Effects of recombinant human serum albumin on ascites in rats with puromycin aminonucleoside-induced nephropathy.

1. Recombinant human serum albumin (rHSA) (1 g/kg) significantly decreased the weight of ascites in rats with puromycin aminonucleoside-induced nephropathy. 2. Furosemide (1-30 mg/kg) did not significantly reduce the weight of ascites in this model. 3. A combination of rHSA (1 g/kg) with furosemide (5 mg/kg) significantly decreased the weight of ascites in this model compared with furosemide alone. 4. In consideration of these results, rHSA can be a substitute for human serum albumin products prepared from human plasma in therapy for ascites or edema in furosemide-resistant nephrotic syndrome.

Preparation and characterization of a disulfide-linked bioconjugate of annexin V with the B-chain of urokinase: an improved fibrinolytic agent targeted to phospholipid-containing thrombi.

A conjugate of annexin V and the B-chain of urokinase was prepared and its fibrinolytic properties were studied. First, a mutant of annexin V was constructed with an N-terminal extension of six amino acids (Met-Ala-Cys-Asp-His-Ser) and with Cys316 mutated to Ser; this molecule was expressed in Escherichia coli. The urokinase B-chain was prepared by limited reduction of the interchain disulfide bond between the A- and B-chains of urokinase. These two molecules were then then connected by a disulfide bond and purified to yield a 1:1 stoichiometric conjugate. The conjugate had the same catalytic activity as urokinase against a synthetic substrate, Glt-Gly-Arg-MCA, and a similar plasminogen activating activity. The conjugate showed the same binding affinity for phosphatidylserine-containing membranes as annexin V. The in vitro fibrinolytic activity of the conjugates on clots prepared from platelet-rich plasma was comparable to that of urokinase. However, the conjugate showed 3-4-fold stronger in vivo thrombolytic activity than urokinase in a rat pulmonary embolism model, while having essentially the same plasma clearance rate as urokinase or B-chain. These results show that annexin V is a useful agent for targeting plasminogen activators to phospholipid-containing thrombi.

Functional-morphological effect of the synthetic atrial natriuretic factor (ANF 101-126) on the zona glomerulosa cells of adrenal cortex.

The effects of atrial natriuretic factor (ANF) on the adrenal steroidogenesis were examined in the zona glomerulosa of rats in vivo. ANF administration provoked a significant decrease in the width of zona glomerulosa (P < 0.01) and a significant enlargement of nuclei of zona glomerulosa cells (P < 0.01). Ultrastructurally conspicuous change was observed in mitochondria of the cells of the zona glomerulosa treated with ANF, representing elongated mitochondria with "parallel arrays" of tubules with pipe-like structure. In vitro autoradiography 125I-labelled ANF binding displayed a significant decrease of a specific receptor site in the zona glomerulosa treated with ANF. A significant lowering in plasma levels of aldosterone was observed in ANF-treated rats. These findings could be interpreted as an inhibitory effect of ANF on adrenal steroidogenesis of the glomerular zone by action through a specific ANF-receptor.

Positive effect of GAC gene product on the mRNA level of glyoxalase I gene in Saccharomyces cerevisiae.

A DNA fragment carrying a part of the structural gene for yeast (Saccharomyces cerevisiae) glyoxalase I was cloned from a lambda gt11 expression library using anti-glyoxalase IIgG as a probe. By Northern blotting analysis, the amount of glyoxalase I mRNA was found to increase in yeast cells containing plasmids carrying the GAC gene, which is a positive regulator for yeast glyoxalase I activity. This suggests that the GAC gene product may accelerate the transcription of glyoxalase I gene or may have some positive effects on the accumulation of glyoxalase I mRNA in yeast cells.

Analysis of 2-amino-N6-hydroxyadenine-induced mutagenesis in phage M13mp2.

The mechanism of mutagenesis induced by 2-amino-N6-hydroxyadenine (AHA) and its deoxyriboside (AHAdR) was studied by determining the nucleotide sequences of phage M13mp2 mutant DNA samples. Mutations in the lac promoter-lacZ alpha region of the phage were induced by addition of this agent to culture media in which the phage was growing inside the host bacteria. The spectrum of spontaneous mutation was also investigated. The induced sequence changes were mostly base transitions (80% with AHA and 90% with AHAdR). A few single-base deletions and additions were detected, but they were ascribable to spontaneous mutations. These results are consistent with the incorporation type mechanism proposed by Janion (this issue). In the Ames Salmonella assay, both AHA and AHAdR showed strong mutagenicity in strain TA100 but no activity in TA98.

Effect of the adrenal inhibitor trilostane on the morphology of the adrenocortical cells of Dahl salt-sensitive and Dahl salt-resistant rats.

The effects of trilostane on the adrenal cortex of Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were investigated morphometrically, histochemically, ultrastructurally and biochemically. The statistical analysis indicated that trilostane induced a significant increase in the adrenal weight and the surface area of cells and nuclei in the zona fasciculata (ZF) of the adrenal cortex of DS and DR rats (P less than O.OI). DS rats treated with trilostane revealed marked accumulation of large amounts of lipid droplets and a decrease in the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the ZF. Ultrastructurally, the mitochondria of the ZF in DS rats treated with trilostane revealed swelling of matrix with a loss of cristae and occasional interruption of the membranes of mitochondria. Some of them had a continuity with lipid vacuoles or SER, presenting a characteristic 'feather'-like appearance. Other characteristic findings in DS rats treated with trilostane were a marked villous proliferation of plasma membranes with numerous dense bodies, occasional coated pits, and pinocytic vesicles in the outer portion of the ZF. In DS and DR rats the plasma level of ACTH increased, and corticosterone decreased significantly (P less than O.OI) after treatment with trilostane. These morphological alterations were considered to be an expression of the inhibitory effects of trilostane on the adrenal steroidogenesis in DS and DR rats, more especially in DS rats. Simultaneously there were confirmed morphological alterations in the cells of the ZF, reflecting the feedback stimulation of endogenous ACTH. The cells of the ZF of DS rats were more responsive to suppression by trilostane than those of DR rats.

Immunoreactivity and receptor expression of insulinlike growth factor I and insulin in human adrenal tumors. An immunohistochemical study of 94 cases.

Using immunoperoxidase methods, 94 human adrenal tumors were examined for evidence of immunoreactivity and receptor expression of insulinlike growth factor I (IGF-I) and insulin. The frequency of IGF-I in adrenocortical carcinomas was significantly higher than that in adenomas of the adrenal glands. The adrenocortical carcinomas showed strong intensity of staining for IGF-I, IGF-I receptors, and insulin receptors. A significant correlation between immunoreactivity and receptor expression of both IGF-I and insulin was found only in the adrenocortical carcinomas. The adrenocortical adenomas with Cushing's syndrome and pheochromocytomas, more than adrenocortical adenomas with Conn's syndrome, also stained strongly for insulin receptors. Thus the IGF-I and insulin probably play a role in the growth of adrenocortical carcinoma tissues, possibly through autocrine mechanisms. The expression of insulin receptors in adrenocortical adenomas in the presence of Cushing's syndrome and pheochromocytomas may be associated with functions.

Partial characterization of insulin-like growth factor I in primary human lung cancers using immunohistochemical and receptor autoradiographic techniques.

We investigated primary human lung cancers resected surgically or obtained at autopsy. Included were squamous cell carcinoma (SQC) (five cases), adenocarcinoma (ADC) (six cases), large cell carcinoma (LCC) (four cases), and small cell carcinoma (SCC) (two cases). The objective of the study was to search for the presence of insulin-like growth factor I (IGF-I)-like immunoreactivity using immunohistochemical staining and for the localization of IGF-I binding sites, using in vitro quantitative receptor autoradiographic techniques. IGF-I-like immunostaining was present in all cases of SQC, ADC, and LCC, but not in cases of SCC. Strong immunostaining was observed in cases of SQC. On the other hand, ADC and LCC tissues showed a moderate or weak staining. Specific binding sites for IGF-I were present in all cases of SQC, ADC, LCC, and SCC examined. High densities of 125I-IGF-I binding sites were localized in cases of SQC and SCC. Low to high densities of the binding sites were found in LCC. Cases of ADC showed low densities of 125I-IGF-I binding sites. Specific binding obtained at a concentration of 80 pM 125I-IGF-I was competitively displaced by unlabeled IGF-I, with a 50% inhibitory concentration value of 1.84 +/- 0.31 x 10(-10) mol, whereas human insulin was much less potent in displacing the binding. This specificity profile is consistent with characteristics of IGF-I receptors. Scatchard analysis showed the presence of a single class of high affinity binding sites for IGF-I, with a Kd of approximately 1 nmol. Thus, the possibility that IGF-I may play a role in the growth of human lung cancers would have to be considered.

Immunohistochemical expression of epidermal growth factor receptors in human adrenocortical carcinoma.

Epidermal growth factor (EGF) receptors were examined immunohistochemically in 64 adrenocortical carcinomas obtained at autopsy, and in 23 adrenocortical adenomas and seven pheochromocytomas obtained during surgery. In the nonneoplastic adrenal gland, EGF receptors were scattered to the zona glomerulosa, zona fasciculata, and zona reticularis. Adrenocortical carcinomas (63 of 64), more than adrenocortical adenomas (10 of 23) or pheochromocytomas (four of seven), stained positively for EGF receptors (P less than .01). The immunoreactivity was limited to the cytoplasm, cell membrane, and chromatin. When the antibody was immunoabsorbed with an excess of immunogen peptide, there was no evidence of immunostaining. The adrenocortical carcinomas could be classified into 16 cases of the well-differentiated type, 33 cases of the moderately differentiated type, and 15 cases of the poorly differentiated type. There was no relationship between histologic grading and staining intensity of the EGF receptors. On the other hand, more than 80% of the cases of adrenocortical carcinomas revealed a moderate to high intensity for EGF receptors. In 62 of the 64 patients, there was already metastases to other organs. We conclude that the expression of EGF receptors is associated with tumor growth and/or metastatic potential in adrenocortical carcinoma.

Specific [125I]brain natriuretic peptide-26 binding sites in rat and pig kidneys.

Specific binding sites for porcine brain natriuretic peptide-26 (BNP-26), a member of the atrial natriuretic peptide family (ANPs), were investigated in the kidney by using receptor autoradiographic and membrane binding techniques with [125I]BNP-26. The binding sites were discretely localized in rat and porcine kidney areas corresponding anatomically to the glomeruli and inner medulla. There were no differences between the localization of [125I]BNP-26 and [125I]alpha-rat ANP binding sites in the kidney. [125I]BNP-26 binding to solubilized membranes from isolated glomeruli of the rat kidney was saturable, and a single class of high-affinity sites was labeled with a KD of 372 pM. The radioligand bound to two sites in solubilized inner medullary membranes of the rat, a low-affinity site with a KD of 30 nM, and a high-affinity site with a KD of 33 pM. The rank order of potency to inhibit binding was BNP-26 = alpha-rat ANP-(1-28) greater than atriopeptin III (ANP-(103-126)) much greater than atriopeptin I (ANP-(103-123)) greater than des-Cys105,Cys121- ANP-(104-126). Thus, [125I]BNP-26 presumably recognizes ANP receptors in the kidney. The possibility that BNP-26 regulates, as a circulating hormone, kidney functions by binding to ANP receptors would have to be considered.

A case of deoxycorticosterone-producing benign adrenocortical tumor.

A case of deoxycorticosterone-producing benign adrenocortical tumor is presented. A review of the English literature revealed that this is the second case of deoxycorticosterone-producing adrenocortical adenoma.

[Two cases of amylase-producing lung cancer].

In 1951, Weiss et al. first reported a case of an amylase-producing lung cancer. Since then, reports on immunohistochemical, ultrastructural, and biochemical studies have been published. We recently have experienced two autopsy cases of lung cancer that manifested high amylase activity in the serum, urine, and pleural effusions. Histologically, both lung cancers were classified as being well differentiated adenocarcinomas, and immunohistochemically, the tumor cells showed positive immunoreactivity for amylase. This paper discusses the histogenesis of amylase-producing lung cancers and cases found in the literature.

"Parallel array" of tubules with pipe-like structure in the mitochondria of glomerulosa cells of adrenal cortex. Ultrastructural and freeze-fracture replica studies.

We found "parallel array" of tubules with a pipe-like structure in the mitochondria of glomerulosa cells of the adrenal cortex of the rats treated with atrial natriuretic factor (ANF) that inhibits aldosterone production. This structure was characterized by the regular parallel arrays of tubules running straight along the mitochondrial long axis, representing a pipe-like structure. The cross section exhibited the round shape with an electron lucent core measuring about 40 nm and dual thick membranes measuring 10 nm in thickness. Some of these tubules were connected with the mitochondrial inner membrane. Freeze fracture replica represented round particles distributed on the surface membrane of these tubules, indicating protein particles. This particular structure was considered to be one of the changes occurring in the mitochondrial inner membrane on which structural proteins were localized. These tubules probably appeared in connection with the impaired mitochondrial protein synthesis in the process of the biosynthesis of mineralcorticoid, because this structure was found only in the glomerulosa cells under the decreased and increased aldosterone production.

Receptor autoradiographic localization of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands.

We report here the first evidence of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands, obtained at autopsy. Sections of tissue were incubated with 0.1 nM [125I]IGF-I and analyzed using [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry. Specific binding sites of [125I]IGF-I were found to be localized in the definitive zone, fetal zone, and fetal medulla of the fetal adrenal glands. In the adult adrenal glands, the entire cortex and medulla were specifically labeled with [125I]IGF-I. Specific binding obtained at a concentration of 0.1 nM [125I]IGF-I to areas in the fetal and adult human adrenal glands was competitively displaced by unlabeled IGF-I, with an IC50 value of 0.34-2.54 nM, and 0.38-0.73 nM, respectively, whereas insulin was much less potent in displacing the binding. Acquisition of this knowledge will aid in studies on cell growth and steroid-catecholamines biosynthesis of the human adrenal gland.

Osteoclast-type giant cell tumor of the endometrium. An immunohistochemical study.

We present a rare case of a 55 year old female with an osteoclast-type giant cell tumor of the endometrium associated with leiomyoma and adenomyosis. Multinucleated giant cells and mononuclear stromal cells reacted with vimentin and alpha-1-antichymotrypsin (AACT) using the immunoperoxidase method. Epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), and keratin exhibited negative immunoreaction in these tumor cells. Our immunohistochemical results do not support the epithelial origin of an osteoclast-type giant cell tumor and mesenchymal derivation appeared more likely, suggesting histiocytic origin.

Receptor autoradiographic evidence of specific brain natriuretic peptide binding sites in the porcine subfornical organ.

Specific binding sites of brain natriuretic peptide (BNP), a newly discovered peptide in the subfornical organ (SFO) of porcine brain were investigated, following incubation of related tissue sections with 125I-BNP, then using autoradiography and an image analysis coupled with computer-assisted microdensitometry. Specific 125I-BNP binding sites were found to be localized in the SFO, an area densely labeled by 125I-alpha-rat atrial natriuretic peptide and 125I-(Sar1,Ile8)-angiotensin II. Specific 125I-BNP binding to the SFO was displaced by unlabeled BNP, with a high affinity, and was calculated to be Ka = 0.385 x 10(-9) M and Bmax = 40.1 fmol/mg using a LIGAND computer program. Acquisition of these present findings enhances our knowledge of the physiology of BNP, atrial natriuretic peptides and angiotensin II system in the SFO.

Preparation of anti-ras Mr 21,000 protein monoclonal antibodies and immunohistochemical analyses on expression of ras genes in human stomach and thyroid cancers.

Sixteen clones (RASK-1 to -16) of murine monoclonal antibodies were raised against ras Mr 21,000 protein (p21). The p21 produced by Escherichia coli with inserted v-Ki-ras genes was used as immunogen. RASK-1 was found to be specific for Ki-ras p21, whereas RASK-2 to -16 reacted with the p21s of Ki-, N-, and Ha-ras genes in both enzyme-linked immunosorbent and immunoblotting assays. Binding inhibition assays with biotinylated monoclonal antibodies by enzyme-linked immunosorbent assay showed that the monoclonal antibodies of the 16 clones included those binding to several mutually distinct sites on p21. The expressions of ras p21 in human stomach and thyroid tissues were examined with RASK-3, which reacted with all the Ki-, N-, and Ha-ras p21s immunohistochemically by the avidin-biotin peroxidase complex method. Formalin-fixed, paraffin-embedded tissues of 101 cases of stomach cancer, 53 cases of noncancerous stomach, 74 cases of cancer of the thyroid, and 59 cases of noncancerous thyroid were analyzed. In both the stomach and thyroid, cancer cells expressed p21 predominantly. Cells of cases with various noncancerous disorders as well as certain types of normal cells were also p21 positive. These findings suggest that precaution is required in use of p21 as a cancer marker. Expression of p21 was noted in moderately to well-differentiated stomach cancer, intestinal metaplasia, and atypical hyperplasia. This finding suggests that the appearance of p21 in stomach cancer may be initiated before cytological transformation.

Eosinophilic globules in pheochromocytoma of the adrenal medulla. A histochemical, immunohistochemical and ultrastructural study.

Histochemical, immunohistochemical, and ultrastructural characteristics of eosinophilic globules in pheochromocytoma of the adrenal medulla are described. The globules were observed in 7 (63.5%) out of 11 cases of pheochromocytoma of the adrenal medulla. These globules were eosinophilic, PAS-positive with and without diastase predigestion, phosphotungstic acid hematoxylin (PTAH) positive, and autofluorescent under ultraviolet illumination. Although almost all globules were not stained with neuron specific enolase (NSE) using the immunoperoxidase method, a few globules were stained positive. The lectins of Triticum vulgaris (WGA) and Ricinus communis (RCA-120) were weakly bound to most of the eosinophilic globules. Immunohistochemical reactions for alpha-fetoprotein (AFP), alpha-l-antichymotrypsin (ACT), and human chorionic gonadotropin (HCG) revealed negative reaction in these globules. An ultrastructural study revealed no relationship between these globules and chromaffin secretory granules. These findings suggested that the eosinophilic globules in pheochromocytoma of the adrenal medulla were not related to the chromaffin secretory granules and might be some kind of complex protein.

Malacoplakia of the endometrium. An unusual case studied by electron microscopy and a review of the literature.

A rare case of malacoplakia (MKP) of the endometrium in an 88-year-old Japanese woman is presented. By light microscopy, typical Michaelis-Gutmann bodies (M-G bodies) could be identified. Electron microscopic findings revealed M-G bodies in various stages of development. These features suggested that M-G bodies were derived from round electron-dense granules that coalesced to form phagolysosomes. Early-stage M-G bodies were manifested as small, irregular, needle-shaped areas of calcification in phagolysosomes. The present paper reviews 6 cases of endometrial MKP, 5 of which have previously been reported in the literature, and this case being the sixth, to our knowledge.

Analysis of ras gene expression in stomach cancer by anti-ras p21 monoclonal antibodies.

Using anti-ras p21 monoclonal antibodies, RASK-3, which reacts with all of Ki-, N-, and Ha-ras p21, we examined by immunohistochemistry the expression of p21 in human gastric cancer (80 cases) and benign gastric lesions (32 cases). Ten percent formalin fixed tissues were studied. Ras p21 was positive in 51 cases (64%) out of 80 cases and partially positive in 12 cases (15%) at the cancerous areas. Ras p21 was partially positive in 7 cases (9%) at the noncancer areas of the same slides. Intestinal metaplasia and normal parietal cells were also often positive. In the study of 32 cases of benign stomach lesions, 2 out of 3 cases of atypical hyperplasia (ATP) and 3 out of 11 cases of stomach ulcer with regenerating epithelials were positive. Ras p21 was more dominantly expressed in the well-differentiated type of stomach cancer than the poorly differentiated type. Expression of ras p21, however, was not correlated either with the grades of cancer invasion or with the types of cancer infiltration.