Collagenolysis-dependent DDR1 signalling dictates pancreatic cancer outcome.

Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic, aggressive cancer that frequently progresses and spreads by metastasis to the liver1. Cancer-associated fibroblasts, the extracellular matrix and type I collagen (Col I) support2,3 or restrain the progression of PDAC and may impede blood supply and nutrient availability4. The dichotomous role of the stroma in PDAC, and the mechanisms through which it influences patient survival and enables desmoplastic cancers to escape nutrient limitation, remain poorly understood. Here we show that matrix-metalloprotease-cleaved Col I (cCol I) and intact Col I (iCol I) exert opposing effects on PDAC bioenergetics, macropinocytosis, tumour growth and metastasis. Whereas cCol I activates discoidin domain receptor 1 (DDR1)-NF-κB-p62-NRF2 signalling to promote the growth of PDAC, iCol I triggers the degradation of DDR1 and restrains the growth of PDAC. Patients whose tumours are enriched for iCol I and express low levels of DDR1 and NRF2 have improved median survival compared to those whose tumours have high levels of cCol I, DDR1 and NRF2. Inhibition of the DDR1-stimulated expression of NF-κB or mitochondrial biogenesis blocks tumorigenesis in wild-type mice, but not in mice that express MMP-resistant Col I. The diverse effects of the tumour stroma on the growth and metastasis of PDAC and on the survival of patients are mediated through the Col I-DDR1-NF-κB-NRF2 mitochondrial biogenesis pathway, and targeting components of this pathway could provide therapeutic opportunities.

SERTAD3 induces proteasomal degradation of ZIKV capsid protein and represents a therapeutic target.

Zika virus (ZIKV) is a mosquito-borne RNA virus that belongs to the Flaviviridae family. While flavivirus replication is known to occur in the cytoplasm, a significant portion of the viral capsid protein localizes to the nucleus during infection. However, the role of the nuclear capsid is less clear. Herein, we demonstrated SERTA domain containing 3 (SERTAD3) as an antiviral interferon stimulatory gene product had an antiviral ability to ZIKV but not JEV. Mechanistically, we found that SERTAD3 interacted with the capsid protein of ZIKV in the nucleolus and reduced capsid protein abundance through proteasomal degradation. Furthermore, an eight amino acid peptide of SERTAD3 was identified as the minimum motif that binds with ZIKV capsid protein. Remarkably, the eight amino acids synthetic peptide from SERTAD3 significantly prevented ZIKV infection in culture and pregnant mouse models. Taken together, these findings not only reveal the function of SERTAD3 in promoting proteasomal degradation of a specific viral protein but also provide a promising host-targeted therapeutic strategy against ZIKV infection.

Structural basis for GPCR-independent activation of heterotrimeric Gi proteins.

Heterotrimeric G proteins are key molecular switches that control cell behavior. The canonical activation of G proteins by agonist-occupied G protein-coupled receptors (GPCRs) has recently been elucidated from the structural perspective. In contrast, the structural basis for GPCR-independent G protein activation by a novel family of guanine-nucleotide exchange modulators (GEMs) remains unknown. Here, we present a 2.0-Å crystal structure of Gαi in complex with the GEM motif of GIV/Girdin. Nucleotide exchange assays, molecular dynamics simulations, and hydrogen-deuterium exchange experiments demonstrate that GEM binding to the conformational switch II causes structural changes that allosterically propagate to the hydrophobic core of the Gαi GTPase domain. Rearrangement of the hydrophobic core appears to be a common mechanism by which GPCRs and GEMs activate G proteins, although with different efficiency. Atomic-level insights presented here will aid structure-based efforts to selectively target the noncanonical G protein activation.

Long non-coding RNA CASC19 facilitates non-small cell lung cancer cell proliferation and metastasis by targeting the miR-301b-3p/LDLR axis.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a lethal tumor resulting in a large number of cancer-related deaths globally. Long noncoding RNAs (lncRNAs) may modulate tumor initiation and metastasis. Although dysregulation of lncRNA cancer susceptibility 19 (CASC19) is validated in NSCLC, further exploration of the CASC19-regulated mechanism in NSCLC is still needed. METHODS: CASC19 expression was examined in NSCLC cells by a quantitative reverse transcriptase-polymerase chain reaction. The specific role of CASC19 in NSCLC was analyzed by cell counting kit-8, EdU, Transwell and western blot assays. The interaction between miR-301b-3p and CASC19 or low-density lipoprotein receptor (LDLR) was confirmed by luciferase reporter and RNA immunoprecipitation assays. RESULTS: CASC19 is markedly overexpressed in NSCLC. Its deficiency impairs cell proliferation, as well as metastasis in NSCLC. Molecular mechanism experiments indicated that CASC19 negatively modulates the expression of miR-301b-3p and miR-301b-3p can bind with CASC19 in NSCLC. In addition, miR-301b-3p binds to LDLR to impair its expression in NSCLC. Finally, rescue experiments showed that miR-301b-3p inhibition or LDLR overexpression counteracted the CASC19 knockdown-mediated function on cell proliferation and metastasis in NSCLC. CONCLUSIONS: CASC19 facilitates NSCLC cell proliferation and metastasis by targeting the miR-301b-3p/LDLR axis, offering a possible strategy for lncRNA-targeted treatment in NSCLC.

DKK1 is epigenetically downregulated by promoter methylation and inhibits bile acid-induced gastric intestinal metaplasia.

Dickkopf-related protein 1 (DKK1) is essential to gastric cancer as an inhibitor of Wnt signaling. Gastric intestinal metaplasia (GIM) is an important precancerous lesion of gastric cancer that can be activated by bile acid reflux and chronic inflammation. However, the exact mechanism of DKK1 in bile acid-induced GIM has not been completely elucidated. We aimed to explore the epigenetic alterations and biological functions of DKK1 in the development of GIM. In the present study, bile acid was found to induce the expression of intestinal markers in gastric epithelial cells, whereas DKK1 was downregulated in response to bile acid stimulation. The mRNA and protein expression levels of DKK1 were decreased in GIM tissues as evidenced by qRT-PCR and immunohistochemical staining. Surprisingly, the methylation of the DKK1 promoter increased in GIM tissues, and we discovered 28 differential methylation sites of the DKK1 promoter in GIM tissues. Bile acid was able to induce the partial methylation of the DKK1 promoter, while 5-aza could increase DKK1 expression as well as decrease intestinal markers expression in gastric epithelial cells. In conclusion, the promoter methylation and downregulation of DKK1 might play important roles in the development of GIM, especially bile acid-induced GIM.

Azithromycin Protects against Zika virus Infection by Upregulating virus-induced Type I and III Interferon Responses.

Azithromycin (AZM) is a widely used antibiotic, with additional antiviral and anti-inflammatory properties that remain poorly understood. Although Zika virus (ZIKV) poses a significant threat to global health, there are currently no vaccines or effective therapeutics against it. Herein, we report that AZM effectively suppresses ZIKV infection in vitro by targeting a late stage in the viral life cycle. Besides that, AZM upregulates the expression of host type I and III interferons and several of their downstream interferon-stimulated genes (ISGs) in response to ZIKV infection. In particular, we found that AZM upregulates the expression of MDA5 and RIG-I, pathogen recognition receptors (PRRs) induced by ZIKV infection, and increases the levels of phosphorylated TBK1 and IRF3. Interestingly, AZM treatment upregulates phosphorylation of TBK1, without inducing phosphorylation of IRF3 by itself. These findings highlight the potential use of AZM as a broad antiviral agent to combat viral infection and prevent ZIKV associated devastating clinical outcomes, such as congenital microcephaly.

SOX2 interferes with the function of CDX2 in bile acid-induced gastric intestinal metaplasia.

BACKGROUND: Intestinal metaplasia (IM) is a premalignant lesion associated with gastric cancer. Both animal and clinical studies have revealed that bile acid reflux and subsequent chronic inflammation are key causal factors of IM. Previous studies indicated that SOX2, the key transcription factor in gastric differentiation, was downregulated during IM development while CDX2, the pivotal intestine-specific transcription factor was upregulated significantly. However, it remains unclear whether the downregulation of SOX2 promotes gastric IM emergence or is merely a concomitant phenomenon. In addition, the underlying mechanisms of SOX2 downregulation during IM development are unclear. METHODS: Gastric cell lines were treated with deoxycholic acid (DCA) in a dose-dependent manner. The expression of CDX2 and miR-21 in gastric tissue microarray were detected by immunohistochemistry and in situ hybridization. Coimmunoprecipitation and immunofluorescence were performed to ascertain the interaction of SOX2 and CDX2. Luciferase reporter assays were used to detect the transcriptional activity of CDX2, and confirm miR-21 binding to SOX2 3'-UTR. The protein level of SOX2, CDX2 and downstream IM-specific genes were investigated using western blotting. mRNA level of miR-21, SOX2, CDX2 and downstream IM-specific genes were detected by qRT-PCR. RESULTS: Bile acid treatment could suppress SOX2 expression and simultaneously induce expression of CDX2 in gastric cell lines. Furthermore, we demonstrated that SOX2 overexpression could significantly inhibit bile acid- and exogenous CDX2-induced IM-specific gene expression, including KLF4, cadherin 17 and HNF4α expression. In contrast, SOX2 knockdown had the opposite effect. A dual-luciferase reporter assay demonstrated that SOX2 overexpression could significantly suppress CDX2 transcriptional activity in HEK293T cells. CDX2 and SOX2 could form protein complexes in the nucleus. In addition, bile acid induced the expression of miR-21. The inhibition of SOX2 in bile acid-treated gastric cell lines was rescued by miR-21 knockdown. CONCLUSIONS: These findings suggested that SOX2 can interfere with the transcriptional activity of CDX2 in bile acid-induced IM and that miR-21 might play a key role in this process, which shed new lights in the prevention of gastric cancer.

Factors associating with the presence of residual thrombosis after 3-month treatment of acute pulmonary embolism.

The present study aimed to investigate the factors associating with the presence of residual thrombosis in patients with acute pulmonary embolism (APE) after at least 3-month anticoagulant therapy. Demographic and clinical data of 180 cases in the affiliated hospital of Qingdao University from January 2005 to June 2015 were retrospectively analyzed. APE in all patients were confirmed by computed tomography pulmonary angiography (CTPA). Patients were then detected for the presence of residual thrombosis according to a second CTPA. After appropriate comparison test, multivariate logistic regression analysis was performed to identify predictors for residual thrombosis. Among 180 patients, complete clearance of thrombosis occurred in 115 (63.9%) patients. Residual thrombosis remained in 65 (36.1%) patients. The independent factors associating with residual thrombosis include unprovoked APE (OR 0.231, 95% CI 0.062-0.861) and fibrinogen level in acute phase (OR 1.958, 95% CI 1.282-2.911). Furthermore, these two variables were both associated with the presence of residual thrombosis in patients receiving different parenteral anticoagulants (unfractionated heparin or low-molecular-weight heparin). Pulmonary thrombosis in some patients with APE are not completely dissolved after at least 3-month treatment. Additionally, unprovoked APE is positive predictor of decreased residual thrombosis and fibrinogen level in acute phase is a risk factor of the presence of residual thrombosis.

Determinants of substrate and cation transport in the human Na+/dicarboxylate cotransporter NaDC3.

Metabolic intermediates, such as succinate and citrate, regulate important processes ranging from energy metabolism to fatty acid synthesis. Cytosolic concentrations of these metabolites are controlled, in part, by members of the SLC13 gene family. The molecular mechanism underlying Na(+)-coupled di- and tricarboxylate transport by this family is understood poorly. The human Na(+)/dicarboxylate cotransporter NaDC3 (SLC13A3) is found in various tissues, including the kidney, liver, and brain. In addition to citric acid cycle intermediates such as α-ketoglutarate and succinate, NaDC3 transports other compounds into cells, including N-acetyl aspartate, mercaptosuccinate, and glutathione, in keeping with its dual roles in cell nutrition and detoxification. In this study, we construct a homology structural model of NaDC3 on the basis of the structure of the Vibrio cholerae homolog vcINDY. Our computations are followed by experimental testing of the predicted NaDC3 structure and mode of interaction with various substrates. The results of this study show that the substrate and cation binding domains of NaDC3 are composed of residues in the opposing hairpin loops and unwound portions of adjacent helices. Furthermore, these results provide a possible explanation for the differential substrate specificity among dicarboxylate transporters that underpin their diverse biological roles in metabolism and detoxification. The structural model of NaDC3 provides a framework for understanding substrate selectivity and the Na(+)-coupled anion transport mechanism by the human SLC13 family and other key solute carrier transporters.

An amplified fluorescence detection of T4 polynucleotide kinase activity based on coupled exonuclease III reaction and a graphene oxide platform.

A novel amplified fluorescence graphene oxide (GO) sensing platform for sensitive detection of T4 polynucleotide kinase (PNK) activity and inhibition was developed based on the exonuclease III (ExoIII) reaction. The efficient digestion capacity of ExoIII and the super quenching ability of GO both contribute to the detection sensitivity.

The aldehyde dehydrogenase ALDH1B1 exerts antiviral effects through the aggregation of the adaptor MAVS.

Type I interferons (IFNs) are produced by almost all cell types and play a vital role in host defense against viral infection. Infection with an RNA virus activates receptors such as RIG-I, resulting in the recruitment of the adaptor protein MAVS to the RIG-I-like receptor (RLR) signalosome and the formation of prion-like functional aggregates of MAVS, which leads to IFN-ß production. Here, we identified the aldehyde dehydrogenase 1B1 (ALDH1B1) as a previously uncharacterized IFN-stimulated gene (ISG) product with critical roles in the antiviral response. Knockout of ALDH1B1 increased, whereas overexpression of ALDH1B1 restricted, the replication of RNA viruses, such as vesicular stomatitis virus (VSV), Zika virus (ZIKV), dengue virus (DENV), and influenza A virus (IAV). We found that ALDH1B1 localized to mitochondria, where it interacted with the transmembrane domain of MAVS to promote MAVS aggregation. ALDH1B1 was recruited to MAVS aggregates. In addition, ALDH1B1 also enhanced the interaction between activated RIG-I and MAVS, thus increasing IFN-ß production and the antiviral response. Furthermore, Aldh1b1-/- mice developed more severe symptoms than did wild-type mice upon IAV infection. Together, these data identify an aldehyde dehydrogenase in mitochondria that functionally regulates MAVS-mediated signaling and the antiviral response.

Interferon-stimulated gene PVRL4 broadly suppresses viral entry by inhibiting viral-cellular membrane fusion.

BACKGROUND: Viral infection elicits the type I interferon (IFN-I) response in host cells and subsequently inhibits viral infection through inducing hundreds of IFN-stimulated genes (ISGs) that counteract many steps in the virus life cycle. However, most of ISGs have unclear functions and mechanisms in viral infection. Thus, more work is required to elucidate the role and mechanisms of individual ISGs against different types of viruses. RESULTS: Herein, we demonstrate that poliovirus receptor-like protein4 (PVRL4) is an ISG strongly induced by IFN-I stimulation and various viral infections. Overexpression of PVRL4 protein broadly restricts growth of enveloped RNA and DNA viruses, including vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whereas deletion of PVRL4 in host cells increases viral infections. Mechanistically, it suppresses viral entry by blocking viral-cellular membrane fusion through inhibiting endosomal acidification. The vivo studies demonstrate that Pvrl4-deficient mice were more susceptible to the infection of VSV and IAV. CONCLUSION: Overall, our studies not only identify PVRL4 as an intrinsic broad-spectrum antiviral ISG, but also provide a candidate host-directed target for antiviral therapy against various viruses including SARS-CoV-2 and its variants in the future.

Genetic basis of an elite wheat cultivar Guinong 29 with harmonious improvement between multiple diseases resistance and other comprehensive traits.

Fungal diseases, such as powdery mildew and rusts, significantly affect the quality and yield of wheat. Pyramiding diverse types of resistance genes into cultivars represents the preferred strategy to combat these diseases. Moreover, achieving collaborative improvement between diseases resistance, abiotic stress, quality, and agronomic and yield traits is difficult in genetic breeding. In this study, the wheat cultivar, Guinong 29 (GN29), showed high resistance to powdery mildew and stripe rust at both seedling and adult plant stages, and was susceptible to leaf rust at the seedling stage but slow resistance at the adult-plant stage. Meanwhile, it has elite agronomic and yield traits, indicating promising coordination ability among multiple diseases resistance and other key breeding traits. To determine the genetic basis of these elite traits, GN29 was tested with 113 molecular markers for 98 genes associated with diseases resistance, stress tolerance, quality, and adaptability. The results indicated that two powdery mildew resistance (Pm) genes, Pm2 and Pm21, confirmed the outstanding resistance to powdery mildew through genetic analysis, marker detection, genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), and homology-based cloning; the stripe rust resistance (Yr) gene Yr26 and leaf rust resistance (Lr) genes Lr1 and Lr46 conferred the stripe rust and slow leaf rust resistance in GN29, respectively. Meanwhile, GN29 carries dwarfing genes Rht-B1b and Rht-D1a, vernalization genes vrn-A1, vrn-B1, vrn-D1, and vrn-B3, which were consistent with the phenotypic traits in dwarf characteristic and semi-winter property; carries genes Dreb1 and Ta-CRT for stress tolerance to drought, salinity, low temperature, and abscisic acid (ABA), suggesting that GN29 may also have elite stress-tolerance ability; and carries two low-molecular-weight glutenin subunit genes Glu-B3b and Glu-B3bef which contributed to high baking quality. This study not only elucidated the genetic basis of the elite traits in GN29 but also verified the capability for harmonious improvement in both multiple diseases resistance and other comprehensive traits, offering valuable information for breeding breakthrough-resistant cultivars.

Nonsteroidal anti-inflammatory drugs and other anthranilic acids inhibit the Na(+)/dicarboxylate symporter from Staphylococcus aureus.

The Na(+)/dicarboxylate symporter from Staphylococcus aureus, named SdcS, is a member of the divalent anion sodium symporter (DASS) family that also includes the mammalian SLC13 Na(+)/dicarboxylate cotransporters, NaDC1 and NaCT. The mammalian members of the family are sensitive to inhibition by anthranilic acid derivatives such as N-(p-amylcinnamoyl)anthranilic acid (ACA), which act as slow inhibitors. This study shows that SdcS is inhibited by ACA as well as the fenamate nonsteroidal anti-inflammatory drugs, flufenamate and niflumate. The inhibition was rapid and reversible. The IC(50) for ACA was approximately 55 µM. Succinate kinetics by SdcS were sigmoidal, with a K(0.5) of 9 µM and a Hill coefficient of 1.5. Addition of ACA decreased the V(max) and increased the Hill coefficient without affecting the K(0.5), consistent with its activity as a negative modulator of SdcS activity. ACA inhibition was not correlated with the K(0.5) for succinate in SdcS mutants, and ACA did not affect the reactivity of the N108C mutant to the cysteine reagent, MTSET. We conclude that ACA and other anthranilic acid derivatives are effective allosteric inhibitors of SdcS. Furthermore, the mechanism of inhibition appears to be distinct from the mechanism observed in human NaDC1.

Functional characterization of SdcF from Bacillus licheniformis, a homolog of the SLC13 Na⁺/dicarboxylate transporters.

The SdcF transporter from Bacillus licheniformis (gene BL02343) is a member of the divalent anion sodium symporter (DASS)/SLC13 family that includes Na⁺/dicarboxylate transporters from bacteria to humans. SdcF was functionally expressed in Escherichia coli (BL21) and assayed in right side out membrane vesicles. ScdF catalyzed the sodium-coupled transport of succinate and α-ketoglutarate. Succinate transport was strongly inhibited by malate, fumarate, tartrate, oxaloacetate and L-aspartate. Similar to the other DASS transporters, succinate transport by SdcF was inhibited by anthranilic acids, N-(p-amylcinnamoyl) anthranilic acid and flufenamate. SdcF transport was cation-dependent, with a K0.5 for sodium of ~1.5 mM and a K0.5 for Li⁺ of ~40 mM. Succinate transport kinetics by SdcF were sigmoidal, suggesting that SdcF may contain two cooperative substrate binding sites. The results support an ordered binding mechanism for SdcF in which sodium binds first and succinate binds last. We conclude that SdcF is a secondary active transporter for four- and five-carbon dicarboxylates that can use Na⁺ or Li⁺ as a driving cation.

[Incidence of pulmonary embolism in patients with acute exacerbations of chronic obstructive pulmonary disease: a meta-analysis].

OBJECTIVE: To evaluate the incidence of pulmonary embolism in patients with acute exacerbations of chronic obstructive pulmonary disease (COPD). METHODS: Comprehensive searches as of June 2012 were performed in PubMed (1966-), Embase (1974-), Chinese Biomedical Literature Database (1978-), Chinese Journal Full-text Database (1979-) and VIP Database (1989-) for literatures on the incidence of pulmonary embolism in patients with acute exacerbations of COPD. Meta-analysis was conducted with Stata version 11.0. RESULTS: Among 2273 articles identified, 5 studies met the inclusion criteria (4 in English, 1 in Chinese). The total sample size was 762 patients, among whom 145 were diagnosed with pulmonary embolism. The incidence of pulmonary embolism ranged from 3.3% to 33.0%. Meta-analysis showed that the combined incidence was 15.8% (95%CI: 5.1%-26.4%). Among patients with acute exacerbations of COPD of unknown etiology, the incidence was 29.0% (95%CI: 20.8%-37.1%). CONCLUSIONS: There is a high incidence of pulmonary embolism in patients with acute exacerbations of COPD, especially among those of an unknown etiology. More attention should be paid to this population.

Ensuring access to justice: the need for community paralegals to end AIDS by 2030.

INTRODUCTION: The HIV response has long recognized that certain "key populations" such as individuals in detention, adolescent girls and young women, sex workers, people who use drugs, LGBTQ individuals, migrants and others face higher barriers to access to, uptake of, and retention in HIV prevention and treatment services. One approach to addressing these barriers is the training of community paralegals to advocate for the rights of individuals and to address discrimination in health settings. DISCUSSION: Community paralegal programmes have been able to successfully address rights violations that impact access to health services and underlying determinants of health across a range of countries and populations, focusing upon issues such as discrimination and the denial of health services; unlawful detention of outreach workers, sex workers, persons who use drugs and men who have sex with men; and harmful traditional practices and gender-based violence. In addition to resolving specific cases, evaluations of paralegal programmes have found that these programmes increased legal literacy among key populations at risk of HIV and increased understanding of human rights among healthcare providers, resulting in improved access to HIV services. Some evaluations have noted challenges related to the sustainability of paralegal programmes similar to those raised with community health worker programmes more broadly. CONCLUSIONS: To achieve global HIV goals, funding for legal literacy and paralegal programmes should be increased and interventions should be rigorously evaluated. Efforts should target discrimination in access to HIV prevention and treatment and criminalization of key populations, two key barriers to ensuring access to HIV prevention and treatment services.

Regulation of DNA damage response by trimeric G-proteins.

Upon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of the two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DSBs hinges on GIV/Girdin, a guanine nucleotide-exchange modulator of heterotrimeric Giα•ßγ protein. GIV suppresses HR by binding and sequestering BRCA1, a key coordinator of multiple steps within the HR pathway, away from DSBs; it does so using a C-terminal motif that binds BRCA1's BRCT-modules via both phospho-dependent and -independent mechanisms. Using another non-overlapping C-terminal motif GIV binds and activates Gi and enhances the "free" Gßγ→PI-3-kinase→Akt pathway, which promotes survival and is known to suppress HR, favor NHEJ. Absence of GIV, or loss of either of its C-terminal motifs enhanced cell death upon genotoxic stress. Because GIV selectively binds other BRCT-containing proteins suggests that G-proteins may fine-tune sensing, repair, and survival after diverse types of DNA damage.

Transmembrane helix 7 in the Na+/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function.

Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased Vmax for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer.