Freeze-fracture studies of nexuses between smooth muscle cells. Close relationship to sarcoplasmic reticulum

The freeze-fracture appearance of the nexus was compared in the smooth muscle of guinea pig sphincter pupillac, portal vein, pulmonary artery, taenia coli, uretzr, and vas diferens, mouse vas deferens, chicken gizzard and anterior mesenteric artery, and toad stomach. Nexuses are particularly numerous in the guinea pig sphincter pupillae; they are usually oval and their average area is 0.15 mum2, although some as large as 0.6 mum2 were seen. Small aggregations of particles were observed which would not be recognizable as nexuses in thin section. What constitutes the minimum size of a nexus is discussed. It is estimated that the number of nexuses per cell in this preparation is of the order of tens rather than hundreds. All nexuses examined had 6-9-nm particles in the PF face, with corresponding 3-4-nm pits on the EF face forming a polygonal tending towards a hexagonal lattice. The nexuses are arranged in rows parallel to the main axis of the cell, usually alternating with longitudinal rows of plasmalemmal vesicles. Many nexuses in the guinea pig sphincter pupillae, chicken gizzard, and toad stomach show a close relationship with sarcoplasmic reticulum. The possibility that this may have some role in current flow across this specialized junction is discussed.     

CYTOTOXIC EFFECTS OF COLCEMID OR HIGH SPECIFIC ACTIVITY TRITIATED THYMIDINE ON CLONOGENIC CELL SURVIVAL IN B6CF1 MICE

High specific activity tritiated thymidine (HSA-[³H]TdR) and colcemid were given in cytotoxic doses and regimens to B6CF₁/Anl mice. The number of cells per intestinal crypt was reduced by the S-phase-specific HSA-[³H]TdR and the metaphase blocking and cytotoxic effect of multiple injections of colcemid. In 50-day old mice, the cytotoxic effect of multiple injections of colcemid reduced both the number of cells per crypt and the clonogenic cell survival. However, the number of surviving intestinal clonogenic or stem cells, assayed by the micro-colony technique, did not change in 110–130-day old mice. These data suggest that most of the cells at risk from these cytotoxic agents are not clonogenic in adult 110–130-day old mice but are the cells in amplification division. However, since the stem cells of young mice are more susceptible to colcemid, they are apparently in a more rapid cell cycle than those of older mice. The clonogenic cell survival measured in 110–130-day old mice after a single radiation dose of 14 Gy (1400 rad) responded in a non-linear way to increasing time of continuous colcemid cytotoxicity. These data suggest that the intestinal stem cells can respond to amplification compartment cell death by a shortening of their cell cycle and thus, over time, the number of stem cells at risk to colcemid cytotoxicity increases.     

The effect of salts on the kinetics of iron release from N-terminal and C-terminal monoferrictransferrins

The kinetics of iron release from N-terminal and C-terminal monoferric human transferrins has been studied using EDTA as the accepting chelate. In the absence of added salts iron release from the N-terminal site is more facile but the relative lability can be reversed by the addition of NaClO₄, NaCl and LiCl. The results indicate that both anions and cations can affect the lability of the two sites. Since the relative lability of the two monoferrictransferrins is affected by fairly moderate concentrations of NaCl and NaClO₄ we suggest that the ionic composition serum may play an important role in determining the observed distribution of iron among the sites. A new method for the preparation of N-terminal monoferrictransferrin is described.     

Cell numbers and in vitro responses of leucocytes and lymphocyte subpopulations following maximal exercise and interval training sessions of different intensities.

In vitro lymphocyte function and the mobilisation of peripheral blood leucocytes was examined in eight trained subjects who undertook an incremental exercise test to exhaustion and a series of interval training sessions. Venous blood samples were obtained before the incremental test, immediately after, and 30, 60, and 120 min after the test. Interval training sessions were undertaken on separate days and the exercise intensities for each of the different sessions were 30%, 60%, 90% and 120% of their maximal work capacity respectively, as determined from the incremental exercise test. There were 15 exercise periods of 1-min duration separated by recovery intervals of 2 min in each session. Venous blood samples were obtained immediately after each training session. Significant increases in lymphocyte subpopulations (CD3+, CD4+, CD8+, CD20+, and CD56+) occurred following both maximal and supramaximal exercise. This was accompanied by a significant decrease in the response of cultures of peripheral blood lymphocytes to Concanavalin A (ConA), a T-cell mitogen. The state of lymphocyte activation in vivo as measured by CD25+ surface antigen was not, however, affected by acute exercise. The total number of lymphocytes, distribution of lymphocyte subpopulations and in vitro lymphocyte response to ConA had returned to pre-exercise levels within half an hour of termination of exercise but serum cortisol concentrations had not begun to fall at this time. There was a significant decrease in the CD4+:CD8+ cell ratio following exercise; this was more the result of increases in CD3-CD8+ cells (CD8+ natural killer cells) than to CD3+CD8+ cells (CD8+ T-lymphocytes). Decreased responsiveness of T-cells to T-cell mitogens, postexercise, may have been the result of decreases in the percentage of T-cells in postexercise mixed lymphocyte cultures rather than depressed cell function. The cause of this was an increase in the percentage of natural killer cells which did not respond to the T-cell mitogen. The results indicated that while a substantial immediate in vitro "immunomodulation" occurred with acute exercise, this did not reflect an immunosuppression but was rather the result of changes in the proportions of reactive cells in mononuclear cell cultures. We have also demonstrated that the degree of the change in distribution of lymphocyte subpopulation numbers and responsiveness of peripheral blood mononuclear cells in in vitro mitogen reactions increased with increasing exercise intensity. Plasma volume changes may have contributed to some of the changes seen in leucocyte population and subpopulation numbers during and following exercise.     

Characterization of an infective molecular clone of the B-tropic, ecotropic BL/Ka(B) murine retrovirus genome.

Using molecular cloning techniques, we amplified the unintegrated, linear proviral DNA of the BL/Ka(B) virus, a non-leukemogenic retrovirus of mouse strain C57BL/Ka. Two independent clones in lambda phage vector 607 and one subclone in pBR322 were infective when transfected into mouse fibroblasts. Analysis of the progeny virus revealed biological properties and a restriction map identical to those of the parental viral shock. Comparison of the restriction map with the maps of other ecotropic murine viruses reveals many similarities. Particularly interesting is the comparison of the N-tropic Akv virus and the B-tropic BL/Ka(B) virus. The long terminal repeats of the two viruses are virtually identical, as are 22 of 23 restriction sites located outside of the region which spans from 1.8 to 3.8 kilobases from the left end of the genome. Within this region, however, only three of nine sites examined are shared. This suggests that the BL/Ka(B) virus was derived from an endogenous N-tropic virus closely related to Akv by recombinational events which altered the sequence in the last half of the gag gene and the first third of the pol gene. This change is probably responsible for the observed difference in the Fv-1 tropism of the two viruses.     

Further studies on the charge-related alterations of methotrexate transport in Ehrlich ascites tumor cells by ionic liposomes: Correlation with liposome-cell association

Summary Interaction of positively (phosphatidylcholine/stearylamine 51) or negatively (phosphatidylcholine/stearic acid 51) charged liposomes with Ehrlich ascites tumor cells for 1–5 min increases or decreases, respectively, the bidirectional fluxes of the folic acid analog, methotrexate. These effects on influx and efflux appear to be symmetrical since the liposomes do not change the intracellular level of methotrexate at the steady state. Influx kinetics show that these alterations result from an increase or decrease in theV _max with no change in theK _m ⁱⁿ . These effects appear to be specific for the methotrexate-tetrahydrofolate carrier system since the transport of other compounds which utilize this carrier, aminopterin, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate, is affected similarly to methotrexate, whereas, the transport of folic acid, a compound similar in structure and charge but not significantly transported by this carrier is unaffected by liposomes. Once cells are exposed to charged liposomes, the effects on methotrexate transport cannot be reversed by washing the cells free of the extracellular liposomes. If, however, cells are exposed to liposomes of one charge, washed and then exposed to liposomes of the opposite charge, methotrexate influx is reversed to control rates. The effects of charged liposomes on methotrexate influx were not abolished by treating the cells with neuraminidase, metabolic inhibitors or lowering the temperature to 4°C. Studies on the uptake of [¹⁴C] liposomes show that these effects are not proportional to the total amount of lipid associated with the cell but result from an initial rapid liposome-cell association that is not dependent on temperature or energy metabolism nor related to cell surface charge.     

Phenolic components of the primary cell wall. Feruloylated disaccharides of D-galactose and L-arabinose from spinach polysaccharide.

1. Cell walls from rapidly growing cell suspension cultures of Spinacia oleracea L. contained ferulic acid and p-coumaric acid esterified with a water-insoluble polymer. 2. Prolonged treatment with trypsin did not release may feruloyl esters from dearabinofuranosylated cell walls, and the polymer was also insoluble in phenol/acetic acid/water (2:1:1, w/v/v). 3. Treatment of the cell walls with the fungal hydrolase preparation "Driselase' did liberate low-Mr feruloyl esters. The major esters were 4-O-(6-O-feruloyl-beta-D-galactopyranosyl)-D-galactose and 3?-O-feruloyl-alpha-L-arabinopyranosyl)-L-arabinose. These two esters accounted for about 60% of the cell-wall ferulate. 4. It is concluded that the feruloylation of cell-wall polymers is not a random process, but occurs at very specific sites, probably on the arabinogalactan component of pectin. 5. The possible role of such phenolic substituents in cell-wall architecture and growth is discussed.     

Late‐life exercise mitigates skeletal muscle epigenetic aging

There are functional benefits to exercise in muscle, even when performed late in life, but the contributions of epigenetic factors to late‐life exercise adaptation are poorly defined. Using reduced representation bisulfite sequencing (RRBS), ribosomal DNA (rDNA) and mitochondrial‐specific examination of methylation, targeted high‐resolution methylation analysis, and DNAge™ epigenetic aging clock analysis with a translatable model of voluntary murine endurance/resistance exercise training (progressive weighted wheel running, PoWeR), we provide evidence that exercise may mitigate epigenetic aging in skeletal muscle. Late‐life PoWeR from 22–24 months of age modestly but significantly attenuates an age‐associated shift toward promoter hypermethylation. The epigenetic age of muscle from old mice that PoWeR‐trained for eight weeks was approximately eight weeks younger than 24‐month‐old sedentary counterparts, which represents ~8% of the expected murine lifespan. These data provide a molecular basis for exercise as a therapy to attenuate skeletal muscle aging.     

Protein kinases in control of the centrosome cycle.

The centrosome is the major microtubule nucleating center of the animal cell and forms the two poles of the mitotic spindle upon which chromosomes are segregated. During the cell division cycle, the centrosome undergoes a series of major structural and functional transitions that are essential for both interphase centrosome function and mitotic spindle formation. The localization of an increasing number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling many of these transitions. Here, we focus on two protein kinases, the polo-like kinase 1 and the NIMA-related kinase 2, for which recent data indicate key roles during the centrosome cycle.     

Genetic identification of Southern Ocean octopod samples using mtCOI

East Antarctic octopods were identified by sequencing mtCOI and using four analytical approaches: Neighbor-joining by Kimura-2-Parameter-based distances, character-based, BLAST, and Bayesian Inference of Phylogeny. Although the distance-based analytical approaches identified a high proportion of the sequences (99.5% to genus and 88.1% to species level), these results are undermined by the absence of a clear gap between intra- and interspecific variation. The character-based approach gave highly conflicting results compared to the distance-based methods and failed to identify apomorphic characters for many of the species. While a DNA independent approach is necessary for validation of the method comparisons, crude morphological observations give early support to the distance-based results and indicate extensive range expansions of several species compared to previous studies. Furthermore, the use of distance-based phylogenetic methods nevertheless group specimens into plausible species clades that are highly useful in non-taxonomical or non-systematic studies.