食管淋巴管的超微结构

目的:探讨食管淋巴管内皮的大分子物质转运方式。方法:用透射电镜方法观察家兔食管淋巴管的超微结构。结果:发现食管毛细淋巴管内皮细胞间连接有4种,即插入连接、端端连接、重叠连接和复杂连接。毛细淋巴管内皮的质膜小泡分为无壁小泡和有壁大泡。并且质膜小泡的数量很多。结论:我们认为在组织液和大分子转运途径中,食管毛细淋巴管可通过内皮质膜小泡(乔饮)和内皮间通道两种方式来完成。     

Production of heterokaryons of Candida albicans by protoplast fusions: Effects of differences in proportions and regenerative abilities of fusion partners

Production of heterokaryons of Candida albicans by polyethylene glycol-induced fusion of complementing auxotrophic protoplasts was assayed in two series of fusion crosses designed to test the significances of the in-put ratios and regenerative abilities of parental protoplasts. The findings indicate that productive fusions require interaction of multiple protoplasts of each complementing type and can occur equally well with either regenerable or non-regenerable protoplasts.     

Efficient translation of the UAG termination codon in Candida species

Summary Clinical isolates of the dimorphic fungus Candida albicans encode a tRNA that, in a cell-free translation system prepared from the yeast Saccharomyces cerevisiae, efficiently translates the amber (UAG) termination codon. Unusually, the efficiency of this UAG read-through in the heterologous cell-free system is not further enhanced by polyamines. The suppressor tRNA is also able to efficiently translate the UAG codon in the rabbit reticulocyte cell-free system and with efficiencies approaching 100% in a homologous (C. albicans) cell-free system. That the suppressor tRNA is nuclear-encoded is demonstrated by the lack of activity in purified C. albicans mitochondrial tRNAs. Finally, UAG suppressor tRNA activity is also demonstrated in three other pathogenic Candida species, C. parapsilosis, C. guillermondii and C. tropicalis. These results suggest that some, but not all, Candida species have evolved an unusual nuclear genetic code in which UAG is used as a sense codon.     

Inflammation Induced by Inoculation of the Joint with Candida albicans

In humans Candida albicans is the most frequently isolated opportunistic fungal pathogen. In immunocompromized host the balance with the commensal fungus easily turns to life-threatening disseminated infection. The asymptomatic Candida persistence in organs and the recurrent infections suggest continuous circulation of yeast cells and their degradation products. Under certain conditions, joints might become one of the infectious sites. More easily a reactivation and destructive process can be provoked in individuals with established arthritis. We have investigated the joint inflammation caused by inoculation of the paw with live C. albicans, in intact mice and mice with collagen-induced arthritis (CIA). The results demonstrate that C. albicans infection when localized into the joints caused rapidly progressing septic arthritis. The effect was associated with a strong swelling, a rapid influx of polymorphonuclear (PMN) cells, and an elevated secretion of TNF-alpha and IFN-gamma by lymph node cells. Joint infection exacerbated the established CIA which correlated with an increased level of anti-collagen antibodies.     

Transport properties of a C. albicans amino-acid permease whose putative gene was cloned and expressed in S. cerevisiae

Using a gene bank of C. albicans, the lysine-permease deficiency in a strain of S. cerevisiae was complemented, and the restriction map of the corresponding C. albicans DNA fragment was constructed. Its expression in S. cerevisiae showed that the permease of C. albicans actively transports arginine (K_T=18 mol/l, J_max=26 nmol/min per mg dry weight), lysine (K_T=12 mol/l, J_max=18 nmol/min per mg dry weight), histidine (K_T=37 mol/l, J_max=9.7 nmol/min per mg dry weight), as well as their toxic analogues canavanine and thialysine, with high affinity. The intracellular concentration of basic amino acids transported into S. cerevisiae by the C. albicans permease reaches more than a thousand-times-higher value compared to the external concentration in the medium. Accumulated amino acids do not leave the cells. The uptake is strongly reduced by the protonophores and inhibitors of plasma membrane H⁺-ATPase.     

Temperature-dependent internuclear transfer of genetic material in heterokaryons of Candida albicans

Summary Heterokaryons (hets) of Candida albicans are produced by fusing protoplasts of complementing auxotrophic strains and can be propagated continuously on minimal medium despite their tendency to assort nuclei into monokaryotic blastospores. Most mono-karyons have parental-type nuclei, but some are nuclear hybrids with DNA contents between one and two times that of their parental strains. Evidence is presented that hybrids arise by transfer of a portion of the genetic material of one bet nucleus to another, and that the amount of material conveyed during transfer increases with increasing het growth temperatures over the range 25°C to 41°C. This partial hybridization is a general property of hets and is not determined by the wild-type strain backgrounds of their parental components or by the kinds of auxotrophies forcing heterokaryosis. Frequencies of mitotic recombinants induced in partial hybrids by ultraviolet radiation indicate that nuclei of C. albicans are naturally diploid.     

The isolation of osmotic-remedial conditional lethal mutants of Candida albicans

Summary We have developed a method for the isolation of a broad range of conditional lethal mutants of the pathogenic fungus Candida albicans. The method substantially alleviates the problems posed by the diploid nature, and the biased mutant spectrum, exhibited by most strains of this organism. We have used the method to isolate 560 temperature-sensitive mutants which grew at 30°C but not at 42°C. We identified amongst them a number of osmotic-remedial strains which, at the restrictive temperature, show phenotypes indicating defects in cell wall biosynthesis.     

Multilocus sequence typing of Candida albicans isolates from animals

Multilocus sequencing strain types of a panel of 43 Candida albicans isolates from animals, including mammals and avian species, were compared with strain types for human isolates. The clade distribution of the animal isolates was significantly different from that of the human isolates, in both a comparison involving a total of 1580 isolates from multiple geographical sources and a comparison restricted to 675 human isolates from the same geographical regions as the animal isolates. A nearest-neighbour analysis involving the 43 animal isolates and 67 human isolates, randomly selected to give a proportionate distribution of geographical sources, showed a strong statistical trend towards genetic selection of different C. albicans strain types adapted to non-human animal hosts, but without complete genetic separation.     

Partial nucleotide sequence of a single ribosomal RNA coding region and secondary structure of the large subunit 25 s rRNA of Candida albicans

A rDNA cistron of Candida albicans strain WO-1 was cloned and the ITS1, ITS2, 5.8 s rDNA and 25 s rDNA coding regions sequenced in their entirety. These sequences were compared to those of three related yeast species (Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Thermomyces lanuginosus), and the 5.8 s rDNA was compared to seven additional 5.8 s rDNAs from organisms ranging in complexity from D. discoideum to H. sapiens. The C. albicans ITS regions are shorter than those of most other eukaryotes. The 25 s and 5.8 s rDNA sequences were folded into a secondary structure model based on comparative methods. In a comparison of regional similarities between the large subunit rDNAs of C. albicans, the three related yeasts and other eukaryotes, it is demonstrated that the additional sequences not present in the E. coli 23 s rDNA are more variable than the regions present in both prokaryotes and eukaryotes.     

Identification,characterization and sequence of Candida albicans repetitive DNAs Rel-1 and Rel-2

Two moderately repetitive DNA elements, Rel-1 and Rel-2, were identified in a screen for clones that hybridized to a Candida albicans minichromosome. Rel-1, a 223-bp sequence, is C. albicans-specific. The 2789-bp Rel-2 sequence hybridizes weakly to C. stellatoidia DNA but not to DNA from several other yeast species. Genomic Southern-blot analysis indicated that Rel-1 and Rel-2 are often closely associated in the genome, suggesting that they may be subsequences of a larger repetitive element. Small subrepeats are located in the nucleotide sequence of both clones. Hybridization demonstrated that Rel-2 contains both repetitive and unique DNA sequences. The repetitive DNA is present on most, and perhaps all, C. albicans chromosomes. The unique sequence maps to chromosome 7; however, in some strains, it is also present on additional chromosomes.     

Ultrastructure of multinucleated giant cell apoptosis in foreign-body granuloma

To elucidate the role of apoptosis in the disappearance of multinucleated giant cells from the granulation tissue in cases of foreign-body granuloma, we induced a foreign-body reaction by implanting a collagen sponge into the dorsum of the rat and observed apoptotic changes within the multinucleated giant cells using electron microscopy. Two types of multinucleated giant cells were identified presenting apoptotic characteristics morphologically. One was characterized by apoptosis of only one nucleus, followed by cytoplasmic changes, rupture of the plasma membrane and necrosis evoking an inflammatory reaction. The other showed typical apoptotic changes in the majority or in all of the nuclei, followed by phagocytosis of the apoptotic syncytia. The results of the present study suggest that apoptosis occurring within only one nucleus might be triggered by overexpression of the p53 protein, because DNA abnormalities are confined to this single nucleus. In contrast apoptosis occurring simultaneously in the majority or all of the nuclei is most probably due to cell death caused by senescence.     

The Candida albicans PMM1 gene encoding phosphomannomutase complements a Saccharomyces cerevisiae sec 53-6 mutation

We have constructed an ordered-array genomic DNA library of the pathogenic dimorphic fungus Candida albicans which facilitates the rapid cloning of C. albicans genes by hybridisation. Using the Saccharomyces cerevisiae SEC53 gene encoding phosphomannomutase as a hybridisation probe we have cloned the C. albicans homologue, PMM1, and determined its sequence. This gene shows high similarity, both at the nucleotide (76.2%) and amino-acid (77.7%) level, to the S. cerevisiae SEC53 gene. We have used the C. albicans PMM1 gene, in single copy, to transform temperature-sensitive S. cerevisiae sec53-6 mutant cells, which are defective in PMM activity at 37°C, to growth at 37°C. The C. albicans PMM1 gene is thus the structural and functional equivalent of the SEC53 gene.     

Segregant-defective heterokaryons of Candida albicans

Summary Heterokaryons (hets) of the asexual, pathogenic yeast Candida albicans obtained by fusing protoplasts of complementing auxotrophic strains generate large numbers of parental-type auxotrophic monokaryons by random assortment of single nuclei into blastospores, and smaller numbers of monokaryons bearing hybrid nuclei formed through either karyogamy or the transfer of genetic material from one het nucleus to another. Het populations grown at 30 °C or 37 °C contain high frequencies (approx. 5%–10%) of two kinds of stable variants peculiar specifically for segregation of parental-type monokaryons: NS variants produce inviable auxotrophic monokaryons of one or both parental classes while AT variants yield parental-type monokaryons which grow very slowly. Variant frequencies are not affected by the wild-type strain background of hets, or the auxotrophies used to force heterokaryosis. However, both kinds of variants are induced by growth at 25 °C or by treatments with certain chemical or physical metabolic inhibitors. Evidence is presented that variant nuclei of independent origins carry different nutritionally irreparable recessive lethal (NS) or debilitating (AT) defects acquired in the course of actual or potential internuclear transfers of genetic material within het cells. The high incidence of variants, therefore, indicates considerable intrinsic genetic instability among het nuclei. Significances of these observations for parasexual genetic analyses of C. albicans and other yeasts through protoplast fusions are considered.     

Ultrastructure of early cleavage stages and preimplantation in the rabbit

Summary Fertilized rabbit ova were studied in the period from 25 to 144 hr after insemination. Eggs were recovered by flushing the uterine horns and oviducts with Hank's solution. The cells were morphologically alike in the 24 and 48 hr ova. At 661/2 hr blastomeres were differentiated into an inner mass of cells with dense cytoplasm and an outer trophoblastic layer with less dense cytoplasm. Otherwise no morphological differences were seen. Whether the 661/2 hr ova were morulas or blastocysts is discussed. The 96 hr ova were clearly blastocysts. Inner cells and trophoblastic cells at this stage bad the same cytoplasmic density. Mitochondria were increased in number and crystal-like figures were present for the first time. In the 120 and 144 hr ova the cytoplasm of the trophoblastic cells was denser than that of the inner cells. Trophoblastic cells were characterized by their density, crystal-like figures, elongated mitochondria with transverse cristae and many single ribosomes and they were interconnected with well developed junctional complexes. In a few cases a continuity seemed to exist between trophoblastic cells and inner cells. The latter were characterized by cytoplasm of less density than that of the trophoblastic cells, rounded mitochondria and fewer ribosomes. The fine structure of the crystal-like figures, their possible origin and differentiation of the mitochondria are discussed.This investigation was supported by the U.S. Public Health Service NIH International Postdoctoral Fellowship No. 4 FO5 TW 1365-02.     

A gene (Cargl) that regulates tissue resistance toCandida albicansmaps to chromosome 14 of the mouse

Tissue susceptibility and resistance to infection with the yeastCandida albicansis genetically regulated. Analysis of the strain distribution pattern of theC. albicansresistance gene (Carg1) and additional gene and DNA segment markers in the AKXL recombinant inbred (RI) set showed that 13/15 RI strains were concordant forCarg1,TcraandRib1. Therefore,Carg1is probably located within a 17 cM segment of chromosome 14, within approximately 4 cM of the other two genes.     

Shuttle vectors for <Emphasis Type="Italic">Candida albicans</Emphasis>: control of plasmid copy number and elevated expression of cloned genes

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20–40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.     

Virulence modulation of Candida albicans biofilms by metal ions commonly released from orthodontic devices

The installation of metal devices leads to an increase in the salivary concentration of metal ions and in the growth of salivary Candida spp. However, the relationship between released metal ions and Candida virulence has not been previously examined. The objective of this study was to evaluate whether metal ions affect fungal virulence. We prepared culture media containing Ni²⁺, Fe³⁺, Cr³⁺, Co²⁺ or a mixture of these metal ions at concentrations similar to those released in saliva of orthodontic patients. Biofilms of Candida albicans SC5314 were grown for 72 h and their biomasses were determined. The supernatants were analyzed for secretory aspartyl protease (SAP) and hemolysin activities. To verify changes in virulence following treatment with metals, proteolytic and hemolytic activities were converted into specific activities. The results revealed that all ions, except Co²⁺, caused increases in biofilm biomass. In addition, Ni²⁺ caused an increase in SAP activity and Fe³⁺ reduced hemolytic activity. However, the SAP and hemolysin activities in the presence of the mixture of ions did not differ from those of control. These results indicate that metal ions released during the degradation of orthodontic appliances can modulate virulence factors in C. albicans biofilms.     

DNA translocations contribute to chromosome length polymorphisms in Candida albicans

Summary Rotating-gel electrophoresis and DNA hybridization were used to compare the electrophoretic karyotype of six Candida albicans isolates. The hybridization pattern for 22 cloned sequences, including eight previously unmapped genes, indicates that there are eight pair of homologous chromosomes in each strain. However, since homologous chromosomes can differ in length, it is possible to resolve more than eight bands in some strains. The mapping data demonstrate that linkage groups are generally conserved suggesting that, in spite of gross karyotype differences, there is an underlying similarity in the genome organization of different isolates. The hybridization data also provide direct evidence that DNA translocations and reciprocal translocations contribute to chromosome length polymorphisms in C. albicans.