Origin of human triosephosphate isomerase isozymes: Further evidence for the single structural locus hypothesis with Japanese variants

Summary Four electrophoretic variants of human erythrocyte triosephosphate isomerase (TPI) have been studied to investigate the origin of the multiple forms of human TPI, in particular the constitutive TPI-B isozyme and the cell division-associated TPI-A isozyme. The variant phenotype expressed by the constitutive TPI-B isozyme in both erythrocytes and peripheral lymphocytes was also expressed by the cell division-associated isozymes in mitogen-stimulated lymphocytes and hair root cells. These results strongly support the hypothesis of Decker and Mohrenweiser (1981) that TPI-B and TPI-A originated from the same structural gene. We also found that the isozyme e is different from TPI-A with respect to both its electrophoretic mobility and heat stability. This finding is in contrast to the recent conclusion of Yuan et al. (1981) that both the isozyme e and TPI-A are deamidation products of TPI-B.     

Characterization of photoreceptor cell differentiation in the rat retinal cell culture

Photoreceptor cell differentiation in the rat retina was studied in vivo and in vitro, using an immunohistochemical method to demonstrate opsin-like immunoreactivity. Cells in a dissociated monolayer culture expressed some properties characteristic of rat rod cells developing in vivo, including a ciliary structure and opsin-like immunoreactivity. Immunoblot analysis revealed that cultured retinal cells synthesize a polypeptide with the same molecular weight as that synthesized by the intact retina. Although the outer segment (OS) was not present in the culture, immunoreactive cells possessed a ciliary structure. Opsin-like immunoreactivity was found on the plasma membrane, including the cilia. The neuritic extensions were also intensely stained. In mature rod cells of the intact rat retina, opsin was detected only on the OS but, during development, it was found both in the somatic region of the rod cells and on the differentiating OS. During maturation of rod cells opsin immunoreactivity seemed to shift to the OS from other locations. However, some "displaced" photoreceptor cells, found in the inner nuclear layer and extending fibers bipolarly, retained immunoreactivity throughout their structure. The absence of polarized distribution of opsin in these cells is considered to be due to an abnormal environment, which may also be the case with cultured retinal cells. The present culture conditions will offer a useful model system to understand the cellular mechanism of the hereditary retinal dystrophy of rodent animals in which photoreceptor cells selectively degenerate.     

Effects of steroid and thyroid hormones on synthesis of atrial natriuretic peptide by cultured atrial myocytes of rat

The in vitro effects of various steroid and thyroid hormones on synthesis of rat atrial natriuretic peptide (rANP) were studied using new-born rat atrial myocytes in culture. Dexamethasone, testosterone and triiodothyronine markedly stimulated both synthesis and secretion of immunoreactive (IR)-rANP with the same peak after 4-day-culture. Dexamethasone and testosterone dose-dependently (10(-7)-10(-6) M) stimulated synthesis of IR-rANP and were the most potent among various steroids tested. Triiodothyronine (T3) also stimulated synthesis of IR-rANP in a dose-dependent manner (10(-8)-10(-7) M), of which effect was more potent than that of tetraiodothyronine, whereas reverse T3 was ineffective. The present study clearly shows that glucocorticoids, androgens and thyroid hormones directly stimulate synthesis of ANP by atrial myocytes and suggests that ANP may play a potential role in mediating and/or modulating the biological effects by these hormones in the cardiovascular system.     

Purification of a young age-related glycoprotein (Ugl-Y) from normal human urine

Ugl-Y is a glycoprotein that is detected in normal urine samples from young men and women aged 0 to 17 years. It was purified by ammonium sulfate precipitation and various column chromatographies including affinity chromatography using anti-adult urine antibody coupled to Sepharose 4B. The homogeneity of the glycoprotein was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, column chromatography on Sephadex G-75, and the precipitation reaction with anti-Ugl-Y antibody. It was shown to have a molecular weight of 29,000 by gel filtration, and to contain 5.2% neutral sugars (mannose and galactose) and 4% hexosamine (glucosamine). Amino acid analysis of the glycoprotein indicated high contents of acidic and hydroxylic amino acids. Its origin is unknown.     

Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation.

When Saccharomyces cerevisiae cells grown at 23 degrees C were transferred to 36 degrees C, they initiated synthesis of heat shock proteins, acquired thermotolerance to a lethal heat treatment given after the temperature shift, and arrested their growth transiently at the G1 phase of the cell division cycle. The bcy1 mutant which resulted in production of cyclic AMP (cAMP)-independent protein kinase did not synthesize the three heat shock proteins hsp72A, hsp72B, and hsp41 after the temperature shift. The bcy1 cells failed to acquire thermotolerance to the lethal heat treatment and were not arrested at the G1 phase after the temperature shift. In contrast, the cyr1-2 mutant, which produced a low level of cAMP, constitutively produced three heat shock proteins and four other proteins without the temperature shift and was resistant to the lethal heat treatment. The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle.     

Steroidogenic effect of ent-kaur-16-en-15 beta-ol (kaurenol) on isolated rat adrenal cells

In an in vitro bioassay system for adrenocorticotropic hormone using isolated rat adrenal cells, kaurenol, a diterpene alcohol, stimulated corticosterone production and augmented the steroidogenic effect of adrenocorticotropin or forskolin, dose-dependently. Kaurenol had no effect on cyclic AMP production by the cells. The diterpene also had no stimulatory effect on the adrenal adenylate cyclase activity in a cell free system. The results suggest that this particular diterpene exerts a steroidogenic effect through a mechanism independent of cyclic AMP generation.     

Reversible change in light scattering following formation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase modified with N-[p-(2-benzimidazolyl)phenyl]maleimide

An increase in light scattering (3.5 +/- 0.2%) was observed when pig kidney Na+,K+-ATPase preparations modified with N-[p-(2-benzimidazolyl)phenyl] maleimide were phosphorylated by ATP in the presence of 2 M Na+ with Mg2+ to form ADP-sensitive phosphoenzyme (E1P), which had a negative fluorescence intensity (-1.5 +/- 0.3%). Addition of K+ or ouabain to E1P reduced the light scattering to the original level observed in the absence of ATP. Stopped flow measurements showed that the fluorescence change accompanying the E1P formation (t1/2 = 0.1 s) occurred preceding the light-scattering change (t1/2 = 1 s). Oligomycin affected the rate of the scattering increase little, but it diminished the effect of K+ on E1P to reduce the light scattering and increase the fluorescence. The addition of 2 M Na+ to K+-sensitive phosphoenzyme (E2P) immediately decreased the fluorescence (t1/2 = 0.02 s) to form E1P which was followed by a slow increase in the light scattering (t1/2 = 0.25 s). Oligomycin reduced both rates of the above changes accompanying the transition of E2P to E1P. The data suggest the sequential appearance of species of E1P that precede E2P formation during the hydrolysis of ATP.     

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and 1 h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED50s of these antitumor effects after i.v. administration of rHu-TNF were about 50 times as high as ED50s after i.t. administration. ED50 of i.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED50 of i.v. rHu-TNF for vascular effect was only 2-3 times as high as that for cytotoxicity. The whole body autoradiographies with [125I]HSA given i.v. to see the blood influx into tumor tissue and [14C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues. In conclusion, the in vivo antitumor effect of rHu-TNF given i.t. or i.v. appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

Inhibition of atrial natriuretic peptide secretion by forskolin in noncontracting cultured atrial myocytes

Secretory rates for immunoreactive atrial natriuretic peptide (ANP) by 7 - 8 day-old primary cultures of atrial myocytes from adult rats (with myocyte contraction inhibited by tetrodotoxin (TTX)) were (a) constant for at least two hours, and (b) significantly slowed by forskolin (1, 5, and 25 microM), dibutyryl cyclic adenosine monophosphate (1 mM), or isobutylmethylxanthine (100 microM). The substantial rates of ANP secretion which persisted in cells rendered noncontracting either by inhibiting Ca2+ influx via reduction of external [Ca2+] to less than 10(-7) M or by inhibiting sarcoplasmic reticulum Ca2+ release with 100 microM ryanodine were significantly slowed by 25 microM forskolin, but forskolin sensitivity was lost by cells exposed simultaneously to external Ca2+ concentration of less than 10(-7) M and 100 microM ryanodine. Quiescent myocytes whose ANP secretory rate was depressed by forskolin remained responsive to secretory stimulation by phorbol ester.     

Immunohistochemical studies on the localization of fibronectin in human thyroid neoplastic tissues

Fibronectin (FN) has been considered to be involved in the malignant transformation of cells. It was thus of interest to morphologically study the cell surface distribution of FN in various types of thyroid neoplasms employing immunohistochemical techniques. Immunohistochemical staining using the specific anti-human FN antibody was performed by the avidin-biotin-peroxidase complex procedure. Compared to the staining pattern of normal thyroid tissues (control), differences in staining in malignant thyroid lesions and even in fetal and embryonal adenomas were evident, often on the lateral and basal aspects of the cell membrane. Colloid adenomas, however, showed FN staining on the basement membrane, basically similar to the controls. Moreover, in thyroid carcinoma, different staining patterns relating to each of the histological types were observed. The present investigation demonstrates the heterogeneity of the thyroid neoplasms in terms of FN binding.