA cleavage by 25% (CC25 s) demonstrated that quinolones highly active against S. Typhimurium DNA gyrase share a fluorine atom at C-6. The relationships between the minimum inhibitory concentrations (MICs), IC50 s and CC25 s were assessed by estimating a linear regression between two components. MICs measured against S. Typhimurium NBRC 13245 correlated better with IC50 s (R = 0.9988) than CC25 s (R = 0.9685). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test to identify quinolones with promising activity against S. Typhimurium. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and N-1 cyclopropyl substituents are desirable structural features in targeting S. Typhimurium gyrase.