Root colonization by the endophytic fungus Piriformospora indica shortens the juvenile phase of Piper nigrum L. by fine tuning the floral promotion pathways.

Piriformospora indica, the mutualistic biotrophic root colonizing endosymbiotic fungus belonging to the order Sebacinales, offers host plants various benefits and enhances its growth and performance. The effect of colonization of P. indica in Piper nigrum L. cv. Panniyur1 on growth advantages, floral induction and evocation was investigated. Growth and yield benefits are credited to the alteration in the phytohormone levels fine-tuned by plants in response to the fungal colonization and perpetuation. The remarkable upregulation in the phytohormone levels, as estimated by LC- MS/MS and quantified by qRT-PCR, revealed the effectual contribution by the endophyte. qRT-PCR results revealed a significant shift in the expression of putative flowering regulatory genes in the photoperiod induction pathway (FLOWERING LOCUS T, LEAFY, APETALA1, AGAMOUS, SUPPRESSOR OF CONSTANS 1, GIGANTEA, PHYTOCHROMEA, and CRYPTOCHROME1) gibberellin biosynthetic pathway genes (GIBBERELLIN 20-OXIDASE2, GIBBERELLIN 2-OXIDASE, DELLA PROTEIN REPRESSOR OF GA1-3 1) autonomous (FLOWERING LOCUS C, FLOWERING LOCUS VE, FLOWERING LOCUS CA), and age pathway (SQUAMOSA PROMOTER LIKE9, APETALA2). The endophytic colonization had no effect on vernalization (FLOWERING LOCUS C) or biotic stress pathways (SALICYLIC ACID INDUCTION DEFICIENT 2, WRKY family transcription factor 22). The data suggest that P. nigrum responds positively to P. indica colonization, affecting preponement in floral induction as well as evocation, and thereby shortening the juvenile phase of the crop.

A New Technique to Induce Experimental Myointimal Hyperplasia.

BACKGROUND: Arterial myointimal hyperplasia (MIH) has a significant impact on the long-term outcomes of vascular procedures such as bypass surgery and angioplasty. In this study, we describe a new and innovative technique to induce MIH using a dental flossing cachet in Wistar rats. METHODS: The intimal damage in the common carotid artery was induced by inserting the tip of the dental flossing cachet through the external carotid artery into the common carotid artery and turning it on for 3 rounds of 20 s each (n = 10). After 2 weeks, the rats were anesthetized and the common carotid arteries of the experimental side and the contralateral side (control) were harvested and preserved for histopathological studies. RESULTS: The experimental carotid arteries showed significant intimal proliferation and thickening compared to the controls. The intima/media ratio of the experimental and normal (control) common carotid arteries were 1.274 ± 0.162 and 0.089 ± 0.023 (mean ± SEM), respectively (p < 0.001). CONCLUSION: This technique is simple, inexpensive, and highly reproducible and it induces sufficient MIH to study this phenomenon in animal models.

Microsurgical skill assessment: toward skill-based surgical robotic control.

A surgical skill assessment system was developed to quantify microsurgical skills. Infrared optical makers, an inertial measurement unit, and strain gauges were mounted on tweezers to record surgical tasks. In preliminary experiments, the tool tip trajectory, acceleration, and applied force were measured and microsurgery videos were evaluated by three expert surgeons. The preliminary results indicated the feasibility of the system by showing the significant difference between unskilled and skilled surgeons.

Incidence of von Hippel-Lindau disease in hemangioblastoma patients: the University of Tokyo Hospital experience from 1954-1998.

BACKGROUND: Incidence of von Hippel-Lindau disease among hemangioblastomas is important clinical information affecting the management of hemangioblastomas. Studies from Western countries reported 36-40% for the incidence, but no report has been made on the Japanese population. METHOD: To investigate the incidence in Japan, we retrospectively analyzed all hemangioblastoma patients treated at The University of Tokyo Hospital from 1954 to 1998. By reviewing medical records and imaging studies, von Hippel-Lindau disease was diagnosed clinically following the currently suggested diagnostic criteria. FINDINGS: There were 82 hemangioblastoma patients recorded during the period, and 14 cases (17%) were compatible with von Hippel-Lindau disease. However, when the incidence was calculated for each of the three 15-year periods, which are 1954-1968 (first), 1969-1984 (second), and 1985-1998 (third), the number increased dramatically in the later periods: 2 of 33 (6%) during the first, 4 of 26 (15%) during the second, and 8 of 22 (36%) during the third period. Such increase occurred after the introduction of whole body CT to our institution in 1981, suggesting that improvement of imaging techniques contributed to the sensitivity of diagnosis. In addition, one recent patient with multiple hemangioblastomas was found to harbor germline mutation of the VHL, thereby being diagnosed as von Hippel-Lindau disease on the basis of molecular genetics. INTERPRETATION: The 40% incidence of von Hippel-Lindau disease in hemangioblastomas suggests that extensive screening for von Hippel-Lindau disease associated neoplasms, and probably molecular genetic examination, is indicated for all patients with hemangioblastomas, which should aim for earlier diagnosis and better management of this devastating hereditary disease.

[A case of sacral meningeal cyst with Marfan syndrome].

A rare case of sacral meningeal cyst Marfan syndrome is reported. A 40-year-old male who had a past history of Marfan syndrome was admitted to Tokyo Metropolitan Ebara Hospital due to sensory disturbance in the right S1-2 nerve root area on October 1998. On admission, neurological deficits were sensory disturbance and decrease right Achilles reflex. Plain sacral X-ray was normal. Magnetic resonance imaging revealed a cystic mass in the intrasacral space. MR myelography showed the cyst followed the thecal sac. Signal intensity of the cystic mass was the same as the cerebrospinal fluid. In the prone position, laminectomy of the sacrum was carried out. The neck of the meningeal cyst was ligated with the help of an aneurysmal needle. The patient's sensory disturbance disappeared postoperatively. As MRI becomes more frequently used in Marfan syndrome, it is important to keep the presence of such an entity in mind. MR myelography made it easy to diagnose the meningeal cyst.

Precocious puberty in a girl with an hCG-secreting suprasellar immature teratoma. Case report.

Although precocious puberty is common in boys with human chorionic gonadotropin (hCG)-secreting brain tumors, it is extremely rare in girls. The authors describe a 6-year-old girl with an hCG-secreting suprasellar immature teratoma who presented with diabetes insipidus, increased intracranial pressure, and precocious puberty. On admission, breast budding was observed. The serum hCG level was 1230 mIU/ml. Both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) remained below detectable levels, even after gonadotropin-releasing hormone stimulation. Serum estrogen and androgen were moderately elevated. After chemotherapy, breast budding disappeared with normalization of serum hCG. It has been believed that hCG does not produce precocious puberty in girls in the absence of FSH, and this has been used as an explanation for the rarity of precocious puberty in girls with hCG-secreting brain tumors. However, it has also been reported that hCG has not only LH activity but also intrinsic, although weak, FSH-like activity. In the present case, this FSH-like activity was considered to have played a role in the development of precocious puberty. It is speculated that a very high level of serum hCG can produce precocious puberty in girls. The rarity of intracranial germ-cell tumors with a high potential of hCG secretion may be one of the reasons why hCG-induced precocious puberty is uncommon in girls.

A gene tightly linked to CEN6 is important for growth of Saccharomyces cerevisiae.

Transcriptional analysis of the region flanking the left boundary of the centromere of chromosome VI revealed the presence of a gene immediately adjacent to CEN6. The transcription of the gene is directed toward the centromere, and nucleotide sequence analysis showed that the coding region terminates only 50 bp away from CEN6. Our results extend to chromosome VI the observation that centromere-flanking regions of S. cerevisiae are transcriptionally active. Disruption of the coding region of the gene showed that its product, whilst not essential for cell viability, is important for normal cell growth. The gene has been termed DEG1 (DEpressed Growth rate). Comparison of the deduced amino acid sequence of DEG1 with a protein sequence databank revealed homology with the enzyme tRNA pseudouridine synthase I of E. coli.

Preparation of high molecular weight plant DNA and its use for artificial chromosome construction.

The availability of a substantial amount of high molecular weight DNA is an essential prerequisite for the construction of yeast artificial chromosome (YAC) libraries. Parameters concerning protoplast isolation and DNA extraction have been systematically analyzed. Conditions have been established for the obtainment of high molecular weight DNA from Arabidopsis thaliana and Nicotiana plumbaginifolia protoplasts either embedded in agarose plugs or in liquid suspension. Restriction fragments were obtained by partial and total digestion with different endonucleases, and separated by pulsed-field gel electrophoresis. Ligation of partially EcoRI-digested DNA (range 30-300 kbp) followed by transformation of yeast spheroplasts gave rise to YACs with an average size of 60 kbp. The introduction of a DNA size-selection step before ligation led to production of YACs in the range of 100-200 kbp. Clones of up to 460 kbp were obtained by blunt-end ligation of pre-selected unrestricted DNA.

A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes.

A novel procedure is described for the cloning of partial EcoRI fragments of bovine DNA: it reduces the chance of sequence rearrangements due to multiple insertions (co-cloning) of restriction fragments in the resulting YAC. The DNA to be inserted has been dephosphorylated, whereas the matching ends of the vector, pYAC4, have not. The ligation was essentially complete, the transformation efficiency was close to 19 transformants per ng of vector and the frequency of clones carrying YAC, 60-100 kb in size, was close to 70%. The YACs show segregative and replicative stability.

Induction of meiotic chromosomal malsegregation in yeast.

A system to detect chromosome number abnormalities occurring during meiosis in Saccharomyces cerevisiae is described. It is based on selection of spores carrying 2 multi-marked chromosomes V. Each step of the technical procedure is critically analyzed and the origin of some biases discussed. Selection and subsequent genetic analysis allow the estimation of the frequency of spontaneous and induced diploid and aneuploid n + 1 (diplo-V) spores. Data are reported concerning the effect of 53 chemical compounds. The great majority of active chemicals induce diploid clones while a minority cause non-disjunction of chromosome V.

Caffeine interactions with methyl methanesulphonate, hycanthone, benlate, and cadmium chloride in chromosomal meiotic segregation of Saccharomyces cerevisiae.

Interactions of caffeine with chemicals known for their effects on chromosomal segregation during meiosis of Saccharomyces cerevisiae were studied. It appears that caffeine does interfere with the action of other compounds during the different phases of meiosis. Treatments with methyl methanesulphonate (MMS) and cadmium chloride (CdCl2) resulted in a synergistic effect consisting of an increase in the frequency of recombination. The greatest effects were found on the induction of diploid spores: MMS, hycanthone, and distamycin demonstrated strong, benlate little synergistic action. CdCl2 demonstrated antagonism to caffeine by counter-inhibiting its effect on the induction of diploids. Concerning disomic induction: caffeine reduced (or left unchanged) the effect on non-disjunction when MMS and hycanthone were used. Simple additive effects were caused in conjunction with distamycin, benlate, and (in small doses) CdCl2. 2 mg of caffeine/ml in treatments with CdCl2 resulted in a very high frequency of disomic clones.

Disomic and diploid meiotic products induced in Saccharomyces cerevisiae by the salts of 27 elements.

The effects of salts of 27 elements on recombination and on the production of disomic and/or diploid spores during meiosis of Saccharomyces cerevisiae has been investigated. Be(NO3)2, MgSO4, FeSO4, CuSO4, AgNO3, Na2HAsO4 were inactive on the events studied during meiotic cell division. AuCl4, CdCl2, C4H6O4Pb, SnCl2, K2Cr2O7, RbCl induced both disomic and diploid spores. LiCl acted similarly and also affected recombination. Activity in the induction of disomic spores was shown by MnSO4, HgCl2 and SrCl2. CsCl, CaCl2, Na2MoO4, NiCl2, K2PtCl4 increased the frequency of diploid spores, while NaWO4, VOSO4, KCl, BaCl2 already increased recombination frequency. NaBiO3 showed an effect on meiotic recombination only. A decrease in the occurrence of both diploid and disomic spores was suggested by the data obtained with CoCl2.

Effects of some DNA-ligands and of some mutagenic compounds on the induction of meiotic disomic or diploid yeast products.

Data were collected following the administration of various compounds to meiotic yeast cultures to investigate their effects on disomic or diploid induction. Epi chlorohydrin, actinomycin D, and 9-aminoacridine were unable to induce either diploidy or disomy. Ethyl methanesulphonate was active in inducing diploids, while hycanthone induced disomics. beta-Propiolactone, N-methyl-N'-nitro-N-nitro-soguanidine, and beta-naphtylamine were able to increase both disomy and diploidy.

Drug synergism or antagonism in the induction of diploid meiotic products in Saccharomyces cerevisiae.

The induction of yeast diploid meiotic products by treatment with bleomycin and mitomycin C is reduced when sporulating cells are treated in combination with propranolol, and increased when they are treated in combination with caffeine. We show that bleomycin and mitomycin C act by blocking the second meiotic division. The frequency of this event appears to be directly related to the intracellular cAMP concentration which is known to be influenced in opposite ways by caffeine and propranolol.

Disomic and diploid meiotic products in Saccharomyces cerevisiae. Effect of vincristine, vinblastine, adriamycin, bleomycin, mitomycin C and cyclophosphamide.

The effect of some antineoplastic drugs on the induction of disomic and diploid meiotic products in Saccharomyces cerevisiae was evaluated. Vincristine and vinblastine turned out not to be effective on any of the four genetic phenomena studied (sporulation, recombination, disomic induction and diploid induction). Adriamycin showed only slight activity in inducing diploids, particularly during the first meiotic division. Cyclophosphamide was active on both phenomena leading to the formation of disomic and diploid spores. Mitomycin C and bleomycin were effective on the induction of diploids. In all of these inductions, the origin of diploids was due to failure of the second meiotic division. No significant effects were found on recombination frequency. As a general conclusion, one may assume that formation of aneuploid and diploid gametes are two distinct phenomena, not necessarily correlated from the point of view of the mechanism and of the specificity of induction.

Propranolol, atenolol, and trifluoperazine reduce the spontaneous occurrence of meiotic diploid products in Saccharomyces cerevisiae.

The effect of atenolol, propranolol, trifluoperazine, and caffeine on the occurrence of meiotic diploid and disomic products in Saccharomyces cerevisiae was investigated. We demonstrated that atenolol, propranolol, and trifluoperazine reduce the occurrence of meiotic diploid products and that propranolol also slightly decreases the spontaneous frequency of disomics. On the other hand, caffeine appears to be a powerful inducer of diploid meiotic products, but also shows a lesser effect on disomic induction. Since spontaneous or caffeine-induced diploids arise from a failure of the second meiotic division, it appears that the target of these drugs is at the beginning of the second meiotic division. The only common effect of trifluoperazine and propranolol, mainly investigated in mammals, was an inhibition of calmodulin activity via direct interaction. We tend, therefore, to believe that calmodulin activity must be a crucial point for the second meiotic division to begin. The increased induction of diploids, due to caffeine, may be interpreted as a consequence of an increased cyclic AMP level.

Meiotic Diploid Progeny and Meiotic Nondisjunction in SACCHAROMYCES CEREVISIAE.

Abnormalities in chromosome number that occurred during meiosis were evaluated with a specially-constructed diploid strain of Saccharomyces cerevisiae. The strain is heterozygous for six markers of the right arm of chromosome V and heterozygous for cyh2 (resistance to cycloheximide) on chromosome VII.-Selection of meiotic spores on a medium containing cycloheximide and required nutrilites-except those for the markers of the right arm of chromosome V-allows the growth of aberrant clones belonging only to two classes: a) diploid clones, caused by failure of the second meiotic division, with a frequency of 0.54 x 10(-4) per viable spore; and b) diplo V, aneuploids derived from nondisjunctions in meiosis I or meiosis II, with a total spontaneous frequency of 0.95 x 10(-4) per viable spore. About two-thirds of the aneuploids originated during meiosis I, the rest during meiosis II. An investigation of these events in control meioses and after treatment with MMS, Benomyl and Amphotericin B suggests that this assay system is suitable for screening environmental mutagens for their effects on meiotic segregation.

Production of amino acids by analog-resistant mutants of the cyanobacterium Spirulina platensis.

Mutants of Spirulina platensis resistant to 5-fluorotryptophan, beta-3-thienyl-alanine, ethionine, p-fluorophenylalanine, or azetidine-2-carboxylic acid were isolated. Some of these mutants appeared to be resistant to more than one analog and to overproduce the corresponding amino acids. A second group was composed of mutants that were resistant to one analog only. Of the latter mutants, one resistant to azetidine-2-carboxylic acid was found to overproduce proline only, whereas one resistant to fluorotryptophan and one resistant to ethionine did not overproduce any of the tested amino acids.

DNA synthesis catalyzed in vitro by yeast extracts.

A procedure to prepare crude extracts from single colonies of Saccharomyces cerevisiae is described. Partially purified extracts catalyzed DNA synthesis directed by single-stranded fd DNA. Maximum activity requires the presence of ribonucleoside triphosphates although extensive DNA synthesis is observed in the presence of only deoxynucleoside triphosphates. Alkaline sucrose gradient analysis demonstrated that initiation of new DNA chains occurs in vitro and the DNA products synthesized are heterogeneous in size, Isopycnic analysis of the products of ribonucleotide-initiated fd DNA replication showed covalent linkage between the initiator RNA and the newly synthesized DNA. The fd-replicating capacity of extracts prepared from cell-division-cycle mutants, defective in events controlling DNA initiation of elongation, showed an increased thermosensitivity in the DNA replication reaction in vitro.