Vitamin D regulated keratinocyte differentiation

The epidermis is the largest organ in the body. It is comprised primarily of keratinocytes which are arranged in layers that recapitulates their programmed life cycle. Proliferating keratinocytes are on the bottom-the stratum basale. As keratinocytes leave the stratum basale they begin to differentiate, culminating in the enucleated stratum corneum which has the major role of permeability barrier. Calcium and the active metabolite of vitamin D, 1,25(OH)(2)D(3), play important roles in this differentiation process. The epidermis has a gradient of calcium with lowest concentrations in the stratum basale, and highest concentrations in the stratum granulosum where proteins critical for barrier function are produced. Vitamin D is made in different layers of the epidermis, but 1,25(OH)(2)D(3) is made primarily in the stratum basale. Together calcium and 1,25(OH)(2)D(3) regulate the ordered differentiation process by the sequential turning on and off the genes producing the elements required for differentiation as well as activating those enzymes involved in differentiation. Animal models in which the sensing mechanism for calcium, the receptor for 1,25(OH)(2)D(3), or the enzyme producing 1,25(OH)(2)D(3) have been rendered inoperative demonstrate the importance of these mechanisms for the differentiation process, although each animal model has its own phenotype. This review will examine the mechanisms by which calcium and 1,25(OH)(2)D(3) interact to control epidermal differentiation.     

Partial alanine scan of mast cell degranulating peptide (MCD): importance of the histidine- and arginine residues.

The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcepsilonRIalpha binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His13 by Ala o ccurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg7 and His8 resulted in an approximately 40 fold increase, and for Arg16 in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcepsilonRIalpha binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala8 (for His) and Ala16 (for Arg) showed the same binding affinities as MCD, whereas analog Ala7 (for Arg) and analog Ala13 (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.     

Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures

It is known that heterogeneous conditions exist in large-scale animal cell cultures. However, little is known about how heterogeneities affect cells, productivities, and product quality. To study the effect of non-constant dissolved oxygen tension (DOT), hybridomas were subjected to sinusoidal DOT oscillations in a one-compartment scale-down simulator. Oscillations were forced by manipulating the inlet oxygen partial pressure through a feedback control algorithm in a 220-mL bioreactor maintained at a constant agitation. Such temporal DOT oscillations simulate spatial DOT gradients that can occur in large scales. Different oscillation periods, in the range of 800 to 12,800 s (axis of 7% (air saturation) and amplitude of 7%), were tested and compared to constant DOT (10%) control cultures. Oscillating DOT decreased maximum cell concentrations, cell growth rates, and viability indexes. Cultures at oscillating DOT had an increased glycolytic metabolism that was evidenced by a decrease in yield of cells on glucose and an increase in lactate yield. DOT gradients, even several orders of magnitude higher than those expected under practical large-scale conditions, did not significantly affect the maximum concentration of an IgG(1) monoclonal antibody (MAb). The glycosylation profile of the MAb produced at a constant DOT of 10% was similar to that reported in the literature. However, MAb produced under oscillating culture conditions had a higher amount of triantennary and sialylated glycans, which can interfere with effector functions of the antibody. It was shown that transient excursions of hybridomas to limiting DOT, as occurs in deficiently mixed large-scale bioreactors, is important to culture performance as the oscillation period, and thus the time cells spent at low DOT, affected cell growth, metabolism, and the glycosylation pattern of MAb. Such results underline the importance of monitoring protein characteristics for the development of large-scale processes.     

The trypsin-inhibitory, immunostimulatory and antiproliferative activities of a napin-like polypeptide from Chinese cabbage seeds.

A heterodimeric 13.8 kDa napin-like polypeptide has previously been isolated from Chinese cabbage (Brassica parachinensis) seeds with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. In the present study the N-terminal sequence of the 8.8 kDa subunit of the polypeptide (PQGPQQRPPKLLQQQTNEEHE) was found to have pronounced homology to napins, albumins and trypsin inhibitors, but demonstrated little similarity to the 5 kDa subunit. The polypeptide stimulated nitrite production by mouse peritoneal macrophages and reduced the viability of leukaemia (L1210) cells. It inhibited trypsin with a higher potency than it inhibited chymotrypsin, but was devoid of ribonuclease and antifungal activities.     

GABA(B) receptors in 5-HT transporter- and 5-HT1A receptor-knock-out mice: further evidence of a transduction pathway shared with 5-HT1A receptors

The functional properties of GABA(B) receptors were examined in the dorsal raphe nucleus (DRN) and the hippocampus of knock-out mice devoid of the 5-HT transporter (5-HTT-/-) or the 5-HT(1A) receptor (5-HT(1A)-/-). Electrophysiological recordings in brain slices showed that the GABA(B) receptor agonist baclofen caused a lower hyperpolarization and neuronal firing inhibition of DRN 5-HT cells in 5-HTT-/- versus 5-HTT+/+ mice. In addition, [(35)S]GTP-gamma-S binding induced by GABA(B) receptor stimulation in the DRN was approximately 40% less in these mutants compared with wild-type mice. In contrast, GABA(B) receptors appeared functionally intact in the hippocampus of 5-HTT-/-, and in both this area and the DRN of 5-HT(1A)-knock-out mice. The unique functional changes of DRN GABA(B) receptors closely resembled those of 5-HT(1A) autoreceptors in 5-HTT-/- mice, further supporting the idea that both receptor types are coupled to a common pool of G-proteins in serotoninergic neurons.     

Cultured peritoneal mesothelial cells exhibit apical primary cilia

This study examined primary cilia on cultured human and rabbit peritoneal mesothelial cells (PMC) and investigated factors that influence ciliary expression. Primary cilia were examined with indirect immunocytochemistry, laser scanning confocal microscopy and scanning electron microscopy. Ciliary expression was evaluated in cultures with or without l-cysteine (0.25 mM) or exposure to Ca(2+)-free Krebs-Ringer solution supplemented with EGTA, 0.5 mM. This treatment disrupted cell monolayer integrity. Cilia were counted and normalized to total cell counts using NIH image. Primary cilia were identified on both human and rabbit PMC. Cells treated with l-cysteine expressed significantly more cilia compared with monolayers deprived of l-cysteine. Exposure to Ca(2+)-free Krebs-Ringer solution significantly reduced cilia (5.9+/-1.0%, n=7). Although ciliary expression could be augmented with l-cysteine, approximately 60% of human PMC and 84% of rabbit PMC did not exhibit cilia. Together, these data show that monolayers of PMC express apical cilia that can be augmented with l-cysteine, independently of increased cell density.