Comparison of prevalence, phenotype, and antimicrobial resistance of Salmonella serovars isolated from turkeys in Taiwan.

Salmonella spp. is a foodborne pathogen that causes zoonotic disease worldwide. The aim of this study was to investigate the prevalence of antimicrobial resistance of Salmonella isolated from turkey farms in Taiwan. During the past 2 yr, 243 strains of Salmonella were isolated from 2,040 samples (11.9%) from turkey farms, including 32.5% (52/160) from the intestines of 12-day-old turkey poults, 14.2% (119/840) from feces collected from the turkey growing periods, and 6.9% (72/1,040) from finishing periods. S. Albany (35.0%, 85/243), S. Schwarzengrund (23.0%, 56/243), and S. Hadar (19.3%, 47/243) were the most common serovars on turkey farms. For these strains, a high frequency of resistance was observed against florfenicol (97.5%), oxytetracycline (89.3%), doxycycline (78.6%), colistin (77.8%), ampicillin (75.7%), amoxicillin (75.3%), trimethoprim-sulfamethoxazole (73.7%), chloramphenicol (69.1%), and nalidixic acid (67.9%). floR (63.8%), tet (A) (60.5%), blaPSE (57.6%), blaTEM (42.0%), blaCTX-M (34.2%), cmlA (34.2%), and tet (D) (29.2%) were the most common resistance genes found in this study. The int1 gene was identified in 72.4% (176/243) of Salmonella isolates in which the conserved region 3' of class 1 integrons also was amplified, whereas none had the int2 gene. This study demonstrates that imported and fattening turkeys could be a reservoir for Salmonella isolates resistant to multiple antimicrobials. These results also reinforce the need to develop strategies and implement specific control procedures to reduce the development of antimicrobial resistance.

Serotonergic paraneurones in the female mouse urethral epithelium and their potential role in peripheral sensory information processing.

AIM: The mechanisms underlying detection and transmission of sensory signals arising from visceral organs, such as the urethra, are poorly understood. Recently, specialized ACh-expressing cells embedded in the urethral epithelium have been proposed as chemosensory sentinels for detection of bacterial infection. Here, we examined the morphology and potential role in sensory signalling of a different class of specialized cells that express serotonin (5-HT), termed paraneurones. METHODS: Urethrae, dorsal root ganglia neurones and spinal cords were isolated from adult female mice and used for immunohistochemistry and calcium imaging. Visceromotor reflexes (VMRs) were recorded in vivo. RESULTS: We identified two morphologically distinct groups of 5-HT+ cells with distinct regional locations: bipolar-like cells predominant in the mid-urethra and multipolar-like cells predominant in the proximal and distal urethra. Sensory nerve fibres positive for calcitonin gene-related peptide, substance P, and TRPV1 were found in close proximity to 5-HT+ paraneurones. In vitro 5-HT (1 µm) stimulation of urethral primary afferent neurones, mimicking 5-HT release from paraneurones, elicited changes in the intracellular calcium concentration ([Ca2+ ]i ) mediated by 5-HT2 and 5-HT3 receptors. Approximately 50% of 5-HT responding cells also responded to capsaicin with changes in the [Ca2+ ]i . In vivo intra-urethral 5-HT application increased VMRs induced by urethral distention and activated pERK in lumbosacral spinal cord neurones. CONCLUSION: These morphological and functional findings provide insights into a putative paraneurone-neural network within the urethra that utilizes 5-HT signalling, presumably from paraneurones, to modulate primary sensory pathways carrying nociceptive and non-nociceptive (mechano-sensitive) information to the central nervous system.

Molecular heterogeneity and function of EWS-WT1 fusion transcripts in desmoplastic small round cell tumors.

Desmoplastic small round cell tumor (DSRCT) is a primitive sarcoma with a consistent cytogenetic abnormality, t(11;22)(p13;q12). This chromosomal translocation generates a chimeric transcript that is formed by fusion of the 5' region of the Ewing's sarcoma gene, EWS, with the 3' DNA-binding segment of WT1, the Wilms' tumor suppressor gene. We collected 14 DSRCT tumor samples and examined the hybrid transcripts. We identified: (a) combinatorial heterogeneity of EWS exons fused to WT1 including use of EWS exons 7, 8, and 9; (b) subpopulations of variant transcripts in 6 of 14 tumors characterized by aberrant splicing resulting in loss of EWS exon 6 or WT1 exon 9; (c) multiple cDNA products with large internal deletions; and (d) insertion of small stretches of heterologous DNA at the fusion site or exon splice region in transcripts from two tumors. Most of the splice variants were in-frame, and in vitro translated fusion proteins with intact DNA-binding motifs formed complexes with a WT1 response element in gel mobility assays. Each of the chimeric proteins retains the ability to bind to the GC and TC elements of the early transcription factor EGR-1 as well as WT1 consensus sequences. We present evidence that various EWS-WT1 proteins up-regulated EGR-1 promoter activity and that this up-regulation is specifically dependent upon the absence of the exon 9 KTS domain of WT1. The molecular diversity and functionality exhibited by these fusion transcripts may have significant biological implications for their transactivating and tumorigenic potential.

Glycans derived from porcine stomach mucin are effective inhibitors of natural anti-alpha-galactosyl antibodies in vitro and after intravenous infusion in baboons.

The current shortage of donor organs has stimulated investigation of pig-to-human xenotransplantation as a practical alternative to allotransplantation. However, a major obstacle to this xenotransplantation is hyperacute rejection, which is believed to be initiated by the interaction of natural anti-alpha-galactosyl (alphaGal) antibodies with alphaGal epitopes on pig vascular endothelium. Previously, we reported that neutral oligosaccharides derived from porcine stomach mucin (PSM) are effective inhibitors of human anti-alphaGal IgG in vitro. We now report that O-glycans derived from PSM by beta-elimination (PSMO) reduce the cytotoxicity of both baboon and human sera to pig kidney (PK15) cells in vitro. Crude PSM had some inhibitory effect in vitro, but PSMO were more than 100 times more potent. Moreover, 1 microg/ml of beta-eliminated PSMO that bound to an immunoaffinity column of anti-alphaGal antibodies were four times more efficient than total PSMO in protecting PK15 cells from the cytotoxic effect of baboon or human sera. Blood recovered from baboons after intravenous infusion of PMSO also showed significant protection of PK15 cells. We conclude that PSMO eluted from an anti-alphaGal immunoaffinity column demonstrate potent inhibitory effects against baboon and human serum cytotoxicity to PK15 cells in vitro and when administered intravenously. PSM may provide a cheap and readily available source of glycans that will be of therapeutic value in the prevention of hyperacute rejection.

Sequence diversity of a gene encoding a putative primary sigma factor among Borrelia burgdorferi sensu lato strains.

Twenty-six strains of Borrelia burgdorferi sensu lato were subjected to polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis for assessing the sequence divergence of rpoD gene encoding the primary sigma factor. Four and five RFLP patterns were observed from two fragments of rpoD gene. Sequence analysis of a subgenic fragment covering region 1 through 4 from 13 strains of Borrelia burgdorferi s. 1. revealed that 21 of 450 deduced amino acid residues were diverged. These results indicate that the sequence heterogeneity of rpoD is present in different strains of Borrelia burgdorferi s. 1., and agreed well with the current classification of genospecies.

Steady-state disposition of diflunisal: once- versus twice-daily administration.

To evaluate the steady-state bioequivalence of the nonsteroidal antiinflammatory analgesic agent, diflunisal, administered once versus twice daily, 13 healthy volunteers received diflunisal as follows: 1000 mg at 8:00 AM and 500 mg at 8:00 AM and 8:00 PM, each for 14 days in a randomized crossover study. The mean (+/- SD) steady-state peak plasma concentrations were significantly greater after once-daily dosing (186 +/- 25 micrograms/ml vs 150 +/- 37 micrograms/ml; p less than 0.01). The time to peak concentration was also longer after the single-dose regimen (2.5 +/- 0.8 vs 1.9 +/- 0.9 hr; p less than 0.05). The regimens were similar with respect to the mean 24-hour area under the plasma concentration-time curve at steady state (2839 +/- 612 vs 2782 +/- 778 micrograms.hr.ml-1), steady-state plasma concentrations (118 +/- 25 vs 116 +/- 32 micrograms/ml), trough plasma concentration (85 +/- 27 vs 92 +/- 28 micrograms/ml) as well as 24-hour urinary excretion (776 +/- 79 vs 771 +/- 89 mg) of diflunisal. Based on urinary recoveries, the bioequivalence ratio (once vs twice daily) was 1.01 +/- 0.08. These results indicate that diflunisal administered once daily might offer comparable therapeutic effects but be more convenient than a twice-daily regimen.

Body composition in hemodialysis patients--is it different from that of normal subjects?

OBJECTIVE: Body composition assessment is an important method of evaluating nutritional and metabolic status in hemodialysis patients. PATIENTS AND METHODS: To assess the body composition of hemodialysis patients, we used dual-energy X ray absorptiometry to test 40 stable chronic hemodialysis patients and 40 normal subjects. The patients were from 38 to 70 years old, and all without diabetic mellitus. Comparing with the hemodialysis patients, the normal subjects were selected on a one-to-one base with the same sex and about the same age, body weight and height. RESULTS: The lean body mass/body weight (LBM/BW) ratio had no significant statistical difference between hemodialysis patients and normal subjects in this study (63.02 +/- 8.42% vs 64.80 +/- 7.92%, p =0.3308). The male LBM/BW ratio was higher than that of the female (71.07 +/- 4.63% vs 59.30 +/- 6.35%, p < 0.0001). According to the multiple linear regression analyses, the LBM of hemodialysis patients had positive correlation with gender (p < 0.0001), height (p = 0.0360) and weight (p < 0.0001). The total bone mineral density (BMD) of hemodialysis patients was found to be lower than that of the normal subjects (0.90 +/- 0.10 g/cm vs 0.97 +/- 0.08 g/cm, p = 0.0092). The BMD had been found to be low in the hemodialysis patients with serum intact parathyroid hormone (iPTH) > 1,700 pg/ml. The BMD had negative correlation with age in the female hemodialysis patients (r = 0.63, p = 0.0009), but no correlation in the male hemodialysis patients and in the female or male normal subjects. With the multiple linear regression analyses, the BMD of hemodialysis patients had positive correlation with weight (p = 0.0329) and negative correlation with age (p = 0.0183) and serum iPTH (p = 0.0231). CONCLUSION: We concluded that: the LBM/BW ratio of hemodialysis patients was not different from that of normal subjects. Severe secondary hyperparathyroidism hemodialysis patients had low BMD. The BMD had negative correlation with age in the female hemodialysis patients.

Secondary polycythemia associated with membranous nephropathy.

A 61-year-old male patient had secondary polycythemia associated with idiopathic nephrotic syndrome. Renal biopsy revealed membranous nephropathy. Polycythemia did not change in spite of partial remission of proteinuria. Serum erythropoietin determined by an enzyme-linked immunosorbent assay was 7.2 mU/ml. His serum erythropoietin maintained at a constant level during polycythemia was higher than it was before the appearance of renal ischemia, so he was kept in a polycythemic state. Whether decreasing proteinuria can improve renal ischemia requires future study. We must observe the patient for the occurrence of thromboembolism. Renal ischemia possibly induced by nephrotic syndrome is likely to cause secondary polycythemia.

Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors.

PURPOSE: To characterize Ca2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). METHODS: Changes in intracellular Ca2+ ([Ca2+]i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca2+-sensitive dye Fura-PE3. Relative rates of Ca2+ influx were measured employing Mn2+ as a surrogate for Ca2+. RESULTS: Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca2+]i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 microM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn2+ influx following [Ca2+]i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca2+-ATPase. Under Ca2+-free conditions, peak responses were similar to those in the presence of external Ca2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 +/- 18.8% but UTP responses by only 10.6 +/- 9.5%. Repeated exposures to UTP or msATP reduced [Ca2+]i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 microM) with temporal characteristics of [Ca2+]i mobilization similar to that of cultured cells. CONCLUSION: BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+]i response, subsequently caused capacitative Ca2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.

Absorbance- and light-based solid-phase assays for CMPNeuAc:Galbeta1-4GlcNAc-R alpha-2,3-sialyltransferase.

We now report a solid-phase assay for CMPNeuAc: Galbeta1-3/4GlcNAc-R alpha-2,3-sialyltransferase (alpha2,3ST) that is nonradioactive and allows specific identification of the sialylated product. An acceptor glycoprotein, desialylated fetuin, is immobilized on a microtiter plate. The transfer of sialic acid from CMPNeuAc generates the product NeuAc alpha2-3Gal beta1-4GlcNAc-R that is specifically bound by biotinylated Maackia amurensis leukoagglutinin (MAL). The binding of biotinylated MAL is measured with either an absorbance-based reagent (streptavidin conjugated to alkaline phosphatase) or a light-based reagent (streptavidin conjugated to the bioluminescent protein aequorin, Aqualite). The rat liver alpha2,3ST was used to optimize the assay. The formation of product is linear with respect to time and dependence on the amounts of CMPNeuAc, enzyme, and acceptor coated on the plates. As little as 0.2 microU of enzyme can be measured using the streptavidin-aequorin reagent. The assay is useful with crude tissue extracts, as demonstrated by the determination of the alpha2,3ST activity in human serum and in microsomes of HL-60 and Chinese hamster ovary cells.

A molecular model for the active site of S-adenosyl-L-homocysteine hydrolase.

S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase, EC 3.3.1.1), a specific target for antiviral drug design, catalyzes the hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) as well as the synthesis of AdoHcy from Ado and Hcy. The enzyme isolated from different sources has been shown to contain tightly bound NAD+. Based on the 2.0 A-resolution X-ray crystal structure of dogfish lactate dehydrogenase (LDH), which is functionally homologous to AdoHcy hydrolase, and the primary sequence of rat liver AdoHcy hydrolase, we have derived a molecular model of an extended active site for AdoHcy hydrolase. The computational mutation was performed using the software MUTAR (Yeh et al., University of Kansas, Lawrence), followed by molecular mechanics optimizations using the programs AMBER (Singh et al., University of California, San Francisco) and YETI (Vedani, University of Kansas). Solvation of the model structure was achieved by use of the program SOLVGEN (Jacober, University of Kansas); 56 water molecules were explicitly included in all refinements. Some of these may be involved in the catalytic reaction. We also studied a model of the complex of AdoHcy hydrolase with NAD+, as well as the ternary complexes of the enzyme, NAD+, and substrate or inhibitor molecules. Our refined model is capable of explaining part of the redox reaction catalyzed by AdoHcy hydrolase and has been used to differentiate the relative binding strength of inhibitors.

Molecular cloning and expression of a novel beta-1, 6-N-acetylglucosaminyltransferase that forms core 2, core 4, and I branches.

Mucin-type O-glycans are classified according to their core structures. Among them, cores 2 and 4 are important for having N-acetyllactosamine side chains, which can be further modified to express various functional oligosaccharides. Previously, we discovered by cloning cDNAs that the core 2 branching enzyme, termed core 2 beta-1,6-N-acetylglucosaminyltransferase-leukocyte type (C2GnT-L), is highly homologous to the I branching beta-1, 6-N-acetylglucosaminyltransferase (IGnT) (Bierhuizen, M. F. A., Mattei, M.-G., and Fukuda, M. (1993) Genes Dev. 7, 468-478). Using these homologous sequences as probes, we identified an expressed sequence tag in dbEST, which has significant homology to C2GnT-L and IGnT. This approach, together with 5'and 3' rapid amplification of cDNA ends, yielded a human cDNA that encompasses a whole coding region of an enzyme, termed C2GnT-mucin type (C2GnT-M). C2GnT-M has 48.2 and 33.8% identity with C2GnT-L and IGnT at the amino acid levels. The expression of C2GnT-M cDNA directed the expression of core 2 branched oligosaccharides and I antigen on the cell surface. Moreover, a soluble chimeric C2GnT-M had core 4 branching activity in addition to core 2 and I branching activities. A soluble chimeric C2GnT-L, in contrast, almost exclusively contains core 2 branching activity. Northern blot analysis demonstrated that the C2GnT-M transcripts are heavily expressed in colon, small intestine, trachea, and stomach, where mucin is produced. In contrast, the transcripts of C2GnT-L were more widely detected, including the lymph node and bone marrow. These results indicate that the newly cloned C2GnT-M plays a critical role in O-glycan synthesis in mucins and might have distinctly different roles in oligosaccharide ligand formation compared with C2GnT-L.

Secretion of alpha1,3-galactosyltransferase by cultured cells and presence of enzyme in animal sera.

Glycosyltransferases are normally synthesized as membrane-anchored proteins. However, we recently found that the murine enzyme UDP-Gal:Gal beta1 -->4GLcNAc (Gal to Gal) alpha1,3 galactosyltransferase (alpha1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46). To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the alpha1,3GT activity. A recombinant and soluble form of the murine alpha1,3GT was produced in H293 cells (H293-alpha1,3GT) to aid in optimizing the assay. Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates. The formation of product was detected by a biotinylated human-derived anti-alpha-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, alpha1,3GT and the time of incubation. The assay was also useful in monitoring alpha1,3GT activity during enzyme enrichment procedures. Using this assay, we found that alpha1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines. Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons. Our results demonstrate the development of a novel assay for the alpha1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon.

Expression cloning of a human alpha1, 4-N-acetylglucosaminyltransferase that forms GlcNAcalpha1-->4Galbeta-->R, a glycan specifically expressed in the gastric gland mucous cell-type mucin.

Among mucus-secreting cells, the gastric gland mucous cells, Brunner's glands, accessory glands of pancreaticobiliary tract, and pancreatic ducts exhibiting gastric metaplasia are unique in that they express class III mucin identified by paradoxical Con A staining composed of periodate oxidation, sodium borohydride reduction, Con A, and horseradish peroxidase reaction. Recently it was shown that these mucous cells secrete glycoproteins having GlcNAcalpha1-->4Galbeta-->R at nonreducing terminals of the carbohydrate moieties. Herein we describe the expression cloning of a cDNA encoding a human alpha1,4-N-acetylglucosaminyltransferase (alpha4GnT), a key enzyme for the formation of GlcNAcalpha1-->4Galbeta1-->R. COS-1 cells were thus cotransfected with a stomach cDNA library and a leukosialin cDNA. Transfected COS-1 cells were screened by using monoclonal antibodies specific for GlcNAcalpha1-->4Galbeta-->R and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of GlcNAcalpha1-->4Galbeta-->R. The deduced amino acid sequence predicts a type II membrane protein with 340 amino acids, showing no significant similarity with any other proteins. The alpha4GnT gene is located at chromosome 3p14.3, and its transcripts are expressed in the stomach and pancreas. An in vitro GlcNAc transferase assay by using a soluble alpha4GnT revealed that alpha1,4-linked GlcNAc residues are transferred most efficiently to core 2 branched O-glycans (Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAc), forming GlcNAcalpha1-->4Galbeta1-->4GlcNAcbeta1-->6(GlcNAca lpha1-->4Galbeta1- ->3)GalNAc. Transfection of alpha4GnT cDNA into gastric adenocarcinoma AGS cells produced class III mucin, indicating that alpha4GnT is responsible for the formation of class III Con A reactivity. These results indicate that the alpha4GnT is a glycosyltransferase that forms alpha1,4-linked GlcNAc residues, preferentially in O-glycans.

Expression cloning of a human sulfotransferase that directs the synthesis of the HNK-1 glycan on the neural cell adhesion molecule and glycolipids.

The HNK-1 carbohydrate is expressed on various adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substratum interactions. Here we describe the isolation and functional expression of a cDNA encoding a human sulfotransferase that synthesizes the HNK-1 carbohydrate epitope. A mutant Chinese hamster ovary cell line, Lec2, which stably expresses human neural cell adhesion molecule (N-CAM) (Lec2-NCAM), was first established. Lec2-NCAM was co-transfected with a human fetal brain cDNA library, a cDNA encoding the rat glucuronyltransferase that forms a precursor of the HNK-1 carbohydrate, and a vector encoding the polyoma large T antigen. The transfected Lec2-NCAM cells expressing the HNK-1 glycan were enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA encoding a sulfotransferase, HNK-1ST, that directs the expression of the HNK-1 carbohydrate epitope on the cell surface. The deduced amino acid sequence indicates that the enzyme is a type II membrane protein. Sequence analysis revealed that there is a short amino acid sequence in the presumed catalytic domain, which is highly homologous to the corresponding sequence in other Golgi-associated sulfotransferases so far cloned. The amount of HNK-1ST transcript is high in fetal brain compared with fetal lung, kidney, and liver. Expression of HNK-1ST resulted in the formation of the HNK-1 epitope on N-CAM and a soluble chimeric form of HNK-1ST was shown to add a sulfate group to a precursor, GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, forming sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R. The results combined together indicate that the cloned HNK-1ST directs the synthesis of the HNK-1 carbohydrate epitope on both glycoproteins and glycolipids in the nervous tissues.

Renal biopsy in pregnancies complicated by undetermined renal disease.

BACKGROUND: The aim of this retrospective study was to verify the role of renal biopsy in pregnancies complicated by renal dysfunction. METHODS: A series of 15 percutaneous renal biopsies performed in 15 pregnant women with renal disease presenting during pregnancy over the past 10 years (1990-1999) were reviewed. RESULTS: All the patients underwent renal biopsy before 30 weeks of gestation. The indications for renal biopsy were renal dysfunction of unknown cause or symptomatic nephrotic syndrome (NS). Patients with toxemia were excluded. Eight women had lupus nephritis, including five with diffuse crescenteric changes and three with a mesangial proliferative pattern. Three had chronic glomerulonephritis (CGN), two had mesangial proliferative glomerulonephritis and one each had diabetic nephrosclerosis and endocapillary proliferative glomerulonephritis. There were no significant complications except in one patient who experienced gross hematuria. Early induction of labor was recommended for the four patients with diabetic nephrosclerosis or CGM. The other 11 patients received intravenous pulse methylprednisolone or high dose oral prednisolone therapy. The responses to steroid therapy in these 11 patients were as follows: five achieved complete remission of NS, three achieved incomplete remission, and three achieved partial remission. After 2 years' follow-up, seven mothers achieved complete remission, three had died, three developed chronic renal failure (CRF), and two progressed to end stage renal failure (ESRF) requiring chronic hemodialysis. Fourteen of the 15 pregnancies resulted in live births and the other child was stillborn. CONCLUSIONS: Renal biopsy performed during pregnancy is not contraindicated. The results of histopathological studies are extremely useful in counseling regarding continuation or termination of pregnancy, potential maternal and fetal outcome, and recommending specific therapeutic modalities.

Site-specific N-glycosylation and oligosaccharide structures of recombinant HIV-1 gp120 derived from a baculovirus expression system.

We report the complete structures of the N-linked oligosaccharides and the site-specificity of the N-glycosylation of recombinant gp120 (rgp120) of the HIV-1 BH8 isolate produce by a baculovirus expression system. Glycopeptides derived from the tryptic digests of intact rgp120 or of cyanogen bromide-generated fragments of rgp120 were isolated by their binding to concanavalin A-Sepharose and were purified by reversed-phase HPLC. The isolated glycopeptides were treated with PNGase F, releasing the carbohydrate moiety while converting Asn to Asp, and identified by amino acid analysis and/or peptide sequencing. Our results indicate that all 22 potential N-glycosylation sites in the rgp120 sequence are utilized. We did not detect N-acetylgalactosamine in rgp120, indicating that the glycoprotein lacks typical O-linked oligosaccharides. To investigate the oligosaccharide structures at the sites of glycosylation, we determined the carbohydrate composition for each site and characterized the oligosaccharides by 1H-NMR spectroscopy and by oligosaccharide mapping using high pH anion-exchange chromatography. Mannose and N-acetylglucosamine were the only sugars observed in the intact rgp120 and likewise in individual glycopeptides. All glycopeptides derived from rgp120 contained high mannose-type N-linked oligosaccharides, ranging from GlcNAc2Man5 to GlcNAc2Man9. However, different glycosylation sites showed varied degrees of processing of the high mannose-type oligosaccharides, as characterized by the ratio of GlcNAc2Man8-9 to GlcNAc2Man5-7. These results demonstrate that N-glycosylation of rgp120 in the baculovirus expression system occurs at all potential sites and is site specific in terms of oligosaccharide structures.