Desensitization protocol in highly HLA-sensitized and ABO-incompatible high titer kidney transplantation.

BACKGROUND: A positive crossmatch indicates the presence of donor-specific alloantibodies and is associated with a graft loss rate of >80%; anti-ABO blood group antibodies develop in response to exposure to foreign blood groups, resulting in immediate graft loss. However, a desensitization protocol for highly HLA-sensitized and ABO-incompatible high-titer kidney transplantation has not yet been established. METHODS: We treated 6 patients with high (≥1:512) anti-A/B antibody titers and 2 highly HLA-sensitized patients. Our immunosuppression protocol was initiated 1 month before surgery and included mycophenolate mofetil (1 g/d) and/or low-dose steroid (methylprednisolone 8 mg/d). Two doses of the anti-CD20 antibody rituximab (150 mg/m(2)) were administered 2 weeks before and on the day of transplantation. We performed antibody removal with 6-12 sessions of plasmapheresis (plasma exchange or double-filtration plasmapheresis) before transplantation. Splenectomy was also performed on the day of transplantation. Postoperative immunosuppression followed the same regimen as ABO-compatible cases, in which calcineurin inhibitors were initiated 3 days before transplantation, combined with 2 doses of basiliximab. RESULTS: Of the 8 patients, 7 subsequently underwent successful living-donor kidney transplantation. Follow-up of our recipients showed that the patient and graft survival rates were 100%. Acute cellular rejection and antibody-mediated rejection episodes occurred in 1 of the 7 recipients. CONCLUSIONS: These findings suggest that our immunosuppression regimen consisting of rituximab infusions, splenectomy, plasmapheresis, and pharmacologic immunosuppression may prove to be effective as a desensitization protocol for highly HLA-sensitized and ABO-incompatible high-titer kidney transplantation.

SspA, an outer membrane protein, is highly induced under salt-stressed conditions and is essential for growth under salt-stressed aerobic conditions in Rhodobacter sphaeroides f. sp. denitrificans.

We have previously shown that an outer membrane protein, SspA, is prominently induced by salt stress in a photosynthetic bacterium, Rhodobacter sphaeroides f. sp. denitrificans IL106 (R. sphaeroides). In this study, we investigated the physiological role of SspA under various stress conditions. Using recombinant SspA expressed in Escherichia coli as an antigen, the polyclonal antiserum of SspA was prepared. Western blot analysis demonstrated that SspA was highly induced by salt stress under both anaerobic and aerobic conditions. SspA was also induced, but to a lesser extent, by osmotic and acid stress. It is reduced under heat and cold compared to non-stressed conditions. While sspA-disrupted R. sphaeroides grew normally under anaerobic conditions in either the presence or absence of stress, it displayed significantly retarded growth under aerobic conditions in the dark, especially when osmotic or salt stress were imposed. In addition, the sspA disruptant, but not the wild type, formed cell aggregates when grown under both anaerobic and aerobic conditions, and this phenotype was significantly enhanced under salt-stressed aerobic conditions. Together, our findings suggest that SspA is critical under salt-stressed, aerobic growth conditions.

Molecular cloning, functional expression and characterization of p15, a novel fungal protein with potent neurite-inducing activity in PC12 cells.

p15 is a novel fungal protein which induces neurite outgrowth and neuronal differentiation of PC12 cells. In the present study, we report molecular cloning, functional expression and characterization of the gene encoding p15. The deduced amino acid sequence suggested that p15 is synthesized as a precursor with 31 extra amino-terminal amino acids including a putative signal sequence, and 20 carboxy-terminal amino acids, in addition to the 118 amino acids-long mature region with neurite-inducing activity. From the poly(A)(+) RNA prepared from the producing fungal strain, a cDNA fragment encoding the mature region of p15 was amplified and His(6)-tagged recombinant p15 was produced in Escherichia coli. The recombinant protein purified by a single step on Ni(2+) agarose column chromatography exhibited comparable specific activity as native p15 in the PC12 neurite extension assay. The effect of His(6)-p15 was blocked by nicardipine, suggesting that Ca(2+) influx through the L-type Ca(2+) channels is essential for its neurite-inducing activity. In addition, mutational analysis of His(6)-p15 demonstrated that both intramolecular disulfide bonds are essential for its biological activity.

Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae.

Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (Deltacmp1 Deltacmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5.

Cloning and sequencing of a gene encoding a novel salt stress-induced membrane protein from Rhodobacter sphaeroides f. sp. dentrificans.

The gene encoding a membrane protein, SspA, induced under salt stress conditions was cloned and sequenced from a photosynthetic bacterium, Rhodobacter sphaeroides f. sp. denitrificans IL106. A single open reading frame consisting of 972 base pairs that encoded a polypeptide composed of a signal peptide of 24 amino acids and a mature protein of 300 amino acids (Mr 33,386) was found. A database search failed to detect any highly homologous sequences, indicating that SspA is a novel protein. The protein was present in the outer membrane as a transmembrane protein and was specifically induced by salt stress, but not by heat shock.

Visualization of nuclei in Aspergillus oryzae with EGFP and analysis of the number of nuclei in each conidium by FACS.

Aspergillus oryzae has been reported to form conidia with multinuclei. In order to analyze nuclei in living cells, we developed an expression system of the A. nidutans histone H2B protein tagged by EGFP (H2B::EGFP). In both A. oryzae niaD300 and A. nidulans FGSC89 transformants expressing H2B::EGFP, fluorescence was detected in nuclear regions of hyphae and conidia. While a conidium contained only one fluorescent spot in the A. nidulans transformant, approximately 66% of conidia had two, 24% had one, and 10% had three or more in the A. oryzae transformant. The conidia expressing H2B::EGFP were put through FACS (fluorescence-activated cell sorting) analysis and two sharp peaks, corresponding to one and two nuclei in each conidium, were noted in the A. oryzae transformant. In addition, the A. oryzae uninucleate conidia that were successfully isolated by FACS reproduced conidia with almost the same number distribution of nuclei as that of the original. Conidia of five A. oryzae strains used in sake brewing were scored for the number of nuclei, showing that a varied number of nuclei existed in each conidium and some strains had a small number of uninucleate conidia.

Cloning and characterization of the cpyA gene encoding intracellular carboxypeptidase from Aspergillus nidulans.

Carboxypeptidase Y (CPY) has been used as a maker enzyme for investigations on intracellular transport of vacuolar proteins and on vacuolar biogenesis in Saccharomyces cerevisiae. We describe the cloning and characterization of the CPY homologue encoding gene (cpyA) from the filamentous fungus Aspergillus nidulans. The cpyA gene has one intron and encodes a protein with 552 amino acids containing a putative signal sequence and pro-sequence. The predicted CpyA protein is highly similar in sequence with carboxypeptidases from several yeast species and contains a catalytic triad (Asp-His-Ser) like that of serine carboxypeptidase. The cpyA disruptant cells showed reduced levels of intracellular carboxypeptidase. These results suggest that the cpyA gene encodes a vacuolar carboxypeptidase in A. nidulans.

Cloning and characterization of Aspergillus nidulans vpsA gene which is involved in vacuolar biogenesis.

In Saccharomyces cerevisiae, vacuoles play very important roles in pH and osmotic regulation, protein degradation and storage of amino acids, small ions as well as polyphosphates. In filamentous fungi, however, little is known about vacuolar functions at a molecular level. In this paper, we report the isolation of the vpsA gene from the filamentous fungus Aspergillus nidulans as a homologue of the VPS1 gene of S. cerevisiae which encodes a dynamin-related protein. The vpsA gene encodes a polypeptide consisting of 696 amino acids that is nearly 60% homologous to the S. cerevisiae Vps1. Similar to Vps1, VpsA contains a highly conserved tripartite GTPase domain but lacks the pleckstrin homology domain and proline-rich region. The vpsA disruptant shows poor growth and contains highly fragmented vacuoles. These results suggest that A. nidulans VpsA functions in the vacuolar biogenesis.

Guanabenz, an antihypertensive centrally acting alpha2-agonist, suppresses morning elevations in aggregation of human platelets.

To determine whether the antihypertensive agent guanabenz affects the circadian rhythm in the hemorheologic properties of the platelet, we evaluated the aggregability of platelets collected from 11 healthy subjects in the morning and the evening after the oral administration of this agent, daily for 2 weeks. We analyzed platelet aggregation by the turbidimetric method. In an in vitro study, guanabenz, 10 nM-100 microM, did not affect platelet aggregation, whereas epinephrine induced platelet aggregation at an EC50 of 1.5 microM. The healthy volunteers demonstrated a diurnal variation in platelet aggregability that was high in the morning and low in the evening (66 +/- 10% and 56 +/- 11% respectively, of the percent platelet aggregation induced by epinephrine). The same variation was seen with the platelet aggregation induced by adenosine diphosphate (ADP) (62 +/- 8% [morning] vs. 51 +/- 7% [evening]). After the administration of guanabenz, platelet aggregability was significantly reduced in the morning compared with that before drug administration, when platelet aggregation was induced by epinephrine (49 +/- 9%, p < 0.05) or ADP (48 +/- 7%, p < 0.05), although the plasma levels of catecholamine were unchanged. A suppressive effect of guanabenz on platelet aggregability was observed in the evening, as the platelets were stimulated by epinephrine (38 +/- 9%, p < 0.05), but not by ADP (49 +/- 5%). Findings suggest that guanabenz mainly suppressed the morning enhancement in platelet aggregability, which contributes to the formation of intravascular thrombi. Thus, in addition to its antihypertensive actions, guanabenz may help to reduce the risk of vascular accidents, which frequently occur in the morning.

Deletion analysis of the enolase gene (enoA) promoter from the filamentous fungus Aspegillus oryzae.

The enolase gene (enoA) is one of the most strongly expressed genes in Aspergillus oryzae. To elucidate the transcription regulatory element for this strong expression and the process of glucose induction, the transcription activity of a series of truncated enoA promoters was measured by using the Escherichia coli uidA gene as a reporter. Deletion of a 104-bp region located -224 nt to -121 nt upstream of the translation initiation site caused both a drastic decrease in the beta-glucuronidase (GUS) activity and a loss of glucose induction. Northern blot analysis confirmed that the decrease in GUS activity was achieved at the transcriptional level. In addition, electrophoretic gel mobility shift assays indicated that the 104-bp region contained a 15-bp element, to which one or more A. oryzae cellular factors specifically bind. These results suggest that the 15-bp element between -195 nt and -181 nt includes the sequence essential for the transcription regulation of the A. oryzae enoA gene.

Genetic analysis of growth inhibition of yeast cells caused by expression of Aspergillus oryzae RNase T1.

Even though most fungal hydrolytic enzymes have been successfully secreted in S. cerevisiae cells by expression of corresponding cDNA, overexpression of A. oryzae RNase T1 causes severe growth inhibition in yeast. We observed that yeast strains carrying RNase T1 cDNA under control of the GAL1 promoter with a single-copy vector were able to grow on galactose medium while those with a multi-copy vector were not. It was found that overexpression of three mutated versions of RNase T1 with low enzymatic activity did not affect the growth. We also observed that expression of RNase T1 without a signal sequence severely inhibited growth of the transformant even on the single-copy plasmid. Subcellular fractionation showed that overexpressed myc-tagged RNase T1 was localized in the membrane fraction. In the yeast secretory pathway, while the mutants defective in translocation into the ER, ER-Golgi trafficking and vacuole formation had severe growth inhibition during expression of RNase T1 from the single-copy plasmid. These results suggest that a mislocalization of active RNase T1 in cytosol by overflow from the secretory apparatus has toxic effects on the host cells.

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae.

A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of L-glutamine but also of D-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137-143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5'-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae.

Purification and characterization of isoamyl acetate-hydrolyzing esterase encoded by the IAH1 gene of Saccharomyces cerevisiae from a recombinant Escherichia coli.

The IAH1 gene of Saccharomyces cerevisiae encodes an esterase that preferentially acts on isoamyl acetate; however, the enzyme has not yet been completely purified from the yeast S. cerevisiae. We constructed the IAH1 gene expression system in Escherichia coli, and purified the IAH1 gene product (Iah1p). The amount of Iah1p produced by recombinant E. coli was more than 40% of total cellular proteins. The molecular size of Iah1p was 28 kDa by SDS-polyacrylamide gel electrophoresis. Judging from the molecular weight estimation by gel filtration of purified Iah1p, the enzyme was thought to be a homodimer. The Km values for isoamyl acetate and isobutyl acetate were 40.3 mM and 15.3 mM, respectively. The enzyme activity was inhibited by Hg2+, p-chloromercuribenzoate, and diisopropylfluorophosphate.

Production and product quality assessment of human hepatitis B virus pre-S2 antigen in submerged and solid-state cultures of Aspergillus oryzae.

Pre-S2 is a diagnostically important antigen of human hepatitis B virus (HBV). In order to produce pre-S2 antigen in Aspergillus oryzae, the gene [pre-S2]3, which encodes a tandemly triplicated repeat of pre-S2 polypeptides was fused with the partial glaA gene encoding glucoamylase lacking the starch-binding domain. In submerged culture, A. oryzae transformants carrying glaA-[pre-S2]3 secreted a heterogeneously glycosylated form of the fusion protein that was partially degraded. Contrarily, utilization of a wheat brain solid-state culture system resulted in the secretion of a homogeneous glycosylated form of the whole fusion protein. This is the first report of a dissimilarity in glycosylated modification between submerged and solid-state culture conditions in heterologous protein production in A. oryzae.

Characterization of a novel fungal protein, p15, which induces neuronal differentiation of PC12 cells.

In our previous paper, we reported that a 15 kDa protein (p15) produced by a fungus, genus Helicosporium, enhanced NGF-induced neurite outgrowth from PC12 cells. Here we further characterized the actions of p15. The complete amino acid sequence of p15 was determined and it was shown to be a hydrophilic protein composed of 118 amino acid residues with two intramolecular disulfide bridges. p15-induced neurite outgrowth was blocked by the depletion of extracellular Ca(2+) in the culture medium and was significantly inhibited by L-type Ca(2+) channel inhibitor nicardipine. p15 stimulated Src kinase and MAPK activities, and neurite outgrowth was not observed in srcDN2, a dominant negative c-src(K295R)-expressing cell line, and was significantly reduced in RasN17-expressing cells. These results suggest that p15 stimulates neurite outgrowth through the potentiation of L-type Ca(2+) channels, thereby activating the Src-Ras-MAPK cascade.

Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system.

Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.

Hypoalbuminemia accelerates erythrocyte membrane lipid peroxidation in chronic hemodialysis patients.

BACKGROUND: Persistent hypoalbuminemia is a long-term poor prognostic factor in chronic hemodialysis patients. PATIENTS AND METHODS: We investigated the correlation between the degree of peroxidation of erythrocyte membrane lipids, erythrocyte alpha tocopherol content, erythrocyte glutathione peroxidase activity and serum albumin concentration in twelve patients with uremia not undergoing hemodialysis and fifteen patients on maintenance hemodialysis. RESULTS: The glutathione peroxidase activity in erythrocytes was higher in patients of uremia not undergoing hemodialysis than in chronic hemodialysis patients. A significant negative correlation was observed between the erythrocyte alpha tocopherol content and the degree of erythrocyte membrane lipid peroxidation in chronic hemodialysis patients. There was a statistically significant difference in the degree of erythrocyte membrane lipid peroxidation between patients with chronic hemodialysis-associated hypoalbuminemia and chronic hemodialysis patients having normal serum albumin levels. CONCLUSION: This study suggested that serum albumin inhibits peroxidation of erythrocyte membrane lipids and that hemodialysis induces recovery of serum reductivity. We conclude that persistent hypoalbuminemia worsens the serum antioxidant activity in chronic hemodialysis patients and may contribute to increased oxidative cell damage.

The protease activity of a calpain-like cysteine protease in Saccharomyces cerevisiae is required for alkaline adaptation and sporulation.

Abstract Saccharomyces cerevisiae has only one putative gene (designated CPL1) for a cysteine protease with a protease domain similar to that of calpain. This gene product shows significant sequence similarity to PalBp, a fungal (Emericella nidulans) calpain-like protease that is responsible for adaptation under alkaline conditions, both in the protease domain and the domain following the protease domain. CPL1 disruptant strains show impaired growth at alkaline pH, but no obvious growth defects under acidic pH conditions. This phenotype is complemented by the wild-type CPL1 gene, and its protease activity is essential for complementation. Disruption of CPL1 also causes reduced sporulation efficiency and promotes the degradation of the transcription factor Rim101p, which is involved in the sporulation pathway and has been shown to accumulate in a C-terminally truncated, active form under alkaline conditions. Furthermore, expression of the C-terminally truncated Rim101p suppressed the alkaline sensitivity associated with CPL1 disruption. These results indicate that a calpain-like cysteine protease, Cpl1p, plays an important role in alkaline adaptation and sporulation processes, via regulation of the turnover and processing of the transcription factor Rim101p.

Cloning and characterization of a gene (arpA) from Aspergillus oryzae encoding an actin-related protein required for normal nuclear distribution and morphology of conidiophores.

We have isolated the arpA gene from Aspergillus oryzae as a homologue of the Neurospora crassa ro-4 gene. In N. crassa, mutations in the ro-4 gene, which encodes a major component of the dynactin complex Arp1, causes curling of hyphae and abnormalities in nuclear distribution. The arpA gene contains two introns and encodes a polypeptide of 381 amino acids, with a 78% sequence identity to the N. crassa Arp1. Overexpression of the arpA gene causes a defect in nuclear migration into elongating hyphae of germlings in A. oryzae. We constructed arpA disruptant strains of A. oryzae. The arpA null mutants showed poor growth and hyper-branched mycelia, as well as a nuclear distribution defect. Scanning electron microscopy revealed that the arpA null mutant has an aberrant conidiophore morphology with irregular phialides.