The absence of confronting cisternae following thymidine-induced synchronization in HeLa cells

This is the first report of the elimination of confronting cisternae (CC), prominent organelles in dividing HeLa cells, upon experimental manipulation of the cells. CC are lost when a double thymidine block is employed to synchronize cell division. This observation is consistent with the hypothesis that the occurrence of CC in some fetal cells and in selected tumor cells depends on the speed at which the cells cycle through mitosis. If thymidine blocks DNA synthesis and arrests cells at the G₁/S interface, then thymidine probably has an indirect effect on CC. This paper reports the effect of thymidine on the occurrence of CC and briefly discusses how inhibitors of membrane synthesis or microtubule polymerization may affect the occurrence of CC during mitosis.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

The confronting cisternae in neurons in the substantia gelatinosa of the spinal cord of rats and Japanese monkeys

The neurons in the substantia gelatinosa of the spinal cord of the rat and Japanese monkey were investigated electron microscopically and ultracytochemically. The confronting cisternae observed in the cytoplasm of the majority of gelatinosal neurons in both species were composed of closely apposed parallel cisternae with electron-dense flocculent material, and a continuity with the cisternae of the rough endoplasmic reticulum was often observed. Ultracytochemically, the p-nitrophenylphosphatase (p-NPPase) activity was present in the confronting cisternae, but thiamine monophosphatase (TMPase) and thiamine pyrophosphatase (TPPase) activities were absent. These results indicate that the confronting cisternae are a variant of the rough endoplasmic reticulum.     

Heterogeneity of tubulin epitopes in mouse fetal tissues

Summary A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves.Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.     

Heterogeneity of tubulin epitopes in mouse fetal tissues

A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves. Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.     

Homocysteine in tissues of the mouse and rat

A method for the determination of L-homocysteine (Hcy) in tissues is described, which involves adsorption of adenosine and S-adenosyl-L-homocysteine (AdoHcy) in the tissue extract to dextran-coated charcoal, while leaving Hcy in solution. Sufficient dilution of the tissue homogenates and the presence of a reducing agent during the adsorption step are required to obtain high recovery of Hcy. Hcy is condensed with radioactive adenosine, and labeled AdoHcy is quantified by high performance liquid chromatography on a 3-micron reversed phase column. The amount of Hcy was determined in several tissues (liver, kidney, brain, heart, lung, and spleen) of mice and rats, and the concentrations of Hcy were in the range 0.5-6 nmol/g, wet weight. Hcy concentration was about 1 microM in mouse plasma. In mice, liver contained the highest amount of Hcy, and kidneys were also rich in Hcy. Similar concentrations were found in rat tissues. S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), the enzyme which is believed to catalyze the only pathway leading to Hcy formation in vertebrates, was nearly completely inactivated in mice injected with the drug combination 9-beta-D-arabinofuranosyladenine plus 2'-deoxycoformycin. This treatment induced a massive accumulation of AdoHcy in all tissues (Helland, S., and Ueland, P. M. (1983) Cancer Res. 43, 1847-1850). The amount of Hcy increased several-fold in kidney, whereas no change was observed in liver, heart, brain, lung, and spleen.     

Localization of lipoprotein lipase mRNA in selected rat tissues

Measurements of enzymatic activity have demonstrated that lipoprotein lipase (LPL), the principal enzyme responsible for hydrolysis of circulating triglyceride, is present in a number of tissues including brain, kidney, and adrenal gland. To determine the sites of synthesis of LPL in these tissues, in situ hybridization studies were performed using a non-sense 35S-labeled RNA probe produced from a 624-bp mouse LPL cDNA fragment. Control studies were performed with a sense RNA strand. Using 5-10-micron sections of 5-day-old rat brain, strong hybridization was found in pyramidal neurons of the hippocampus. Positive hybridization, indicating the presence of LPL mRNA, was also found in brain cortex and in the intermediate lobe of adult rat pituitary gland. Specific areas of adrenal and kidney medulla showed hybridization with the probe. LPL mRNA is, therefore, present in a number of specific regions of the body. LPL in these areas may not be important in regulating circulating levels of lipoproteins, but may be essential for cellular uptake, binding, and transfer of free fatty acids or other lipophilic substances.     

The role of activity in anticipating and confronting stressful situations.

This paper is an analytical review of studies comparing stress reactions in threatening situations during which the person can or cannot take action. The studies reviewed were conducted mainly in real-life situations and involved diverse forms of coping activities under various conditions of danger and distress. To achieve a better understanding of the effect of activity on stress reactions, several distinctions are made: between various types of stress reactions; between various indices and measurements of stress reactions; between activity which is directly related to the source of threat and activity which is not; and between various personality dimensions. The adaptive potential of activity in stressful situations is discussed in terms of: the sense of mastery and control provided by activity; activity as a means of attention diversion; and activity as a means of discharging energy generated by mobilization. It is suggested that taking action under stressful conditions, as opposed to remaining passive, is a powerful coping tool.     

Dual origin of mesenchymal tissues participating in mouse mammary gland embryogenesis

By the 14th day of gestation, two different mesenchymes can be identified which affect mouse mammary gland embryogenesis: the fibroblastic mammary mesenchyme (MM) closely surrounding the epithelial rudiment, and a condensed mesenchymal tissue (FP) appearing separately, posterior to the mammary rudiment, the precursor tissue of the fat pad. Late on the 16th day, the mammary epithelium (ME), surrounded by MM, starts to elongate, puts out branches, and penetrates the FP. A fatty substance appears in the FP at this stage. Interaction between ME and FP is necessary for typical mammary morphogenesis. When 17-day ME is combined with 14- or 17-day FP, the resulting mammary gland has the normal mammary pattern, but when 17-day ME is combined with 12- to 17-day MM, a ductal hyperplasia is formed by frequent branching, without the “stretching out” of these ducts. All the glands formed by combining ME with either FP or MM will lactate, if the mice carrying the grafts are allowed to mate and give birth. Adult ME also shows a different response to MM and FP.     

Presence of D-aspartate oxidase in rat liver and mouse tissues

Rat liver D-aspartate oxidase activity, which had been reported to be undetectable, was found to be well detectable in dialyzed liver homogenate. The requirements of the enzyme for activity and its sensitivity to inhibitors were identical with the known properties of the enzyme from other sources. We also demonstrated for the first time the presence of the enzyme activity in mouse tissues and some other rat tissues using dialyzed tissue homogenates.     

Hydroxyurea (HU)-induced apoptosis in the mouse fetal tissues

Hydroxyurea (HU), a ribonucleotide reductase inhibitor, induces morphological anomalies in the central nervous system (CNS), craniofacial tissues and limb buds in animals, and neonatal respiratory distress in humans. In the present study, pregnant mice were treated with 400 mg/kg of HU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue to clarify the mechanisms of HU-induced fetotoxicity and teratogenecity. At 6 and 12 HAT, a moderate to marked increase in the number of pyknotic cells was detected in the CNS and lung. A mild increase in the number of pyknotic cells was also found in the craniofacial mesenchymal tissues, limb buds and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results suggest that the HU-induced fetotoxicity is characterized by excess apoptotic cell death in the fetal tissues, and that such excess cell death in the fetal CNS, lung, craniofacial tissue and limb bud may have a certain relation to the later occurrence of morphological or functional anomalies reported in these tissues following HU-administration.     

Lipid peroxidation in mitochondria and microsomes from adult and fetal rat tissues

Pregnant female Wistar rats that received a control (100 ppm Zn) or a Zn-deficient diet (1.5 ppm Zn) from d 0 to 21, or nonpregnant normally fed female rats without or with five daily oral doses of 300 mg/kg salicylic acid were used for the experiments. In isolated mitochondria or microsomes from various maternal and fetal tissues, lipid peroxidation was determined as malondialdehyde formation measured by means of the thiobarbiturate method. Zn deficiency increased lipid peroxidation in mitochondria and microsomes from maternal and fetal liver, maternal kidney, maternal lung microsomes, and fetal lung mitochondria. Lipid peroxidation in fetal microsomes was very low. Zn deficiency produced a further reduction of lipid peroxidation in fetal liver microsomes. Salicylate increased lipid peroxidation in liver mitochondria and microsomes after addition in vitro and after application in vivo. The increase of lipid peroxidation by salicylate may be caused by two mechanisms: an increased cellular Fe uptake that, in turn, can increase lipid peroxidation and chelating Fe, in analogy to the effect of ADP in lipid peroxidation. The latter effect of salicylate is particularly expressed at increased Fe content.     

Immunohistochemical localization of superoxide dismutases in fetal and neonatal rat tissues.

We investigated the developmental profile of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in tissue sections obtained from fetal (Day 12 to 21 of gestation) and neonatal (Day 0 and 6) rats. Tissues were stained immunohistochemically with specific antisera against the respective rat SODs. There was a general trend towards richness of SODs in the epithelial linings and metabolically active sites, although differential distribution between the two SODs also existed. At Day 12 of gestation, immunoreactivity for both SODs was detected in the cardiomyocytes but not in other tissues. Hepatocytes expressed CuZnSOD at Day 14 and MnSOD at Day 17. By Day 18 CuZnSOD was detected in the epithelial cells of the gastrointestinal tract, respiratory tract, pancreatic islets, kidneys, and adrenals. These tissues exhibited MnSOD staining at Day 19. CuZnSOD occurred in the epithelia of the thyroid, thymus, and salivary glands at Day 19, while MnSOD was seen at Day 21. The increase in intensity of the staining for SODs occurred no later than postnatal Day 0, indicating that most tissues accumulated SODs during late gestation. Breathing atmospheric oxygen during early extrauterine life did not appreciably intensify the SOD staining. These results suggest that perinatal increase in SODs occurs as a general mechanism of preparation for birth.     

Presence of transforming growth factor-beta-like activity in multiple fetal rat tissues

The presence and ontogeny of transforming growth factor-beta (TGF-beta)-like bioactivity in rat tissues was studied. Eight separate tissues were extracted in acetic acid and assayed for TGF-beta-like activity by NRK-49F cell colony formation in soft agar. Bioactivity was present in each tissue during late fetal life, being most abundant in skeletal muscle, liver and lung (7.6-87.3 ng equivalents TGF-beta/g tissue), but fell to barely detectable levels from 12 days after birth. Gel filtration (pH 2.5) on Sephadex G75 of extracted fetal skeletal muscle, or culture medium conditioned by isolated fetal myoblasts demonstrated bioactivity in the molecular weight range 25-40 Kd. The results show that TGF-beta-like activity is selectively present in multiple fetal and neonatal, but not adult, rat tissues. Expression may therefore be developmentally regulated.