A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Characterization of a Helicobacter pylori vaccine candidate by proteome techniques

In a previous two-dimensional (2D) gel electrophoretic study of protein antigens of the gastric pathogen, Helicobacter pylori recognized by human sera, one of the highly and consistently reactive antigens, a protein with M_r of approximately 30?000 (Spot 15) seemed to be of special interest because of low yields on N-terminal protein sequencing. This suggested possible N-terminal modification, as the N-terminal sequence analysis of this 30?000 protein (Spot 15) did not provide a definitive match within the H. pylori genomic database. This protein was isolated by 2D polyacrylamide gel electrophoresis, evaluated by liquid chromatography–mass spectrometry, and found to consist of two related species of approximately 28?100 and 26?500. In parallel, the proteins within this spot were digested in situ with the endoprotease Lys-C. Analysis of the Lys-C digest by matrix-assisted laser desorption time-of-flight mass spectrometry, peptide mapping, and sequence analysis was conducted. Comparison of the mass and sequence of the Lys-C peptides with those derived from a H. pylori genomic library identified an open reading frame of approximately 300 base pairs as the source of the Spot 15 protein. This corresponded to HP0175 in the recently reported H. pylori genome sequence, an open reading frame with some homology to Campylobacter jejeuni cell binding protein 2. Mass spectral and sequence analysis indicated that Spot 15 was a processed product generated by proteolytic cleavage at both the carboxy and amino termini of the 34 open reading frame precursor.     

A model for the regulation of cerebral oxygen delivery

On the basis of the assumption that oxygen delivery across theendothelium is proportional to capillary plasmaPO₂, a model is presented that linkscerebral metabolic rate of oxygen utilization(CMR_O2) to cerebral blood flow(CBF) through an effective diffusivity for oxygen (D) of the capillarybed. On the basis of in vivo evidence that the oxygen diffusivityproperties of the capillary bed may be altered by changes in capillaryPO₂, hematocrit, and/or bloodvolume, the model allows changes in D with changes in CBF. Choice inthe model of the appropriate ratio of   (D/D)/(CBF/CBF)determines the dependence of tissue oxygen delivery on perfusion.Buxton and Frank (J. Cereb. Blood Flow. Metab. 17: 64-72, 1997) recently presented alimiting case of the present model in which  = 0. In contrast to thetrends predicted by the model of Buxton and Frank, in the current modelwhen > 0, the proportionality between changes in CBF andCMR_O2 becomes more linear, and similardegrees of proportionality can exist at different basal values ofoxygen extraction fraction. The model is able to fit the observedproportionalities between CBF and CMR_O2 for a large range ofphysiological data. Although the model does not validate any particularobserved proportionality between CBF andCMR_O2, generally values of(CMR_O2/CMR_O2)/(CBF/CBF) close to unity have been observed across ranges of graded anesthesia inrats and humans and for particular functional activations in humans.The model's capacity to fit the wide range of data indicates that theoxygen diffusivity properties of the capillary bed, which can bemodified in relation to perfusion, play an important role in regulatingcerebral oxygen delivery in vivo.

     

Retention of NO_3^ - in an upland streamenvironment: A mass balance approach

Models of the effects of atmosphericN deposition in forested watersheds have notadequately accounted for the effects of aquatic andnear-stream processes on the concentrations and loadsof NO in surface waters. This studycompared the relative effects of aquatic andnear-stream processes with those from the terrestrialecosystem on the retention and transport ofNO in two contrasting stream reaches ofthe Neversink River, a forested watershed in theCatskill Mountains of New York that receives among thehighest load of atmospheric N deposition in thenortheastern United States. Stream water samples werecollected every two hours and ground-water andtributary samples were collected daily at base flowconditions during four 48-hour periods from April toOctober 1992, and NO mass balances werecalculated for each site. Results indicated diurnalvariations in stream NO concentrations inboth reaches during all four sampling periods; this isconsistent with uptake of NO byphotoautotrophs during daylight hours. Mass-balanceresults revealed significant stream reach losses ofNO at both sites during all samplingperiods. The diurnal variations in NO concentrations and the retention of NO relative to terrestrial contributions to the streamreaches were greater downstream than upstream becausephysical factors such as the head gradients ofinflowing ground water and the organic matter contentof sediment are more favorable to uptake anddenitrification downstream. The mass retention ofNO increased as the mean 48-hr streamdischarge increased at each site, indicating that theresponsible processes are dependent onNO supply. Low stream temperatures duringthe April sampling period, however, probably reducedthe rate of retention processes, resulting in smallerlosses of NO than predicted from streamdischarge alone. Water samples collected from thestream, the hyporheic zone, and the alluvial groundwater at sites in both reaches indicated that the neteffect of hyporheic processes on downstreamNO transport ranged from conservativemixing to complete removal by denitrification. Therelative effects of biological uptake anddenitrification as retention mechanisms could not bequantified, but the results indicate that bothprocesses are significant. These results generallyconfirm that aquatic and near-stream processes causesignificant losses of NO in the NeversinkRiver, and that the losses by these processes atdownstream locations can exceed the NO contributions to the stream from the terrestrialenvironment during summer and fall base-flowconditions. Failure to consider these aquatic andnear-stream processes in models of watershed responseto atmospheric N deposition could result inunderestimates of the amount of NO leaching from forested ecosystems and to an inabilityto unequivocally relate geographic differences inNO concentrations of stream waters tocorresponding differences in terrestrial processes.     

Mate Choice by Chemical Cues in Western Redback (Plethodon vehiculum) and Dunn's (P. dunni) Salamanders

Western redback and Dunn's salamanders (Plethodon vehiculum and Plethodon dunni, respectively) can distinguish between potential mates by using chemical cues. In laboratory choice tests, adult males of both species showed significant discrimination between chemical cues of gravid females over non-gravid females of equal body size. Furthermore, males of both species differentiated the odour of paired gravid females that differed by ? 5 mm snout-vent length (SVL). Given that clutch size is related to female body size in these species, adult males may be able to distinguish between females via cues that signal potentially high female reproductive success. In choice tests, P. vehiculum females did not discriminate between two relatively large males that differed by ? 5 mm SVL. However, females of P. vehiculum did discriminate between two relatively small males that differed by the same amount. Apparently, P. vehiculum females ranked males by both absolute and relative body size using chemical cues. This pattern could reflect a female preference for large males or that females avoid mating with the smallest males.     

Metabolism of exogenous auxin by Arabidopsis thaliana: Identification of the conjugate Nα-(indol-3-ylacetyl)-glutamine and initiation of a mutant screen

The accumulation of conjugates of indole-3-acetic acid (IAA) in Arabidopsis thaliana was studied by incubating tissues with high concentrations of exogenous IAA, followed by reverse phase HPLC analysis of the extracts. Using fluorescence detection, indole-3-acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-acetyl-glucose were observed and quantitated in extracts of tissue after 24 h incubation with 500 μ M IAA. In addition, a new metabolite was detected and positively identified as indole-3-acetyl-glutamine by fast atom bombardment mass spectrometry, exact mass measurement, and tandem mass spectrometry in comparison with a synthetic standard. The amounts of individual conjugates formed differed between leaves, shoot axes and roots. In all three tissues, indole-3-acetyl-aspartate was the most abundant conjugate, the highest level being observed in roots. Highest levels of indole-3-acetyl-glutamine were observed in leaves, where it was the second most abundant conjugate and comprised approximately 12% of the fluorescent metabolites. Accumulation of the three amide conjugates was dramatically inhibited by cycloheximide, whereas accumulation of indole-3-acetyl-glucose was little affected. Based on these data, a screen for Arabidopsis mutants altered in the IAA-inducible system for auxin conjugate formation was initiated. The first mutant to be isolated and characterized produces more indole-3-acetyl-glutamine and less indole-3-acetyl-aspartate than wild-type, and is allelic to an existing class of photorespiration mutants ( gluS ) deficient in chloroplastic glutamate synthase.