Etienne J. Nouwen

Heat-stable alkaline phosphatase as a marker for human and monkey type-I pneumocytes

SummaryThe expression of the heat-stable isoenzyme of alkaline phosphatase in the human and monkey (Macaca mulatta, M. fascicularis) lung was investigated at the light- and electron-microscopic level, using cytochemical techniques and immunocytochemical procedures based on monoclonal and polyclonal antibodies against human term-placental alkaline phosphatase. Both in man and monkey, the enzyme was present in type-I pneumocytes. In the monkey, the enzyme was found in all type-I cells. In man, strong staining was observed only in some type-I cells and in certain cuboidal respiratory bronchiolar cells. Staining was localized on the apical and basal plasma membrane, in apical and basal caveolae, and in the underlying basement membrane. The level of heat-stable alkaline phosphatase expression in the human lung was 10-fold lower than in the monkeys studied. In human fetal lung, the onset of heat-stable alkaline phosphatase expression was associated with the development of the alveolar epithelium from 17–20 weeks gestation onward. It is concluded that: (1) heat-stable alkaline phosphatase is a specific constitutent of type-I pneumocytes in man and monkeys; and (2) its subcellular localization may explain its rapid appearance in the circulation under certain conditions.

The cytosolic glutathione S-transferase isoenzymes in the dog kidney cortex as compared with the corresponding MDCK renal cell line.

Cytosolic glutathione S-transferase (GST) (EC 2.5.1.18) isoenzymes of dog kidney and MDCK (an established dog renal cell line) were purified and studied. Specific GST activity was 248 and 317 nmol/min/mg protein, for dog and MDCK, respectively. Cytosolic GST was only partially purified by glutathione affinity chromatography, a substantial amount (43% and 84% for dog kidney and MDCK, respectively) of the GST activity was found in the flow-through fraction. Affinity bound GST was separated into 6 and 3 isoenzymes by anionic chromatofocusing for dog and MDCK, respectively. Flow-through GST was purified by gel filtration, anion exchange chromatography and anionic chromatofocusing showing only one GST isoenzyme, with distinct features from the affinity bound GST, for both dog and MDCK. The isoenzymes were characterized by their kinetic properties, subunit composition, specific substrates and inhibitors and immunoblot. The major dog GSTs (DII, DIV and DVI) correspond to the MDCK isoenzymes (MI, MII and MIII). Comparable pI values, a comparable affinity towards GSH and comparable sensitivities towards the inhibitors N-ethylmaleimide (NEM), triphenyltin chloride, cibacron blue and hematin were observed for the corresponding isoenzymes: DII and MI, DIV and MII, DVI and MIII. Co-electrophoresis showed that the subunit composition was identical for DII and MI, and for DIV and MII. Inhibitor and substrate sensitivities showed that the affinity bound GSTs belong to class pi and mu, the presence of class pi was confirmed by immunoblot analysis. One homodimeric GST isoenzyme was observed in the dog kidney and MDCK flow-through. Both dog and MDCK isoenzyme have a nearly neutral pI, a high affinity towards CDNB and an equal sensitivity towards triphenyltin chloride, cibacron blue and hematin. However, based on inhibitor studies and immunoblot, this isoenzyme could not be attributed to an identified GST class. The overall isoenzyme pattern of dog and MDCK affinity bound and flow through GST is comparable. The dog and MDCK affinity bound GSTs have similar characteristics and all belong to class mu or pi.

5-Aminosalicylic acid and chronic interstitial nephritis

For decades, sulphasalazine, an azo-compound derived from sulphapyridine and 5-aminosalicylic acid (5-ASA) (Fig. 1), has been the only valuable non-cortico-steroid drug in the treatment of inflammatory bowel disease. Azad Kahn et al. [1] showed that the pharmacologically active moiety in sulphasalazine for the treatment of these diseases was 5-ASA. Moreover, it has been shown that the metabolite sulphapyridine was largely responsible for the side-effects of sulphasalazine. Consequently, this resulted in a number of new 5-ASA formulations (mesalazine, olsalazine, balsalazine) for topical and oral use. 5-ASA is partially resorbed, particularly in the acetylated form and eliminated as such in the urine. The colon is the predilected place for this acetylation since in the small bowel there is a lack of the responsible bacterial flora. Hence, 5-ASA is readily absorbed as such in the small bowel. How far this may form a rationale for a possible difference in nephrotoxicity for the different preparations reamins to be determined.

Isolation and characterisation of the class alpha, mu and pi glutathione transferases in LLC-PK1 and pig kidney

Glutathione S-transferase (GST) isoenzymes from pig kidney cortex and LLC-PK1 (an established cell line derived from the pig proximal tubule) were purified by affinity chromatography, anionic and cationic chromatofocusing. Purification revealed nine isoenzymes in the pig kidney cortex and five isoenzymes in the LLC-PK1 cell line. SDS-polyacrylamide gel electrophoresis showed that the pig kidney cortex isoenzymes were homo- or heterodimeric; LLC-PK1 isoenzymes, however, were homodimeric. Isoenzymes from pig and LLC-PK1 showed a higher affinity towards glutathione. The isoenzymes were further characterised and divided into the different GST classes by studying specific inhibitors, specific substrates and immunological properties. Pig GSTs belong to class alpha, mu and pi. The GSTs in LLC-PK1 cells, on the other hand, belong to class pi and mu. The isoenzyme pattern in LLC-PK1 cells indicates the dedifferentiation of this particular cell line compared with the pig kidney cortex.

Detection and Immunohistochemical Localisation of Hplap and CA 125 in Sera and Tissues of Patients with Ovarian, Nonovarian Tumors, and Liver Disorders

Abstract Cancer Antigen 125 (CA 125) was compared to human placental alkaline phosphatase (hPLAP) and CEA in sera of patients with different types of ovarian tumors, non ovarian tumors and patients with liver disorders. In ovarian tumor cases the sensitivity of hPLAP was 44%, 69% for CA 125 and only 13% for CEA. 45 nonovarian tumor patients were examined on hPLAP, CA 125 and CEA. Serum hPLAP and CA 125 levels were almost equally increased (22% and 24% respectively), whereas CEA was increased in 44%. hPLAP was not increased in sera of patients with benign liver pathologies, in contrast to CA 125 and CEA, which were increased in 54% and 24% respectively. Cirrhotic icteric patients had the highest prevalence of CA 125 (89%) and CEA (44%) in their serum. hPLAP, CA 125 and CEA were localized immunhistochemically in paraffin sections of ovarian tumors. A positive reaction was observed in 90%, 99% and 45% respectively. Immunohistochemical staining of CA 125 in normal liver tissue of a patient with a metastatized pancreatic adenocarcinoma and a high CA 125 level in the serum was observed in organelles compatible with the lysosomes of hepatocytes. Normal fetal liver was immunohistochemically negative for hPLAP, CA 125 or CEA, and one fetal ovary studied was positive only for hPLAP.