Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Influence of dietary-induced weight changes on serum luteinizing hormone, estrogen and progesterone in the bovine female

The working hypothesis in the present study was that changes in concentrations and secretory patterns of luteinizing hormone (LH), 17 beta estradiol (E2), and progesterone in sexually mature beef heifers fed diets deficient in energy are related to changes in body weight of the animals. Another important component of the study was to determine if concentrations and secretion patterns of the reproductive hormones changed over time as feeding of the experimental diets continued. Twelve Red Angus X Hereford heifers (20 mo of age; 355 +/- 7 kg) were assigned randomly to receive a low- (L, n = 7) or high- (H, n = 5) energy diet for 100 days (Day 0 = day of initiation of dietary treatment). All heifers were exhibiting estrous cycles at regular intervals when the experiment was initiated and continued to exhibit estrous cycles at regular intervals throughout the study. Stage of the estrous cycle was synchronized in all 12 heifers by administration of prostaglandin F2 alpha (PGF2 alpha) on two occasions (Days 45 and 75) during the experiment. Serial blood samples (taken at 12-min intervals for 4 h) were collected at 0, 12, 24, 36, 48, and 60 h after the PGF2 alpha injections (Days 45-47 and 75-77) to determine patterns of LH secretion during the follicular phase of the estrous cycle. In addition, serial blood samples (taken at 20-min intervals for 18 h) to monitor LH secretion during the luteal phase of the estrous cycle, in which the stage of the cycle was standardized between heifers, were obtained (Days 59 and 89).(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Losing the desire: selection can promote obligate asexuality

Whilst parthenogenesis has evolved multiple times from sexual invertebrate and vertebrate lineages, the drivers and consequences of the sex-asex transition remain mostly uncertain. A model by Stouthamer et al. recently published in BMC Evolutionary Biology shows a pathway by which obligate asexuality could be selected for following endosymbiont infection.     

Dynamic changes in brain functional connectivity during concurrent dual-task performance

This study investigated the spatial, spectral, temporal and functional proprieties of functional brain connections involved in the concurrent execution of unrelated visual perception and working memory tasks. Electroencephalography data was analysed using a novel data-driven approach assessing source coherence at the whole-brain level. Three connections in the beta-band (18-24 Hz) and one in the gamma-band (30-40 Hz) were modulated by dual-task performance. Beta-coherence increased within two dorsofrontal-occipital connections in dual-task conditions compared to the single-task condition, with the highest coherence seen during low working memory load trials. In contrast, beta-coherence in a prefrontal-occipital functional connection and gamma-coherence in an inferior frontal-occipitoparietal connection was not affected by the addition of the second task and only showed elevated coherence under high working memory load. Analysis of coherence as a function of time suggested that the dorsofrontal-occipital beta-connections were relevant to working memory maintenance, while the prefrontal-occipital beta-connection and the inferior frontal-occipitoparietal gamma-connection were involved in top-down control of concurrent visual processing. The fact that increased coherence in the gamma-connection, from low to high working memory load, was negatively correlated with faster reaction time on the perception task supports this interpretation. Together, these results demonstrate that dual-task demands trigger non-linear changes in functional interactions between frontal-executive and occipitoparietal-perceptual cortices.     

Influence of season and estradiol on secretion of luteinizing hormone in ovariectomized cows

The hypotheses that secretion of luteinizing hormone (LH) varies with season and that estradiol may modulate the seasonal fluctuation in secretion of LH in cows were investigated. Seven mature cows were ovariectomized approximately 30 days before initiation of the experiment. Three of the ovariectomized cows (OVX-E2) were administered a subcutaneous estradiol implant that provided low circulating levels of 17 beta-estradiol. The remaining 4 cows (OVX) were not implanted. From December 21, 1982, to September 20, 1984, blood samples were collected sequentially (at 10-min intervals for 6 h) at each summer and winter solstice, and each spring and autumn equinox. In addition, from March 17, 1983, to March 17, 1984, sequential samples were collected midway between each solstice and equinox. Concentration of LH was measured in all samples, and concentration of estradiol was measured in pools of samples. An annual cycle in mean serum concentration of LH and amplitude of LH pulses was detected in both groups of cows. The seasonal pattern did not differ in the two treatment groups. Serum concentration of LH and amplitude of LH pulses were highest around the spring equinox, decreased gradually to the autumn equinox, and then increased and peaked again during the following spring equinox. Frequency of LH pulses and concentration of estradiol in serum did not vary with season. Circulating concentrations of LH and amplitude of pulses tended to be higher in OVX-E2 than OVX cows throughout the experimental period. Frequency of pulses of LH was lower in OVX-E2 than OVX cows throughout the experiment. Concentrations of estradiol were higher in the implanted cows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormone secretion by preimplantation embryos in a dynamic in vitro culture system.

Bovine embryos recovered from superovulated donors on Days 8-18 postestrus were cultured in vitro in a tissue perifusion system to quantify hormone secretion. Embryos were cultured for 24 h at 37 degrees C in Ham's F-10 medium supplemented 5% v/v with heat-treated, charcoal-stripped calf serum; 100 IU/ml penicillin; and 100 micrograms/ml streptomycin. The medium was saturated with 5% CO2 in air and perifused at 50 microliters/min (3 ml/h). Estrone (E1) estradiol (E2), progesterone (P4), prostaglandin E2 (PGE2), and prostacyclin (PGI2) were quantified by RIA in 6-h pools of perifusate fractions. Estrone was measurable (pg/h/embryo; mean +/- SE) on Days 13 (10.80 +/- 4.56) and 15 (34.80 +/- 9.80); E2 on Days 11 (36.80), 12 (81.28 +/- 29.80), 13 (11.75 +/- 4.09), 15 (157.20 +/- 112.60), and 16 (30.26 +/- 8.76); and P4 (ng/h/embryo) on Days 13 (0.5-1.0) and 17 (approximately 1.5). PGE2 was secreted by Day 10 bovine embryos during the last 6 h of culture (19-24 h) and throughout culture for Day 11-18 embryos. The rate of PGE2 secretion increased (p less than 0.05) over the previous days(s) at Days 13 and 17. The mean (+/- SE) secretion rates (pg/h/embryo) for the 24-h culture by embryonic ages were as follows: Day 11 (63.39 +/- 14.61), 12 (172.10 +/- 30.90), 13 (3094.08 +/- 283.35), 14 (1633.89 +/- 49.98), 15 (3739.23 +/- 1082.79), 16 (4955.37 +/- 1381.83), 17 (11893.23 +/- 1188.48), and 18 (13827.99 +/- 3587.88).(ABSTRACT TRUNCATED AT 250 WORDS)     

A similar distribution of gonadotropin isohormones is maintained in the pituitary throughout sexual maturation in the heifer.

Our working hypotheses for this study were that 1) the profile of intrapituitary LH and FSH isoforms would be shifted toward acidic forms as sexual maturation progresses in the bovine female; and 2) concentration of 17 beta-estradiol (E2) in circulation during sexual maturation would be a major factor modulating the percentage of the more acidic isoforms. In addition, the biological-immunoreactive (B:I) ratios of each isoform of LH were evaluated at selected stages of sexual maturation. Heifers (7 mo of age) were assigned to one of three treatment groups: 1) ovariectomized (OVX; n = 16); 2) OVX and administered E2 (OVXE; n = 16); or 3) ovary-intact (INTACT; n = 14). Pituitaries were collected from heifers in each group at an estimated 120 days (prepubertal) of 25 days before puberty (peripubertal). A fourth group of 6 heifers remained intact (postpubertal INTACT) to determine time of puberty during the experimental period. Pituitaries of heifers assigned to the postpubertal INTACT group were collected during the follicular phase of the first or second estrous cycle postpuberty. Pituitaries were used for determination of relative amounts of gonadotropin isohormones. Tissue extracts of the pituitaries were chromatofocused on pH 10.5-4.0 gradients. The LH of all pituitaries resolved into thirteen isoforms that were designated isoforms A-L, and S, with isoform A the most basic form. Isoforms F and G (basic pH range) were the predominant isoforms of each chromatofocusing profile and comprised 50-60% of the immunoreactive LH. Isoforms J and K were the major isoforms eluting in the acidic pH range.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pathology of embryo death caused by the male-killing Spiroplasma bacterium in Drosophila nebulosa

Background

Inherited bacteria that kill male offspring, male-killers, are known to be common in insects, but little is understood about the mechanisms used by male-killing bacteria to kill males. In this paper we describe the tempo and changes that occur during male-killing by Spiroplasma bacteria in the host Drosophila nebulosa.

Results

Spiroplasma infected D. nebulosa males were developmentally retarded from 6–8 h into embryonic development at 25°C, and arrested at between stages 12 and 13 of embryogenesis (10–12 h). Dying males were characterized by a failure to form segments, and ultimately disintegration of the normal oval embryonic shape. Prior to death, dying males exhibited widespread apoptosis, as testified by TUNEL staining.

Conclusion

The Spiroplasma kills male Drosophila in a narrow developmental period, shortly after the formation of the host dosage compensation complex that is required for male-killing. Male death is preceded by widespread apoptosis, but it is uncertain if this is primary or secondary apoptosis.