Active site mutants of the "non-hydrolyzing" UDP-N-acetylglucosamine 2-epimerase from Escherichia coli

The "non-hydrolyzing" bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This homodimeric enzyme is allosterically activated by its substrate, UDP-GlcNAc, and it is thought that one subunit plays a regulatory role, while that of the other plays a catalytic role. In this work, five active site mutants were prepared (D95N, E117Q, E131Q, K15A, and H213N) and analyzed in terms of their effects on binding, catalysis, and allosteric regulation. His213 appears to play a role in UDP binding and may also assist in catalysis and/or regulation, but is not a key catalytic residue. Lys15 appears to be quite important for binding. All three of the carboxylate mutants showed dramatic decreases in the value of k(cat) but relatively unaffected values of K(M). Thus, these residues are playing key roles in catalysis and/or regulation. In the case of E117Q, the reaction intermediates are released into solution at a rate comparable to that of the overall catalysis. This may indicate that Glu117 plays the role as an acid/base catalyst in the second step of the UDP-GlcNAc epimerization reaction. All three carboxylate mutants were found to exhibit impaired allosteric control.     

Possible involvement of genes on the Q chromosome of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> in expression of hybrid lethality and programmed cell death during interspecific hybridization to <Emphasis Type="Italic">Nicotiana debneyi</Emphasis>

Hybrid seedlings from the cross between Nicotiana tabacum, an allotetraploid composed of S and T subgenomes, and N. debneyi die at the cotyledonary stage. This lethality involves programmed cell death (PCD). We carried out reciprocal crosses between the two progenitors of N. tabacum, N. sylvestris and N. tomentosiformis, and N. debneyi to reveal whether only the S subgenome in N. tabacum is related to hybrid lethality. Hybrid seedlings from reciprocal crosses between N. sylvestris and N. debneyi showed lethal characteristics identical to those from the cross between N. tabacum and N. debneyi. Conversely, hybrid seedlings from reciprocal crosses between N. tomentosiformis and N. debneyi were viable. Furthermore, hallmarks of PCD were observed in hybrid seedlings from the cross N. debneyi × N. sylvestris, but not in hybrid seedlings from the cross N. debneyi × N. tomentosiformis. We also carried out crosses between monosomic lines of N. tabacum lacking the Q chromosome and N. debneyi. Using Q-chromosome-specific DNA markers, hybrid seedlings were divided into two groups, hybrids possessing the Q chromosome and hybrids lacking the Q chromosome. Hybrids possessing the Q chromosome died with characteristics of PCD. However, hybrids lacking the Q chromosome were viable and PCD did not occur. From these results, we concluded that the Q chromosome belonging to the S subgenome of N. tabacum encodes gene(s) leading to hybrid lethality in the cross N. tabacum × N. debneyi.     

Electrostatic switches that mediate the pH-dependent conformational change of "short" recombinant human pseudocathepsin D

Human cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatD(lo)) [Baldwin, E. T., Bhat, T. N., Gulnik, S., Hosur, M. V., Sowder, R. C., Cachau, R. E., Collins, J., Silva, A. M., and Erickson, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6796-6800] and an inactive, high pH (pH 7.5) form (CatD(hi)) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatD(hi) and CatD(lo) and that Y10 stabilizes CatD(hi) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. To test these hypotheses, we generated single point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. E180Q, Y10F, and D187N exhibit significantly higher kcat/Km values (2-, 3-, and 6-fold, respectively) at pH 3.7 and 4.75 compared to srCatD, indicating that these residues are important in stabilizing the CatD(hi). E5Q exhibits a 2-fold lower kcat/Km compared to srCatD at both pH values, indicating the importance of E5 in stabilizing the CatD(lo). Accordingly, full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD(hi) to CatD(lo) compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatD(lo) to CatD(hi) and Y10 stabilizes CatD(hi) by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD(lo).     

Diabetes mellitus associated changes in glomerular glycocompounds: A fluorescence microscopical study

Summary Renal biopsy specimens showing histological alterations typical of advanced diabetic glomerulopathy were studied for changes in glomerular glycoconjugates, using fluorochrome-coupled lectins as probes. All samples studied showed a marked reduction in the binding ofTriticum vulgaris (WGA) lectin in the glomerular basement membranes. On the other hand, new glomerular binding sites for the lectins ofDolichos biflorus (DBA),Helix pomatia (HPA) andArachis hypogaea (PNA), recognizing galactosyl moieties of glycoconjugates and giving no reaction in normal glomeruli, were seen in all samples studied. In addition,Wistaria floribunda lectin (WFA), recognizing galactosyl and.N-acetylgalactosaminyl configurations in glycoconjugates, gave a typical linear binding along the glomerular basement membranes, differing markedly from its reaction with normal kidney.Ulex europaeus (UEA I) showed reduced binding in the glomeruli of diabetic nephropathy.The results show that changes in glomerular glycoconjugates may appear in diabetic nephropathy, suggesting a disturbance in the turnover of the non-reducing terminal saccharide residues. In addition, the results show that lectins are useful probes for studying these changes further.     

Increased nitrogen deposition alleviated the adverse effects of drought stress on <Emphasis Type="Italic">Quercus variabilis</Emphasis> and <Emphasis Type="Italic">Quercus mongolica</Emphasis> seedlings

The plasticity response of Quercus variabilis and Quercus mongolica seedlings to combined nitrogen (N) deposition and drought stress was evaluated, and their performance in natural niche overlaps was predicted. Seedlings in a greenhouse were exposed to four N deposition levels (0, 4, 8, and 20 g N m^?2 year^?1) and two water levels (80 and 50 % field-water capacity). Plant traits associated with growth, biomass production, leaf physiology, and morphology were determined. Results showed that drought stress inhibited seedling performance, altered leaf morphology, and decreased fluorescence parameters in both species. By contrast increased N supply had beneficial effects on the nutritional status and activity of the PSII complex. The two species showed similar responses to drought stress. Contrary to the effects in Q. mongolica, N deposition promoted leaf N concentration, PSII activity, leaf chlorophyll contents, and final growth of Q. variabilis under well-watered conditions. Thus, Q. variabilis was more sensitive to N deposition than Q. mongolica. However, excessive N supply (20 g N m^?2 year^?1) did not exert any positive effects on the two species. Among the observed plasticity of the plant traits, plant growth was the most plastic, and leaf morphology was the least plastic. Therefore, drought stress played a primary role at the whole-plant level, but N supply significantly alleviated the adverse effects of drought stress on plant physiology. A critical N deposition load around 20 g N m^?2 year^?1 may exist for oak seedlings, which may more adversely affect Q. variabilis than Q. mongolica.     

Acetylcholine receptor gating at extracellular transmembrane domain interface: the "pre-M1" linker

Charged residues in the beta10-M1 linker region ("pre-M1") are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational "wave." We examined the effects of mutations to all five residues in pre-M1 (positions M207-P211) plus E45 in loop 2 in the mouse alpha(1)-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (K(eq)), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in K(eq) (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Phi values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Phi values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only -0.33 kcal/mol (for both alpha subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.     

原始阔叶红松林下红松、蒙古栎混合凋落叶分解特征及相互作用研究

采用凋落物网袋（litter bag）分解法,模拟红松（Pinus koraiensis）(以下用P表示)和蒙古栎（Quercus mongolica）（以下用M表示）在不同演替阶段可能的组成比例(P、M/P1∶3、M/P1∶1、M/P3∶1、M）进行野外分解实验。分析不同比例的两种凋落叶混合的分解特征、相互作用及机理。结果表明：1.从质量损失率来看,与单种凋落叶分解情况相比,蒙古栎和红松凋落叶混合对凋落物分解具有促进作用,其中蒙古栎和红松（M/P）按1∶3混合分解最快,且随着蒙古栎凋落叶在混合比例中的增加,混合分解速率先减小后增大。2.从C、N元素动态看,C素在各处理的凋落叶主要表现为净释放,而N素在各处理中变现比较复杂,在各处理的红松凋落叶中表现为富集,而在各处理蒙古栎凋落叶中则表现为释放。蒙古栎凋落叶可以促进红松凋落叶C元素释放和N元素的富集,降低红松凋落叶的C/N比,促进红松凋落叶的分解。红松凋落叶能促进蒙古栎凋落物C元素释放,但对蒙古栎凋落叶N元素的释放作用不明显,对蒙古栎凋落叶的C/N比影响也并不一致。     

Secretion and N-linked glycosylation are required for prostatic acid phosphatase catalytic and antinociceptive activity

Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6-7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This "cellular" construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia--a yeast strain that is used to generate biologics for therapeutic purposes.     

Glomerular Endothelial Cell Injury and Focal Segmental Glomerulosclerosis Lesion in Idiopathic Membranous Nephropathy

Background

Focal segmental glomerulosclerosis (FSGS) lesions have often been discussed as a negative predictor in idopathic membranous nephropathy (MN). The mechanism of the development of FSGS lesion in MN is still uncertain.

Methods

From 250 cases of MN, 26 cases contained FSGS lesion. We compared the clinicopathological characteristics between MN cases with FSGS lesion [MN-FSGS(+)] and MN without FSGS lesion [MN-FSGS(−)], matched for gender, age, stage of MN.

Results

The glomerular filtration rate (eGFR) was significantly lower in MN-FSGS(+) cases compared to MN-FSGS(−), although nephrotic syndrome, hematuria, and systolic blood pressure levels were not significantly different between the two groups. Pathologically, glomeruli in MN-FSGS(+) cases showed narrowing and loss of glomerular capillaries with separating from GBM or disappearance of CD34+ endothelial cells, and accumulation of extracellular matrix (ECM) in capillary walls, indicating the development of glomerular capillary injury. These findings of endothelial injury were seen even in MN-FSGS(−) cases, but they were more prominent in MN-FSGS(+) than MN-FSGS(−) by computer assessed morphometric analysis. In MN-FSGS(+) cases, 44 out of 534 glomeruli (8.2%) contained FSGS lesions (n = 31, NOS lesion; n = 13, perihilar lesion). Significant thickness of GBM with ECM accumulation was evident in MN-FSGS(+) cases. Podocyte injury with effacement of foot processes was also noted, but the expression of VEGF on podocytes was not different between the two groups, which suggests that the significant thickness of capillary walls may influence the function of VEGF from podocyte resulting in the glomerular capillary injury that contribute to the development of FSGS lesion in MN.

Conclusion

Glomerular capillary injury was seen in all MN cases. Furthermore, the prominent injuries of glomerular capillaries may be associated with the deterioration of eGFR and the formation of FSGS lesions in MN.     

Alpha-actinin-4 and CLP36 protein deficiencies contribute to podocyte defects in multiple human glomerulopathies

Genetic alterations of α-actinin-4 can cause podocyte injury through multiple mechanisms. Although a mechanism involving gain-of-α-actinin-4 function was well described and is responsible for a dominantly inherited form of human focal segmental glomerulosclerosis (FSGS), evidence supporting mechanisms involving loss-of-α-actinin-4 function in human glomerular diseases remains elusive. Here we show that α-actinin-4 deficiency occurs in multiple human primary glomerulopathies including sporadic FSGS, minimal change disease, and IgA nephropathy. Furthermore, we identify a close correlation between the levels of α-actinin-4 and CLP36, which form a complex in normal podocytes, in human glomerular diseases. siRNA-mediated depletion of α-actinin-4 in human podocytes resulted in a marked reduction of the CLP36 level. Additionally, two FSGS-associated α-actinin-4 mutations (R310Q and Q348R) inhibited the complex formation between α-actinin-4 and CLP36. Inhibition of the α-actinin-4-CLP36 complex, like loss of α-actinin-4, markedly reduced the level of CLP36 in podocytes. Finally, reduction of the CLP36 level or disruption of the α-actinin-4-CLP36 complex significantly inhibited RhoA activity and generation of traction force in podocytes. Our studies reveal a critical role of the α-actinin-4-CLP36 complex in podocytes and provide an explanation as to how α-actinin-4 deficiency or mutations found in human patients could contribute to podocyte defects and glomerular failure through a loss-of-function mechanism.     

Primary structure of the "fast" component of avenin (Avena sativa L

A procedure for the isolation and sequence analysis of the "fast" avenin component (N9) from the oat (Avena sativa L., cv. Narymsky 943) is described. Component N9 was prepared by an ion-exchange high-performance liquid chromatography on a strong cation exchange column type Mono S (Pharmacia, Sweden) in 4 M urea, pH 3.5, with a linear gradient of NaCl. A polypeptide chain of avenin N9 was reconstructed by the CNBr and tryptic peptides on a model 470A protein gas-phase sequencer (Applied Biosystems, USA). A good yield of tryptic peptides were obtained by an enzymatic hydrolysis of avenin N9 preliminary immobilized on Thiopropyl-Sepharose 6B (Pharmacia, Sweden) at cysteine residues. Avenin N9 consists of 182 amino acid residues end exhibits the features common for all the known prolamins.     

Expression of nestin in the podocytes of normal and diseased human kidneys

The complex cyto-architecture of the podocyte is critical for glomerular permselectivity. The present study characterizes the expression of nestin, an intermediate filament protein, in human kidneys. In normal kidneys, nestin was detected at the periphery of glomerular capillary loops. Colabeling showed nestin was expressed in WT1-positive cells. Within the podocyte, nestin immunoreactivity was present in the cell body and primary process. This was supported by immunoelectron microscopy. Nestin also colocalized with vimentin in the periphery of capillary loops but not in the mesangium. Nestin was not detected in other structures of the adult human kidney. To determine the potential role of nestin in proteinuria, nestin was examined in kidney biopsies from patients with or without proteinuria. These patients were diagnosed with IgA nephropathy with mild mesangial expansion but without proteinuria, IgA nephropathy with proteinuria, membranous nephropathy (MN), and focal segmental glomerular sclerosis (FSGS). The distribution of nestin in these biopsies was similar to that in the normal kidney. Semiquantitative analysis of immunostaining showed that glomerular nestin expression in IgA nephropathy without proteinuria was not different from normal kidney; however, nestin expression in kidneys of patients with IgA nephropathy and proteinuria, or MN and FSGS with proteinuria was significantly reduced compared with normal kidney (P < 0.01). Reduced nestin mRNA expression in the patients with IgA nephropathy with proteinuria and FSGN was also observed by quantitative real-time PCR. These studies suggest that nestin may play an important role in maintaining normal podocyte function in the human kidney.     

Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-deficient mouse

Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.     

Glomerular hemodynamic and structural alterations in experimental diabetes mellitus

Elevated glomerular filtration rate (GFR) is a frequent finding in patients with early insulin-dependent diabetes mellitus (IDDM). The mechanisms responsible for this glomerular hyperfiltration in IDDM are unclear. Rats made diabetic with alloxan or streptozotocin, and treated daily with supplemental insulin, have moderate hyperglycemia and elevated GFR, and thus have been used to study mechanisms of glomerular hyperfiltration in diabetes. Renal micropuncture techniques have shown that single-nephron GFR (SNGFR) is elevated in moderately hyperglycemic diabetic rats. In some cases, this is because of elevated glomerular capillary pressure (Pgc), but in other cases, Pgc is normal despite elevated SNGFR. Several potential mediators of increased SNGFR have been examined, including hyperglycemia, increased glomerular prostaglandin production, and decreased sensitivity of the tubuloglomerular feedback mechanism. Renal failure is a common complication of human IDDM. Diabetic rats with long-term moderate hyperglycemia have been used to study the mechanism by which glomerular injury develops in diabetes mellitus. It has been postulated that glomerular hyperfiltration or some determinant of elevated GFR in early diabetes may ultimately cause glomerular damage, leading to a progressive loss of renal function (diabetic nephropathy). Diabetic rats with long-term moderate hyperglycemia, however, do not develop characteristic glomerular lesions of human diabetic nephropathy and, in fact, develop only minimal glomerular injury even after 1 year of diabetes. Thus, although the diabetic rat with moderate hyperglycemia may be useful to study the mechanisms of glomerular hyperfiltration in early diabetes, it may not be an appropriate model of renal failure in IDDM.     

Genes in S and T subgenomes are responsible for hybrid lethality in interspecific hybrids between Nicotiana tabacum and Nicotiana occidentalis

Background

Many species of Nicotiana section Suaveolentes produce inviable F₁ hybrids after crossing with Nicotiana tabacum (genome constitution SSTT), a phenomenon that is often called hybrid lethality. Through crosses with monosomic lines of N. tabacum lacking a Q chromosome, we previously determined that hybrid lethality is caused by interaction between gene(s) on the Q chromosome belonging to the S subgenome of N. tabacum and gene(s) in Suaveolentes species. Here, we examined if hybrid seedlings from the cross N. occidentalis (section Suaveolentes)×N. tabacum are inviable despite a lack of the Q chromosome.

Methodology/Principal Findings

Hybrid lethality in the cross of N. occidentalis×N. tabacum was characterized by shoots with fading color. This symptom differed from what has been previously observed in lethal crosses between many species in section Suaveolentes and N. tabacum. In crosses of monosomic N. tabacum plants lacking the Q chromosome with N. occidentalis, hybrid lethality was observed in hybrid seedlings either lacking or possessing the Q chromosome. N. occidentalis was then crossed with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT), to reveal which subgenome of N. tabacum contains gene(s) responsible for hybrid lethality. Hybrid seedlings from the crosses N. occidentalis×N. tomentosiformis and N. occidentalis×N. sylvestris were inviable.

Conclusions/Significance

Although the specific symptoms of hybrid lethality in the cross N. occidentalis×N. tabacum were similar to those appearing in hybrids from the cross N. occidentalis×N. tomentosiformis, genes in both the S and T subgenomes of N. tabacum appear responsible for hybrid lethality in crosses with N. occidentalis.     

Insights into the Uses of Traditional Plants for Diabetes Nephropathy: A Review

Diabetic nephropathy (DN) is a serious kidney illness characterized by proteinuria, glomerular enlargement, reduced glomerular filtration, and renal fibrosis. DN is the most common cause of end-stage kidney disease, accounting for nearly one-third of all cases of diabetes worldwide. Hyperglycemia is a major factor in the onset and progression of diabetic nephropathy. Many contemporary medicines are derived from plants since they have therapeutic properties and are relatively free of adverse effects. Glycosides, alkaloids, terpenoids, and flavonoids are among the few chemical compounds found in plants that are utilized to treat diabetic nephropathy. The purpose of this review was to consolidate information on the clinical and pharmacological evidence supporting the use of a variety of medicinal plants to treat diabetic nephropathy.     

Effect of deletion polymorphism of angiotensin converting enzyme gene on progression of diabetic nephropathy during inhibition of angiotensin converting enzyme: observational follow up study.

OBJECTIVE: To evaluate the concept that an insertion/deletion polymorphism of the angiotensin converting enzyme gene predicts the therapeutic efficacy of inhibition of angiotensin converting enzyme on progression of diabetic nephropathy. DESIGN: Observational follow up study of patients with insulin dependent diabetes and nephropathy who had been treated with captopril for a median of 7 years (range 3-9 years). SETTING: Outpatient diabetic clinic in a tertiary referral centre. PATIENTS: 35 patients with insulin dependent diabetes and nephropathy were investigated during captopril treatment (median 75 mg/day (range 12.5 to 150 mg/day)) that was in many cases combined with a loop diuretic, 11 patients were homozygous for the deletion allele and 24 were heterozygous or homozygous for the insertion allele of the angiotensin converting enzyme gene. MAIN OUTCOME MEASURES: Albuminuria, arterial blood pressure, and glomerular filtration rate according to insertion/deletion polymorphism. RESULTS: The two groups had comparable glomerular filtration rate, albuminuria, blood pressure, and haemoglobin A1c concentration at baseline. Captopril induced nearly the same reduction in mean blood pressure in the two groups-to 103 (SD 5) mm Hg in the group with the deletion and 102 (8) mm Hg in the group with the insertion-and in geometric mean albumin excretion-573 (antilog SE 1.3) micrograms/min and 470 (1.2) micrograms/min, respectively. The rate of decline in glomerular filtration rate (linear regression of all glomerular filtration rate measurements during antihypertensive treatment) was significantly steeper in the group homozygous for the double deletion allele than in the other group (mean 5.7 (3.7) ml/min/year and 2.6 (2.8) ml/min/year, respectively; P = 0.01). Multiple linear regression analysis showed that haemoglobin A1c concentration, albuminuria, and the double deletion genotype independently influenced the sustained rate of decline in glomerular filtration rate (R1 (adjusted) = 0.51). CONCLUSION: The deletion polymorphism in the angiotensin converting enzyme gene reduces the long term beneficial effect of angiotensin converting enzyme inhibition on the progression of diabetic nephropathy in patients with insulin dependent diabetes.     

Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2

ABSTRACT

The placental syncytiotrophoblast, which is formed by the fusion of cytotrophoblast cells, is indispensable for the establishment and maintenance of normal pregnancy. The human endogenous retrovirus envelope glycoprotein syncytin-2 is the most important player in mediating trophoblast cell-cell fusion as a fusogen. We constructed expression plasmids of wild-type and 21 single-amino-acid substitution mutants of syncytin-2, including 10 N-glycosylation sites individually silenced by mutagenizing N to Q, 1 naturally occurring single-nucleotide polymorphism (SNP) N118S that introduced an N-glycosylation site, and another 10 non-synonymous SNPs located within important functional domains. We observed that syncytin-2 was highly fusogenic and that the mutants had different capacities in merging 293T cells. Of the 21 mutants, N133Q, N312Q, N443Q, C46R (in the CXXC motif) and R417H (in the heptad repeat region and immunosuppressive domain) lost their fusogenicity, whereas N332Q, N118S, T367M (in the fusion peptide), V483I (in the transmembrane domain) and T522M (in the cytoplasmic domain) enhanced the fusogenic activity. We also proved that N133, N146, N177, N220, N241, N247, N312, N332 and N443 were all glycosylated in 293T cells. A co-immunoprecipitation assay showed compromised interaction between mutants N443Q, C46R, T367M, R417H and the receptor MFSD2A, whereas N118S was associated with more receptors. We also sequenced the coding sequence of syncytin-2 in 125 severe pre-eclamptic patients and 272 normal pregnant Chinese women. Surprisingly, only 1 non-synonymous SNP T522M was found and the frequencies of heterozygous carriers were not significantly different. Taken together, our results suggest that N-glycans at residues 133, 312, 332 and 443 of syncytin-2 are required for optimal fusion induction, and that SNPs C46R, N118S, T367M, R417H, V483I and T522M can alter the fusogenic function of syncytin-2.