Effective wound bed preparation using maggot debridement therapy for patients with critical limb ischaemia.

OBJECTIVE: Patients with critical limb ischaemia (CLI) lack sufficient blood flow in to the limb, which leads to difficulties in the normal wound healing process. Therefore, maggot debridement therapy (MDT) has not generally been recommended for CLI patients. We evaluated the effectiveness of wound bed preparation by MDT in CLI patients who had undergone mid-foot amputation. METHODS: Patients who underwent mid-foot amputation after angioplasty between April 2014 and October 2016 were retrospectively investigated by classifying them into an MDT group or a conventional treatment group. The primary outcome was defined as achievement of wound healing. Secondary outcomes were the proportions of amputation-free survival (AFS) and successful ambulatory improvement. Propensity scores were used to evaluate treatment outcomes based on five factors: ankle-brachial index, skin perfusion pressure of the foot, nutritional status, experience with dialysis and age. RESULTS: A total of 39 patients (39 legs) were included, seven within the MDT group and 32 in the conventional treatment group. Clinical backgrounds of the two groups showed no significant differences except for higher albumin levels for the MDT group (3.5±0.4g/dl; p=0.014). The wound healing proportion was significantly higher in the MDT group (86%) than in the control group (38%) (p=0.035). At 6 months after amputation, no significant differences were found between the two groups for AFS (71% versus 47%; p=0.41) or ambulatory capability (43% versus 28%; p=0.65). This result was also similar to the propensity score adjustment analysis. CONCLUSIONS: The efficacy of MDT with favourable wound bed preparation was shown in our CLI patients based on effective debridement and granulation formation by maggots, avoiding the loss of their heels. Wound-healing rates after MDT were higher for patients than for those receiving conventional treatment. MDT is considered a valid adjuvant treatment strategy for patients with CLI after revascularisation treatment is conducted. More favourable wound bed preparation and successful graft take were achieved in the MDT group, suggesting the effectiveness of MDT for wound healing in CLI patients.

A simple high performance liquid chromatography method for quantitatively determining the reduced form of peroxiredoxin 2 and the mass spectrometric analysis of its oxidative status.

Peroxiredoxins (Prxs) are a family of thiol peroxidases, which have been suggested to serve as biomarkers for diseases caused by oxidative stress. In this study, we established a high performance liquid chromatography (HPLC) method for quantifying the amount of Prx2 in red blood cells (RBCs). RBC proteins were separated using HPLC, and a single peak was detected that matched that produced by recombinant Prx2. Under improved conditions, the calibration curve for Prx2 (reduced form) was linear over the range of 0.5-20.0µg with a correlation coefficient of 0.999. The minimum detectable level of the recombinant Prx2 was 0.2µg, with a signal-to-noise ratio of 3 per 20µl of injection volume. SDS-PAGE and mass spectrometric analysis showed that the proteins comprising the peak were almost exclusively Prx2. Further high-resolution analysis using nanoLC-MS/MS demonstrated that the oxidation sensitive, Cys-51 was carbamidomethylated by iodoacetamide-alkylation during in-gel digestion but was not modified with sulfinic acid (-SO2H) or sulfonic acid (-SO3H). These results indicated that the separated Prx2 was the reduced form and not the hyperoxidized form. These basic experiments allowed us to determine the relative amounts of native Prx2 in RBCs taken from healthy subjects. The average levels of Prx2 in male and female subjects were 7.28ng/mg and 8.29ng/mg, respectively, and no significant difference was observed between the sexes. Therefore, the HPLC method with UV detection described herein offers a convenient method to quantitatively determine the levels of reduced form of Prx2 and its oxidative decrease in human RBCs.

Irreversible hyperoxidation of peroxiredoxin 2 is caused by tert-butyl hydroperoxide in human red blood cells.

Peroxiredoxin 2 (Prx2) is the third most abundant protein in red blood cells (RBCs). In this study, we have succeeded in implementing the rapid and simultaneous detection of the hyperoxidized (Prx2-SO2/3) and reduced (Prx2-SH) forms of Prx2 in human RBCs using reverse phase high-performance liquid chromatography. The detection of a peak corresponding to Prx2-SO2/3 was clearly observed following treatment of tert-butyl hydroperoxide (t-BHP), but not H2O2, and was found to be dose-dependent. The identity of the peak was confirmed as Prx2 by immunoblotting and mass spectrometry analysis. Our results suggest that t-BHP hyperoxidizes cysteine residues in Prx2 more readily than H2O2, and that accumulation of hyperoxidized Prx2 might reflect disruption of redox homeostasis in RBCs.

Copper chaperone for superoxide dismutase co-aggregates with superoxide dismutase 1 (SOD1) in neuronal Lewy body-like hyaline inclusions: an immunohistochemical study on familial amyotrophic lateral sclerosis with SOD1 gene mutation.

The copper chaperone for superoxide dismutase (CCS) interacts with Cu/Zn-binding superoxide dismutase 1 (SOD1) specifically and delivers copper to SOD1. To determine the role of the CCS-SOD1 interaction in the pathogenesis of SOD1-mutated familial amyotrophic lateral sclerosis (FALS) patients, we produced an affinity-purified rabbit antibody against CCS and investigated the immunohistochemical localization of both CCS and SOD1 in neuronal Lewy body-like hyaline inclusions (LBHIs) in the spinal cords of two FALS patients with a two-base pair deletion at codon 126 in the SOD1 gene and three FALS patients with an Ala to Val substitution at codon 4. The LBHIs in anterior horn cells from the five FALS patients showed identical immunoreactivities for CCS: the reaction product deposits with the antibody against CCS were generally restricted to the periphery of the core and halo-type LBHIs. The localizations of the immunoreactivities for CCS and SOD1 were similar in the inclusions: both CCS and SOD1 colocalized in neuronal LBHIs in the five mutant SOD1-linked FALS patients. Our results suggest that the specific interaction and aggregation of CCS-SOD1 (probably CCS-mutant SOD1) in SOD1-mutated FALS patients may amplify the formation of inclusions and emphasize a more marked mutant SOD1-mediated toxicity.

Specific cytotoxicity of antibody to YAL-198, a sperm antigen peptide, to murine zygote.

Active immunization with the peptide segments rSMP-230 and YAL-198, corresponding to the hydrophilic extracellular domain of two human sperm antigens (rSMP-B and YWK-II, respectively), reduced fertility in female rats by different mechanisms. The anti-rSMP-230 antibody interferes with human and murine fertilization, and the anti-YAL-198 antibody blocks the development of mouse embryos. The authors examined in vitro at which stage the antibodies to rSMP-230 and YAL-198 were cytotoxic to murine embryos up to morula/blastocyst stage. Anti-rSMP-230 antibody was not cytotoxic to any stages. On the other hand, the anti-YAL-198 antibody arrested the growth of embryos at the 2-cell stage but not at more advanced developmental stages. When the anti-YAL-198 antibody was used, spotty staining was observed only on the surfaces of embryos that had arrested at the 2-cell stage. Unstained embryos, however, continued to develop normally. In contrast, the anti-rSMP-230 antibody stained murine sperm but failed to stain murine ova and embryos. The present results suggest that the human sperm components rSMP-B and YWK-II play important roles in sperm-egg interaction and early development of the embryo, respectively.

The immunohistochemical expression of stress-response protein (srp) 60 in human brain tumours: relationship of srp 60 to the other five srps, proliferating cell nuclear antigen and p53 protein.

This study analyzed the expression of stress-response (heat-shock) protein 60 (srp 60) in a series of 158 human brain tumours. Immunohistochemical procedures were employed; cells of the human cervical cancer line HeLa S3 exposed to hyperosmolar stress served as positive controls. Deposits of reaction products were found in the cytoplasm. Approximately half of the glioblastomas multiforme (17/31), breast carcinoma metastases (6/10), and lung carcinoma metastases (5/11) as well as about one-third of the astrocytomas (5/13) and meningiomas (8/23) had tumour cells that expressed srp 60. A positive reaction for srp 60 was also seen in some medulloblastomas (2/16), primitive neuroectodermal tumours (PNETs) (2/11), schwannomas (2/21), and pituitary adenomas (2/7), but no positive reactions were observed with oligodendrogliomas and ependymomas. Compared with srp 60-negative tumours, srp 60-positive tumours coexpressed one or more stress-related proteins, among which srp 90, srp 72, srp 27, alphaB-crystallin and ubiquitin occurred with higher frequencies; a high correlation between srp 60 and the other five srps (0.88 - 0.97, p<0.01, Pearson correlation coefficient) was observed in srp 60-positive tumours. In contrast, the correlation coefficient in srp 60-negative tumours was not significant (-0.26 - 0.71). There was a tendency for the proliferating cell nuclear antigen (PCNA)-labeling index to be higher in glioblastomas, astrocytomas, medulloblastomas, PNETs, and breast and lung carcinoma metastases that expressed srp 60 than in those that did not. No significant immunohistochemical reactions of srp 60, PCNA and p53 protein were seen with sections of normal brain tissues. We conclude that primary and metastatic tumours of the brain produce srp 60 and that srp 60 in certain brain tumour cells may coexpress the other five srps. In addition, srp 60 expression might depend, in part, on proliferating potential.

Evaluation of two sperm antigens, rSMP-B and YWK-II, as targets for immunocontraception.

To determine whether sperm membrane components, rSMP-B and YWK-II, are suitable candidates as immunocontraceptives in humans, antifertility activities of the antibodies to the peptide fragments, rSMP-229 and rSMP-230 of rSMP-B and YAL-198 of YWK-II, were examined. In a previous report, anti-rSMP-230 antibody was shown to immobilise human sperm and to block human fertilisation, and the antigen (rSMP-230) to interact with antisperm antibodies found in sera of infertile women. Antibody to the second synthetic peptide, rSMP-229, corresponding to a different segment of rSMP-B, mimicked the biological activities of the anti-rSMP-230 antibody. Anti-YAL-198 antibody significantly, although weakly, inhibited human fertilisation. In the murine model, the anti-rSMP-B antibodies blocked in vitro fertilisation of mouse eggs but had no influence on embryo growth. Anti-YAL-198 antibody, however, arrested the growth of zygotes. In conclusion, rSMP-B, a human sperm protein, is a promising candidate in the development of an immunocontraceptive for human application. A second sperm protein, YWK-II, is effective as an antifertility immunogen in experimental animals.

Formation of advanced glycation end-product-modified superoxide dismutase-1 (SOD1) is one of the mechanisms responsible for inclusions common to familial amyotrophic lateral sclerosis patients with SOD1 gene mutation, and transgenic mice expressing human SOD1 gene mutation.

Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) are morphological hallmarks of certain familial amyotrophic lateral sclerosis (FALS) patients with superoxide dismutase-1 (SOD1) gene mutations, and transgenic mice expressing the human SOD1 gene mutation. The ultrastructure of inclusions in both diseases is identical: the essential common constituents are granule-coated fibrils approximately 15-25nm in diameter and granular materials. Detailed immunohistochemical analyses have shown that the essential common protein of the inclusions in both diseases is an SOD1 protein. This finding, together with the immunoelectron microscopy finding that the abnormal granule-coated fibrils comprising the inclusions are positive for SOD1, indicates that these granule-coated fibrils containing SOD1 are important evidence for mutant SOD1-linked disease in human and mouse. For immunoelectron microscopy, the granule-coated fibrils are modified by advanced glycation endproducts (AGE) such as N(epsilon)-carboxymethyl lysine, pyrraline and pentosidine (Maillard reaction). Based on the fact that AGE themselves are insoluble molecules with direct cytotoxic effects, the granule-coated fibrils and granular materials are not digested by the lysosomal and ubiquitin systems. The neurons and astrocytes of the normal individuals and non-transgenic mice show no significant immunoreactivity for AGE. Considered with the mutant-SOD1 aggregation toxicity, a portion of the SOD1 comprising both types of the inclusion is modified by the AGE, and the formation of the AGE-modified SOD1 (probably AGE-modified mutant SOD1) is one of the mechanisms responsible for the aggregation (i.e. granule-coated fibril formation).

Temporospatial relationship between the expressions of superoxide dismutase and nitric oxide synthase in the developing human brain: immunohistochemical and immunoblotting analyses.

To clarify a significant relationship between superoxide dismutase (SOD) and nitric oxide synthase (NOS) in the developing human brain temporospatially, we demonstrate immunohistochemical expression of Cu/Zn-binding SOD1 (SOD1), Mn-containing SOD2 (SOD2), neuronal NOS (nNOS), inducible NOS (iNOS), and nitrotyrosine in human brains from 13 weeks of gestation to 2 years after birth. The immunoreactivities of both SOD1 and SOD2 were detected in fetal neuroblasts at 13 weeks' gestation, as well as mature neurons at the age of 2 years. By contrast, nNOS neurons could be recognized only at 28 and 33 weeks of gestation in the cerebrum, and only at 15, 18, and 23 weeks of gestation in the brain stem. No significant immunoreactivity for iNOS or nitrotyrosine was detected in any type of cell in any region during any stage examined. Immunoblotting analysis using frontal tissue homogenates at 15, 28, 40 weeks of gestation and 18 months of age revealed single band corresponding to SOD1 molecular weight, observed at all stages examined; a single band compatible with the nNOS molecular mass was detected only at the 28th week of gestation. Together with the fact that nitric oxide (NO) plays a potential role in neuronal differentiation, and that large amounts of NO have cytotoxicity from the reaction of NO with superoxide anions, our data suggested that the expressions of both SOD1 and SOD2, as scavengers of superoxide anions, were maintained from an early developmental stage to prepare stage-specific nNOS expression for a potential differentiation role and to elude NO cytotoxicity.

Midkine, a new neurotrophic factor, is present in glial cytoplasmic inclusions of multiple system atrophy brains.

The glial cytoplasmic inclusion (GCI) is a histological hallmark for multiple system atrophy (MSA): these inclusions are found in oligodendrocytes and consist of abnormal granule-coated fibrils of approximately 24- to 40-nm diameter. To clarify the significance of the presence of midkine (MK) in these GCIs, we carried out immunohistochemical, electron and immunoelectron microscopical, and Western blot analyses of MSA brains using a monoclonal antibody against the C-terminal region of human MK. Immunohistochemically, most of the GCIs were intensely stained by the antibody to MK. Electron and immunoelectron microscopy showed that the GCIs were composed of MK-positive granule-coated fibrils that were essential constituents of these inclusions. No significant MK immunoreactivity was observed in oligodendrocytes, astrocytes and neurons of the normal control subjects. The presence of MK in MSA brain but not in normal brain was confirmed by Western blotting. Together with the fact that MK is associated with fetal morphogenesis during the midgestation period, the presence of MK immunoreactivity in oligodendroglial GCIs may suggest the existence of a repair mechanism on the basis of morphogenesis in the degenerated oligodendrocytes themselves as well as the affected neurons and their axons through the oligodendrocyte-axon-neuron relationship.

Advanced glycation endproduct-modified superoxide dismutase-1 (SOD1)-positive inclusions are common to familial amyotrophic lateral sclerosis patients with SOD1 gene mutations and transgenic mice expressing human SOD1 with a G85R mutation.

To clarify the biological significance of the neuronal Lewy body-like hyaline inclusions and astrocytic hyaline inclusions characteristically found in patients with familial amyotrophic lateral sclerosis with superoxide dismutase-1 (SOD1) gene mutations and in transgenic mice expressing human SOD1 with G85R mutation, the detailed protein composition in both types of inclusions was immunohistochemically analyzed using 45 different antibodies. Both types of inclusions had very strong immunoreactivity for SOD1. The SOD1-positive inclusions in both cell types were also immunoreactive for the insoluble advanced glycation endproducts (AGEs) such as Nepsilon-(carboxymethyl)lysine (CML), pyrraline and pentosidine: both inclusions in both conditions were ultrastructurally composed of the granule-coated fibrils that had immunoreactivities to CML and pyrraline. Both types of inclusions were negative for stress-response proteins (SRPs), 4-hydroxy-2-nonenal (HNE), acrolein, nitric oxide synthases (NOSs) and nitrotyrosine as representative markers of oxidative stress. The neurons and astrocytes of the normal individuals and non-transgenic mice showed no significant immunoreactivity for SOD1, AGEs, SRPs, HNE, acrolein, NOSs or nitrotyrosine. Our results suggest that a portion of the SOD1 composing both type of inclusions, probably toxic mutant SOD1, is modified by the AGEs, and that the formation of the AGE-modified SOD1 is one of the mechanisms responsible for the aggregation involving no significant oxidative mechanisms.

Suppression of GD1alpha ganglioside-mediated tumor metastasis by liposomalized WHW-peptide.

GD1alpha ganglioside-replica peptides were recently isolated from a phage-displayed random pentadecapeptide library by assaying for inhibition of adhesion of RAW117-H10 lymphosarcoma cells to hepatic sinusoidal microvessel endothelial (HSE) cells. We show here that the Trp-His-Trp (WHW) peptide was identified as a minimal sequence of the GD1alpha-replica peptide WHWRHRIPLQLAAGR. The addition of WHW peptide-attached liposomes displayed efficient inhibition of liver metastasis of RAW117-H10 cells as well as of GD1alpha-mediated adhesion of RAW117-H10 cells to HSE cells in vitro. These results suggest that engineered liposomes for peptide delivery are applicable to treatment for metastasis.

New consensus research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 (SOD1) gene mutations: inclusions containing SOD1 in neurons and astrocytes.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily involves the motor neuron system. Approximately 5-10% of ALS is familial. Superoxide dismutase 1 (SOD1) gene mutations are shown to be associated with about 20% of familial ALS (FALS) patients. The neuronal Lewy-body-like hyaline inclusion (LBHI) and astrocytic hyaline inclusion (Ast-HI) are morphological hallmarks of certain SOD1-linked FALS patients with SOD1 gene mutant and transgenic mice expressing human SOD1 with G85R mutation. From the detailed immunohistochemical analyses, the essential common protein of both inclusions is SOD1. Ultrastructurally, both inclusions consist of granule-coated fibrils 15-25 nm in diameter. Based on the immuno-electron microscopical finding that these abnormal granule-coated fibrils are positive for SOD1, the formation (or aggregation) of the abnormal fibrils containing SOD1 would be essential evidence in diseases caused by various SOD1 mutations. The granule-coated fibrils are also modified by advanced glycation end products (AGEs). The AGEs themselves are insoluble molecules with direct toxic effects on cells. AGE formation of SOD1 composing the granule-coated fibrils (probable AGE-modified mutant SOD1) may amplify their aggregation and produce a more marked toxicity.

Identification of the human sperm protein that interacts with sperm-immobilizing antibodies in the sera of infertile women.

OBJECTIVE: To identify the target antigen of sperm-immobilizing antibodies present in the circulation of infertile women. DESIGN: Laboratory research. SETTING: Academic research laboratory. PATIENT(S): Twenty-nine infertile women with sperm-immobilizing antibodies, 22 infertile women with other disorders, and 20 fertile women. INTERVENTION(S): Titers of antibodies to the sperm protein, rSMP-B, were determined by ELISA using as substrate the synthetic peptide segment (rSMP-230) that corresponds with the hydrophilic domain of rSMP-B. Tests for sperm immobilization and zona pellucida penetration were performed using the human IVF system. MAIN OUTCOME MEASURE(S): Human sera with sperm-immobilizing activity were assayed for the presence of antibodies to rSMP-230. Polyclonal antibodies to rSMP-230 were assessed for the same biologic activities as sperm-immobilizing antibodies. RESULT(S): Antibodies to rSMP-230 were detected in 10 (34%) of 29 sera obtained from women with immunologic infertility. In contrast, only one serum sample (2%) from women without sperm-immobilizing activity had a low titer of antibodies to rSMP-230. Polyclonal antibodies to rSMP-230 completely immobilized human sperm in the presence of complement and blocked sperm penetration across the zona pellucida. CONCLUSION(S): The human sperm protein, rSMP-B, probably is the target antigen of sperm-immobilizing antibodies.

beta-Microseminoprotein/prostatic secretory protein is a member of immunoglobulin binding factor family.

Human seminal plasma contains a factor that binds human IgG, designated as immunoglobulin binding factor (IgBF). Under reducing condition IgBF interacts with anti-Leu-11b, a murine monoclonal antibody raised against human FcgammaRIII/CD16. IgBF shows no binding activity under non-reducing condition. Three components having IgBF activity were separated by HPLC and their amino acid sequences determined. The main IgBF showed structural identity to beta-microseminoprotein (beta-MSP), prostatic secretory protein of 94 amino acids (PSP94) and beta-inhibin. The slight variation in the reported sequences of these proteins has been attributed to analytical error. In the present study the molecular masses of main IgBF and beta-MSP/PSP94 were found to be identical by mass spectrometry. In addition, a large component of IgBF and a shorter beta-MSP consisting of 93 amino acids were identified. The binding of beta-MSP for human IgG and anti-Leu-11b antibody is demonstrable only under reducing condition, determined by Western blot analysis. The present data clearly show that IgBF is a family composed of at least three isoforms. One of the members is beta-MSP/PSP94. This family should be designated as IgBF.

IL-1 alpha induces inducible nitric oxide synthase in uterine smooth muscle.

In a previous report, we have identified interleukin-1 alpha (IL-1 alpha) in amniotic fluid of women with premature rupture of the amniotic membrane (PROM), and suggested that this cytokine may play a role in the development of the fetus. To clarify the functional role of amniotic IL-1 alpha, we studied its effect on the induction of nitric oxide (NO) synthesis in the uterus. IL-1 alpha administered intraperitoneally to rats induced an increase in the amount of mRNA encoding the macro-phage-type iNOS which was determined by reverse transcription-polymerase chain reaction. NO measurement by NO selective electrode revealed that NO production in the rat and human uterus treated with IL-1 alpha was promoted by the addition of 1 mM L-arginine to the culture medium. These findings suggest that in women with PROM, amniotic IL-1 alpha may reduce uterine contractions, in part, by inducing of iNOS.

Complement-inhibiting activity of human seminal plasma and semen quality.

Human seminal plasma (SP) contains potent complement inhibitors. This study examined the complement-inhibiting activity of individual SP samples from 118 patients with infertility and analyzed them in relation to various semen parameters. When 25% complement-inhibiting activity was considered the cut off value, less than 1 SD unit from the mean percentage of inhibition of SP samples with normal semen quality, 32 samples (27%) showed low inhibiting activity. Among the lower group, incidences of patients with asthenozoospermia (66%) and oligozoospermia (31%) were significantly (p < .01) higher than those (36 and 10%) in the group whose SP showed significant inhibiting activity. Partial characterization revealed that the component responsible for complement inhibition was heat labile, trypsin resistant, high molecular weight (>10 kD) glycoprotein that can inhibit alternative as well as classical complement pathways. Furthermore, since in the majority of SP samples the anticomplementary activity was blocked by monoclonal antibody against membrane cofactor protein (MCP) or decay accelerating factor (DAF), the complement-inhibiting factors that were identified are likely to be MCP and/or DAF, which are known to be present in human SP. These results suggest that complement-regulatory proteins in SP such as MCP and DAF may protect sperm cells against complement attack in the male reproductive tract.

Quantitative analysis of hypothalamic-hypophyseal-testicular system: why testosterone can act under negative feedback control.

Quantitative analysis of the positive and negative feedback actions of testosterone (T) was carried out using intact rats, and orchidectomized rats implanted with T-filled Silastic capsules. Target organ weights and serum levels of LH and T were examined 7 days later, and response ratios of positive and negative actions were plotted against logarithm of serum T. From x-intercepts and slopes of regression lines, thresholds and responsiveness of reactions were calculated, respectively. Maintenance T levels necessary to maintain onset weights of the organ were also calculated from the regression lines. We compared 5 parameters at various ages, 1) thresholds for lowering serum LH, 2) mean T concentration of intact animals, 3) upper limit of serum T (mean + 2SD), 4) maintenance T levels, and 5) lower 95% limit of the thresholds for target organ response in orchidectomized animals. Though T can act between thresholds for target organ response and upper limit of serum T, the action range of T to induce organ growth over the onset organ weight (growth-inducing range) should be the area between the maintenance T level and the upper limit for serum T. At 3 weeks of age, the threshold for serum LH was very low, which makes the upper limit of serum T lower than maintenance T, allowing no growth-inducing range. From 5 weeks of age, the threshold for serum LH increased, and the serum T level with its upper limit also increased over the maintenance T level, allowing the presence of a growth-inducing range of T to make the growth of target organs possible.

Structure of the D-mannan of the pathogenic yeast, Candida stellatoidea ATCC 20408 (type II) strain, in comparison with that of C. stellatoidea ATCC 36232 (type I) strain.

Acid treatment of the cell-wall D-mannas of Candida stellatoidea strains ATCC 36232 (Type I, A3 strain) and ATCC 20408 (Type II, A2 strain) gave (1----2)-linked beta-D-manno-oligosaccharides (dp 2-5), whereas treatment with alkali gave the (1----2)-linked alpha-D-mannobiose. Conventional acetolysis of the acid- and alkali-treated D-mannan of the A3 strain gave oligosaccharides consisting of (1----2)- and (1----3)-linked alpha-D-mannopyranose residues, similar to those of Candida albicans serotype B strain. Mild acetolysis of the acid- and alkali-treated D-mannan of the A2 strain gave higher oligosaccharides that were digested by the Arthrobacter GJM-1 strain exo-alpha-D-mannosidase. The results of 1H- and 13C-NMR analyses indicated this D-mannan to contain branches with the following structures: beta-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp++ +-(1----2)-alpha-D-Manp- (1----2)-D-Man, beta-D-Manp-(1----2)-beta-D-Manp-(1----2)-alpha-D-Manp -(1----2)- alpha-D-Manp-(1----2)-D-Man, and beta-D-Manp-(1----2)-beta-D-Manp-(1----2)-beta- D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp-(1- ---2)-alpha-D-Manp- (1----2)-D-Man, in common with the D-mannans of C. albicans serotype A strains.