The MACHO Project is a search for dark matter in the form of massive compact halo objects (MACHOs). Photometric monitoring of millions of stars in the Large Magellanic Cloud (LMC), Small Magellanic Cloud (SMC), and Galactic bulge is used to search for gravitational microlensing events caused by these otherwise invisible objects. Analysis of the Ðrst 2.1 yr of photometry of 8.5 million stars in the LMC reveals eight candidate microlensing events. This is substantially more than the number expected (D1.1) from lensing by known stellar populations. The timescales (t) of the events range from 34 to 145 days. We estimate the total microlensing optical depth toward the LMC from events with days to be based upon our eight event sample. This exceeds the 2 \ tü \ 200 q 2 200 \ 2.9~0 .9 1.4 ] 10~7 optical depth, expected from known stars, and the di †erence is to be compared q backgnd \ 0.5 ] 10~7, with the optical depth predicted for a "" standard ÏÏ halo composed entirely of MACHOs : q halo \ 4.7 To compare with Galactic halo models, we perform likelihood analyses on the full eight-event ] 10~7. sample and a six-event subsample (which allows for two events to be caused by a nonhalo "" background ÏÏ). This gives a fairly model-independent estimate of the halo mass in MACHOs within 50 kpc of which is about half of the "" standard halo ÏÏ value. We also Ðnd a most prob-2.0~0 .71.2 ] 1011 M _ , able MACHO mass of although this value is strongly model dependent. In addition, the 0.5~0 .2 0.3 M _ , absence of short duration events places stringent upper limits on the contribution of low-mass MACHOs : objects from 10~4 to 0.03 contribute of the "" standard ÏÏ dark halo.

Accumulation of radiolabelled naphthalene-1-acetic acid (1-NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-acetic acid (IAA) has been measured in suspension-cultured tobacco (Nicotiana tabacum) cells. In this paper is presented a simple methodology allowing activities of the auxin influx and efflux carriers to be monitored independently by measuring the cellular accumulation of [H]NAA and [C]2,4-D. We have shown that 1-NAA enters cells by passive diffusion and has its accumulation level controlled by the efflux carrier. By contrast, 2,4-D uptake is mostly ensured by the influx carrier and this auxin is not secreted by the efflux carrier. Both auxin carriers contribute to IAA accumulation. The kinetic parameters and specificity of each carrier have been determined and new information concerning interactions with naphthylphthalamic acid, pyrenoylbenzoic acid, and naphthalene-2-acetic acid are provided. The relative contributions of diffusion and carrier-mediated influx and efflux to the membrane transport of 2,4-D, 1-NAA, and IAA have been quantified, and the data indicate that plant cells are able to modulate over a large range their auxin content by modifying the activity of each carrier.

The vacuolar membrane protein a-TIP is a seedspecific protein of the Major Intrinsic Protein family. Expression of ac-TIP in Xenopus oocytes confered a 4-to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that a-TIP forms water channels and is thus a new aquaporin. a-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in a-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of a-TIP in oocytes, suggesting that phosphorylation of a-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of a-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating a-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing a-TIP, as well as for in vitro phosphorylation of a-TIP. These findings demonstrate that the a-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99. To our knowledge, this is the first evidence of aquaporin regulation via phosphorylation and we propose this process as a mechanism for regulating water permeability of biological membranes.

We report the detection of 45 candidate microlensing events in fields toward the Galactic bulge. These come from the analysis of 24 fields containing 12.6 million stars observed for 190 days in 1993. Many of these events are of extremely high signal to noise and are remarkable examples of gravitational microlensing. The distribution of peak magnifications is shown to be consistent with the microlensing interpretation of these events. Using a sub-sample of 1.3 million "Clump Giant" stars whose distance and detection efficiency are well known, we find 13 events and estimate the microlensing optical depth toward the Galactic Bulge as τ bulge = 3.9 +1.8 −1.2 × 10 −6 averaged over an area of ∼ 12 square degrees centered at Galactic coordinates ℓ = 2.55 • and b = −3.64 • . This is similar to the value reported by the OGLE collaboration, and is marginally higher than current theoretical models for τ bulge . The optical depth is also seen to increase significantly for decreasing |b|. These results demonstrate that obtaining large numbers of microlensing events toward the Galactic bulge is feasible, and that the study of such events will have important consequences for the structure of the Galaxy and its dark halo.

Since July 1992, the MACHO project has been carrying out long-term photometric monitoring of over 20 million stars in the Magellanic Clouds and Galactic Bulge. Our aim is to search for the very rare gravitational microlensing events predicted if the dark halo of our Galaxy is comprised of massive compact halo objects (hereafter Machos). We have now analysed most of the rst year's LMC data, comprising 9.5 million light curves of stars with an average of 235 observations each. Automated selection procedures applied to this sample show 3 events consistent with microlensing, of which one is very striking (Alcock et al. 1993) and two are of modest amplitude. We have evaluated our experimental detection e ciency using a range of detailed Monte-Carlo simulations, including addition of arti cial stars to real data frames. Using a`standard' halo density pro le we nd that a halo comprised entirely of Machos in the mass range 3 10 4 to 0:06 M would predict > 15 detected events in this dataset, and objects around 3 10 3 M would predict 25 events; thus a standard spherical halo cannot be dominated by objects in this mass range. Assuming all three events are microlensing of halo objects and tting a naive spherical halo model to our data yields aMacho halo fraction f = 0:19 +0:16 0:10 , a total mass in Machos (inside 50 kpc) of 7:6 +6 4 10 10 M , and a microlensing optical depth 8:8 +7 5 10 8 (68% CL). We have explored a wide range of halo models and nd that, while our constraints on the Macho fraction are quite model-dependent, constraints on the total mass in Machos within 50 kpc are quite secure. Future observations from this and other similar projects and accurate measurements of the Galactic mass out to large radii should combine to give much improved constraints on the Macho fraction of the halo. * A similar calculation was carried out by Petrou (1981), but was not published.

Oligomeric degradation products of alginate elicited a respiratory and oxidative burst in the sporophytes of the kelp Laminaria digitata. The generation of activated oxygen species (AOS), O 2 Ϫ , and H 2 O 2 was detected at the single cell level, using nitroblue tetrazolium precipitation and a redox-sensitive fluorescent probe, respectively. The oxidative burst involved diphenyleneiodonium-sensitive AOS-generating machinery and its amplitude depended on the type of tissue. After a first elicitation plants were desensitized for about 3 h. The activity of alginate oligosaccharides was dose dependent, saturating around 40 m. It was also structure-dependent, with homopolymeric blocks of ␣-1,4-l-guluronic acid, i.e. the functional analogs of oligogalacturonic blocks in pectins, being the most active signals. The perception of oligoguluronate signals resulted in a strong efflux of potassium. Pharmacological dissection of the early events preceding the emission of AOS indicated that the transduction chain of oligoguluronate signals in L. digitata is likely to feature protein kinases, phospholipase A 2 , as well as K ϩ , Ca 2ϩ , and anion channels.

Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date.

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