Bacterial production and purification of recombinant human prolactin.

Escherichia coli cells transformed with a recombinant plasmid (pT7L) containing the coding sequence of human prolactin (hPrl) expressed a new protein. This protein, comigrating with human Prl on sodium dodecyl sulfate (SDS)-polyacrylamide gels, represented 50% of the total bacterial extract. Immunoprecipitation of [35S]methionine-labeled bacterial lysate with a rabbit antiserum to hPrl followed by SDS-polyacrylamide gel electrophoresis (PAGE) analysis showed that the major component had a Mr identical to that of standard hPrl. The majority of the recombinant hPrl (r-hPrl) accumulated in inclusion bodies. Analysis of these inclusion bodies by SDS-PAGE under nonreducing conditions showed that they are composed mostly of fully reduced monomers. Solubilization of the inclusion bodies and protein denaturation were performed in 8 M urea. Refolding during the renaturation procedure was confirmed by SDS-PAGE under nonreducing conditions. r-hPrl was further purified by gel permeation chromatography on a fast protein liquid chromatography column. More than 95% of the molecules were recovered as oxidized monomeric forms. The refolded molecule was tested for its bioactivity in the Nb2 lymphoma mitogenic assay. The dose-response curves obtained with either r-hPrl or pituitary-derived hPrl showed a complete parallelism. Furthermore, Nb2 cell proliferation was completely blocked by addition of hPrl antiserum to both preparations. Recombinant hPrl is identical to natural hPrl except for an additional methionine group at the amino terminal end.

Immunohistochemical localization of 54,000 dalton sialoglycoprotein in the epididymal duct of the germ cell-free W/Wv mouse.

A monoclonal antibody T21 specifically recognizes the mouse epididymal sialoglycoprotein of 54,000 dalton (SGP54). The localization of SGP54 was studied in the epididymal duct of germ cell-free WBB6F1W/Wv mutant mice (W/Wv mice) by avidin biotin complex (ABC) immunohistochemistry using T21. None of the testis cells showed immunoreaction. No spermatozoa were present in the epididymal duct lumen. The duct luminal fluid was stained weakly in the proximal corpus epididymidis, and strongly in the cauda epididymidis. Degenerated cells appeared in the duct lumen. The degenerated cells located at the corpus epididymidis showed strong immunostaining in the cytoplasmic region, while the degenerated cells located at the cauda epididymidis showed weak immunostaining. Immunoreaction was also detected between and on microvilli along the epididymis, the intensity being very strong at the distal caput and proximal corpus epididymidis. Invaginations and coated vesicles at the luminal surface of the principal cells were frequently immunostained at the corpus epididymidis. Giant inclusions frequently occurred in the principal cells of the distal caput and corpus epididymidis, with these being very intensely immunostained. These inclusions are ultrastructurally confirmed to be giant multivesicular bodies reported by ABE et al. (1984) in the mouse with the efferent duct cutting. These results suggest that the majority of excess SGP54 are absorbed by the principal cells at the distal caput to corpus epididymidis and catalyzed in the giant multivesicular bodies.

Intracytoplasmic crystalline inclusions in the hepatocytes of humans and chimpanzees.

Intracytoplasmic crystalline inclusions (CCIs) were observed in liver biopsy specimens taken from three patients and three chimpanzees. CCIs are rare in human hepatocytes and have never been described in chimpanzees. All three human cases were connected with alcoholic liver disease; no viral infection was noted. The histologies of the two liver biopsy specimens obtained from healthy chimpanzees were normal, but hepatitis B virus (HBV) infection was demonstrated in the third case by immunohistochemistry and radioimmunoassay. In the last biopsy specimen a large accumulation of HBV core particles was present in the nuclei and among the filaments of CCIs of the hepatocytes. This finding suggests that crystallizations of viral proteins may form intracytoplasmic inclusions in hepatocytes, whereas in other cases toxic effects (alcohol) or normal conditions (storage) may lead to the formation of similar structures.

Immunocytochemical detection of the 70-kd heat shock protein in alcoholic liver disease.

Exposure of cells to physical (eg, heat) or chemical (eg, alcohol) stress results in increased synthesis of a set of highly conserved polypeptides termed heat shock proteins (HSPs), among which the 70-kd protein (HSP 70) is one of the most consistently inducible and highly conserved. This HSP has adenosine triphosphate-binding properties and is known to associate strongly with cytoskeletal structures that are usually disrupted on injury by heat or alcohol. Some HSPs apparently function as accessories to a nonlysosomal, adenosine triphosphate-dependent proteolytic system that binds and digests away stress-generated abnormal or denatured proteins after their conjugation with ubiquitin, a small HSP. Ubiquitin has been demonstrated immunocytochemically in Mallory bodies, which represent mainly degenerated intermediate filaments accumulated in hepatocytes of alcoholic-diseased liver. We immunostained histologic sections from patients with alcoholic liver disease using a polyclonal antibody raised against HSP 70. Strong diffuse cytoplasmic immunoreactivity was observed in many hepatocytes, including cells without Mallory bodies or fatty degeneration. Positive immunoreactivity for HSP 70 points to a possible involvement of this HSP in the pathogenesis of alcoholic liver disease. It also suggests that immunocytochemical detection of HSP 70 may serve as a more sensitive indicator of hepatocellular injury.

The nature of hyaline (eosinophilic) globules and vascular slits of Kaposi's sarcoma.

Ultrastructural studies of Kaposi's sarcoma (KS) from skin biopsies of 24 patients (eight with acquired immunodeficiency syndrome (AIDS) and 16 without) were performed to delineate the nature of hyaline globules and vascular slits. These structures have been regarded as one of the important criteria for the recognition of KS under light microscopy. Histochemical and immunochemical studies were also performed to correlate with the electron microscopic (EM) observations. The most remarkable EM findings of KS were the intracytoplasmic lumen formation and erythrophagocytic activities of the neoplastic cells, particularly in the mature nodular, or neoplastic stage. The spindle-shaped or ovoid neoplastic cells frequently contained one to several intact and fragmented red blood cells. The intracellular and extravasated erythrocytes were often arranged in single files, giving these vascular slits an elongated appearance on longitudinal sections. The phagocytic activities of the neoplastic cells were demonstrated by the presence of membrane-bound lysosomes containing phagocytized erythrocytes and their partially digested forms (erythrophagosomes) adjacent to pinocytotic vesicles, prominent rough endoplasmic reticulum, and Golgi apparatus, as well as scattered, small, membrane-bound lysosomal granules, some of which were attached to the erythrophagosomes. The erythrophagosomes underwent various stages of disintegration. The partially digested red cells varied from 0.4 to 10 microns in diameter. The results of histochemical and immunochemical findings also strongly suggested that erythrophagosomes were most likely the hyaline globules (bodies) seen in light microscopy. The exact mechanism of erythrophagocytosis is uncertain. However, its consequences, erythrophagosomes, and intracytoplasmic lumen formation, particularly in the nodular or neoplastic stage in patients with and without AIDS, are among the important histologic features of KS.

Identification of basement membrane components in eosinophilic globules in a case of Spitz's nevus.

A case of Spitz's nevus with eosinophilic globules was examined using antibodies for several components of the basement membrane. Aggregated tumor cells revealed the same characteristics as normal nevocytic nevi, that is, they were surrounded by laminin and type-IV collagen, whereas type-VII collagen was absent. All of these components of basement membranes, including type-VII collagen, were also found in eosinophilic globules, which were densely stained by these antibodies. It is assumed that these eosinophilic globules are essentially composed of basement membrane components, which are probably synthesized by epidermal and possibly also by melanocytic tumor cells.

Detection of the surface-exposed 18-kilodalton binding protein in Chlamydia trachomatis by immunogold staining.

Dot-blot analysis of Chlamydia trachomatis elementary bodies (EBs) with monospecific polyclonal antibodies demonstrated that the 18-kilodalton binding protein is surface exposed. Immunoelectron microscopy with whole serovar L2 EBs and ultrathin sections confirmed this finding. In addition, only the extracellular EBs and not the intracellular reticulate bodies were labeled with immunogold.

Ultrastructural localization of Charcot-Leyden crystal protein (lysophospholipase) to intracytoplasmic crystals in tumor cells of primary solid and papillary epithelial neoplasm of the pancreas.

Charcot-Leyden crystals (CLC), composed of a single protein with lysophospholipase activity, have been traditionally associated with eosinophil-rich disorders. Atypically shaped and typical CLC were noted by electron microscopy in the surgical sample from a patient with solid and papillary epithelial neoplasm of the pancreas. This rare primary tumor of the pancreas with limited aggressive behavior also contained damaged and partially or completely degranulated eosinophils in the tumor stroma. We localized CLC protein by ultrastructural immunogold staining to the CLC within vacuolar structures and in vesicles of tumor cell cytoplasm as well as to the cytoplasm and nucleus of eosinophils in the tumor stroma. These findings provide evidence that epithelial tumor cells contain Charcot-Leyden crystal protein that most likely originates from tumor stroma eosinophils. Tumor stroma eosinophils have generally been associated with improved prognoses in a wide variety of epithelial neoplasms. The role of tumor CLC protein (lysophospholipase) in these settings deserves further investigation.

Influence of fixation procedures on the microanalysis of lead-induced intranuclear inclusions in rat kidney.

Using Laser Microprobe Mass Analysis (LAMMA), we studied the chemical composition of lead-induced intranuclear inclusions in rat kidney tissue prepared by three different wet chemical fixation procedures for transmission electron microscopy. Fixation with glutaraldehyde-Na2S gave the same results as fixation with glutaraldehyde only: a high lead concentration could be detected. Therefore, for lead strongly bound to proteins, precipitation procedures are not essential. Post-fixation with osmium tetroxide drastically changed the composition of the inclusions: the lead concentration decreased substantially, while sodium, calcium, and barium were introduced. The osmium tetroxide fixative was found to be the source of the contamination. It also contained aluminum, and we suggest that other proteins (e.g., in neurofibrillary tangles) might be able to take up Al out of solution and that care must be exercised in interpreting the microanalytical results of osmium-fixed material. For the microanalysis of the lead inclusions, fixation with glutaraldehyde only provides a good compromise between preservation of the ultrastructure and maintenance of the element distribution.

Cytologic, ultrastructural and immunologic features of intracytoplasmic hyaline bodies in fine needle aspirates of hepatocellular carcinoma.

Intracytoplasmic hyaline bodies in malignant cells from an aspirate of a liver mass are suggestive of hepatocellular carcinoma. Such inclusions were studied by light and electron microscopy and by immunocytochemistry in fine needle aspirates from five cases of hepatocellular carcinoma. Seen by light microscopy, the inclusions were round or ovoid and were surrounded by a prominent halo. By both light and electron microscopic immunocytochemistry, the hyaline bodies showed negative staining for alpha-fetoprotein, alpha-1-antitrypsin and cytokeratin. Ultrastructurally, they were not membrane bound and were composed of filamentous, finely granular material, resembling the early stages of Mallory bodies.

Neoplastic embryoid bodies of embryonal carcinoma C44 as a source of blastocele-like fluid.

There is a cytotoxic activity in blastocele fluid that kills embryonal carcinoma cells with trophectodermal potential but spares those with embryonic potential. This activity is present when programmed cell death occurs in the inner cell mass (ICM), and the ICM loses its trophectodermal potential. Because of the paucity of blastocele fluid, cystic embryoid bodies of embryonal carcinoma C44 were examined ultrastructurally and in tissue culture to determine if they corresponded to late blastocysts and if their fluid corresponded to blastocele fluid. No trophectoderm was demonstrated in the embryoid bodies, but embryonal carcinoma and endoderm were present, leading to the conclusion that the embryonal carcinoma corresponded to late ICM that had expressed endodermal potential. As a result the cyst fluid might have contained the toxic activity of blastocele fluid. The cyst fluid of C44 embryoid bodies did contain a soluble, low-molecular-weight, cytotoxic activity that preferentially killed embryonal carcinoma cells with trophectodermal potential while sparing those with embryonic potential. Enough of this fluid was available to determine the chemical nature of this toxic activity.

Immunocytochemical and lectin-binding studies on Lafora bodies.

Antibodies to a range of intermediate filaments and lectins specific for several different carbohydrates were used to study Lafora bodies in two cases of Lafora disease. Positive staining of Lafora bodies was found with antibodies to 160 KD and 200 KD neurofilaments and to desmin. Positive staining with concanavalin A showed different patterns of inhibition with glucose and mannose, suggesting that the latter hexose contributes to the 10% non-glucose carbohydrate content of these bodies.

Characterization of gastrin-releasing peptide immunoreactivity in distinct storage particles in guinea pig myenteric and Torpedo electromotor neurones.

Using high resolution centrifugal density-gradient separation of cytoplasmic extracts of guinea pig myenteric plexus and Torpedo electric tissue, we have succeeded in isolating fractions of storage particles rich in gastrin-releasing peptide (GRP). In extracts of myenteric plexus and gradients derived therefrom, the 10-amino acid GRP peptide (GRP-10) was the sole form present; this was bimodally distributed in the gradients, one peak copurifying with Golgi membranes and apparently consisting of immature storage particles, the other with other synaptophysin-rich neuropeptide-containing particles. In extracts of electric organ, a tissue rich in cholinergic electromotor nerve terminals, and gradients derived therefrom, GRP-like immunoreactivity behaved in gel permeation and reversed phase high performance liquid chromatography like the 27-amino acid peptide (GRP-27). About half of the immunoreactivity sedimented in the centrifugal gradient to a region rich in particles containing vasoactive intestinal polypeptide-like immunoreactivity; the remainder was recovered in a very dense region of the gradient containing larger membrane fragments, including synaptosomes. The electromotor nerves and cell bodies also contained GRP-27-like immunoreactivity in relatively high concentration as did the Torpedo gut. It is concluded that this GRP-like peptide is packaged in dense storage particles in the electromotor neurones.

Asbestos body formation and iron accumulation in mouse peritoneal granulomas after the introduction of crocidolite asbestos fibers.

This report describes the cell biology of the development of asbestos bodies after a single intraperitoneal injection of a suspension of crocidolite asbestos fibers into the mouse peritoneal cavity. The majority of the infected fibers were found in aggregates of peritoneal macrophages, exudate cells, and fibrous tissue. These aggregates developed into granulomas containing not only numerous asbestos fibers, but also cells of various types, including macrophages, multinucleated giant cells, fibroblasts, plasma cells, granulocytes, and mast cells. Cytoplasmic ferritin was abundantly present in macrophages and giant cells. In addition, iron-rich inclusion bodies were detected. The results of this study show that asbestos body formation can occur outside the pleural cavity. Asbestos body formation occurred in the granulomas after periods of 1 month and longer. On the basis of morphologic criteria, various types of asbestos body were distinguished. X-ray microanalysis showed that variations in the density of the coat could attributed to the presence of chemical elements in various concentrations. Evidence is presented that asbestos body formation is an extracellular phenomenon.

A case of testosterone-secreting adrenal cortical adenoma with spironolactone body-like inclusion.

Testosterone-secreting adrenal adenoma is rare. We recently experienced a 17-year-old pubertal girl who showed signs of virilization and a high serum level of testosterone. The excised adrenal gland showed a 3.5 x 3 x 3-cm cortical adenoma. Light and electron microscopic findings together with the high serum level and high tumor tissue contents of testosterone and dehydroepiandrosterone (DHA) indicated that the tumor was a testosterone-secreting adrenal cortical adenoma. This appears to be a rather rare tumor from a review of the literature. Interestingly, in this case, the cytoplasm of the tumor cells contained structures resembling spironolactone bodies. From the results of enzyme histochemistry, the steroidogenetic pathways in this tumor were speculated.

Sequence of a human MAP-2 region sharing epitopes with Alzheimer neurofibrillary tangles.

Microtubule-associated protein 2 (MAP-2), an abundant neuronal protein, consists of a short microtubule-binding domain and a long projection arm. MAP-2 shares epitopes with Alzheimer neurofibrillary tangles (NFT). However, most anti-MAP-2 antibodies do not stain detergent-extracted NFT, and the role of MAP-2 in NFT formation has therefore been unclear. We have determined the sequence of a 1.7 kb partial MAP-2 cDNA encoding at least three NFT epitopes. The epitopes are not removed by detergent extraction of tangle preparations, suggesting that they are integral components of NFT. Expression vectors containing restriction fragments of the cDNA were used to assign the epitopes to a 51-amino-acid region near the end of the MAP-2 projection arm, distal to the microtubule.

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.

Inhibition of growth of Chlamydia trachomatis by the calcium antagonist verapamil.

Treatment of BGM (African Green Monkey kidney) cells with the calcium antagonist Verapamil resulted in a reduced yield of chlamydial infectious particles. The inhibitory effect was concentration-dependent, the maximal effect being achieved at 200 microM-Verapamil, which produced a 99.99% reduction of infectious particle yield. Electron microscopy showed that control Chlamydia trachomatis-infected BGM cells contained typical large inclusions in which most of the particles were elementary bodies, whereas Verapamil-treated infected cells contained small inclusions consisting predominantly of reticulate bodies. The findings indicate a possible therapeutic use of this calcium antagonist as an anti-chlamydial drug.

Verifying copper toxicosis in Bedlington terriers by analytical electron microscopy of needle biopsies of liver.

Diagnosis of copper toxicosis in Bedlington terriers was performed by analytical electron microscopy of needle biopsy of the liver. The animals were four Bedlington terriers clinically suspected of hereditary copper toxicosis, and four healthy control animals of the same breed. Light microscopy of the livers of the diseased animals revealed dense bodies in the hepatocytes which stained histochemically for copper. Analytical electron microscopy showed that the intrahepatocytic bodies consisted almost entirely of copper.