Compatible Solutes in the Thermophilic Bacteria Rhodothermus marinus and "Thermus thermophilus"

(sup13)C nuclear magnetic resonance spectroscopy and (sup1)H nuclear magnetic resonance spectroscopy were used to identify and quantify the organic solutes of several strains of halophilic or halotolerant thermophilic bacteria. Two strains of Rhodothermus marinus and four strains of "Thermus thermophilus" grown in complex medium containing NaCl were examined. 2-O-Mannosylglycerate was a major compatible solute in all strains: the Thermus strains accumulated the (beta)-anomer only, whereas both anomers were found in R. marinus. 2-O-(beta)-mannosylglycerate and 2-O-(alpha)-mannosylglycerate were the major compatible solutes in R. marinus. The former was the predominant solute in cells grown in 2.0 and 4.0% NaCl-containing medium, while the latter was the predominant compatible solute at higher salinities. Glutamate, trehalose, and glucose were also present as minor components. The intracellular K(sup+) concentration, as determined by (sup39)K nuclear magnetic resonance spectroscopy, in R. marinus increased with salinity and was sufficient to balance the negative charges of the mannosylglycerate. In addition to 2-O-(beta)-mannosylglycerate, trehalose was a major compatible solute of "T. thermophilus." 2-O-(beta)-Mannosylglycerate was the main solute in medium containing 1.0 or 2.0% NaCl, while trehalose predominated in cells grown in medium supplemented with 3.0 or 4.0% NaCl. Glycine betaine, in lower concentrations, was also detected in two "T. thermophilus" strains. This is the first report of mannosylglycerate as a compatible solute in bacteria.     

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.Abbreviations IEC immature-embryo calluses - PAs polyamines - PEC pollen-embryo calluses - PH insoluble conjugated PA fraction - Put putrescine - S free PA fraction - SH soluble conjugated PA fraction - Spd spermidine - Spm spermine 2,4d-2,4 dichlorophenoxyacetic acid     

Further insights on chromosomal pairing of autopolyploids: a triploid and tetraploids of rye

Chromosomal pairing of one triploid and three tetraploid plants of rye, Secale cereale, was analyzed by electron microscopy in surface-spread prophase I nuclei and compared with light microscopic observations of metaphase I cells. Prophase I is characterized by: (i) the weak alignment showed by the three or four unsynapsed or partially homologous synapsed axes; (ii) the low number ber of pairing partner switches (PPSs) displayed by both trivalents and quadrivalents; and (iii) the existence of complex multivalents in which up to 13 chromosomes in the triploid and 22 chromosomes in the tetraploids were involved. However, only few heterologous chromosomal associations were maintained at metaphase I. The results obtained are discussed under the assumptions of the random end pairing model with some modifications.     

Modulation of cisPlatin cytotoxicity by interleukin-1α and resident tumor macrophages

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1α) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1α in SCC-7 solid tumors. Neither IL-1α nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1α had no direct effect on tumor cell growthin vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC₉₀=6.0 μM), but, the addition of IL-1α (500–2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC₉₀=3.1 μM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1α. The modulation of cisPlatin cytotoxicity by IL-1α exhibited a biphasic dose response that paralleled the IL-1α dose dependent release of H₂O₂by resident tumor macrophages. Further, IL-1α modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H₂O₂ (50 μM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1α treatment for 24 hrs, before cisPlatin, produced drug resistance (IC₉₀=11.1 μM). Our study shows that IL-1α can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explanation for the synergistic antitumor activity of cisPlatin and IL-1αin vivo.     

Preliminary investigation of feeding performance of larvae of early red-spotted grouper, Epinephelus coioides, reared with mixed zooplankton

Larvae of red-spotted grouper, Epinephelus coioides, were reared inoutdoor tanks with nauplii of copepods (mainly Pseudodiaptomus annandaleiand Acartia tsuensis) and/or rotifers, Brachionus rotundiformis. Grouperlarvae successfully started feeding on early stage nauplii even though theirabundance was as low as approximately 100 individuals l^–1 andshowed better survival and growth thereafter compared to those fed withrotifers only. Incidence of feeding reached 100% on day 4 whennauplii were available and only on day 9 when rotifers were given alone.Larvae seemed to be poor feeders at the onset of feeding, attempting tocapture any food organisms in the tank water. Selective feeding ability oflarvae started from day 4 and the larvae then preferred to feed on medium-and large-size nauplii rather than on rotifers as they grew. Larvae appearedto have a better chance at surviving in the presence of early stage nauplii,which were probably caught more easily than rotifers.     

Neocentromere-mediated Chromosome Movement in Maize

Neocentromere activity is a classic example of nonkinetochore chromosome movement. In maize, neocentromeres are induced by a gene or genes on Abnormal chromosome 10 (Ab10) which causes heterochromatic knobs to move poleward at meiotic anaphase. Here we describe experiments that test how neocentromere activity affects the function of linked centromere/kinetochores (kinetochores) and whether neocentromeres and kinetochores are mobilized on the spindle by the same mechanism. Using a newly developed system for observing meiotic chromosome congression and segregation in living maize cells, we show that neocentromeres are active from prometaphase through anaphase. During mid-anaphase, normal chromosomes move on the spindle at an average rate of 0.79 μm/min. The presence of Ab10 does not affect the rate of normal chromosome movement but propels neocentromeres poleward at rates as high as 1.4 μm/min. Kinetochore-mediated chromosome movement is only marginally affected by the activity of a linked neocentromere. Combined in situ hybridization/immunocytochemistry is used to demonstrate that unlike kinetochores, neocentromeres associate laterally with microtubules and that neocentromere movement is correlated with knob size. These data suggest that microtubule depolymerization is not required for neocentromere motility. We argue that neocentromeres are mobilized on microtubules by the activity of minus end–directed motor proteins that interact either directly or indirectly with knob DNA sequences. C urrent models suggest that chromosomes move by a combination of forces generated by microtubule disassembly (Inoue and Salmon, 1995; Waters et al., 1996) and the activity of molecular motors (Vernos and Karsenti, 1996; Yen and Schaar, 1996). Microtubule disassembly generates a constant poleward force; while molecular motors can generate force in either poleward or away-from-pole directions, depending on the characteristics of the motor protein. Both plus and minus end–directed microtubule-based motors are localized to kinetochores (Hyman and Mitchison, 1991). Immunolocalization experiments indicate that mammalian kinetochores contain the minus end– directed motor dynein throughout metaphase and anaphase (Pfarr et al., 1990; Steuer et al., 1990). The kinesin-like proteins CENP-E, which has a transient kinetochore localization in animals, and MCAK, which is localized between the kinetochore plates of mammalian chromosomes, are also thought to generate and/or regulate chromosome movement (Yen et al., 1992; Lombillo et al., 1995; Wordeman and Mitchison, 1995).In addition to the molecular motors on kinetochores, several kinesin-like proteins are localized to chromosome arms (Vernos and Karsenti, 1996). Two subfamilies of arm-based motors have been identified in animals: the NOD subfamily (Afshar et al., 1995; Tokai et al., 1996) and the Xklp1/chromokinesin subfamily (Vernos et al., 1995; Wang and Adler, 1995). Both Nod and Xklp1 are required for positioning chromosomes on the metaphase plate, suggesting that they encode plus end–directed motors (Afshar et al., 1995; Vernos et al., 1995). Other evidence suggests that minus end–directed motors interact with chromosome arms. In the plant Haemanthus, a poleward force acts along chromosome arms during metaphase (Khodjakov et al., 1996), and forces propelling chromosome arms poleward have been detected during anaphase in crane fly spermatocytes (Adames and Forer, 1996). Little is known about how poleward arm motility at metaphase–anaphase affects the fidelity or rate of chromosome segregation.The neocentromeres of maize (Rhoades and Vilkomerson, 1942) provide a particularly striking example of poleward chromosome arm motility. In the presence of Abnormal chromosome 10 (Ab10),¹ heterochromatic DNA domains known as knobs are transformed into neocentromeres and mobilized on the spindle (Rhoades and Vilkomerson, 1942; Peacock et al., 1981; Dawe and Cande, 1996). Knobs are primarily composed of a tandem 180-bp repeat (Peacock et al., 1981) which shows homology to a maize B centromere clone (Alfenito and Birchler, 1993). A characteristic feature of neocentromeres is that they arrive at the spindle poles in advance of centromeres; in extreme cases the neocentromere-bearing chromosome arms stretch towards the poles (Rhoades and Vilkomerson, 1942; Rhoades, 1952). A recently identified mutation (smd1) demonstrates that a trans-acting factor(s) encoded on Ab10 is essential for converting the normally quiescent heterochromatic knobs into active neocentromeres (Dawe and Cande, 1996).Here we use neocentromeres as a model for understanding the mechanisms and importance of nonkinetochore chromosome movement. As a part of our analysis, we developed a four-dimensional system for observing chromosome segregation in living meiocytes. Our experiments were designed to determine (a) how poleward arm motility affects the rate and fidelity of chromosome segregation; and (b) whether the mechanism of neocentromere motility is comparable to the mechanism of kinetochore motility.     

Ventilation and metabolism among rat strains

Strohl, Kingman P., Agnes J. Thomas, Pamela St. Jean, EvelynH. Schlenker, Richard J. Koletsky, and Nicholas J. Schork. Ventilation and metabolism among rat strains. J. Appl. Physiol. 82(1): 317-323, 1997.We examinedventilation and metabolism in four rat strains with variation in traitsfor body weight and/or blood pressure regulation.Sprague-Dawley [SD; 8 males (M), 8 females (F)], BrownNorway (BN; 10 M, 11 F), and Zucker (Z; 11 M, 12 F) rats were comparedwith Koletsky (K; 11 M, 11 F) rats. With the use of noninvasiveplethysmography, frequency, tidal volume, minute ventilation(E),O₂ consumption, andCO₂ production were derived atrest during normoxia (room air) and during the 5th minute of exposureto each of the following: hyperoxia (100% O₂), hypoxia (10%O₂-balanceN₂), and hypercapnia (7%CO₂-balance O₂). Statistical methods probedfor strain and sex effects, with covariant analysis by body weight,length, and body mass. During resting breathing, strain effects werefound with respect to both frequency (BN, Z > K, SD) and tidal volume(SD > BN, Z) but not to E. Sexinfluenced frequency (F > M) alone. Z rats had higher values forO₂ consumption,CO₂ production, and respiratoryquotient than the other three strains, with no independent effect bysex. During hyperoxia, frequency was greater in BN and Z than in SD orK rats; SD rats had a larger tidal volume than BN or Z rats; Z rats hada greater E than K rats; and M had alarger tidal volume than F. Strain differences persisted duringhypercapnia, with Z rats exhibiting the highest frequency andE values. During hypoxic exposure,strain effects were found to influenceE (SD > K, Z), frequency (BN > K), and tidal volume (SD > BN, K, Z). Body mass was only amodest predictor of E during normoxia, of both E and tidal volume withhypoxia, hypercapnia, or hyperoxia, and of frequency duringhypercapnia. We conclude that strain of rats, more than their body massor sex, has major and different influences on metabolism, the patternand level of ventilation during air breathing, and ventilation duringacute exposure to hypercapnia or hypoxia.