The effect of monensin on gametogenesis and zoosporogenesis in the aquatic fungus,Allomyces macrogynus

Summary Cytoplasmic cleavage in the gametangia and zoosporangia ofA. macrogynus was studied using monensin, an ionophore known to disrupt several endomembrane functions in plant and animal cells. Monensin interfered with normal gamete and zoospore formation in a dose dependent manner such that at a 20 M concentration very abnormal cells were released from the reproductive structures. It was evident that monensin's effect was most pronounced during the first 25 minutes of gametogenesis and parallels in time the onset and continuation of the cytoplasmic cleavage events. Observations using fluorescence and differential interference contrast microscopy indicated that the ionophore inhibited normal cytoplasmic cleavage resulting in the production of multinucleate cells, many of which had either no flagella or multiple flagella. Transmission electron microscopy showed that the monensin-treated gametangia had many large vacuoles which contained amorphous electron-opaque material. X-ray microprobe analysis demonstrated that the elemental composition of the large vacuoles was identical to that of the dense globular inclusions seen in untreated gametangia, and morphological analysis confirmed the relationship between these endomembrane structures. Thus this swollen endomembrane component probably is not involved in the cleavage process. Single endomembrane cisternae which were very common in untreated gametangia were seldom seen in monensin-treated preparations. Instead, many smaller electron-transparent vacuoles were observed. These swollen cisternae may both represent monensin-modified Golgi apparatus equivalents and/or play a critical role during the process of gametogenesis and zoosporogenesis inA. macrogynus.     

Solvation properties of ubiquinone-10 in solvents of different polarity

The solvation properties of ubiquinone-10 and ubiquinol-10 in a wide variety of solvents of polarity varying from alkanes to water are reported. Greatest solubility is observed in solvents of intermediate polarity and particularly where low polarity is combined with a pronounced tendency to interact with the benzoquinone substituent of the ubiquinone molecule. This includes solvents like chloroform and benzene. Ubiquinone-10 is somewhat less polar than ubiquinol-10 as judged by comparative solubilities of the two molecules. Proton-NMR chemical shift measurements and aggregation studies in selected solvents indicate that in ubiquinone-10 in the liquid phase and in solution in hydrocarbons like dodecane the molecules have a preferred association possibly involving stacking of the benzoquinone rings. Surface balance studies indicated that the surface-active character of ubiquinone-10 is relatively weak and only in a comparatively polar and highly structured solvent, formamide, was there evidence of an effect on surface tension of the solvent. The critical micelle concentratiom in this solvent was estimated to be about 5 M on the basis of surface tension measurements. Ubiquinone-10 is well known to form virtually insoluble monolayers at the air/water interface. Studies of the partition of ubiquinone-10 in binary mixtures of solvents suggest that the interaction of the benzoquinone ring substituent with structured polar solvents is considerably weaker than the internal cohesion between molecules of the solvent. No evidence on the basis of wide-angle X-ray diffraction measurements was obtained to indicate that solvent molecules were a component of the crystal lattice of ubiquinone-10 that had precipitated from solvent mixtures.     

Effects of light and nitrogen limitation on the cell cycle of the dinoflagellate Amphidinium carteri

Cell cycle phase durations of cultures of Amphidinium carteriin light- or nitrogen-limited balanced growth were determinedusing flow cytometry. For both types of growth rate limitation,the increases in generation time caused by increasing degreesof limitation were due solely to expansion of the G₁ phase ofthe cell cycle. The durations of the S and G₂ + M phases wereindependent of growth rate. Furthermore, when cells were deprivedcompletely of light and nitrogen, they arrested in the G₁ phaseof the cell cycle. The results indicate that light- and nitrogen-dependentprocesses are heavily concentrated in the early part of thecell cycle, while DNA replication and cell division, once initiated,are independent of light or nitrogen supply.     

Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver.

Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states.     

Expression by human fetal organs of organ-specific cancer neoantigens as measured by leukocyte adherence inhibition

Summary Human cancers express organ-specific cancer neoantigens (OSN) as determined by in vitro leukocyte responses to extracts of cancers by the tumor host. In this study, we determined whether the OSNs were normal developmental proteins that were expressed by fetal organs and re-expressed with oncogenesis. Fetal extracts, principally of lung and colon but also of liver and kidney, were tested for their ability to induce leukocyte adherence inhibition (LAI) as compared to extracts from adult tissues of the same organ. Leukocytes from lung cancer patients showed positive LAI responses to 13- and 19-week fetal lung tissue. Likewise, leukocytes from colon cancer patients showed positive LAI responses to 14- and 19-week fetal colon tissue, whereas leukocytes from control subjects did not. Neither group responded positively to 21-week fetal organs. Criss-cross experiments showed that the fetal antigen was organ specific. Multiparous pregnant women showed positive LAI responses to cancer extracts but not to extracts from normal tissues of the same organ. The pattern of the LAI response was bell-shaped. Positive LAI responses to lung and breast cancer were detected at 4 to 7 months gestation and peaked at 5 months. To the fetal colon, LAI positive responses were detected at 5 to 8 months gestation, with the peak response at 6 months. The results indicate that OSN of cancers are also expressed by fetal organs and sufficient antigen is shed by fetal organs to sensitize pregnant women. Older fetal organs (21 weeks) and adult organs do not express an immunogenic or antigenic OSN.Supported by a grant from the National Cancer Institute of Canada     

The level of nerve growth factor (NGF) as a function of innervation : A correlative radio-immunoassay and bioassay study of the rat iris

A two-site radio-immunoassay for βNGF demonstrated 5–10 pg of NGF in the normal, adult rat iris. Ciliarectomy or sympathectomy did not significantly alter the amount of NGF after 10 days. However, denervation including all sensory axons (stereotactic lesion distal to the trigeminal ganglion) increased the level to about 100 pg of NGF. Total denervation resulting from homologous transplantation of the iris gave a similar increase after only 2 days. Fibre outgrowth responses evoked by corresponding iris explants in an NGF bioassay supported the results and suggested in addition that sympathetic denervation may cause a moderate transient increase in NGF after 3 days. It seems that sensory nerves in particular influence the level of NGF in a terminal field, either by a high capacity for uptake and removal of NGF or by exerting a negative feed-back on the production or processing of this growth factor.     

Mutagen sensitivity of Neurospora meiotic mutants

The Neurospora meiotic mutants, mei-1, mei-2, mei-3, and mei-4 were tested for cross sensitivity to mutagens. Mei-2 and mei-3 are sensitive to MMS, gamma-irradiation and histidine. Mei-2 is not sensitive to ultraviolet (UV) at 20 degrees C. Tests with recombinants with crossovers to either side of mei-2 or mei-3 show that these traits are the pleiotropic properties of a single gene which also determines meiotic behavior. The mei-2 gene maps to the right of al-3 on linkage group V. It is not allelic to mus-11. Upon backcrossing, the originally dominant meiotic effect of mei-2 becomes recessive to partially dominant. The histidine sensitivity is recessive.     

Structure of tomato busy stunt virus IV. The virus particle at 2.9 A resolution

The structure of tomato bushy stunt virus has been determined crystallographically to 2.9 A resolution. Details are presented of both the molecular structure and the methods by which it has been solved. The icosahedrally symmetric viral shell is composed of 180 protein subunits (Mr 43,000), with three similar but distinct modes of subunit bonding. This capacity for alternative packing is due to localized flexibility in the folded polypeptide (hinges between domains) and to multiple conformations for surface side-chains. The polypeptide backbone has an essentially invariant fold within a compact domain. A mechanism for correct positioning of the different modes of subunit interaction is evident from the structure of the TBSV particle. Thirty-five residues of the polypeptide chain fold in an ordered way on 60 of the 180 subunits, forming an internal framework. Interaction of folded domains with this framework permits accuracy of long-range geometry (correct curvature and closure) to be determined by unambiguous switching between alternative local contact angles. RNA packs tightly into the particle interior. Protein-RNA interactions occur through parts of the subunit that are flexibly linked to the well-ordered domains of the shell. This variable interaction imposes minimum restrictions on the folding of the RNA chain.     

Stimulation of ovine myometrial activity by 6-keto prostaglandin E1

6-keto prostaglandin E1 (6KE) is a metabolite of PGI2, which we have shown previously inhibits spontaneous myometrial activity. In the present study we examined the effects of 6KE on uterine electrical and mechanical activity in non-pregnant ovariectomized sheep. 6KE stimulated uterine activity in a dose-dependent fashion. The effect was enhanced by pre-treatment with estradiol (E2). It was not influenced by pre-treatment with meclofenamic acid and was not associated with significant changes in the concentrations of 13,14 dihydro 15-keto PGF2 alpha in vena cava plasma. After E2 treatment, 6KE had 0.2-0.3 of the stimulatory activity of PGF2 alpha. In the absence of E2, the uterine response to both 6KE and PGF2 alpha was decreased. In animals in which spontaneous myometrial activity was inhibited by PGI2, the uterus remained responsive to 6KE. We conclude that in the ovariectomized non-pregnant sheep 6KE stimulates uterine activity, and that the effect is independent of endogenous PG production.     

Serum progesterone concentration in pyometritic and normal postpartum dairy cows

Two studies were conducted to evaluate the concentration of serum progesterone in pyometritic cows and relate it to palpation of ovarian structures per rectum . In Trial 1, serum samples from 34 pyometritic cows were assayed for progesterone. Mean serum progesterone concentrations were 6.8 +/- 0.7 ng/ml. In Trial 2, each of 54 pyometritic cows was paired with a control cow on the basis of days post partum (18-50 days). Mean concentration of progesterone was 9.7 +/- 1.0 ng/ml for the pyometritic cows and 5.7 +/- 0.8 ng/ml in control cows (P<0.005). Progesterone concentration was greater (P<0.005) in both groups of cows with palpable corpora lutea (CLs). Ninety-six percent of the pyometritic cows had palpable CLs compared to 57% of the control group. Comparing serum progesterone only in cows with a palpable CL, the mean concentration was still greater (P<0.005) in the pyometritic group (10.6 +/- 1.0 ng/ml) than the control group (6.6 +/- 1.0 ng/ml). Compatability of rectal palpation findings and concentrations of serum progesterone were 92.6% for the pyometritic group and 72.2% for the control group. Progesterone concentration increased (P<0.05) by increased days post partum in Trial 2 (n=54) but not in Trial 1 (n=23). In both Trials 1 and 2, uterine size due to pyometra increased (P<0.05) with increased days post partum. No other associations were found.