Acid hydrolases of the rat uterus in relation to pregnancy, post-partum involution and collagen breakdown

1. The rat uterus contains acid cathepsin, beta-glucuronidase, beta-galactosidase, acid phosphatase and deoxyribonuclease II at concentrations comparable with those found in liver. Two non-hydrolytic uterine enzymes, cytochrome c oxidase and aspartate aminotransferase, display only 2-6% of the activity found in liver. 2. The concentrations of acid cathepsin and beta-glucuronidase are significantly decreased in pregnancy and increase 3-4-fold during post-partum involution. 3. The concentrations of beta-galactosidase and acid phosphatase are not decreased in pregnancy and increase only 2-3-fold during involution. 4. The concentrations of these four acid hydrolases increase linearly during the first 4 days post partum and reach their peak values at the same time that wet weight and collagen content fall to their lowest point. 5. The concentration of deoxyribonuclease is depressed in pregnancy but does not rise above normal in the post-partum period. 6. Only a small proportion of each hydrolytic activity can be isolated in the mitochondrial-lysosomal fraction of sucrose homogenates of the rat uterus. This proportion increases during involution. However, the extensive mitochondrial rupture occurring during homogenization indicates that the technique is probably too harsh to obtain a true measure of the proportion of lysosomes present in the intact tissue. 7. There are no significant changes in either the concentration or subcellular distribution of the five acid hydrolases in the livers of the experimental rats during pregnancy or involution. In each case the largest proportion of the activity is found in the mitochondrial-lysosomal fraction of liver homogenates. 8. The results are interpreted in terms of the lysosomal theory of intracellular digestion.     

Binding of kinesin to stress fibers in fibroblasts under condition of microtubule depolymerization

The localization of kinesin in EBTr (bovine embryonic trachea fibroblast) cells was studied by indirect immunofluorescence microscopy using an affinity-purified antibody against bovine adrenal kinesin. It has already been shown that in interphase cells a part of kinesin is located on microtubules and the rest diffusely distributed throughout the cytoplasm [Murofushi et al., 1988]. When microtubules were depolymerized with cold or colchicine treatment, antikinesin antibody-stained fibrous components distinct from microtubules. These fibrous structures were considered to be stress fibers because they were stained with rhodamine-phalloidin and because the fibrous staining with antikinesin antibody was completely lost by treating the cells with cytochalasin D along with colchicine. When cold-treated cells in which a major part of kinesin had been localized on stress fibers were incubated at 37 degrees C, kinesin reappeared on reconstituted microtubules. These observations strongly suggest that kinesin has affinity not only to microtubules but also to stress fibers in culture cells.     

Alternative splicing mechanism in a cytochrome P-450 (P-450PB-1) gene generates the two mRNAs coding for proteins of different functions

Two mRNAs for P-450PB-1 and P-450PB-1(ps) are about 2 kilobase pairs long and have identical sequences with each other except for one short region of high variability (Kimura, H., Yoshioka, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1988) J. Biol. Chem. 263, 701-707). To clarify the origin of the short replacement block between the two mRNAs, we isolated several genomic clones containing relevant gene sequences. Sequence analysis of these genomic clones revealed that the two short segments specific for the two mRNAs are tandemly arranged in a genomic sequence and form exonic sequences equipped with AG and GT sequences on their 5' and 3' ends, respectively, and the putative consensus sequences for the lariat formation. The two short sequences lie between the two exonic sequences coding for the common part of the two mRNAs. Taken together with the structure of the related P-450(M-1) gene (Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285), all these results clearly demonstrate that the two mRNAs are generated from a single gene by alternative splicing at the eighth exons. The synthesis of the two mRNAs is regulated temporally in livers of male and female rats and brains of the female animals. One of the two mRNAs codes for a monooxygenase of P-450PB-1, and the other (P-450PB-1(ps) mRNA) lacks the sequence coding for the heme-binding site conserved among all species of P-450 molecules, and, therefore, it cannot function as a monooxygenase. The immunoblot analysis using an antibody specific for the 15-mer peptide uniquely encoded by P-450PB-1(ps) mRNA shows that the P-450PB-1(ps) peptide is synthesized at least in rat livers of both sexes in temporally regulated manners and is bound to the microsomal membranes. The function of this peptide remains to be seen.     

Purification and characterization of a Ca2+-dependent actin filament severing protein from bovine adrenal medulla

We describe the purification of an actin regulatory protein from bovine adrenal medulla. This protein caused a dose-dependent decrease of the specific viscosity of actin solution within 30 s of its addition in a Ca2+-sensitive way. Sedimentation assays and the observation by electron microscopy showed that this effect was ascribable to the fragmentation of actin filaments. This protein apparently promoted nucleation of actin polymerization and increased the critical concentration of actin for polymerization nearly 5-fold, suggesting its binding to the barbed end of actin filaments. The inhibitory effect of this protein on the elongation of actin from the barbed end of the myosin subfragment S1-labeled actin seeds confirmed this suggestion. These properties are similar to those of gelsolin. However, the physicochemical properties of this protein having a single polypeptide chain with a molecular weight of 74,000, a Stokes radius of 3.9 nm, a sedimentation coefficient (s0(20),w) of 4.5 S, and an immunological characterization showed that this protein is different from gelsolin.     

Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2

We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.     

The existence of activin A/erythroid differentiation factor and its inhibitor in human serum: comparison of normal and chronic renal failure sera.

Activin A/EDF, initially found as a differentiation inducer of murine Friend erythroleukemia, also has a stimulatory effect on erythropoiesis in vitro and in vivo. Here we proved activin A/EDF activity in human serum. The activin A/EDF level in 18 normal human serum samples was measured by a specific bioassay and was found to be 8.3 +/- 4.6 ng/ml, indicating that there exists sufficient activity to affect erythropoiesis in normal serum. In contrast, activin A/EDF activity was reduced in the chronic renal failure patients and 23/26 serum samples examined showed levels below 1.2 ng/ml. Further analysis using HPLC revealed that chronic renal failure serum actually contained as much activin A/EDF as normal serum, and that the difference between normal and patient serum existed in the content of a specific inhibitor of activin A/EDF. This observation suggests the possibility that the inhibitor is participating in the regulation of activin A/EDF activity in vivo in chronic renal failure patients and also the possibility of activin A/EDF could be utilized in the therapy of the anemia of such patients.     

Gene expression of metalloproteinase and its inhibitor in mesangial cells exposed to high glucose.

To clarify the roles of metalloproteinases and their inhibitor (TIMP) in diabetic glomerulopathy, we studied the effect of a high glucose concentration on the gene expression of metalloproteinase transin and TIMP as well as collagen type IV and laminin in cultured rat mesangial cells (MCs). In the high glucose group, collagen type IV, laminin, and TIMP mRNA levels were all elevated in a concentration-dependent manner, whereas transin expression was suppressed. Osmotic control of high glucose with mannitol selectively stimulated TIMP expression. We hypothesize that high glucose decreases matrix-degrading activity as well as increases matrix productivity in MCs.     

Comparison of muscle force,muscle endurance,and electromyogram activity during an expedition at high altitude

Handgrip force (HF), maximal pinch force (MF), muscle endurance (ME), and the median power frequency (MdPF) of the activity shown in the electromyogram (EMG) were studied at various altitudes in eight normal healthy subjects. MF and ME were measured between the index finger and thumb, and all measurements were obtained at altitudes ranging from 610 to 4860 m during an expedition in the Qinghai Plateau in China. With the change in altitude HF, ME, and MF showed no significant change. Compared to the MdPF at 2260 m on ascent, the MdPF at other altitudes showed a significant decrease (P<0.01). Thus, we conclude that muscle performance (HF, MF, and ME) was not affected by the environment at high altitude. However, MdPF was affected and the mean MdPF at 610 m after the expedition did not recover to initial values of MdPF. We suggest these results may have been affected by fatigue and chronic exposure to the hypobaric hypoxic environment, since the members of the expedition party expressed feelings of sluggishness and fatigue after the expedition.     

Intradermal administration of lipopolysaccharide in treatment of human cancer

 Lipopolysaccharide (LPS) has been recognized as a potent antitumor agent in animal tumor models; however, its use in human cancer therapy has been limited to only one trial, in which LPS from Salmonella was given intravenously. It was not very successful because of poor tumor response and was also toxic. We originally developed LPS prepared from Pantoea agglomerans (LPSp), and this was a well-purified, small-molecular-mass (5 kDa) agent. We chose intradermal rather than intravenous administration in the hope that the former would release LPS slowly into the bloodstream, and thus be less toxic while preserving antitumor activity. In our animal tumor models, intradermal administration was indeed less toxic and more beneficial for tumor regression than intravenous administration. We made a pilot study with intradermal administration of LPSp on the treatment of ten advanced cancer patients. Five of them had evaluable tumor, which had failed earlier to respond to conventional chemotherapy. Cyclophosphamide was also administered in this trial, in anticipation of its synergistic effect with LPSp. In this study LPSp was injected intradermally into each patient twice a week, starting with an initial dose of 0.4 ng/kg, and raising it to 600 or 1800 ng/kg. A 400-mg/m² dose of cyclophosphamide was given intravenously every 2 weeks. After completion of the dose escalation, the treatment was continued for at least 4 months, and it was found that 1800 ng/kg LPSp was well tolerated. A significant level of cytokines was observed in the sera for at least 8 h. These results indicate higher tolerable doses and remarkably more continuous induction of the cytokines than were reported in a previous study by others using intravenous administration. Three of the five evaluable tumors showed a significant response to our combined therapy. Intradermally administered, LPS was less toxic and elicited a tumor response in combination with cyclophosphamide; it can thus can be applied to cancer treatment even in humans. Received: 3 August 1995 / Accepted: 2 April 1996