Vitamin D regulated keratinocyte differentiation: role of coactivators

1,25 Dihydroxyvitamin D (1,25(OH)(2)D) regulates the differentiation of keratinocytes. 1,25(OH)(2)D raises intracellular free calcium (Cai) as a necessary early step toward stimulating differentiation. 1,25(OH)(2)D induces the calcium sensing receptor (CaR) in keratinocytes and enhances the calcium response of these cells. Activation of the CaR by calcium increases intracellular free calcium by a mechanism involving phospholipase C (PLC) cleavage of phosphatidylinositolbisphosphate into inositoltrisphosphate (IP(3)) and diacylglycerol (DG). 1,25(OH)(2)D induces the family of PLCs. PLC-gamma1 has a DR6 VDRE in its promoter which binds and is activated by VDR/RAR rather than VDR/RXR. The involucrin gene, which encodes a critical component of the cornified envelope, contains a DR3 VDRE in its promoter that acts in conjunction with a nearby AP-1 site. The sequential regulation of these genes is critical for the differentiation process. In undifferentiated keratinocytes, the VDR binds preferentially to the DRIP complex of coactivators. However, with differentiation DRIP 205 is no longer produced, and the VDR switches partners to the SRC family (SRC2 and 3). These studies suggest that at least part of the sequential activation of genes required during keratinocyte differentiation is regulated by the change (availability) of these different coactivator complexes.     

Contribution of a single hydrogen bond between betaHis81 of MHC class II I-E(k) and the bound peptide to the pH-dependent thermal stability

To determine the energetic contribution of the hydrogen bond between betaHis81 of the major histocompatibility complex class II (MHC II) molecule, I-E(k), and the bound hemoglobin peptide (Hb), we analyzed the thermal stability of the hydrogen bond-disrupted mutant, I-E(k)-Hb betaH81Y, in which the betaHis81 residue was replaced with Tyr, by differential scanning calorimetry. The thermal stability of the I-E(k)-Hb betaH81Y mutant was lower than that of the I-E(k)-Hb wild-type, mainly due to the decreased enthalpy change. The difference in the denaturation temperature of the I-E(k)-Hb betaH81Y mutant as compared with that of the I-E(k)-Hb wild-type at pH 5.5 was only slightly smaller than that at pH 7.4, in agreement with the increased stability at an acidic pH, a unique characteristic of MHC II. Thus, the hydrogen bond contributed by betaHis81 is critical for peptide binding, and is independent of pH, which can alter the hydrophilicity of the His residue.     

Depletion of intracellular NAD(+) and ATP levels during ricin-induced apoptosis through the specific ribosomal inactivation results in the cytolysis of U937 cells

Our previous studies demonstrated that ricin induces the apoptotic death of U937 cells as evidenced by DNA fragmentation, nuclear morphological changes, and increases in caspase-like activities. In this study, we have found that intracellular NAD(+) and ATP levels decrease in ricin-treated U937 cells and that this decrease is followed by the ricin-mediated protein synthesis inhibition. The PARP inhibitor, 3-aminobenzamide (3-ABA), prevents the depletion in NAD(+) and ATP levels and concomitantly protects U937 cells from the lysis that follows ricin treatment. Hence, the protective action of 3-ABA is due to the inhibition of PARP and does not result from its other pharmacological side effects. Moreover, the enzymatic activity of PARP gradually increases and reaches a maximum level after ricin exposure for 3 h, whereas no significant change in activity was observed in untreated cells. However, 3-ABA has no effect on ricin-mediated DNA fragmentation. In addition, immunoblot analysis revealed that significant PARP cleavage occurred more than 12 h after ricin addition, while DNA fragmentation reached a maximum level within 6 h of incubation. Thus, in the case of ricin-induced apoptosis, it appears that PARP cleavage is not an early apoptotic event associated with the onset of apoptosis. Our results suggest that multiple apoptotic signaling pathways may be triggered by ricin-treatment. Probably, the pathway leading to cell lysis via PARP activation and NAD(+) depletion is independent of the pathway leading to DNA fragmentation in which caspases may be profoundly involved. Other protein synthesis inhibitors, including diphtheria toxin and cycloheximide, were less effective in terms of inducing DNA fragmentation and cytolysis, even at concentrations that cause significant inhibition of protein synthesis. Thus, a specific ricin action mechanism through which ribosomes are inactivated may be responsible for the apoptotic events induced by ricin.     

Human renin activation by protease from the renin granule fraction of the dog kidney cortex

To clarify the possible conversion of prorenin in renin granules where conversion reportedly occurred, we investigated whether the renin granule fraction of the kidney could activate prorenin to the active form. Renin granules were isolated from the dog kidney cortex by discontinuous sucrose density gradient centrifugation. Human active renin was quantified by immunoradiometric assay which could detect only the human active renin but not the inactive human renin or dog renin. Inactive renin from human amniotic fluid was incubated with the subcellular fraction of the dog kidney cortex. The renin granule fraction that showed the highest renin activity stimulated the inactive renin to become the active form. The membrane preparation obtained from the renin granule fraction by freezing and thawing the fraction in low osmolarity retained the activity of renin activation. Other subcellular fractions showed less renin activation. The optimal pH for renin activation by the membrane was pH 5.0 to 6.0. The activation depended on the time of incubation and concentration. The activation was inhibited by N-ethylmaleimide but not by EDTA or serine protease inhibitors. These results suggest that renin is processed by a membrane bound protease in renin granules.     

Stimulation of pulmonary surfactant secretion by activating neutrophils in rat type II pneumocytes culture

The influence of activating neutrophils on the secretion of phosphatidylcholine (PC), the predominant component of pulmonary surfactant, was examined using primary culture of rat type II pneumocytes. Simultaneous addition of neutrophils and opsonized zymosan, but not neutrophils or opsonized zymosan alone, to type II pneumocytes caused a significant increase in PC secretion without affecting the release of lactate dehydrogenase, a marker of cytotoxicity. The increase in PC secretion was dependent on the number of activating neutrophils. In addition, pretreatment of culture with the combination of superoxide dismutase and catalase inhibited the increase in PC secretion. These findings indicate that activating neutrophils stimulate the secretion of pulmonary surfactant and that the stimulation is mediated by oxygen radicals.     

Production of low temperature active lipase from the pink snow mold, Microdochium nivale (syn. Fusarium nivale)

Summary The pink snow mold, Microdochium nivale (syn. Fusarium nivale) SUF 1377 strain produced an extracellular low temperature active lipase during growth at 4°C. The lipase had the highest activity at 20°C, and retained 19% of its maximum activity at 0°C.     

Cross-correlation of bilateral differences in fatigue during sustained maximal voluntary contraction

Maximal isometric force and electromyograph (EMG) activity of biceps brachii muscle during bilateral sustained elbow flexion were followed in 25 right-handed oarsmen. The percentage decline in force was greater for the left than for the right arm. Also, the mean power frequency (MPF) and the root mean square (rms) value of the EMG amplitude decreased more for the left than for the right arm. It was hypothesized that a common drive would indicate that the two forces curves would be highly correlated during the nonfatigued period, but the level of cross-correlation would decline during muscle fatigue. For the first 4 s of the contraction, the cross-correlation between the right and left force was high (r = 0.99), but thereafter it declined rapidly to a constant level. The decline of the cross-correlation was accompanied by a similar decrease in the correlation between the right and left EMG activations (MPF and rms). Thus, the decline in the cross-correlation level of force accompanied by a similar decrease in the correlation level of EMG would suggest a fatigue-induced neural derangement of the common drive.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

Complete amino acid sequence of Scytalidium lignicolum acid protease B

The acid protease B (SLB) of Scytalidium lignicolum was reduced and carboxymethylated and then subjected to tryptic digestion. Five fragments were isolated and some of them were further digested with alpha-chymotrypsin, thermolysin, and dilute acetic acid. The sequence analysis of these fragments and the peptides by conventional methods established the complete amino acid sequence of SLB. The enzyme was composed of 204 amino acid residues with threonine and valine as its amino- and carboxyl-termini, respectively. Locations of three disulfide bridges were also established to be Cys47-126, Cys140-163, and Cys192-201 by enzymatic fragmentation of the denatured and unmodified SLB. Only a slight homology was found in the sequences of SLB and other acid proteases hitherto reported.