Serum Gc system in liver cirrhosis and hepatoma

Summary Serum Gc polymorphism was studied in 85 patients with liver cirrhosis, in 65 with cirrhosis plus hepatoma, and in 40 with hepatoma without cirrhosis. Six hundred unrelated healthy Greeks served as controls. The Gc 1-1 phenotype was found more frequently in patients who had cirrhosis with or without hepatoma but the incidence of the Gc¹ gene was significantly higher in patients with cryptogenic-HBsAg negative cirrhosis. On the other hand, the Gc 2-2 phenotype was found about three times more frequently in patients who had hepatoma without cirrhosis, and the incidence of the Gc² gene was significantly higher in the same group than in the controls. Consequently, it could be surmised that the Gc¹ and the Gc² genes predispose, under the influence of various factors, the development of cryptogenic cirrhosis and hepatoma without cirrhosis, respectively.     

Serum protein groups (Hp, GC, C3) in patients with gastric carcinoma.

The phenotypes and gene frequencies of 3 serum protein systems (Hp, GC and C3) were studied in 114 consecutive patients from all over Greece with gastric carcinoma. Healthy Greeks studied previously in our Department served as controls. No significant differences were found between patients and controls concerning Hp. Significant differences were found in the GC and C3 systems; GC 2-1 and C3F phenotype as well as C3*F gene frequencies were significantly higher in patients than in controls.     

Oxytocin analogs with oxygen-containing side chains in position 3

We have synthesized three oxytocin analogs containing an oxygen atom in the amino acid side chain in position 3 to determine the influences of increased side chain length and of hydrophilicity on the potencies and specificities of the resulting analogs. These three analogs: [3-O-methylhomoserine] oxytocin, [3-O-ethylserine] oxytocin, and [3-O-methylthreonine] oxytocin - have the following activities in U/mg: 490, 208, 265, milk ejection; 125, 129, 63, uterus in vivo; 0.2, 16, 0.03, antidiuretic; and 0.1, 0.5, 0.1, pressor. The results show that a longer side chain, [3-O-methylhomoserine] and [3-O-ethylserine] vs. [3-O-methylthreonine], tends to increase all activities. Moving the hydrophilic oxygen farther away from the peptide backbone, on the other hand, decreases vasopressin-like activities but increases or has no effect on oxytocin-like activities.     

Localization of putative gonadotrophin releasing hormone receptor protein in the anterior pituitary

Specific binding of a fully biologically active 125I-gonadotrophin releasing hormone (GnRH) to isolated anterior pituitary cells is time dependent, saturable and the concentration dependent binding curves exhibit positive cooperativity. Binding to intact or solubilized plasma membranes and an affinity purified GnRH receptor protein reveals in all instances multiple high affinity binding sites. Thus, GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles. To investigate the latter, the binding of 125I-GnRH to various subcellular fractions was studied and its affinity and time requirements determined. GnRH binding to plasma membranes and secretory granules was to multiple high affinity sites, while that to nuclei and microsomes was to a single high affinity site. Binding was 1.83 +/- 0.07, 0.78 +/- 0.04, 0.31 +/- 0.03 and 0.27 +/- 0.03 fmol micrograms-1 protein for isolated plasma membranes, secretory granules, microsomes and nuclei, respectively, after 30 min incubation with 10(-9) M GnRH. The magnitude of binding to microsomes did not change during the incubation period. It did not show any decrease (p greater than 0.05) in isolated nuclei and plasma membranes, except for the 24 h time period, when a significant drop (p less than 0.001) was seen. Binding to the secretory granule fraction culminated at 15 min and then decreased (p less than 0.001) steadily to a non-detectable level at 24 h. Thus GnRH receptor protein or its portion may be an integral part of some membranous particles in the anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microtubule-interacting drugs induce moderate and reversible damage to human bone marrow mesenchymal stem cells

Objectives:  This study aimed to investigate molecular and cellular changes induced in human bone marrow mesenchymal stem cells (hMSCs) after treatment with microtubule-interacting agents and to estimate damage to the bone marrow microenvironment caused by chemotherapy.
Materials and methods:  Using an in vitro hMSC culture system and biochemical and morphological approaches, we studied the effect of nocodazole and taxol® on microtubule and nuclear envelope organization, tubulin and p53 synthesis, cell cycle progression and proliferation and death of hMSCs isolated from healthy donors.
Results and conclusions:  Both nocodazole and taxol reduced hMSC proliferation and induced changes in the microtubular network and nuclear envelope morphology and organization. However, they exhibited only a moderate effect on cell death and partial arrest of hMSCs at G₂ but not at M phase of the cell cycle. Both agents induced expression of p53, exclusively localized in abnormally shaped nuclei, while taxol, but not nocodazole, increased synthesis of β-tubulin isoforms. Cell growth rates and microtubule and nuclear envelope organization gradually normalized after transfer, in drug-free medium. Our data indicate that microtubule-interacting drugs reversibly inhibit proliferation of hMSCs; additionally, their cytotoxic action and effect on microtubule and nuclear envelope organization are moderate and reversible. We conclude that alterations in human bone marrow cells of patients under taxol chemotherapy are transient and reversible.     

Normal and Ha-ras-1 oncogene transformed Buffalo rat liver (BRL) cells show differential resistance to cytoskeletal protein inhibitors.

In the present study, using immunofluorescence microscopy, we have demonstrated that normal and Ha-ras-1 transformed Buffalo rat liver (BRL) cells which were exposed to cytoskeletal protein inhibitors, showed a differential resistance of their microfilament and microtubule networks. One hour exposure of normal BRL cells to 10(-5) M cytochalasin B provoked a clear and already total breakdown of actin filaments. However, at this concentration of cytochalasin B, the microfilaments of transformed BRLHO6T1-1 cells were not seriously affected; a higher cytochalasin B concentration (> or = 2 x 10(-5) M) was required to induce a significant breakdown of microfilaments in these transformed cells. The two cell lines also demonstrated differential microtubule stability when they were treated with either colchicine or triethyllead. Three hours exposure to 10(-6) M of either antimicrotubule agents was sufficient to disrupt the microtubules of normal BRL cells, without affecting their counterparts in the transformed BRLHO6T1-1 cells. A 10-fold higher drug concentration (10(-5) M) was required to induce microtubular breakdown in the transformed BRL cells. The differential stability of microfilaments and microtubules in normal and transformed BRL cells that was observed could not be attributed to a differential internalization of the agents, as shown by experiments on the uptake of [3H]-cytochalasin B and triethyllead. In addition, the transformed BRLHO6T1-1 cells did not express altered actin and tubulin isoforms, as demonstrated by isoelectric focusing followed by immunoblotting analysis. We conclude that the transformation of BRL cells with the Ha-ras-1 oncogene results in a greater stability of microfilaments and microtubules, leading to a structurally firmer cell shape.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decrease of acid phosphatase activity in murine peritoneal macrophages infected with Leishmania donovani

The effect of infection with Leishmania donovani on the activity and isoenzyme composition of acid phosphatase within individual murine peritoneal macrophages maintained in vitro was studied. Concentrations of acid phosphatase activity and number of intracellular parasites were quantitated using a computer-assisted cytospectrophotometry system. Changes in the isoenzyme composition of macrophages during infection with L. donovani were detected by comparing the patterns of acid phosphatase levels between macrophages treated in the absence and presence of an enzyme inhibitor. It was observed that the concentration levels of acid phosphatase activity in macrophages were decreased significantly by infection with L. donovani. An inverse relation existed between concentration of acid phosphatase activity and the number of intracellular L. donovani. Reduced concentrations of acid phosphatase activity were also observed in macrophages uninfected but exposed to L. donovani. The isoenzyme composition in macrophages did not change during the course of infection with L. donovani. These results demonstrate that L. donovani reduces the acid phosphatase activity of macrophages.     

Differences in the G/total actin ratio and microfilament stability between normal and malignant human keratinocytes

The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0·30 ± 0·03 (mean ± SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0·49 ± 0·03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0·5 ± 0·07 (n = 4), indicating a 1·7-fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress-fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10^?6 and 10^?5 M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10^?4 M ), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted. We concluded that the G/total actin ratio was significantly increased in malignant keratinocytes. This seems to be correlated with altered microfilament morphology and resistance to cytochalasin B treatment. Our results suggest that the process of malignant transformation may be characterized by changes in the state of the polymerization of actin and in the stability of the microfilament network indicating that both features could potentially serve as markers determine the transformed state of keratinocytes.