Orbit, a novel microtubule-associated protein essential for mitosis in Drosophila melanogaster.

We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner.

Murine DNA polymerase beta gene: mapping of transcription initiation sites and the nucleotide sequence of the putative promoter region.

The nucleotide sequence of the region (total, 2,512 base pairs [bp]) from intron 2 to the 5'-flanking region was determined for the mouse DNA polymerase beta genomic clone, and the 300-bp region from intron 1 to the 5'-flanking region was also sequenced for the rat clone. At 51 bp upstream from the ATG codon which was previously suggested to be the translation initiation codon for the rat cDNA sequence, we found another ATG in the same reading frame in both mouse and rat genes. Three major transcription initiation sites (cap sites) each for rat and mouse DNA polymerase beta mRNAs were localized precisely by primer extension analysis at 51, 41, and 0 bp upstream from the first ATG codon, suggesting that this codon is used for translation initiation. The 400-bp region around exon 1 was extremely G + C rich (about 70%). Although neither a TATA box nor a CAAT box was found within the 500-bp region upstream of the 5'-most cap site, triple repeats of 5'-CCGCCC were found within the 100-bp region flanking the cap site.

A distinct function of the retinoblastoma protein in the control of lipid composition identified by lipidomic profiling.

Here, by combining lipidomics with transcriptome analysis, we demonstrate that Rb depletion in mouse embryonic fibroblastss induces significant alterations in their lipid composition. We discovered that Rb depletion induced increase in lysophosphatidylserine, diacylglycerol (DAG), fatty acid (FA), acylcarnitine, phosphatidylcholine (PC), arachidonoyl ethanolamine, and decrease in phosphatidylglycerol, monoacylglycerol, without change in total lipid per protein levels. Analysis of the acyl chain composition of DAG, PC and phosphatidylserine revealed increase of saturated and mono-unsaturated acyl chains with specific carbon chain length. Consistently, we observed that Rb depletion increased the levels of fatty acids with the corresponding carbon chain length and number of carbon-carbon double bondssuch as myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0) and all forms of FA 18:1. Microarray analysis revealed that Rb depletion induced significant upregulation of enzymes involved in elongation and desaturation of fatty acids. Among these, we found that elongation of long chain fatty acid family member 6 (Elovl6) and stearoyl-CoA desaturase 1 (Scd1) are the most robustly controlled by Rb possibly through E2F and sterol regulatory element-binding protein transcription factors. Depletion of Elovl6 or Scd1 significantly suppressed colony formation, sphere formation and xenograft tumor growth of Rb-deficient tumor cells. Suppression of self-renewal by the SCD1 inhibitor was rescued upon supplementation of the mono-unsaturated fatty acids generated by this enzyme. This study suggests a novel role for Rb in suppressing the malignant progression of tumors by controlling the lipid composition.

The RB-IL-6 axis controls self-renewal and endocrine therapy resistance by fine-tuning mitochondrial activity.

Retinoblastoma (RB) protein inactivation during tumor progression is often associated with acquisition of immature phenotypes and resistance to therapy. Determination of an RB inactivation signature in a context of gaining undifferentiated phenotype in a p53-null sarcoma system revealed a critical role for interleukin (IL)-6. Using a Gene Set Enrichment Analysis (GSEA), we discovered that poorly differentiated breast cancers are enriched for this RB inactivation signature. Accelerated IL-6 secretion following RB inactivation in an RB-intact luminal-type breast cancer cell line MCF-7 promoted a positive feed forward loop between IL-6 and STAT3 driving tumor growth and endocrine therapy resistance. In addition, some of RB-intact basal-like type breast cancer cell lines exhibited a similar phenotype following RB depletion. The mechanism whereby RB inactivation increases IL-6 production in MCF-7 cells appeared to involve fatty acid oxidation (FAO)-dependent mitochondrial metabolism and c-Jun NH(2)-terminal kinase (JNK). In addition, IL-6, via STAT3-mediated feedback to mitochondria, autonomously adjusts mitochondrial superoxide to levels suitable to maintain stem cell-like activity. The gene expression profile of luminal-type breast cancer patients with low RB expression revealed high enrichment of genes involved in mitochondrial respiration and downstream targets of IL-6. These findings unveiled an unexpected strategy whereby RB suppresses malignant features of cancer cells through metabolic reprogramming and cell-autonomous inflammation.

Relationship between real-time monitoring of the graft motility and mucosal histology in swine intestinal transplantation.

We studied the correlation between the motility and the mucosal histology of the small bowel seeking to detect rejection in an early stage by real-time monitoring using a swine model. Intestinal transplantation (ITx) was performed orthotopically using FK506 immunosuppression. The distal about 20 cm segment of the allograft was exteriorized as a Thiry-Vella stoma for biopsies. Strain gauge (SG) force transducers were attached to the graft for real-time monitoring of graft motility. Pigs without ITx were used as controls (group 1). Rejection was classified into four groups by histologic findings: nonrejection (group 2), mild rejection (group 3), moderate rejection (group 4), and severe rejection (group 5). Migrating motor complex (MMC) phase III was analyzed for the following parameters: duration, amplitude, interval, motility index, velocity, and frequency of propagation. In group 2, all parameters were almost the same as those for group 1. In contrast, groups 4 and 5 showed most parameters significantly lower than those in group 1. In group 3, the contractility of the MMC was not significantly altered, but the frequency of the propagation was decreased significantly. In conclusion, graft motility detected by a real-time SG method correlated with the grade of mucosal histology. This method is useful to detect rejection at an early stage by examining the frequency of MMC propagation.

Effects of hyperthermia on cell surface charge and cell survival in mastocytoma cells.

Changes in the structure of the surface of mastocytoma cells were induced by hyperthermia and were investigated by means of cell electrophoresis. A decrease in the cell electrophoretic mobility was detected as early as 15 min after treatment at 42 degrees and progressed more rapidly under hypoxic conditions than under oxic conditions. Subsequent recovery of electrophoretic mobility at 37 degrees was dependent on the length of heat treatment and oxygenation. The surviving fraction of cells detected by their colony-forming ability and the fraction of electrophoretically recovered cells 24 hr after exposure to hyperthermia showed a good statistical correlation. It was suggested that the mechanism of electrophoretic mobility reduction by heating was the vertical translocation of hyaluronidase-sensitive charge from the peripheral layer into a deeper layer by combined use of specific enzymes and stepwise different ionic strengths. These results suggest the importance of irreparable changes of membrane conformation in the loss of colony-forming ability of heated tumor cells.

[Quality control (QC) of CT on rail system (FOCAL Unit) with a micro-multi leaf collimator (mMLC) using new GafChromic film for stereotactic radiotherapy].

PURPOSE: Recent years, CT on rail system was reported to be useful as a tool for image-guided radiotherapy (IGRT). This system was clinically developed with the aim of stereotactic irradiation (STI) for brain, lung, liver, prostate and other sites. Quality assurance and quality control (QC) is an important issue in CT on rail system to assure geometric accuracies. The purpose of this study is to estimate the geometric accuracies of our CT on rail system using a detachable micro-multi leaf collimator (mMLC) with new type radiochromic films. Carrying out our original QC program, translational errors, setup reproducibility, beam misalignment and beam characteristics were evaluated. METHODS AND MATERIALS: We have studied with CT on rail system (FOCAL unit, Toshiba Medical systems, Tokyo, Japan) and mMLC unit (Accuknife, Direx Inc., Tokyo, Japan). We have developed original alignment phantom and small steel markers (2 mm phi) were implanted on its surface at certain intervals. Firstly, we have evaluated the accuracy of self-moving CT gantry and CT resolutions for cranio-caudal directions by changing slice thickness. And then using the phantom, we have measured the accuracy and reproducibility of geometric isocenter of the linac side and the CT gantry side by scanning the phantom. We have also measured the geometric changes of the common treatment couch by weight-loaded test (up to 135 kgw). To estimate dosimetric and geometric accuracies with the mMLC unit, the misalignment of the beam axes (gantry, collimator and couch rotation axis), mMLC leaf positions, and dose distributions for the verification plan were measured with new type GafChromic films (GafChromic-RTQA, ISP Inc., USA) and cylindrical phantom. The dose characteristics of the GafChromic film were also evaluated. RESULTS: The reproducibility of the self-moving CT gantry have a good agreement within 1 mm. Weight-load test have shown a good reliability within 2 mm at the common treatment couch. The translational precision of the common treatment couch was 0.0 +/- 0.1 mm at linac side and -0.2 +/- 0.5 mm at CT gantry side. The misalignments of beam axes have been kept within 0.4 mm at maximum. Gap test have shown the accuracies of the mMLC leaf positions, which is needed to keep within 1 mm by a routine calibration. CONCLUSIONS: To practice quality control program for the FOCAL unit and the mMLC unit is essential for a regular interval to reduce systematic errors. New type radiochromic film would be useful for a verification tool as alternative to conventional film.

Depth dependence of the perturbing effect of placing a bulky group (oxazolidine ring spin labels) in the membrane on the membrane phase transition.

Electron paramagnetic resonance (EPR) and differential scanning calorimetry (DSC) have been used to study the effect on the phase transition of dimyristoylphosphatidylcholine membranes of incorporating various stearic acid spin labels (SASL's) that contain the bulky oxazolidine ring at various positions along the stearyl chain. SASL's lowered the phase transition temperature and decreased the size of the cooperative unit, with the effects stronger in the order of 9- > 12- > 5- > 16-SASL > stearic acid (no label). Incorporation of stearic acid without the spin label slightly increases the phase transition temperature. Incorporation of 9-SASL (3 mol% of lipid) decreased the transition temperature by 1.8 degrees C and the cooperative unit to 1/5 of that without the spin label, while the effect of 16-SASL was slight. The effect on transition enthalpy was small. It is concluded that the perturbing effect of placing a bulky group on the alkyl chain on phase transition is through inducing packing defects in the gel-phase.

Analysis of the fatty acid components in a perchloric acid-soluble protein.

We had previously found that a perchloric acid-soluble protein (PSP1) occurs in rat liver, and that this novel protein inhibits protein synthesis in a rabbit reticulocyte lysate system (T. Oka, H. Tsuji, C. Noda, K. Sakai, Y.-H. Hong, I. Suzuki, S. Muñoz, Y. Natori, J. Biol. Chem. 270 (1995) 30060-30067). In the present study, we analyzed lipid components bound to PSP1. Native PSP1 was purified from rat liver using Sephadex G-75, DE-52 cellulose and IgGPSP-affinity chromatography, and the lipid components were extracted. The components obtained from the purified PSP1 were shown to be free fatty acids by thin-layer chromatography. By GC-MS, six major fatty acids were identified as 14:0, 16:0, 18:0, 18:1, 18:2 and 20:4. 1 mol of PSP1 contained 1.26 mol of total fatty acid components. The fatty acid-binding assay of PSP1 showed that the Bmax was 1.25 mol fatty acid/mol PSP1 and the Kd value for palmitic acid was 6.03 microM. The concentration of PSP1 mRNA in rat liver increased 2.3-fold by the administration of peroxisome proliferator, bezafibrate. These findings show that PSP1 is a fatty acid-binding protein-like protein, which is involved in the intracellular metabolism of fatty acid and is quite different from the known fatty acid-binding proteins.

Enhanced secretion of insulin plays a role in the development of atherosclerosis and restenosis of coronary arteries: elective percutaneous transluminal coronary angioplasty in patients with effort angina.

OBJECTIVES: We investigated the relation between insulin and coronary atherosclerosis and restenosis of the coronary arteries, by performing elective percutaneous transluminal coronary angioplasty (PTCA). BACKGROUND: Insulin is known to promote atherosclerosis of the arteries and has been implicated in the development of restenosis after PTCA. METHODS: Of 210 angina patients who underwent PTCA, newly detected lesions in 35 consecutive nondiabetic subjects without previous intervention on the same main coronary arteries were analyzed after a 75-g oral glucose tolerance test (OGTT) and follow-up coronary angiography. Atherosclerotic lesions were evaluated by pattern, severity and extent. Restenosis was defined as loss of gain, the percentage of loss of the initial gain in the coronary diameter achieved by PTCA > or = 50%. RESULTS: Patients with restenosis had a significantly higher extent index (a marker of atherosclerosis), insulin area, ratio of insulin area to glucose area, insulinogenic index and minimal lumen diameter after PTCA than those without restenosis (p=0.001, 0.011, 0.002, 0.016 and 0.041, respectively). Simple regression analysis revealed that only the ratio of insulin area to glucose area (a relative marker of enhanced insulin secretion) significantly correlated with the extent index (p=0.035). Extent index, insulin area, the ratio of insulin area to glucose area and insulinogenic index significantly correlated with loss of gain (p=0.001, 0.010, 0.002 and 0.032, respectively). Stepwise multiple regression analyses revealed that extent index and the ratio of insulin area to glucose area significantly correlated with loss of gain. CONCLUSIONS: Enhanced secretion of insulin during the OGTT might be useful as a predictor of coronary atherosclerosis and of restenosis after elective PTCA in nondiabetic patients with effort angina.

Crystallization of 31 kDa C-terminal fragment of rat DNA polymerase beta.

A 248 residue C-terminal fragment of rat DNA polymerase beta (335 amino acid residues), a eukaryotic DNA repair enzyme, has been crystallized from polyethylene glycol 6000 solution. The crystals are orthorhombic, space group P2(1)2(1)2 with cell dimensions a = 120.3 A, b = 64.2 A, c = 39.4 A, and contain a single 31 kDa fragment in an asymmetric unit. The crystals diffract to 2.8 A resolution with laboratory X-ray source, and to 2.3 A resolution with synchrotron X-ray source, and are suitable for detailed structural analysis.

Diabetes mellitus and its vascular complications in Japanese migrants on the Island of Hawaii.

Japanese migrants and their offspring on the island of Hawaii and Japanese living in Hiroshima were examined for diabetes mellitus and its vascular complications. the same methods and investigators were used in both locations. Death certificates of Japanese and Caucasians dying on the island during the past 26 yr were analyzed. Diabetes, defined as a venous serum glucose concentration of at least 200 mg/dl 2 h after a 50-g oral glucose load, was significantly more common in the Hawaiian Japanese than in the Hiroshima Japanese subjects. This suggests that diabetes is more prevalent in Japanese in Hawaii than in Japan, although lack of knowledge about the total population of Japanese migrants in Hawaii makes this generalization uncertain. The proportion of deaths attributed to diabetes was much higher in Japanese migrants and their offspring in Hawaii than in Japan. During the 1950s, the proportional death rate from diabetes was about half as large in Japanese Hawaiians as in Caucasian Hawaiians, but it increased to become 1.6 times the Caucasian rate during the 1970s. A nutritional study revealed that the total caloric intake was similar in Japanese in Hawaii and Hiroshima, although the estimated level of physical activity was less in the Hawaiian subjects. Consumption of animal fat and simple carbohydrates (sucrose and fructose) were at least twice as high in Hawaiian as in Hiroshima Japanese. Conversely, Hiroshima Japanese consumed about twice the amount of complex carbohydrate as the Hawaiian Japanese. These observations support the hypothesis that a high fat, high simple carbohydrate, low complex carbohydrate diet and/or reduced levels of physical activity increase risk of diabetes. The proportion of deaths attributed to ischemic heart disease was higher in both diabetic and nondiabetic Japanese Hawaiians than in diabetic subjects in Japan. The rates were similar for Japanese and Caucasians in Hawaii. There was no evidence of an environmental influence on the development of microangiopathy (retinopathy) in diabetes, as the prevalence of diabetic retinopathy (stratified for diabetes duration) was similar in Japanese subjects in Hawaii and in Japan, and it was similar to previous reports from England. On the other hand, diabetes alone did not appear to account for the greater prevalence of macroangiopathy in Hawaiian Japanese than in Hiroshima. Thus environmental factors, possibly including diet, appear to be involved in the development of macrovascular complications of diabetes.

Usefulness of anti-GQ1b IgG antibody testing in Fisher syndrome compared with cerebrospinal fluid examination.

Fisher syndrome (FS), a variant of Guillain-Barré syndrome (GBS), is a rare disorder, and there are few reported studies of a large number of patients with FS. Cerebrospinal fluid (CSF) albuminocytological dissociation was found in 59% of 123 FS patients during the first 3 weeks of illness, while serum anti-GQ1b IgG antibody was positive in 85%. Whereas the incidence of CSF albuminocytological dissociation increased from the first to second weeks in FS, anti-GQ1b IgG antibody peaked in the first week, but there was no CSF albuminocytological dissociation. Statistically, anti-GQ1b antibody testing was superior to a CSF examination in supporting a diagnosis of FS during the first 3 weeks of illness, especially in the first week.

Coprecipitation of 3'----5' exonuclease and DNA polymerase gamma with anti-DNA polymerase gamma antibody.

Highly purified preparations of chick embryo DNA polymerase gamma contained 3'----5' exonuclease activity which might be responsible for the exonucleolytic proofreading during DNA synthesis [Kunkel, T.A. & Soni, A. (1988) J. Biol. Chem. 262, 4450-4459]. A rabbit antibody produced against highly purified chick DNA polymerase gamma precipitated 3'----5' exonuclease activity to the same extent as DNA polymerase gamma activity. Furthermore, the antibody neutralized the two enzyme activities to an equal extent. However, the exonuclease activity was more resistant than DNA polymerase gamma activity to thermal treatment at 50 degrees C, although both activities were partially protected with polynucleotides. The results obtained suggest that these two enzymes are associated as a single enzyme complex or that the two activities reside in a single molecule, and the active site of DNA polymerase gamma and 3'----5' exonuclease are, although not identical, closely correlated.

Preoperative cisplatin for initial treatment of limb osteosarcoma: its local effect and impact on prognosis.

PURPOSE: The significance of preoperative cisplatin (CDDP) as a single agent has not been assessed in terms of its effect on prognosis. The purpose of this multi-institution study was to assess the local effect of preoperative CDDP as a single agent as well as its impact on the prognosis of limb osteosarcoma. PATIENTS AND METHODS: The study group comprised 44 patients with stage IIB limb osteosarcoma who were treated with single-agent CDDP as initial preoperative chemotherapy. Two to five courses of CDDP (mean 2.4 courses) were administered intravenously and/or intraarterially as an initial preoperative treatment. The mean dose of CDDP was 3.0 mg/kg (2.5-3.4 mg/kg). The effect of the treatment was evaluated clinically and histologically. RESULTS: The clinical and histological response rates to preoperative CDDP were 56.8% and 47.6%, respectively. The survival rate was 59.1% among all patients in the study, 64.0% among those with a grade III or IV clinical response, and 52.6% among those with a grade I or II clinical response, with no significant differences between the groups. The survival rate was 70% among patients with a grade III or IV histological response, and 54.5% among those with a grade I or II histological response, with no statistical differences between the groups. CONCLUSIONS: We consider that CDDP is a useful chemotherapeutic agent for preoperative induction therapy for osteosarcoma because of the excellent local effect observed. Good responders to preoperative CDDP showed a better survival rate, but a correlation between the local response to CDDP and the survival rate was not demonstrated statistically. Systemic multidrug chemotherapy should follow preoperative CDDP to diminish the microscopic foci of metastatic disease.

Uncoupling of incorporation of ascorbic acid and apoptosis induction.

Exposure of human promyelocytic leukemic HL-60 cells to millimolar concentration of sodium ascorbate induced apoptotic cell death. The extent of apoptosis induction was a positive function of temperature at the time of exposure. The incorporation of [1-14C] ascorbic acid into the cytosolic fraction of HL-60 cells was also temperature-dependent, and competitively inhibited by active analogs (L-ascorbic acid, sodium L-ascorbate, D-isoascorbic acid, sodium 6-beta-O-galactosyl-L-ascorbate, sodium 5,6-benzylidene-L-ascorbate), but not by inactive analogs (L-ascorbic acid-2-phosphate magnesium, L-ascorbic acid 2-sulfate). Calcium depletion, which had considerably reduced the apoptosis-inducing activity of sodium ascorbate, did not affect the intracellular incorporation of [14C] ascorbic acid. These data suggests that cell death might not be simply induced by the intracellular incorporation of ascorbate, but rather initiated by the rapid elevation of intracellular Ca2+ concentration, possibly mediated by an as yet unidentified temperature-sensitive mechanism.

Calcium mobilization during nicotine-induced cell death in human glioma and glioblastoma cell lines.

Nicotine dose-dependently induced cytotoxicity in human glioma (KG-1-C) and glioblastoma (GBS-1, T98G) cell lines, but could not induce internucleosomal DNA cleavage, in contrast to apoptosing human myelogenous leukemic cell lines. Human glioma/glioblastoma cell lines thus might have a chromatin structure resistant to endonuclease digestion. Nicotine induced a rapid increase in the intracellular calcium concentration. Confocal experiments with Fluo-3 fluorescence revealed that nicotine elevated the free Ca2+ concentration in both nuclear and cytoplasmic regions of the cells, and the elevation of Ca2+ in the nuclear region was more pronounced than that of the cytoplasmic region. The present study suggests that nuclear accumulation of Ca2+ is an important initial step for cell death induction by nicotine.

The development of a pollen information system for the improvement of QOL.

The Kochi Prefecture Japanese Cedar and Cypress Pollen Information System (P-Net Kochi) was established in 1991 on the initiative of the Pharmaceuticals and Sanitation Division to improve the quality of life of people in Kochi Prefecture, particularly patients with pollinosis. Kochi Prefecture has the highest forests percentage (84%) in Japan. In addition, 40% of the population of the prefecture is concentrated in Kochi City. The average pollen count at 10 observation points an one year was about 20,000/cm2 during the observation period, but it exceeded 110,000/cm2 in 1995, when it was also high nationwide. Kochi Prefecture organized a system to promote people's understanding of pollinosis in connection with environmental problems, and to enlighten people on appropriate preventive measures. The system has been improved in the rapid and efficient transmission of information over the years with technological advances. The introduction of an automatic monitoring system not dependent on human labor and the support of the users proved to be indispensable for the maintenance of the system.

Protective effect of sulfobutyl ether beta-cyclodextrin on DY-9760e-induced hemolysis in vitro.

The hemolytic behavior of a novel cytoprotective agent, DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) was investigated using rabbit erythrocytes. Further, the effects of water-soluble cyclodextrin derivatives, such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and sulfobutyl ether of beta-cyclodextrin (SBE-beta-CyD), on the hemolytic activity of DY-9760e were studied. DY-9760e induced hemolysis at concentrations >0.2-0.3 mM in phosphate buffered saline (PBS) of pH 4.0 and 6.0, where DY-9760e is predominantly in dicationic and monocationic forms, respectively. The hemolytic activity of the monocationic DY-9760e was higher than that of the dicationic species, and the hemolysis at pH 4.0 involved the formation of methemoglobin. DY9760e induced the morphological change of erythrocytes towards membrane invagination at both pH 4.0 and 6.0. SBE7-beta-CyD significantly suppressed the DY-9760e-induced hemolysis and morphological change at both pH 4.0 and 6.0, as well as the formation of methemoglobin at pH 4.0. On the other hand, HP-beta-CyD suppressed only the hemolysis, but neither the morphological change nor the formation of methemoglobin. In addition, the inhibitory effect of SBE7-beta-CyD on the hemolysis was greater than that of HP-beta-CyD. The superior inhibitory effect of SBE7-beta-CyD on the DY-9760-induced hemolysis, the morphological change, and the formation of methemoglobin may be attributable to the formation of a stable inclusion complex with DY-9760e and to the weaker hemolytic activity of SBE7beta-CyD than HP-beta-CyD. These results suggest potential use of SBE7-beta-CyD as a parenteral carrier for DY-9760e.

A preferential isolation procedure for asporogenous Gram-positive bacteria.

A preferential isolation procedure was devised for asporogenous (Asp), Gram-positive (Gp), aerobic or facultative anaerobic bacteria which included the genera Arthrobacter, Corynebacterium, Brevibacterium, Microbacterium, Mycobacterium, and Micrococcus (Asp-Gp bacteria). An antibiotics-mixture agar which contained 5 to 10 micrograms per ml of colistin, 10 to 20 micrograms per ml of nalidixic acid and 30 micrograms per ml of cycloheximide was used in the isolation. Using this technique 47 Asp-Gp bacteria representing 26 subgroups of coryneform bacteria and Micrococcus were isolated from 3 soil samples. The method was far more efficient than the standard dilution-plate technique. This preferential method is available to isolate Asp-Gp bacteria from a sample containing about 500-fold more of other Gram-positive and negative bacteria.