Clinically significant micafungin resistance in Candida albicans involves modification of a glucan synthase catalytic subunit GSC1 (FKS1) allele followed by loss of heterozygosity.

OBJECTIVES: To determine the mechanism of intermediate- and high-level echinocandin resistance, resulting from heterozygous and homozygous mutations in GSC1 (FKS1), in both laboratory-generated and clinical isolates of Candida albicans. METHODS: The DNA sequences of the entire open reading frames of GSC1, GSL1 (FKS3) and RHO1, which may contribute to the beta-1,3-glucan synthase of a micafungin-susceptible strain and a resistant clinical isolate, were compared. A spontaneous heterozygous mutant isolated by selection for micafungin resistance, and a panel of laboratory-generated homozygous and heterozygous mutants that possessed combinations of the echinocandin-susceptible and -resistant alleles, or mutants with individual GSC1 alleles deleted, were used to compare levels of echinocandin resistance and inhibition of glucan synthase activity. RESULTS: DNA sequence analysis identified a mutation, S645P, in both alleles of GSC1 from the clinical isolate. GSL1 had two homozygous amino acid changes and five non-synonymous nucleotide polymorphisms due to allelic variation. The predicted amino acid sequence of Rho1p was conserved between strains. Reconstruction of the heterozygous (S645/S645F) and homozygous (S645F/S645F) mutation showed that the homozygous mutation conferred a higher level of micafungin resistance (4 mg/L) than the heterozygous mutation (1 mg/L). Exposure of the heterozygous mutant to micafungin resulted in a loss of heterozygosity. Kinetic analysis of beta-1,3-glucan synthase activity showed that the homozygous and heterozygous mutations gave echinocandin susceptibility profiles that correlated with their MIC values. CONCLUSIONS: A homozygous hot-spot mutation in GSC1, caused by mutation in one allele and then loss of heterozygosity, is required for high-level echinocandin resistance in C. albicans. Both alleles of GSC1 contribute equally and independently to beta-1,3-glucan synthase activity.

Protein serine/threonine kinases in signal transduction for secondary metabolism and morphogenesis in Streptomyces.

A number of proteins in the Gram-positive bacterial genus Streptomyces are phosphorylated on their serine/threonine and tyrosine residues in response to developmental phases. AfsR is one of these proteins and acts as a transcriptional factor in both the regulation of secondary metabolism in Streptomyces coelicolor A3(2) and morphological differentiation in Streptomyces griseus. In S. coelicolor A3(2), AfsR is phosphorylated on its serine and threonine residues by more than three protein kinases whose kinase activity is enhanced by means of autophosphorylation on their serine and threonine residues. The degree of autophosphorylation of AfsK is regulated by KbpA which, by binding directly to the kinase domain of AfsK, inhibits its autophosphorylation. Phosphorylation of AfsR enhances its DNA-binding activity and causes it to bind the promoter elements, including -35, of afsS, thus resulting in activation of afsS transcription. ATPase activity of AfsR is essential for this transcriptional activation, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. afsS, encoding a 63-amino-acid protein, then activates transcription of actII-ORF4, a pathway-specific transcriptional activator in the actinorhodin biosynthetic gene cluster, in an as yet unknown way. Distribution of the afsK- afsR systems in a wide variety of Streptomyces species and the presence of many phosphorylated proteins in a given Streptomyces strain suggest that the signal transduction via not only two-component regulatory systems but also serine/threonine kinases generally regulates secondary metabolism and morphogenesis in this genus.

Autophosphorylation of a bacterial serine/threonine kinase, AfsK, is inhibited by KbpA, an AfsK-binding protein.

A protein serine/threonine kinase, AfsK, and its target protein AfsR globally control physiological and morphological differentiation in the bacterial genus Streptomyces. A protein (KbpA) of 252 amino acids encoded by an open reading frame in a region upstream of afsK in Streptomyces coelicolor A3(2) was identified as an AfsK-interacting protein. The interaction site of AfsK was in the N-terminal portion containing the kinase catalytic domain. KbpA bound a nonphosphorylated form of AfsK and inhibited its autophosphorylation at serine and threonine residues. KbpA in the reaction mixture containing AfsK and AfsR also inhibited the phosphorylation of AfsR by AfsK, presumably because KbpA inhibited the conversion from the inactive, nonphosphorylated form of AfsK to the active, phosphorylated form. kbpA was transcribed throughout growth, and the transcription was enhanced when production of actinorhodin had already started. KbpA thus appeared to play an inhibitory role in a negative feedback system in the AfsK-AfsR regulatory pathway. Consistent with these in vitro observations, kbpA served as a repressor for actinorhodin production in S. coelicolor A3(2); disruption of kbpA greatly enhanced actinorhodin production, and overexpression of kbpA reduced the production.

The acute effects of stellate ganglion block on circulation in human ocular fundus.

PURPOSE: To study the acute effects of local-anesthetic stellate ganglion block (SGB) on tissue circulation in the human fundus. METHODS: Eleven patients with Bell's palsy (age 56+/-6 y, mean+/-SD) who underwent SGB for its treatment participated in the study. Using the laser speckle method, normalized blur (NB) value, a quantitative index of tissue blood velocity, was measured every 0.125 s over an area located halfway between the macula and the optic nerve head (ONH) with no discrete visible vessels and averaged over 3 pulses when fixation was satisfactory (NB(ch-ret)). NB(ch-ret) and intraocular pressure (IOP) in both eyes, blood pressure (BP), and pulse rate (PR) were measured before, and 10, 20, 30, and 60 min after SGB. SGB was induced by injecting 1% mepivacaine hydrochloride (5 ml) into the vicinity of the seventh cervical vertebra on the paralyzed side. RESULTS: The IOP in the blocked side significantly decreased between 20 and 60 min following SGB, compared to the baseline, while IOP in the unblocked side remained unchanged. The NB(ch-ret) was significantly increased after 10 min by about 8% in the blocked side, but its effect almost disappeared at 60 min. There was no significant change in NB(ch-ret) in the unblocked side, BP or PR throughout the experimental period. CONCLUSION: SGB increased tissue circulation in the fundus in the blocked side, but its effect was thought to be small and transient.

Genetic and biochemical characterization of a protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2).

A gene encoding a protein phosphatase (SppA) with a phosphoesterase motif, which was predicted by the genome project of the Gram-positive bacterium Streptomyces coelicolor A3(2), was cloned by PCR in pET32a(+) and expressed in Escherichia coli. SppA fused to thioredoxin (TRX-SppA) showed distinct heat-stable phosphatase activity toward p-nitrophenyl phosphate with optimal pH 8.0 and optimal temperature 55 degrees C. Mn2+ greatly enhanced enzyme activity, as is found with other protein Ser/Thr phosphatases. TRX-SppA was not inhibited by sodium orthovanadate or okadaic acid, both of which are known to be specific inhibitors of protein phosphatases. TRX-SppA showed phosphatase activity toward not only phosphoThr (pThr) and pTyr but also oligopeptides containing pSer, pThr, and pTyr, indicating that SppA is a protein phosphatase with dual substrate specificity. Disruption of the chromosomal sppA gene resulted in severe impairment of vegetative growth. All of these observations show that SppA, a protein phosphatase with dual specificity, plays an important, but not essential, role in vegetative growth of S. coelicolor A3(2). The presence of a single copy of sppA in all the 13 Streptomyces species examined, as determined by Southern hybridization, suggests a common role of SppA in general in Streptomyces species.

Rotational acetabular osteotomy for the treatment of dysplastic hips with end-stage osteoarthrosis--a biological alternative to total hip arthroplasty?

We evaluated the results of rotational acetabular osteotomy (RAO) for the treatment of dysplastic hips with end-stage osteoarthrosis. Sixteen patients, aged 15-45 years at the time of surgery, were reviewed at a mean follow-up of 8 years (range 3-17 years). Remodeling of the hip joint occurred in half of the patients, with significant clinical improvement. A subsequent total hip arthroplasty, however, was done within 2 years after RAO in two other patients who had had large bone cysts in the femoral head and acetabulum. We suggest that RAO may be the procedure of choice for selected young patients, especially teenage patients, to postpone total hip arthroplasty.

Rotational acetabular osteotomy using biodegradable internal fixation.

We used biodegradable poly-L-lactide screws in rotational acetabular osteotomy in 41 hips of 41 patients, and studied the complications after an average follow-up of 4.9 years (range 1.0-7.7 years). There were 39 females and 2 males, their average age at the time of the operation was 32 years (range 12-55 years). A small subcutaneous abscess appeared around the non-absorbable sutures in 2 patients after surgery. There was 1 case of thrombophlebitis and 1 of local dermatitis. The small subcutaneous abscess resolved after the removal of the suture material in the 2 cases, and the thrombophlebitis resolved with aspirin. The local dermatitis persisted but was cured by local steroid therapy over 5.8 years. The incidence of local dermatitis after the use of biodegradable implants should be further investigated.

Long-term outcome of rotational acetabular osteotomy: 145 hips followed for 10-23 years.

We report the long-term outcome of rotational acetabular osteotomy in 145 dysplastic hips of 131 patients after an average follow-up of 13 (10-23) years. The mean age at operation was 28 (11-52) years. The radiographic severity of osteoarthrosis before operation, according to the criteria of the Japanese Orthopaedic Association, was stage I (no degenerative change) in 63 hips, stage II (early degenerative stage) in 49, stage III (progressive stage) in 21 and stage IV (end stage) in 12. The clinical outcome based on the Merle d'Aubigné and Postel score was excellent or good for 90 (80%) of the 112 hips which had stage I or II osteoarthrosis preoperatively, and was excellent or good for only 9 of the 33 hips which had stage III or IV osteoarthrosis (p < 0.001, chi-square test). The radiographic severity of osteoarthrosis at the most recent review was stage I or II for 79 (70%) of the 112 hips which had stage I or II osteoarthrosis preoperatively. The long-term outcome of rotational acetabular osteotomy was satisfactory for a dysplastic hip with little, if any, osteoarthrosis, but was unsatisfactory for a hip with more advanced osteoarthrosis.

[Aspiration pneumonia during the endotracheal intubation on the occasion of bladder perforation by TUR].

We report on two cases of aspiration pneumonia which developed during the endotracheal intubation after bladder perforation during TUR. The first case was a 79 y.o. male, who underwent TUR-P and lithotripsy under spinal and epidural anesthesia. The second case was a 69 y.o. male, who had undergone TUR-Bt under nitrous oxide-oxygen-enflurane anesthesia. General anesthesia was selected to perform an laparotomy when the diagnosis was made. They vomited a considerable amount of gastric content just after giving the drugs for induction. The chest X-rays revealed signs of aspiration pneumonia. These X-ray findings improved in a week using antibiotic therapy. Although TUR is performed as scheduled, vomiting may occur in the case of unexpected bladder perforation, which can cause aspiration pneumonia. In such emergency, we should insert a nasal tube before induction, press the cricoid (crush induction), or intubate with the patient awake.

Familial occurrence of hyperplastic callus in osteogenesis imperfecta.

There is a hypothesis that hyperplastic callus (HC) in osteogenesis imperfecta (OI) is not merely a rare complication but could actually be inherited, although this idea has not yet been investigated. We described two cases, a mother and son, with mild OI, normal scleral colour and no dentinogenesis imperfecta, who repeatedly had HC in their femur. Familial occurrence of HC was found in 13 cases in 5 families among 21 cases in 7 families with a familial background of OI in the literature (including this report). This is higher than the reported incidence of HC, 1.5% (5 cases of 333), and the mode of transmission is concomitant with autosomal dominant inheritance in all these families. Since a review of 47 cases in the literature shows that HC occurs independently of scleral colour and the degree of bone fragility, it may be an additional criterion for subdivision within each type of the Sillence classification.

Expression of the Streptomyces coelicolor A3(2) ptpA gene encoding a phosphotyrosine protein phosphatase leads to overproduction of secondary metabolites in S. lividans.

A DNA fragment that caused pigment production in Streptomyces lividans was isolated from a gene library of PstI-digested chromosomal fragments of S. coelicolor A3(2). Subcloning and nucleotide sequencing proved the identity of the cloned gene to ptpA encoding a low-molecular-mass phosphotyrosine protein phosphatase. The S. lividans transformant containing ptpA on pIJ41 with a copy number of 3 4 per genome produced large amounts of undecylprodigiosin and A-factor, in addition to the pigmented antibiotic actinorhodin, whereas the transformant containing ptpA on an SCP2* derivative with a copy number of 1-2 did not. The PtpA protein produced as a fusion to the maltose binding protein in Escherichia coli showed phosphatase activity toward o-phosphotyrosine, but not toward o-phosphoserine or a-threonine. Introduction of a mutant ptpA gene encoding an inactive protein with serine instead of the 9th cysteine caused no pigmentation. Disruption of the chromosomal ptpA gene of S. coelicolor A3(2), however, appeared to cause no detectable effect on the production of the pigmented antibiotics or A-factor and the ptpA disruptants developed aerial mycelium and spores normally.

Microtubule-associated proteins regulate microtubule function as the track for intracellular membrane organelle transports.

Axonal microtubules have two essential roles: providing the track for organelle transport and forming the cytoskeletal framework to maintain axonal morphology. Microtubule-associated proteins (MAPs) are essential for the formation of cytoskeletal architecture. However, they may have additional roles on the regulation of organelle transport by their interaction with motor proteins on the microtubules. We first examined the effects of axonal MAPs on the organelle movement along microtubules in a heterologous system using COS fibroblasts, which express no axonal MAPs, such as tau or MAP2C. Transfection of tau or MAP2C gene suppressed organelle movement almost completely in this cell type, hence interaction of axonal MAPs with microtubules interferes with organelle transports. It is known that the phosphorylation of MAPs reduces their interaction with microtubules. In this sense, phosphorylation of MAPs can be a good candidate for the molecular switch to regulate the organelle transport. As a second set of experiments, we investigated the effects of modulating cAMP dependent protein kinase pathway on organelle transports in primary sensory neurons, where high-molecular-weight tau protein is the major MAP. We found that the application of dibutyryl cAMP enhanced transports of large organelles in the axon. Furthermore, this drug treatment phosphorylated endogenous tau protein and thus reduced the affinity of tau to microtubules. These results indicate that axonal MAPs can work as a phosphorylation-dependent regulator of organelle transport. Local activation of protein kinase pathways in the axon might play an important role on the segregation of microtubules serving for either organelle transport or cytoskeletal architecture.

[Usefulness of NODSS (nasal/oral discriminate sampling system) in end-tidal carbon dioxide gas monitoring].

We investigated the usefulness of nasal/oral discriminate sampling system (NODSS) that had been developed recently in order to obtain the accurate end-tidal carbon dioxide (PET(CO2)) from a spontaneously breathing patient through a nomal airway. Fifty patients were monitored using a capnograph with NODSS following extubation in the postanesthesia unit. PET(CO2) data were collected by means of nasal, oral or nasal/oral sampling. The levels of arterial carbon dioxide (PaCO2) were determined simultaneously. In addition to examining the correlation between PET(CO2) and PaCO2, we investigated the influence of method of anesthesia, age and respiratory rate on the (PaCO2 - PET(CO2)) gradient. In most patients without nasal obstruction breathing through the nostril, PET(CO2) determined by selective nasal sampling was closer to PaCO2 than those by oral or nasal/oral sampling. Furthermore, the mean (PaCO2 - PET(CO2)) gradient was 4.98 mmHg in patients aged over 60, while it was 2.02 mmHg in patients aged under 60, suggesting that PET(CO2) could not be a good index in elderly people. There was no significant difference in the mean (PaCO2 - PET(CO2)) gradient among different methods of anesthesia. In conclusion, NODSS was useful in determining PET(CO2) more accurately and estimating PaCO2 precisely when used in relatively young people by selective nasal or oral sampling.

The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2).

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus. A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus. These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus, either as a member in the A-factor regulatory network or independently of this network.

Intra-arterial infusion therapy following alteration of pelvic blood flow and concurrent radiation therapy for invasive bladder cancer.

Intra-arterial infusion therapy following alteration of pelvic blood flow and concurrent radiation therapy was performed in 13 patients with muscle invading bladder cancer (T2, 2; T3, 6; T4, 5). The internal iliac artery of the opposite side was embolized and the ipsilateral gluteal and obturator arteries were embolized by metallic coils. A catheter was placed in the ipsilateral internal iliac artery. CDDP was administered daily at a dose of 7-9 mg/body over 1 minute. Radiation was done by Microtron using 10 MV x-ray. Total dose was 4500-7060 cGy. Evaluation was done by cystoscopy, radiography and biopsy. Eight patients achieved complete response (CR) histologically. Others had partial response (PR). All CR patients had no recurrence. The observation period was between 3 and 29 months, with a mean of 11 months. This treatment modality is effective for locally advanced bladder cancer.

[Change in serum prostate specific antigen values in men who had no evidence of prostate cancer on initial biopsy].

To determine the need for repeat prostatic biopsy, we retrospectively evaluated 19 men for longitudinal prostate specific antigen (PSA) for more than 20 weeks from initial biopsy. Their initial biopsy results revealed no evidence of prostatic cancer. They had no surgical therapy or hormone therapy after it. If they had a 50% increase in the PSA level from initial biopsy, we performed the second biopsy. Of the 5 men who had the second biopsy, 3 men had prostatic cancer.

Polarity orientation and assembly process of microtubule bundles in nocodazole-treated, MAP2c-transfected COS cells.

Microtubule bundles reminiscent of those found in neuronal processes are formed in fibroblasts and Sf9 cells that are transfected with the microtubule-associated proteins tau, MAP2, or MAP2c. To analyze the assembly process of these bundles and its relation to the microtubule polarity, we depolymerized the bundles formed in MAP2c-transfected COS cells using nocodazole, and observed the process of assembly of microtubule bundles after removal of the drug in cells microinjected with rhodamine-labeled tubulin. Within minutes of its removal, numerous short microtubule fragments were observed throughout the cytoplasm. These short fragments were randomly oriented and were already bundled. Somewhat longer, but still short bundles, were then found in the peripheral cytoplasm. These bundles became the primordium of the larger bundles, and gradually grew in length and width. The polarity orientation of microtubules in the reformed bundle as determined by "hook" procedure using electron microscope was uniform with the plus end distal to the cell nucleus. The results suggest that some mechanism(s) exists to orient the polarity of microtubules, which are not in direct continuity with the centrosome, during the formation of large bundles. The observed process presents a useful model system for studying the organization of microtubules that are not directly associated with the centrosomes, such as those observed in axons.

Changes in cerebral blood flow estimated after stellate ganglion block by single photon emission computed tomography.

The validity of the hypothesis that the cerebral vasculature is under the control of sympathetic innervation was investigated using brain scintigraphy imaging before and after stellate ganglion block (SGB). The experiment with HM-PAO showed a definite increase in the blood flow of the brain on the block side on both by the dynamic images and the SPECT images. The tympanic temperature (Tty) of the block side decreased significantly after SGB, compared to the unblock side in this study, as had been reported before. This change in Tty coinsided with the increase in cerebral blood flow as mentioned above. This study demonstrated that the cerebral vasculature is under the control of sympathetic innervation, the pathway of which is relayed and/or passes through the stellate ganglion. We conclude that SGB increases intracerebral blood flow and can also exert secondary effects systemically due to CNS blood flow changes as have been previously reported.

[Bladder tumor associated with von Recklinghausen's neurofibromatosis: a case report].

A 47-year-old female was admitted to our hospital complaining of macrohematuria. The patient had a history of von Recklinghausen's disease. Her skin showed multiple cafe-au-lait spots and neurofibromatosis. Thorough examinations were done. Urine cytology was positive. Intravenous pyelography and cystography demonstrated an irregular wall of the bladder. A computerized tomographic scan demonstrated a 7 cm nodular mass. Cystoscopy revealed a papillary tumor on the right lateral and anterior wall of the bladder. She was diagnosed as having a bladder tumor in von Recklinghausen's neurofibromatosis. Total cystectomy was performed. Histopathological diagnosis was transitional cell carcinoma with squamous cell carcinoma (Grade III, pT3N2N0). Fifty three cases of von Recklinghausen's disease in the literature were accompanied with malignancy.

[Investigation of ultrasound guided systematic biopsy of the prostate].

From August 1991 to February 1993, a total of 114 men with mean age of 68 years underwent ultrasound guided systematic biopsy of the prostate. These patients were clinically suspected of having prostate cancer by elevated PA or digital rectal examination. Systematic biopsies were performed on two different sites of peripheral zone and transition zone of each lobe, and echographically detected hypoechoic area was also biopsied. There were 42 prostate cancers detected: 7 (30%) in 23 men between 50 and 59 years old, 17 (45%) in 38 men between 70 and 79 years old, 6 (55%) in 11 men more than 80 years old. Three (11%) were detected in 27 men with a PA level of less than 3.1 ng/ml, 39 (45%) were detected in 89 men with a PA level of no less than 3.1 ng/ml. Thirty-three (44%) were detected in 75 men with positive rectal examination. Non-palpable cancers were detected in 9 men. These results suggest that systematic biopsy may be useful and a sensitive means in detecting prostatic cancer.