Dynamic properties of intermediate filaments: disassembly and reassembly during mitosis in baby hamster kidney cells

A morphological analysis of the organizational changes in the type III intermediate filament (IF) system in dividing baby hamster kidney (BHK-21) cells was carried out by immunofluorescence and immunoelectron microscopy. The most dramatic change occurred during prometaphase, when the typical network of long 10-nm-diameter IF characteristic of interphase cells disassembled into aggregates containing short 4-6 nm filaments. During anaphase-telophase, arrays of short IF reappeared throughout the cytoplasm, and, in cytokinesis, the majority of IF were longer and concentrated in a juxtanuclear cap. These results demonstrate that the relatively stable IF cytoskeletal system of interphase cells is partitioned into daughter cells during mitosis by a process of disassembly and reassembly. This latter process occurs in a series of morphologically distinct steps at different stages of the mitotic process.     

Effect of acute and chronic hypernatremia on myoinositol and sorbitol concentration in rat brain and kidney

In animal models of hypernatremia, increases in brain electrolyte content account for the entire increase in osmolality in acute but not chronic hypernatremia, suggesting that there is generation of additional intracellular solutes ("idiogenic osmoles") in chronic hypernatremic states. In the present study, the concentration of the polyols myoinositol and sorbitol and water content were determined in the brain and kidneys of rats made acutely (2 hours) and chronically (72 hours) hypernatremic by intraperitoneal injection of NaCl and water restriction. Both the brain and the kidney responded to chronic hypernatremia with increased levels of myoinositol. Sorbitol levels increased in the kidney in response to both acute and chronic hypernatremia. Water content dropped in acute hypernatremia, but remained unchanged during chronic hyperosmolar challenge. We conclude that the polyols, myoinositol and sorbitol, may play a significant role in cellular osmoregulation in brain and kidney during chronic hypernatremia in the rat.     

Coordinate regulation of protein synthesis and messenger RNA content during growth arrest of suspension Chinese hamster ovary cells

We have found Chinese Hamster Ovary cells, cultured in suspension, are subject to growth control by serum. When suspended in medium containing 0.5% serum the cells become reversibly arrested in the beginning of the G₁ phase of the cell cycle and can be maintained in this viable, nonproliferating state for several days. This system was used to examine the regulation of protein synthesis with growth rate. In particular, the experiments addressed the question whether mRNA content is the principal controlling factor determining the rate of protein synthesis. The rate of leucine incorporation in resting cells in low serum is 2- to 2.5-fold lower than that of cells growing in 10% serum. The steady-state number of cytoplasmic poly A (+) RNA molecules shows a proportional decrease, consistent with it being a determining factor controlling the rate of protein synthesis. Furthermore, the rate of production of poly A (+) and poly A (?) RNA appears to be regulated coordinately. Regulation of the rate of initiation of translation would result in fewer ribosomes bound per active message and/or a lower proportion of total mRNA's being active. Our measurements indicate that the fraction of cytoplasmic poly A (+) mRNA in polyribosomes and the relative degree of loading of each active poly A(+) mRNA with ribosomes is the same in resting and growing cells. Thus these cells resemble 3T6 and translational control does not appear to be an important part of the change in protein synthetic rate with the state of growth.     

A STABLE ISOTOPE METHOD FOR MEASUREMENT OF THYMIDINE INCORPORATION INTO DNA

A method has been developed for the measurement of DNA synthesis in vivo using the incorporation of multilabeled, non-radioactive thymidine. Simultaneous intraperitoneal injection of hexalabeled thymidine and tritiated thymidine into a normal adult rat resulted in the incorporation of both labeled nucleosides into the DNA of cells undergoing replication. The DNA of several tissues and organs was analysed, including liver, thymus, spleen, bone marrow, and small intestine. Following extraction with hot trichloroacetic acid, acid hydrolysis, and thin-layer chromatography of the hydrolysates, the isotopic compositions of the thymine products were determined by field ionization mass spectrometry and by scintillation counting. The relative incorporation of radioactive and stable isotope-labeled thymidine was similar in all tissues, and corresponded to the ratio of the two labeled nucleosides in the injected material. These results indicate the feasibility of utilizing thymidine multilabeled with stable isotopes for measurement of cellular proliferation rates in conjunction with cancer therapy.     

Fascioloides magna (Bassi, 1875) in feral swine from southern Texas.

Between 1971 and 1975, Fascioloides magna was found in 46 of 67 (69%) feral swine (Sus scrofa) in southern Texas. Flukes were recovered from swine in areas where F. magna commonly has been recovered from white-tailed deer and cattle. One to 12 flukes were recovered from each infected animal. Their presence was indicated by black hematin pigment on the liver and various other internal organs. Eggs were not detected in the gallbladder or feces of infected animals although mature flukes and eggs were recovered in the livers suggesting that, like cattle, feral swine can be infected but are aberrant hosts for the parasite and do not disseminate eggs.     

Sex differences in the expression of lupus-associated miRNAs in splenocytes from lupus-prone NZB/W<Subscript>F1</Subscript> mice

Background

A majority of autoimmune diseases, including systemic lupus erythematosus (SLE), occur predominantly in females. Recent studies have identified specific dysregulated microRNAs (miRNAs) in both human and murine lupus, implying an important contribution of these miRNAs to lupus pathogenesis. However, to date, there is no study that examined sex differences in miRNA expression in immune cells as a plausible basis for sex differences in autoimmune disease. This study addresses this aspect in NZB/W_F1 mice, a classical murine lupus model with marked female bias, and further investigates estrogen regulation of lupus-associated miRNAs.

Methods

The Taqman miRNA assay system was used to quantify the miRNA expression in splenocytes from male and female NZB/W_F1 mice at 17–18, 23, and 30 weeks (wks) of age. To evaluate potential estrogen's effect on lupus-associated miRNAs, 6-wk-old NZB/W_F1 male mice were orchidectomized and surgically implanted with empty (placebo) or estrogen implants for 4 and 26 wks, respectively. To assess the lupus status in the NZB/W_F1 mice, serum anti-dsDNA autoantibody levels, proteinuria, and renal histological changes were determined.

Results

The sex differences in the expression of lupus-associated miRNAs, including the miR-182-96-183 cluster, miR-155, miR-31, miR-148a, miR-127, and miR-379, were markedly evident after the onset of lupus, especially at 30 wks of age when female NZB/W_F1 mice manifested moderate to severe lupus when compared to their male counterparts. Our limited data also suggested that estrogen treatment increased the expression of aforementioned lupus-associated miRNAs, with the exception of miR-155, in orchidectomized male NZB/W_F1 mice to a similar level in age-matched intact female NZB/W_F1 mice. It is noteworthy that orchiectomy, itself, did not affect the expression of lupus-associated miRNAs.

Conclusion

To our knowledge, this is the first study that demonstrated sex differences in the expression of lupus-associated miRNAs in splenocytes, especially in the context of autoimmunity. The increased expression of lupus-associated miRNA in female NZB/W_F1 mice and conceivably in estrogen-treated orchidectomized male NZB/W_F1 mice was associated with lupus manifestation. The notable increase of lupus-associated miRNAs in diseased, female NZB/W_F1 mice may be a result of both lupus manifestation and the female gender.

     

Synthesis of biotinylated glycoconjugates and their use in a novel ELISA for direct comparison of HIV-1 Gp120 recognition of GalCer and related carbohydrate analogues

As part of our program directed toward the design and synthesis of high-affinity ligands for the GalCer-binding site on the HIV cell surface glycoprotein, gp120, we required a reliable method for qualitatively assessing relative binding affinities for related analogues. Due to the hydrophilic nature of these synthetic conjugates, difficulties were encountered with typical ELISA methods, which rely upon hydrophobic interactions to anchor the ligand to a microtiter plate. Other types of assays were also problematic due to nonspecific binding of gp120. Therefore, we developed a general method for plating water-soluble ligands on microtiter plates using biotin/NeutrAvidin recognition for adhesion. A water-soluble GalCer analogue was prepared by conjugating psychosine to biotin using a novel tetraethylene glycol linker. In a similar manner, LacCer and GlcCer analogues were prepared and these conjugates were plated into microtiter wells containing NeutrAvidin. Unoccupied sites were blocked using biotin functionalized as a primary amide. Gp120 binding to galactosyl sphingosine, GalSph (19), GlcSph (22), and LacSph (23) conjugates was assessed through incubation with recombinant HRP-gp120. It was determined that LacSph has the strongest interaction with gp120. The binding affinities of GalSph and GlcSph were similar to each other and less strong than LacSph. These data contradict earlier studies where HPTLC showed that LacCer and GlcCer do not significantly bind gp120. They also contradict liposome-based assays that reported psychosine is not recognized by gp120. The extent of plating for each biotinylated molecule was quantified using HRP-biotin, allowing direct comparison of ligand plating efficiencies for the first time. Several other synthetic biotin conjugates were prepared and tested, demonstrating the feasibility of performing ELISA on water-soluble ligands.     

Detection of Toxoplasma gondii parasitemia in experimentally inoculated cats

Toxoplasma gondii B1 gene polymerase chain reaction (PCR) amplification utilizing a flanking and nesting reaction was compared to mouse bioassay on feline whole blood samples collected before and after experimental inoculation with T. gondii. Samples were collected from 5 cats prior to inoculation with T. gondii and on days 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 84, 112, 140, 143, 147, 150, 154, 161, 168, 175, and 182 after inoculation. Cats were challenged with T. gondii orally on day 140. Bioassay was found to be less effective for detection of parasitemia than B1 gene PCR. Parasitemia was detected in all 5 cats by PCR multiple times after primary and challenge inoculation. Detection of T. gondii parasitemia by PCR utilizing the flanking reaction described here may be useful in predicting the oocyst shedding period in individual cats. As none of the cats developed signs of systemic illness, yet were chronically parasitemic, T. gondii whole-blood PCR is not helpful as a diagnostic test for clinical feline toxoplasmosis.     

IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.