Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.     

Recolonization of estuarine organisms: effects of microcosm size and pesticides

Two six-week laboratory experiments were conducted to evaluate effects of pesticides and microcosm size on benthic estuarine macroinvertebrate recolonization. Sediments fortified with the pesticides (fenvalerate: controls, 5 (low) and 50 μg g⁻¹ wet sediment (high); endosulfan: controls, 1 (low) and 10 μg g⁻¹ wet sediment (high)) were fine-grained, organically rich (approximately 3.5% organic carbon and 22% dry weight) material. Relative dominance of the four most abundant taxa in both experiments was consistent among treatments with few exceptions. The amphipod,Corophium acherusicum, dominated abundance in both experiments. In the fenvalerate experiment, large trays (400 cm²) contained significantly (p<0.05) more total number of taxa (TNT) than small microcosms (144 cm²) but tray size was not significantly related to total number of organisms (TNO). When size was adjusted to a common unit area, small trays contained significantly more TNO than large containers. Adjusted abundance of small trays was 2.5 times that of large containers; a ratio close to that of microcosm sizes (i.e., 2.8). This result suggests that larval supply may have been inadequate to ‘aturate’ the available sediment in large containers. Fenvalerate significantly reduced abundance in the high treatment compared to both controls and low treatment but low treatment was not significantly different from controls. The amphipod,Corophium acherusicum, accounted for most of the decrease in abundance in response to fenvalerate. The holothruroid,Leptosynpta sp. and the polychaete,Mediomastus ambiseta, increased in abundance significantly with increased concentration of fenvalerate. Combined effects of actual microcosm size and concentration of endosulfan were not significant for TNO or TNT. As in the fenvalerate experiment, adjusted abundance of small microcosms was 2.6 times that of large trays which approximated the ratio of unit area between microcosm sizes. Abundance of a few taxa responded significantly to adjusted and unadjusted unit area. Abundance of the tunicate,Molgula manhattensis, increased significantly with increased concentration of endosulfan. Abundance was affected by sample location (e.g., interiorvs exterior cores) within microcosms. Abundance adjusted to unit area resulted in significantly greater TNO in externalvs internal cores. This has importance for sequential sub-sampling of microcosms to determine temporal dynamics. Statistically significant effects were measured in benthic community structure associated with microcosm size; however, the magnitude was relatively small. There appears to be no major biological reason to select one microcosm size over the other for screening for contaminant effects. Where feasible, the small trays provide savings in sample preparation and analysis, allow more replicates where laboratory space is limiting and generate less chemical waste. These benefits may be off-set by less ‘artifacts’ associated with edge effects of larger microcosms and the need for a larger mass of sediment to accommodate additional analytical requirements (e.g., thin vertical surficial samples to refine contaminant exposure at the sediment/water interface).     

Habitat use and support preference of two free-ranging saltatory lemurs (Lepilemur edwardsi and Avahi occidentalis)

Lepilemur edwardsi and Avahi occidentalis are two species of nocturnal, folivorous 'vertical clingers and leapers' (VCL). They have a similar body mass and share the same morphological adaptation for leaping. In a field study under sympatric conditions at Ampijoroa, Madagascar, comparison of support use with support availability using Jacobs' D preference values (Jacobs, 1974) showed that both species actively chose or avoided branches with certain qualities. However, while both species showed a preference for small oblique and horizontal branches, they selected them at different heights in the forest and with varying degrees of preference and avoidance for the other available supports. Despite their traditional locomotor assignation, both species showed a surprisingly strong preference for horizontal supports. These striking variations in detail of support preference may aid the maintenance of species segregation and niche difference.     

How did glycogen structure evolve to satisfy the requirement for rapid mobilization of glucose? A problem of physical constraints in structure building

Optimization of molecular design in cellular metabolism is a necessary condition for guaranteeing a good structure–function relationship. We have studied this feature in the design of glycogen by means of the mathematical model previously presented that describes glycogen structure and its optimization function [Meléndez-Hevia et al. (1993), Biochem J 295: 477–483]. Our results demonstrate that the structure of cellular glycogen is in good agreement with these principles. Because the stored glucose in glycogen must be ready to be used at any phase of its synthesis or degradation, the full optimization of glycogen structure must also imply the optimization of every intermediate stage in its formation. This case can be viewed as a molecular instance of the eye problem, a classical paradigm of natural selection which states that every step in the evolutionary formation of a functional structure must be functional. The glycogen molecule has a highly optimized structure for its metabolic function, but the optimization of the full molecule has meaning and can be understood only by taking into account the optimization of each intermediate stage in its formation. Received: 23 October 1996 / Accepted: 21 April 1997     

Electrochemical changes in media due to microbial

Microbial metabolism affected the electrical impedance parameters of a two terminal-measuring cell-containing growth media. The relationship between microbial growth and relative changes in both The capacitive and resistive parts of impedance was examined. Both components of impedance were shown to be indicative of bacterial growth. In low conductivity media the change in the conductance of the media (G_sol) clearly correlated to bacterial growth. In more conductive media the relative changes in G_sol were smaller, and in these media measurements of the changes of polarization capacitance (C_pol) were useful for monitoring bacterial growth.Yeast growth in two media resulted in large changes in C_pol (20–100%) while the changes in G_sol were very small (1–4%). This result indicated that, for some combinations of microorganisms and media, measuring C_pol might be preferable over G_sol for the detection of microbial growth.Microbial metabolism resulted in a change of 2–2.5 units in pH. This pH change resulted in a 40% change in C_pol but less than a 14% change in G_sol.     

500 MHz H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

Mechanism of natural delayed-type hypersensitivity reactions to tumor cells in nonimmunized syngeneic guinea pigs

Summary Nonimmunized 2/N guinea pigs respond to the presence of chemical carcinogen-transformed syngeneic tumorigenic cells with a sustained (delayed-hypersensitivity-type) 4-day intradermal induration consisting of predominantly polymorphonuclear leukocytes on day 1 and mononuclear cells by day 4, which is independent of the presence of tumor-specific antigens on the tumorigenic cells. Chemical carcinogen-induced morphologically transformed but nontumorigenic cells also induce a polymorphonuclear response by day 1, but neither induration nor a mononuclear response is present on day 4, demonstrating the specificity of the 4-day sustained indurative response for tumorigenic cells. Induration and cellular infiltrates are unaltered if tumor cells are treated prior to injection with the cytostatic lymphokine lymphotoxin or with x-irradiation to inhibit cell proliferation. The intradermal polymorphonuclear leukocyte host response on day 1, but not the mononuclear response on day 4, is also induced by mitomycin C-treated cells or a cytokine culture medium from the cells. No response is present on day 1 or day 4 when cell membranes or lyophilized cells are injected. Thus natural delayed-hypersensitivity-type skin reactivity is a mononuclear leukocyte response specifically directed against intact and metabolically active but not necessarily proliferating tumor cells.     

Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Dose-Dependent Cytokinetic Changes Following 1-β-D-Arabinofuranosylcytosine and Hydroxyurea In L1210 and S-180 In Vivo

DNA synthesis inhibition and recovery in L1210 and S-180 ascites tumors following 1-β-D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) were measured autoradiographically as a basis for optimizing drug schedules. Tumor bearing mice, 10⁶ cells day O, were treated on day 4 with 20, 200 or 2000 mg/kg Ara-C or 50, 300 or 1800 mg/kg HU. At various intervals following drug, [³H]thymidine was administered i.p. and mice were killed 1 hr later. Tumor cells were analyzed for labeling index (LI) and grain count (GC) to determine the percentage of cells in S phase and the distribution of DNA synthesis rates among the labeled cells, respectively. Following each dose of HU, DNA synthesis was inhibited completely. Recovery of LI was rapid and approached control values by 6 hr. Following each dose of Ara-C, DNA synthesis was inhibited completely for at least 6 hr. Recovery of LI was first noted 6 hr following 20 mg/kg Ara-C and 9 hr following 200 mg/kg. Following both doses the LI reached 100% of the control value by 26 hr. GC analysis indicated that following Ara-C treatment, DNA synthesis was reinitiated first with cells with low GC from 6 to 12 hr followed by cells with increasing GC from 12 to 20 hr. the labeling intensity reached control values by 20 hr and an ‘overshoot’ occurred by 26 hr. These data suggest that the recovery of DNA synthesis rate is a gradual process. Survival data for mice receiving two doses of Ara-C indicated that the optimal interval for retreatment following the lower dose of Ara-C occurred by 6 hr as compared to 12–16 hr for the higher dose. These times coincided in both instances with recovery of LI to 33–50% of control values. Early recovery of LI may be the best method currently available for estimating the optimal time for retreatment with an S phase specific drug.