GLC determination of l-2-hydroxy-N-cyclopropylmethylmorphinan in plasma and urine.

A specific method was developed for the determination of l-2-hydroxy-N-cyclopropylmethylmorphinan in plasma and urine by GLC, using flame-ionization detection. The method involves the extraction of the compound into ether from plasma or urine at pH 7.4, followed by back-extraction into 1 N HCl. The acid phase is ether washed and made alkaline, and the compound is reextracted into ether. The ether is evaporated to dryness, the residue is dissolved in methanol, and an aliquot is analyzed by GLC. The same method is applicalble to plasma and urine samples following deconjugation of the compound with glucuronidase-sulfatase. The overall recovery is 93.1 +/- 9.4% SD) in the concentration range of 0.020-2.0 microgram/ml. The method was successfully applied to plasma and urine specimens obtained after administering single 25-mg oral doses to humans.     

Acetylcholine release from synaptosomes and phenytoin action

The release of labeled acetylcholine from synaptosomes loaded with methyl-[3H]choline has been measured in Krebs-Ringer-Bicarbonate (KRB) media containing either 5.6 or 56 mM KCl. Experiments have been performed in media containing either 1.0 mM Ca or 0 Ca with 1 mM EGTA. Phenytoin, 2 X 10(-4) M, reduced the depolarization-dependent release of acetylcholine in media containing 1.0 mM Ca and 56 mM KCl. It also significantly increased the release of acetylcholine from undepolarized samples in 5.6 mM KCl irrespective of the Ca concentration. The drug did not affect release from synaptosomes depolarized in Ca-free media. These results confirm the hypothesis that phenytoin has a dual effect on transmitter release.     

Prostaglandins: Bone resorption stimulating factors released from monkey gingiva

Gingival fragments from monkeys have been found to release a factorin vitro into incubation medium which stimulates bone resorption in organ culture. Indomethacin effectively blocks the occurrence of this stimulatory factor in the gingival incubation medium. All of this bone resorptive activity can be accounted for by prostaglandin-like material. The prostaglandins contributing to the bone resorptive activity have been found to be prostaglandins E₁ and E₂.     

Dependency of sister chromatid exchange in T- and B-cells on the incorporation of deoxyribonucleosides into chromosomal DNA.

Human T-cells (CCRF-HSB2) did not incorporate tritiated thymidine ([3H]TDR)--1.0-5.0 muCi/ml--into the nuclei, where.as they readily incorporated tritiated deoxycytidine (E13H]CDR). When contamination with pleuropneumonia-like organisms was ruled out, these findings strongly suggested a deficiency of the enzyme thymidine kinase in the cells. Human B-cells (CCRF-SB) and normal T-lymphocytes (NTL) readily incorporated [3H]CDR, [3H]TDR, and tritiated 5-bromodeoxyuridine, and they clearly exhibited differential staining of the sister chromatids (SCD). When nonisotopic bromodeoxyuridine (BUDR), 10(-6)-10(-4) M, was used with the B-cells and NTL, SCD were clearly evident and sister chromatid exchange (SCE) was relatively infrequent; when the concentration was 10(-7) M, SCD staining was poor but the frequency of SCE was high. SCE frequencies in NTL, measured by autoradiography after incorporation of [3H]CDR, were the same as SCE frequencies measured by staining with BUDR at 10(-4) M. In the case of CCRF-HSB2, 10(-4) M BUDR produced relatively high frequencies of SCE as did 10(-7) M BUDR with the former two cells. However, [3H]CDR with CCRF-HSB2 gave relatively low frequencies of SCE, of the magnitude observed after 10(-4) M BUDR was used with NTL and the B-cells. Thus the high frequency of SCE in CCRF-HSB2 cells may have been due to the staining property of chromosomes that had incorporated low levels of BUDR.     

Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts

Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3sigma were unable to express this factor. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.