Ex Vivo Reconstruction of the Epidermis With Melanocytes and the Influence of UVB

To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm²× 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.     

Organocatalysis with endogenous compounds: Towards novel non-enzymatic reactions

The aldol reaction of the endogeneous compounds acetone and methylglyoxal has been studied using organocatalysis in relation to biologically relevant non-enzymatic reactions. Under preparative conditions, 3-hydroxy-2,5-hexadione, known as Henze’s ketol, is formed in high yield and with enantioselectivities up to 88% ee. Furthermore, Henze’s ketol is also formed under simulated physiological conditions at micromolar scale, indicating that this reaction might take place in living organisms.     

The Proteolytic Potential of Normal Human Melanocytes: Comparison With Other Skin Cells and Melanoma Cell Lines

To understand the contribution of epidermal melanocytes in the proteolytic potential of human skin, we have studied melanocytes grown in a low-serum medium deprived of phorbol esters, cholera toxin, and other non-physiological supplements. We focused on the plasminogen activation system and certain matrix metalloproteinases (gelatinases). Supposing that the proteolytic activity of cells can influence binding to collagen matrix and its reorganization, we have analyzed these parameters as well. We found that human melanocytes secreted tissue-type plasminogen activator and utilised it to generate cell-bound plasmin. No urokinase-type plasminogen activator was detected in the cultures but its receptor was found in cell extracts. Both the 72 kDa and 92 kDa gelatinases were secreted by the cells and in equal amounts. In addition, melanocytes secreted the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Melanocytes cast into collagen matrices retained a rounded morphology, did not extend processes, and were unable to contract collagen lattices. As a control, these parameters were investigated in parallel in cultures of human keratinocytes, dermal fibroblasts, and two melanoma cell lines. The obtained characteristics suggest that normal human melanocytes are proteolytically active cells. This function may pertain to skin physiology and pathophysiology.     

Pardaxin Permeabilizes Vesicles More Efficiently by Pore Formation than by Disruption

Pardaxin is a 33-amino-acid neurotoxin from the Red Sea Moses sole Pardachirus marmoratus, whose mode of action shows remarkable sensitivity to lipid chain length and charge, although the effect of pH is unclear. Here we combine optical spectroscopy and dye release experiments with laser scanning confocal microscopy and natural abundance ¹³C solid-state nuclear magnetic resonance to provide a more complete picture of how pardaxin interacts with lipids. The kinetics and efficiency of release of entrapped calcein is highly sensitive to pH. In vesicles containing zwitterionic lipids (PC), release occurs most rapidly at low pH, whereas in vesicles containing 20% anionic lipid (PG), release occurs most rapidly at high pH. Pardaxin forms stable or transient pores in PC vesicles that allow release of contents without loss of vesicle integrity, whereas the inclusion of PG promotes total vesicle collapse. In agreement with this, solid-state nuclear magnetic resonance reveals that pardaxin takes up a trans-membrane orientation in 14-O-PC/6-O-PC bicelles, whereas the inclusion of 14-0-PG restricts it to contacts with lipid headgroups, promoting membrane lysis. Pore formation in zwitterionic vesicles is more efficient than lysis of anionic vesicles, suggesting that electrostatic interactions may trap pardaxin in several suboptimal interconverting conformations on the membrane surface.     

Simultaneous determination of tolbutamide,omeprazole, midazolam and dextromethorphan in human plasma by LC–MS/MS—A high throughput approach to evaluate drug–drug interactions

Drug–drug interactions involving cytochrome P450 (CYP450s) are an important factor for evaluation of a new chemical entity (NCE) in drug development. To evaluate the potential inhibitory effects of a NCE on the pharmacokinetics of a cocktail of representative probes of CYP enzymes (midazolam for CYP3A4, tolbutamide for CYP2C9, omeprazole for CYP2C19 and dextromethorphan for CYP2D6) and the safety and tolerability of the NCE in the presence of probe substrates, a high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma using tolbutamide-d₉, midazolam-d₄, (±)-omeprazole-d₃, and dextromethorphan-d₃ as the internal standards (ISs). Human plasma samples of 50 μL were extracted by a simple protein-precipitation procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. Reversed-phase HPLC separation was achieved with a Hypersil GOLD AQ column (50 mm × 4.6 mm, 5 μm). MS/MS detection was set at mass transitions of 271 → 172 m/z for tolbutamide, 346 → 198 m/z for omeprazole, 326 → 291 m/z for midazolam, 272 → 171 m/z for dextromethorphan, 280 → 172 m/z for tolbutamide-d₉ (IS), 349 → 198 m/z for (±)-omeprazole-d₃ (IS), 330 → 295 m/z for midazolam-d₄ (IS), and 275 → 171 m/z for dextromethorphan-d₃ (IS) in positive mode. The high throughput LC–MS/MS method was validated for accuracy, precision, sensitivity, stability, recovery, matrix effects, and calibration range. Acceptable intra-run and inter-run assay precision (<10%) and accuracy (<10%) were achieved over a linear range of 50–50,000 ng/mL for tolbutamide, 1–1000 ng/mL for omeprazole, 0.1–100 ng/mL for midazolam and 0.05–50 ng/mL for dextromethorphan in human plasma. Method robustness was demonstrated by the 100% pass rate of 24 incurred sample analysis runs and all of the 50 clinical study samples used for incurred sample reproducibility (ISR) test having met the acceptance criterion (%Diff within 20%). The overall ISR results for all compounds showed that over 95% of the samples had a %Diff of less than 10%. The method is simple, rapid and rugged, and has been applied successfully to sample analysis in support of a drug–drug interaction study.     

OPTIMIZATION OF THE ENUMERATION OF TOTAL AEROBIC BACTERIAL FLORA IN THE FLESH OF SEAFISH

Abstract The total aerobic flora of seafish flesh is weakly halophilic, and requires on average 1.38% NaCl according to statistical studies. Enumeration is optimal on tryptone soya agar or on NaCl supplemented plate count agar (-H₂S), incubated at 20 and 25C, respectively. Plate count agar (-H₂S) was selected because it can also be used for enumeration of hydrogen sulfide-producing bacteria by degradation of sulfur-containing proteins, which are abundant in fish The models employed are sigmoidal. The initial bioburden is too great for there to be a lag phase during storage in ice at 0C. The models show that when the total aerobic microflora count exceeds 100,000 cfu/g, whole or filleted fish stored on ice at 0C are unfit for consumption.     

OPTIMIZATION OF CULTURE CONDITIONS FOR ENUMERATION OF H2S BACTERIA IN THE FLESH OF SEAFISH

H₂S bacteria of seafish flesh are weakly halophilic and require on average 1.68% NaCl according to statistical studies. Enumeration is optimal on PCA-H₂S(a PCA medium supplemented with sulfur sources and increased NaCl concentrations) incubated at 25C. Total aerobic bacteria can be counted simultaneously on this medium. The proportion of H₂S bacteria relative to total aerobic bacteria increased slightly during prolonged storage of the fish, but was highly variable. Models relating H₂S bacterial counts to spoilage of fish are sigmoidal and showed that when the count exceeds 10,000 CFU/g, whole or filleted fish stored in ice at 0C are unfit for consumption. Shewanella putrefaciens accounted for 69% of the H₂S bacteria at the fifth day of storage and 100% at the fifteenth.     

Respiratory-cardiovascular physiology and chloroethane gill flux in the channel catfish, Ictalurus punctatus

A fish respirometer-metabolism chamber was used to obtain in vivo respiratory-cardiovascular and chloroethane gill flux data on transected channel catfish (Ictalurus punctatus). Methods used for spinal transection, attachment of an oral membrane (respiratory mast), placement and attachment of blood cannulas and urine catheters are described. Respiratory physiology, cardiac output and chemical extraction efficiencies for 1,1,2,2-tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE) were determined on 419–990 g catfish. The overall mean values (± s.d.) for ventilation volume (Q_v), effective respiratory volume (Q_w), oxygen consumption (Vo₂ and percentage utilization of oxygen (U) were 17-3 ±4–71 h^?1 kg^?1, 9·8±l·71 h^?1 kg^?1, 71·6±12·5mg h^?1 kg^?1, and 49± 10%, respectively, while cardiac output calculated via the Fick Method was 2·4±0·61 h^?1 kg^?1. Additional measurements were made on ventilation rate (V_r), total plasma protein, haematocrit (Hct), and urine volume; while both arterial and venous blood were analysed for pH, oxygen partial pressure (P02), carbon dioxide partial pressure (Pco₂), total oxygen (To₂), total carbon dioxide (Tco₂) and total ammonia (TAMM). Physiological measurements taken at 24 h were not significantly different from those taken at 48 h and indicated no deterioration of the in vivo preparation. All of these values agreed well with literature values on UTitransected channel catfish, except for Hct which was lower for cannulated animals used in this study. Overall, these data provide strong support for the use of transected channel catfish for in vivo collection of physiological and chemical gill flux data. The mean initial chemical extraction efficiencies for TCE, PCE and HCE were 41, 61 and 73%, respectively. Chemical clearances (Cl_X) for these same three chemicals were 5·9, 9·3 and 10·8 1 h^?1 kg^?1, respectively. The approximate 1: 1 relationship between effective respiratory volume (Q_w) and chemical clearance (Cl_x) indicated that branchial uptake of PCE and HCE was water flow-limited. Chemical gill flux observed for channel catfish and chloroethanes was similar to that observed for rainbow trout in previous studies and provided further support for the flow-limited model of chemical flux across fish gills.