A dust-obscured massive maximum-starburst galaxy at a redshift of 6.34.

Massive present-day early-type (elliptical and lenticular) galaxies probably gained the bulk of their stellar mass and heavy elements through intense, dust-enshrouded starbursts--that is, increased rates of star formation--in the most massive dark-matter haloes at early epochs. However, it remains unknown how soon after the Big Bang massive starburst progenitors exist. The measured redshift (z) distribution of dusty, massive starbursts has long been suspected to be biased low in z owing to selection effects, as confirmed by recent findings of systems with redshifts as high as ~5 (refs 2-4). Here we report the identification of a massive starburst galaxy at z = 6.34 through a submillimetre colour-selection technique. We unambiguously determined the redshift from a suite of molecular and atomic fine-structure cooling lines. These measurements reveal a hundred billion solar masses of highly excited, chemically evolved interstellar medium in this galaxy, which constitutes at least 40 per cent of the baryonic mass. A 'maximum starburst' converts the gas into stars at a rate more than 2,000 times that of the Milky Way, a rate among the highest observed at any epoch. Despite the overall downturn in cosmic star formation towards the highest redshifts, it seems that environments mature enough to form the most massive, intense starbursts existed at least as early as 880 million years after the Big Bang.

Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry.

The DeltaF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and DeltaF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because DeltaF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and DeltaF508 constructs, and the DeltaF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide (1)H/(2)H exchange rates in matched F508 and DeltaF508 constructs reveal that DeltaF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the DeltaF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-DeltaF508 structures but completely solvent exposed in all DeltaF508 structures. These results reinforce the importance of the perturbation DeltaF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.

Development of an spFRET method to measure structure changes in ion exchange proteins.

AIM: Understanding the mechanism of tightly coupled ion exchange proteins, important effectors of cell volume regulation and other physiologically important transport processes requires means to observe dynamic changes in structure during the transport cycle. As a step towards this goal, we have applied single-pair fluorescence resonance energy transfer to a monomeric bacterial oxalate-formate exchanger (OxlT). METHODS: A His-9 tagged OxlT mutant containing two cysteines at positions 17 and 224 was labelled with cyanine dye maleimides (Cy3 donor and Cy5 acceptor) and attached to glass coverslips for measurements of donor and acceptor emission from single molecules, as described (P. Pal et al. Biophys J89, L11, 2005). RESULTS: Time-series data from 20 spots containing donor and acceptor provided evidence for single-pair energy transfer. From the efficiency of energy transfer, the mean donor-acceptor distance was determined to be 44.2 A. Considering the size of the probes, this is in good agreement with the Calpha distance of 39.6 A for the corresponding sites found in the OxlT structural (homology) model (Q. Yang et al. Proc Natl Acad Sci102, 8513, 2005). CONCLUSION: These results demonstrate the feasibility of single-pair fluorescence resonance energy transfer to measure distances between known sites in single OxlT molecules. This technique provides a potential means to test models for transport-related conformational changes, as well as to detect real-time structure alterations during the catalytic transport cycle.

Rare cancer cell analyzer for whole blood applications: microcytometer cell counting and sorting subcircuits.

We demonstrate sorting of rare cancer cells from blood using a thin ribbon monolayer of cells within a credit-card sized, microfluidic laboratory-on-a-card ("lab card") structure. This enables higher cell throughput per minute thereby speeding up cell interrogation. In this approach, multiple cells are viewed and sorted, not individually, but as a whole cell row or section of the ribbon at a time. Gated selection of only the cell rows containing a tagged rare cell provides enrichment of the rare cell relative to background blood cells. We also designed the cell injector for laminar flow antibody labeling within 20s. The approach combines rapid laminar flow cell labeling with monolayer cell sorting thereby enabling rare cell target detection at sensitivity levels 1000 to 10,000 times that of existing flow cytometers. Using this method, total cell labeling and data acquisition time on card may be reduced to a few minutes compared to 30-60 min for standard flow methods.

Rare cancer cell analyzer for whole blood applications: automated nucleic acid purification in a microfluidic disposable card.

One current challenge facing point-of-care cancer detection is that existing methods make it difficult, time consuming and too costly to (1) collect relevant cell types directly from a patient sample, such as blood and (2) rapidly assay those cell types to determine the presence or absence of a particular type of cancer. We present a proof of principle method for an integrated, sample-to-result, point-of-care detection device that employs microfluidics technology, accepted assays, and a silica membrane for total RNA purification on a disposable, credit card sized laboratory-on-card ('lab card") device in which results are obtained in minutes. Both yield and quality of on-card purified total RNA, as determined by both LightCycler and standard reverse transcriptase amplification of G6PDH and BCR-ABL transcripts, were found to be better than or equal to accepted standard purification methods.

Disease Progression of Phytophthora ramorum and Botryosphaeria dothidea on Pacific Madrone.

Infection by Phytophthora ramorum was associated with stem and leaf lesions of Pacific madrone (Arbutus menziesii) seedlings and saplings. In addition, a common and native pathogen, Botryosphaeria dothidea, caused similar leaf and stem lesions. When exposed to natural levels of inoculum in forests infested with P. ramorum, 50 to 66% of madrone saplings used as bait died. Recovery of P. ramorum from colonized plant tissue on culture media was generally low. From initial infection, P. ramorum was not culturable from leaf tissue after a mean of 3.5 weeks or from stem tissue after a mean of 8 weeks. Generally, B. dothidea was recovered more frequently from necrotic stems and leaves than was P. ramorum. Experimental inoculations of madrone seedlings showed that leaf and stem lesion lengths were, on average, greater on tree seedlings inoculated with P. ramorum than on those inoculated with B. dothidea. P. ramorum and B. dothidea appear to coexist in stem and leaf tissue, forming a novel pathogen complex, affecting growth and reproduction of Pacific madrone.

Allelic imbalance at the DNA mismatch repair loci, hMSH2, hMLH1, hPMS1, hPMS2 and hMSH3, in squamous cell carcinoma of the head and neck.

BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) is one of the 10 most frequently occurring cancers in the world. Defective mismatch repair, as exhibited by the phenomenon of microsatellite instability, has been observed in SCCHN although no reports of mismatch repair gene mutations or altered protein expression have been published. In a variety of microsatellite instability (MSI) positive cancers where mutations in the mismatch repair (MMR) genes were not observed, allelic imbalance at the loci of the MMR genes was prevalent. OBJECTIVE: To investigate whether allelic imbalance at the MMR genetic loci contributes to the development of SCCHN. MATERIALS AND METHODS: 35 matched normal/tumour SCCHN pairs were studied using 29 microsatellite markers located within and adjacent to six known DNA mismatch repair genes. In addition, mutational analysis and protein expression of hMSH2 and hMLH1 were investigated. RESULTS AND CONCLUSIONS: We demonstrated that 36 and 17% of the analysed SCCHN specimens exhibited allele imbalance at the hMLH1 and hMSH3 genetic loci, respectively. Allelic instability at these two loci was found to be correlated with the MSI status of the SCCHN tumours. Allelic instability was found to be uncommon at the other MMR gene loci analysed. One mutation was found in hMSH2 and none in hMLH1 in this series of tumours. 23 of 24 (96%) of the examined SCCHN tumours showed reduced expression of either hMSH2 or hMCH1 genes. Allelic instability in the MMR genes, hMLH1 and hMSH3, is proposed to be involved in the aetiology of SCCHN tumours.

First Report of Phytophthora ramorum on Coast Redwood in California.

Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from discolored leaves and cankers on small branches (<0.5 cm in diameter) on 27 coast redwood (Sequoia sempervirens) saplings (2 to17 cm in diameter) at two locations in California (Jack London State Park, Sonoma County and Henry Cowell State Park, Santa Cruz County). Symptoms were observed on branches throughout the crowns of affected trees. Isolates were identified as P. ramorum by their abundant chlamydospores and caducous, semi-papillate sporangia (2) and internal transcribed spacer (ITS) rDNA sequences identical to those of P. ramorum from Quercus spp., Lithocarpus densiflorus, and Rhododendron (1,2). P. ramorum was also detected in dying basal sprouts on mature redwood trees from an additional five locations in coastal California by polymerase chain reaction (PCR) amplification of the ITS region using DNA extracted from symptomatic tissue and P. ramorum-specific PCR primers. To test for pathogenicity, foliage inoculations were conducted on redwood seedlings in two trials by misting 30 leaves per trial (five leaves per seedling plus controls) with sterile distilled water and then pinning inoculum plugs to the upper surface of leaves. Inoculation resulted in lesions of 1 to 20 mm on individual leaves, and P. ramorum was recovered from 43% of inoculated leaves. Symptoms were not restricted to inoculated leaves because 15 inoculations of individual leaves led to discoloration of two or more adjacent leaves. On one inoculation, 60 mm of the adjacent stem was killed. Stems of redwood seedling (approximately 1 cm in diameter) were wound inoculated (1) in two trials consisting of 10 inoculated seedlings per trial plus 10 controls. After 6 weeks, lesion lengths in the cambium caused by P. ramorum averaged 13.7 mm (range 4 to 21 mm). P. ramorum was recovered from 100% of inoculated stems. Entire branches near the inoculation point became chlorotic even though no direct connection was evident between the lesion and the branches. No chlorosis was observed among the control inoculations. Mean lesion lengths of inoculated stems were significantly greater in both trials than those of control inoculations (mean 6.2 mm) at P < 0.05 based on analysis of variance (ANOVA). Redwood saplings (2.5 to 4.5 cm in diameter) were also wound inoculated in a separate trial. No phloem or cambial discoloration was observed after 7 weeks, but necrotic lesions in the xylem had a mean length of 39 mm (range 12 to 73 mm). In addition, narrow streaks, 1 to 2 mm in diameter, were also noted in the xylem extending from the necrotic areas upward to 90 cm. P. ramorum was recovered from 70% of inoculated stems in this trial. Mean lesion lengths of P. ramorum were significantly greater in all trials than those of control inoculations (mean 20 mm) at P < 0.05 based on ANOVA. While P. ramorum causes a lethal canker on Quercus spp. and L. densiflorus (1), we have not observed unusual mortality or disease symptoms on overstory redwoods in natural forests. The impact of infection by P. ramorum on understory redwoods is also unclear. However, the pathogen appears to be able to kill sprouts. References: (1) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.

Loss of heterozygosity on chromosomes 3, 9, 13, and 17, including the retinoblastoma locus, in uveal melanoma.

PURPOSE: To identify tumor-suppressor loci that may contribute to the pathogenesis of uveal melanoma. METHODS: Multiplex fluorescence microsatellite assays were performed on 27 uveal melanomas using markers at 3p25-p26, 3p14.2, 9p21-p23, 13q14, 13q12.3-q13, and 17p13, close to or within the von Hippel Lindau (VHL), fragile histidine triad (FHIT), p16/cyclin-dependent kinase inhibitor 2 (CDKN2A), retinoblastoma (RB1), breast cancer 2 (BRCA2), and p53 tumor suppressor loci, respectively. Further markers on chromosomes 3 and 9 were analyzed individually. RESULTS: Loss of heterozygosity (LOH) was identified in 63% of tumors, most frequently on chromosome 3 (52%), in association with epithelioid cells (P = 0.0002) and microvascular loops (P = 0.0008). In the majority of cases, LOH on chromosome 3 was detected at all informative markers. The second most common alteration was LOH at an RB1 intragenic marker (21% tumors), with retention of a more centromeric 13q marker (near BRCA2). The pattern of LOH on chromosome 9p was consistent with the involvement of a region telomeric to CDKN2A. LOH at TP53 was infrequent. CONCLUSIONS: In the majority of cases, chromosome 3 LOH involves an entire chromosome homologue, which hampers identification of the relevant suppressor loci. This LOH correlates with the presence of microvascular loops and epithelioid cells, two of the recognized histologic indicators of poor prognosis. Data for chromosomes 13 and 9 support a role for RB1 in the pathogenesis of uveal melanoma but also raise the possibility of the involvement of additional loci close to RB1 and CDKN2A.

Projection structure and molecular architecture of OxlT, a bacterial membrane transporter.

The major facilitator superfamily (MFS) represents the largest collection of evolutionarily related members within the class of membrane 'carrier' proteins. OxlT, a representative example of the MFS, is an oxalate-transporting membrane protein in Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional crystals of OxlT, we have determined the projection structure of this membrane transporter. The projection map at 6 A resolution indicates the presence of 12 transmembrane helices in each monomer of OxlT, with one set of six helices related to the other set by an approximate internal two-fold axis. The projection map reveals the existence of a central cavity, which we propose to be part of the pathway of oxalate transport. By combining information from the projection map with related biochemical data, we present probable models for the architectural arrangement of transmembrane helices in this protein superfamily.

A protocol for the management of compound mandibular fractures based on the time from injury to treatment.

PURPOSE: The purpose of this study was to evaluate the validity of a treatment protocol for compound mandibular fractures that is based on the time of injury to treatment. PATIENTS AND METHODS: Fifty-two patients with 71 mandibular fractures were treated in a prospective fashion in conformity with the protocol. Thirty-seven open reductions with rigid fixation were performed on 30 patients. The remaining 22 patients were treated solely with closed reduction and maxillomandibular fixation (MMF). Forty-five patients were treated before 72 hours and 7 after 72 hours. RESULTS: Fifty-one of the 52 patients healed without evidence of infection. One patient developed suppurative osteomyelitis. Thus, the bone infection rate was 1.9% for all patients treated and 3.3% for patients treated with rigid fixation (ORIF). CONCLUSION: These results underscore the validity of the treatment protocol to immobilize compound fractures within 72 hours of injury, if possible. If the initial treatment is delayed for more than 3 days, any infection at the compound fracture site(s) should first be resolved by MMF and intravenous antibiotics before performing an open reduction. This is done to ensure adequate perfusion of blood at the fracture site when the open reduction is performed.

Helix proximity in OxlT, the oalate:formate antiporter of oxalobacter formigenes. Cross-linking between TM2 and TM11.

Experiments were designed to evaluate the proximity of transmembrane helices two (TM2) and eleven (TM11) in the tertiary structure of OxlT, the oxalate:formate exchange transporter of Oxalobacter formigenes. A tandem duplication of the Factor Xa protease cleavage site (IEGRIEGR) was inserted into the central cytoplasmic loop of an OxlT cysteine-less derivative in which an endogenous cleavage site had been eliminated by mutagenesis (R248Q). Using this host, double cysteine derivatives were constructed so as to pair one of seventeen positions in TM2 with one of four positions in TM11. Following treatment of membrane vesicles with Cu(II)(1,10-phenanthroline)(3), molecular iodine, or N,N'-o-phenylenedimaleimide, samples were exposed to Factor Xa, and disulfide bond formation was assessed after SDS-polyacrylamide gel electrophoresis by staining with antibody directed against the OxlT C terminus. In the absence of disulfide bond formation, exposure to Factor Xa revealed the expected C-terminal 22-kDa fragment, a result unaffected by the presence of reductant. By contrast, after disulfide formation, OxlT mobility remained at 35 kDa, and appearance of the 22-kDa fragment required addition of 200 mm dithiothreitol prior to electrophoresis. The four TM11 positions chosen for cysteine substitution lie on a helical face known to interact with substrate. Similarly, TM2 positions supporting disulfide trapping were also confined to a single helical face. We conclude that TM2 and TM11 are in close juxtaposition to one another in the tertiary structure of OxlT.

The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export.

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V(max) = 18 nmol/mg/min; K(m) for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.

Chronic low back pain and depression in a sample of veterans.

The relationship between chronic low back pain and scores on depression was examined in a sample of 31 veterans who completed a depression inventory. Analysis indicated that those with chronic low back pain scored significantly higher on depression than those without.

Transmembrane segment 11 of UhpT, the sugar phosphate carrier of Escherichia coli, is an alpha-helix that carries determinants of substrate selectivity.

In Escherichia coli, transport of hexose 6-phosphates is mediated by the P(i)-linked antiport carrier, UhpT, a member of the major facilitator superfamily. We showed earlier that Lys(391), a member of an intrahelical salt bridge (Asp(388)/Lys(391)) in the eleventh transmembrane segment (TM11) of this transporter, can function as a determinant of substrate selectivity (Hall, J. A., Fann, M.-C., and Maloney, P. C. (1999) J. Biol. Chem. 274, 6148-6153). Here, we examine in detail the role of TM11 in setting substrate preference. Derivatives having an uncompensated cationic charge at either position 388 or 391 (the D388C, D388V, or D388K/K391C variants) are gain-of-function mutants in which phosphoenolpyruvate, not sugar 6-phosphate, is the preferred organic substrate. By contrast, when an uncompensated anionic charge is placed at position 388 (K391C), we observed behavior consistent with an increased preference for monovalent rather than divalent sugar 6-phosphate. Because positions 388 and 391 lie deep within the UhpT hydrophobic sector, these findings suggested that an extended length of TM11 may be accessible to external substrates and probes. To explore this issue, we used a panel of TM11 single cysteine variants to examine the transport of glucose 6-phosphate in the presence and absence of the membrane-impermeant, thiol-reactive agent p-chloromercuribenzosulfonate (PCMBS). Accessibility to PCMBS, together with the pattern of substrate protection against PCMBS inhibition, leads us to conclude that TM11 spans the membrane as an alpha-helix, with approximately two-thirds of its surface lining a substrate translocation pathway. We suggest that this feature is a general property of carrier proteins in the major facilitator superfamily and that for this reason residues in TM11 will serve to carry determinants of substrate selectivity.

Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling.

The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His(9)-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+ linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.

Cancer-specific genomic instability in bronchial lavage: a molecular tool for lung cancer detection.

We examined genomic instability in DNA from 80 bronchial lavage samples from patients with lung cancer and individuals with no malignant lung disease. We used a multiplex assay of eight fluorescent-tagged microsatellite markers that have a very high incidence of allelic imbalance in lung tumors. When genomic instability at individual loci was analyzed statistically against diagnosis, markers D3S1289 (P = 0.033), D3S1300 (P = 0.001), D13S171 (P = 0.009), and D17S2179E (P = 0.017) demonstrated significantly higher frequency of instability in bronchial lavage specimens from lung cancer cases than those with nonmalignant conditions. In contrast, markers D9S157, D9S161, D13S153, and D5S644 demonstrated lower specificity (P > 0.05) for lung tumors. These results suggest that genomic instability in some loci may be related to high proliferation rates but not necessarily to cell commitment to malignancy. When genomic instability was scored with only the four cancer-specific markers, the assay produced a sensitivity of 73.9% and a specificity of 76.5%. On combining the results from the cytological examination and the molecular assay, the sensitivity reached 82.6%. These results indicate that in our efforts to investigate genomic instability as a potential marker for the early detection of lung cancer, we need to identify cancer-specific genomic instability markers. This paper has shown that these first four markers may be considered to form an individual set of cancer-specific genomic instability markers.

Na(+)/glutamine (asparagine) cotransport by Staphylococcus lugdunensis and Corynebacterium amycolatum.

Staphylococcus lugdunensis and Corynebacterium amycolatum each have a Na(+)/glutamine cotransporter that displays an ordered reaction sequence at the extracellular surface, with sodium binding (K(m) of 6.5 mM) before glutamine (K(m) of 50 microM). Asparagine is low-affinity substrate (K(m) approximately 1 mM) for each system.

Structure/function relationships in OxlT, the oxalate-formate transporter of oxalobacter formigenes. Assignment of transmembrane helix 11 to the translocation pathway.

OxlT, the oxalate:formate antiporter of Oxalobacter formigenes, has a lone charged residue, lysine 355 (Lys-355), at the center of transmembrane helix 11 (TM11). Because Lys-355 is the only charged residue in the hydrophobic sector, we tested the hypothesis that lysine 355 contributes to the binding site for the anionic substrate, oxalate. This idea was supported by mutational analysis, which showed that of five variants studied (Lys-355 --> Cys, Gly, Gln, Arg, or Thr), residual function was found for only the K355R derivative, in which catalytic efficiency had fallen 2,600-fold. Further insight came from a study of TM11 single-cysteine mutants, using the impermeant, thiol-specific reagents, carboxyethyl methanethiosulfonate and ethyltrimethylammonium methanethiosulfonate. Of the five reactive positions identified in TM11, four were at the cytoplasmic or periplasmic ends of TM11 (S344C and A345C, and G366C and A370C, respectively), whereas the fifth was at the center of the helix (S359C). Added study with carboxyethyl methanethiosulfonate and ethylsulfonate methylthiosulfonate showed that the attack on S359C could be blocked by the presence of the substrate, oxalate, and that protection could be predicted quantitatively by a kinetic model in which S359C is accessible only in the unliganded form of OxlT. Parallel study showed that the proteoliposomes used in such work contained OxlT of right side-out and inside-out orientations in about equal amounts. Accordingly, full inhibition of S359C by the impermeable methanethiosulfonate-linked probes must reflect an approach from both the cytosolic and periplasmic surfaces of the protein. This, coupled with the finding of substrate protection, leads us to conclude that S359C lies on the translocation pathway through OxlT. Since position 359 and 355 lie on the same helical face, we suggest that Lys-355 also lies on the translocation pathway, consistent with the idea that the essential nature of Lys-355 reflects its role in binding the anionic substrate, oxalate.