Isolation and characterization of biologically active murine interleukin-1 alpha derived from expression of a synthetic gene in Escherichia coli

A murine interleukin-1 alpha (mIL-1 alpha) gene coding for amino acids 115 to 270 of the precursor protein (Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Giri, J.G., Pan, Y.E., Collier, K., Semionow, R., Chua, A.O. and Mizel, S.B. (1984) Nature 312, 458-462) was chemically synthesized and expressed in Escherichia coli. mIL-1 alpha, in the form of insoluble inclusion bodies, accounted for approx. 30% of total cellular protein produced by the recombinant strain. A simple isolation protocol was developed in which inclusion body material was first solubilized in 3 M guanidine hydrochloride, and the mIL-1 alpha was then simultaneously purified and allowed to fold to its active conformation by dialysis against distilled water. This procedure yielded pure, biologically active mIL-1 alpha with 41% recovery of the mIL-1 alpha present in the guanidine hydrochloride extract. The purified preparation had the expected amino acid composition, a molar absorptivity of 28,200 M-1.cm-1 and a pI of 5.2. No methionyl-mIL-1 alpha was detected by N-terminal sequence analysis, and the endotoxin level was less than 10 pg per micrograms of mIL-1 alpha. The specific biological activity was 3.10(7) units/mg in a co-mitogenic thymocyte proliferation assay. In addition to full-length mIL-1 alpha, the preparation contained N-terminally truncated mIL-1 alpha species (mainly des-4 and des-6 amino acid forms). The truncated species were isolated and found to have the same biological activity as the complete polypeptide. Thus, the active fragment of mIL-1 alpha appears to consist of a proteinase-sensitive N-terminal region which is not essential for activity, and a proteinase-resistant core which harbors the essential determinants of its cytokine function.     

H-2 histocompatibility antigens of subcellular membranes of mouse liver

Purified fractions of plasma membrane, Golgi apparatus, rough endoplasmic reticulum vesicles, nuclear envelope, and mitochondria were isolated from mouse liver and the distribution of H-2 histocompatibility antigens determined by indirect radioimmunoassay before and after membrane disruptive treatments. Fractions enriched in plasma membrane (surface membrane) revealed H-2 antigens in highest concentration; disruptive treatments were not necessary to reveal H-2 antigens with surface membranes. In contrast, internal membranes did not possess H-2 antigens which were accessible to antibody. Golgi apparatus fractions or some component of these fractions (e.g. secretory vesicles) possessed the antigens but in a latent form where accessibility was provided by simple rupture of the membrane vesicles. With endoplasmic reticulum, detergent solubilization of the membranes was required before H-2 antigen could be detected. Nuclear envelope preparations contained little or no demonstrable H-2 activity. These results were confirmed by several techniques including immunoprecipitation of labelled solubilized membrane components with anti-H-2 serum and subsequent analysis in SDS-PAGE.     

In vivo binding and uptake of low-density lipoprotein-gold- and albumin-gold conjugates by parenchymal and sinusoidal cells of the fetal rat liver

Summary To elucidate the participation of fetal rat liver cells in the receptor-mediated internalization of low-density lipoproteins (LDL), rat fetuses were injected with either LDL-gold or albumin-gold conjugates. The degree of binding and uptake of LDL-gold and albumin-gold by parenchymal and sinusoidal cells of the fetal rat liver differs markedly. Endothelial cells exhibit low LDL-gold uptake. In contrast, parenchymal cells internalize LDL-gold more actively (45 ± 8 LDL conjugates/100 m² cytoplasm within 60 min). Kupffer cells exceed this value by a factor of 20. The uptake of albumin-gold by endothelial and Kupffer cells is high, whereas it is extremely low in parenchymal cells. Estradiol pretreatment causes a significant doubling (p<0.05) of the LDL-gold particle density/100 m² cytoplasm both in parenchymal and Kupffer cells, whereas estradiol has no effect on the albumin uptake. The results strongly indicate that LDL uptake by parenchymal and Kupffer cells in the fetal rat liver is mediated by estrogen-inducible receptors, which may correspond to B, E receptors in the adult liver.     

The effect of oral glucose loads on tissue metabolism during angiotensin II receptor and beta-receptor blockade in obese hypertensive subjects.

AT1 receptor blockers and ACE inhibitors decrease the risk for new onset diabetes mellitus. The phenomenon could be related to a direct angiotensin II effect on tissue metabolism. To address the issue, we recruited eighteen obese hypertensive patients. Patients were randomized to double-blind treatment with either valsartan (n = 8) or atenolol (n = 10) for thirteen weeks. They underwent an oral glucose tolerance test before and during active treatment, while metabolism was monitored through subcutaneous and intramuscular microdialysis and indirect calorimetry. After glucose ingestion, venous glucose and insulin concentrations increased rapidly while systemic free fatty acid concentrations were suppressed. Dialysate glucose and lactate concentrations increased briskly in adipose tissue and in skeletal muscle. Dialysate glycerol decreased profoundly in both tissues. Respiratory quotient increased markedly after glucose ingestion. These responses were identical at baseline and during active treatment either drug. We conclude that AT1 receptor blockade in obese hypertensive patients has no effect on interstitial glucose supply, lipolysis, and substrate oxidation. One possible explanation is that angiotensin II levels in obese hypertensives are not sufficient to elicit the metabolic changes that have been observed after direct angiotensin II application. The exact mechanism by which inhibition of the renin-angiotensin-aldosterone system decreases the diabetes risk remains unresolved and requires further study.     

Allocation of a transgenic c-myc integration site to mouse chromosome 8B3-C1

A recombinant plasmid containing the mouse c-myc gene was injected into mouse pronuclei. The transgenic line 478 contains about 100 copies of the transgene integrated into one chromosome site. By in situ hybridization, the integration site was localized to chromosome 8B3-C1.     

Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi

A cysteine proteinase, purified to homogeneity from epimastigotes of Trypanosoma cruzi, was strongly inhibited by L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64). The second-order rate constant was 20,800 M-1.s-1, and the reagent could be used for active site titration. The enzyme hydrolysed chromogenic peptides at the carboxyl Arg or Lys; it required at least one more amino acid, preferably Arg, Phe, Val or Leu, between the terminal Arg or Lys and the amino-blocking group. Enzyme activity on azocasein at pH 5.0 was increased by urea, maximal activity being attained at 2 M, and was still as active at 5 M urea as in its absence. Guanidine hydrochloride and KSCN also activated at low concentrations, but caused a strong inhibition above 2 M and 1 M, respectively. When azocasein was tested as a substrate at pH 7.0, there was no activation, and when synthetic substrates were used all chaotropic agents tested were inhibitory. The results suggest that the enzyme, for which we propose the trivial name 'cruzipain', differs in some aspects from all other cysteine proteinases described so far, although it shares several of the properties of mammalian cathepsin L.     

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues II. Concomitant and mutually exclusive synthesis of trichocytic and epithelial cytokeratins in diverse human and bovine tissues (hair follicle, nail bed and matrix, lingual papilla, thymic reticulum)

The hair-forming cells (trichocytes) and the mature hair contain four major trichocytic cytokeratins from each of the subfamilies, basic (Hb1-4) and acidic (Ha1-4); these are related - but not identical - to the epithelial cytokeratins. Here we show, by biochemical methods and immunofluorescence microscopy using antibodies specific for either epithelial or trichocyte cytokeratins, that the same set of hair-type cytokeratins, including two newly identified minor components, designated Hax (type I) and Hbx (type II), are also expressed in cells forming nails, in the filiform papillae of the dorsal surface of human and bovine tongue, and, most surprisingly, in some cells of the epithelial reticulum of bovine and human thymus. By double-label immunofluorescence microscopy, we also show that the expression of the two subsets of cytokeratins, i.e., the epithelial and the trichocytic ones, is not necessarily mutually exclusive, but that certain cells of hair follicles, nail matrix and bed, lingual papillae, and the nonlymphoid cell system of the thymus contain both trichocytic and certain epithelial cytokeratins. This indicates that these cells coexpress representatives of both kinds of cytokeratin. Implications of these findings with respect to problems of regulatory control of cytokeratin synthesis in tissue development and differentiation, and the possible functional meaning of the occurrence of trichocytic cytokeratins in such histologically diverse tissues, are discussed.     

Identification of Merkel cells in human skin by specific cytokeratin antibodies:

Merkel cells are special neurosecretory cells which, in adult human skin, are usually very scarce. By immunofluorescence microscopy using antibodies to human cytokeratin polypeptide no. 18, we localized distinct non-keratinocyte cells in the glandular ridges of human fetal and adult plantar epidermis. Using electron and immunofluorescence microscopy, these cells were identified as Merkel cells containing typical neurosecretory granules as well as bundles of intermediate-sized filaments and desmosomes. Two-dimensional gel electrophoresis of the cytoskeletal fractions of microdissected epidermal preparations highly enriched in Merkel cells indicated the presence of cytokeratin polypeptides nos. 8, 18 and 19 which are typical of diverse simple epithelia of the human body. Double immunofluorescence microscopy showed that these human Merkel cells contain neither neurofilaments nor vimentin filaments. In human fetuses of 18-24 weeks of age, conspicuously high concentrations of Merkel cells, reaching a density of approximately 1,700 Merkel cells/mm2 skin, were found in the glandular ridges of plantar skin. The concentration decreased considerably at newborn and adult stages. Thin cell processes (up to 20 microns long) were observed in many fetal epidermal Merkel cells. In addition, we detected isolated Merkel cells deeper in the dermis (i.e. at distances of, at most, 100 microns from the epidermis) in fetal and newborn plantar skin. Our results show that Merkel cells are true epithelial cells which, however, differ profoundly from epidermal keratinocytes in their cytokeratin expression. The findings are discussed in relation to the much disputed question of the origin of Merkel cells. The present data speak against the immigration of Merkel cells from the neural crest, but rather suggest that they originate from epithelial cells of the skin, although most probably not from differentiated keratinocytes.