Metabolic interdependence of obligate intracellular bacteria and their insect hosts.

Mutualistic associations of obligate intracellular bacteria and insects have attracted much interest in the past few years due to the evolutionary consequences for their genome structure. However, much less attention has been paid to the metabolic ramifications for these endosymbiotic microorganisms, which have to compete with but also to adapt to another metabolism--that of the host cell. This review attempts to provide insights into the complex physiological interactions and the evolution of metabolic pathways of several mutualistic bacteria of aphids, ants, and tsetse flies and their insect hosts.     

Roosting Requirements of Two Frugivorous Bats (Sturnira lilium and Arbiteus intermedius) in Fragmented Neotropical Forest1

As tropical forest fragmentation accelerates, scientists are concerned with the loss of species, particularly those that play important ecological roles. Because bats play a vital role as the primary seed dispersers in cleared areas, maintaining healthy bat populations is critical to natural forest regeneration. Observations of foraging bats suggest that many Neotropical fruit‐eating species have fairly general habitat requirements and can forage in many different kinds of disturbed vegetation; however, their roosting requirements may be quite different. To test whether or not general foraging requirements are matched by equally broad roosting requirements, we used radiotelemetry to locate roost sites of two common frugivorous bat species (Sturnira lilium and Artibeus intermedius) in a fragmented forest in southeastern Mexico. Sturnira lilium roosted inside tree cavities and selected large‐diameter roost trees in remnant patches of mature forest. Fewer than 2 percent of trees surveyed had a mean diameter equal to or greater than roost trees used by . S. lilium, Artibeus intermedius roosted externally on branches and vines and under palm leaves and selected roost trees of much smaller diameter. Compared to random trees, roost trees chosen by A. intermedius were closer to neighboring taller trees and also closer in height to these trees. Such trees likely provide cryptic roosts beneath multiple overlapping crowns, with sufficient shelter from predators and the elements. While males of A. intermedius generally roosted alone in small trees within secondary forest, females roosted in small groups in larger trees within mature forest and commuted more than three times farther than males to reach their roost sites. Loss of mature forest could impair the ability of frugivorous bats to locate suitable roost sites. This could have a negative impact on bat populations, which in turn could decrease forest regeneration in impacted areas.     

COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p.

Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p. A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins. We previously showed that Sec23p binds to the C-terminal region of Sec16p. Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p. In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p. However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro. Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities. The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p. These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.     

Bioenergetics of Carbon Assimilation in Intact Chloroplasts: Coupling of Proton to Electron Transport at the Ratio H+/e=3 Is Incompatible with H+/ATP=3 in ATP Synthesis

During a transition from aerobic to largely anaerobic conditionslight-saturated carbon assimilation of intact chloroplasts wasnot decreased although both the transthylakoid proton gradientand ATP levels declined. After a dark period under anaerobiosis,illumination failed to initiate carbon assimilation. ATP increasedonly transiently in the light and then returned to the darklevel. Under such conditions, the addition of electron acceptorssuch as oxygen, oxalacetate or nitrite resulted in the increaseof ATP levels and carbon assimilation was initiated. Assimilationcontinued under anaerobiosis in the presence of reduced protongradients and reduced ATP levels after electron acceptors addedin addition to bicarbonate were reduced. Cyclic electron transport was inhibited when anaerobiosis didnot permit linear electron transport. It was induced in thissituation by micromolar concentrations of oxygen or when, underanaerobiosis, DCMU decreased PSII activity. Oxygen inhibitedcyclic electron transport by draining electrons from the cyclicpathway only when electron donation from PSII was weak. Theobservations give evidence of the delicate redox balance requiredfor cyclic electron transport. Since H⁺/e=3 in linear electron transport, the observationsof effective carbon reduction under a decreased transthylakoidproton gradient and decreased levels of ATP are incompatiblewith H⁺/ATP=2 or 3. They are compatible with H⁺/ATP=4. (Received May 1, 1995; Accepted October 3, 1995)     

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.     

Correlation between apparent activation state of nitrate reductase (NR), NR hysteresis and degradation of NR protein

Nitrate reductase (NR) activity was measured in extracts fromspinach leaves exposed to light or prolonged darkness, and tovarious treatments provoking an artificial activation of theenzyme in the dark. NR activity was determined immediately eitherin the presence of Mg²⁺, which gives an estimation of the putative(actual) activity in situ (NR_act), or in EDTA without preincubation,which gives an intermediate activity (NR_int), or after a 30min preincubation with EDTA plus AMP plus Pi, which gives themaximum NR activity (NR_max). NR_max is thought to reflect totalNR protein contents. In the dark, NR_act was usually very low. Dark inactivation wasprevented or reversed by feeding AICAR (5-aminoimidazole-4-carboxiamideribonucleoside), or by anaerobiosis, acid treatment or additionof uncoupler. During prolonged darkness, NR_max decreased, indicatingnet protein degradation with a half-time of 21 h. Conditionswhich caused an activation (dephosphorylation) of NR in thedark, slowed down NR protein degradation. This was also confirmedby Western blotting. Blockage of cytosolic protein synthesis with cycloheximide (CHX)did not accelerate NR protein degradation. In contrast, after5 h in the dark, NR_act increased in CHX-treated leaves. As thisincrease was sensitive to PP2A-inhibitors, it was probably dueto NR dephosphorylation. However, extractable NR kinase andNR phosphatase activities were not changed by CHX treatment.Apparently, CHX interacted with the NR regulatory system indirectlyby affecting turnover of another protein. The increase from NR_int to NR_max which occurred during preincubationof the leaf extract with EDTA plus AMP plus Pi was insensitiveto PP2A inhibitors and was interpreted as a hysteretic conversionof NR from an inactive into an active form. Hysteretic activationwas positively correlated to the NR phosphorylation state. Amodel is presented to explain the hysteretic behaviour of NRin relation to NA phosphorylation/ dephosphorylation. Overall, the data indicate that NR protein phosphorylation notonly controls the catalytic activity of NR, but also acts asa signal for NR protein degradation, with phospho-NR probablybeing a better substrate for protein degradation than the dephospho-form. Key words: Enzyme hysteresis, nitrate reductase, posttranslational modification, protein phosphorylation, protein turnover     

Dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior

Mupanomunda, Maria, Jeffrey F. Williams, Charles D. Mackenzie, and Lana Kaiser. Dirofilaria immitis:heartworm infection alters pulmonary artery endothelial cell behavior.J. Appl. Physiol. 82(2): 389-398, 1997.Thepathogenesis of filariasis has generally been attributed to eitherphysical presence of the adult parasites or the host's immune responseto the parasites. However, the spectrum of filariasis cannot beentirely explained by these causes, and other mechanisms must beoperative. It is now evident that factors released by filarialparasites likely contribute to the pathogenesis of filarial diseases.Adult heartworms (Dirofilaria immitis) reside in the rightheart and pulmonary artery, so the pulmonary artery should be exposedto the highest concentration of filarial factors. We tested thehypothesis that endothelium-dependent relaxation is altered in the invitro pulmonary artery from heartworm-infected dogs. Relaxationresponses to endothelium-dependent vasodilators (methacholine,bradykinin, substance P, and A-23187) and the non-endothelium-dependent vasodilator nitroglycerin and contractile responses were measured inrings of pulmonary artery from control and heartworm-infected dogs.Endothelium-dependent relaxation was assessed in the presence andabsence of inhibitors of nitric oxide synthase, cyclooxygenase, andguanylate cyclase. Responses to methacholine, substance P, and A-23187,but not to bradykinin, nitroglycerin, norepinephrine, or KCl, weredepressed in pulmonary artery from heartworm-infected dogs whencompared with control, suggesting that changes in endothelial cell andnot vascular smooth muscle behavior are involved in altered relaxation.The mechanism of endothelium-dependent relaxation in control pulmonaryartery appears to involve nitric oxide in the case of methacholine andboth nitric oxide and a cyclooxygenase product in the case ofbradykinin and A-23187. The mechanism of endothelium-dependentrelaxation in pulmonary artery from heartworm-infected dogs was notclearly elucidated. These data provide no evidence that heartworminfection globally influences either endothelial cell receptor functionor the vascular smooth muscle guanylate cyclase guanosine 3,5-cyclicmonophosphate system, making it likely that changes in intracellularsignaling are primarily responsible for the observed alteration ofendothelium-mediated relaxation. Alteration of endothelial cellfunction by filarial parasites may be an important component inthe pathology associated with filariasis.

     

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.

     

Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus

The social gliding behaviour of Myxococcus xanthus has previously been associated with the presence of polar pili. A Tn 5 transposon insertion was isolated which introduces a defect in social gliding and is genetically linked to a known sgl locus; this insertion was found also to cause a piliation defect. A 2.7 kb section of DNA was isolated from either side of this transposon and sequenced, revealing three genes which encode amino acid sequences with substantial similarity to components of the Type IV pilus biogenesis pathway in Pseudomonas aeruginosa . The myxococcal pilA gene encodes a putative pilin precursor with a short signal sequence and processing site similar to those of other Type IV pilins. Myxococcal pilS and pilR encode amino acid sequences with similarity to PilS and PilR of P. aeruginosa , as well as to other members of the NtrB/C family of two-component regulators. Mutations within pilR and pilA that have no polar effect were demonstrated to be responsible for pilus and social motility defects. These results indicate that the pili of M. xanthus belong to the Type IV family of pili, and demonstrate that these pili are actually required for social motility.