无绿藻的研究进展:从基础到临床

无绿藻是一种直径约3 ～ 30 μm的单细胞生物,广泛存在于自然界和动物体表及体内,属于条件致病性真菌.目前主要通过直接镜检、真菌培养、组织病理学检查及分子生物学等手段对无绿藻进行鉴定.现已发现无绿藻属包括五个种,其中对人有致病性的仅为中型无绿藻基因型2、小型无绿藻和P.blaschkeae,其致病机制可能与外伤和免疫力低下有关.随着研究的深入,越来越多的无绿藻病被临床确诊.根据不同的类型及其临床表现,对无绿藻病的治疗也有所区别.为了提高对无绿藻这一条件真菌及其致病性的认识,该文对其生物学特性、鉴定方法、致病性、临床表现等研究进展做一简要综述.     

小型无绿藻的分离及形态特征

无绿藻（Ｐｒｏｔｏｔｈｅｃａ）是一种少见的条件致病菌^［１］,可引起人畜共患的无绿藻病。在其所致人类疾病中,主要有三种临床类型：皮肤损害、局限性感染、系统性无绿藻病^［２］。本文于１９９７年从一名女患者面部丘疹处的皮肤结节活检,分离培养出一株无绿藻菌,并进行了分离培养鉴定,利用光学显微镜、电子显微镜对其形态特征和超微结构进行观察,并通过直接组织压片和组织病理切片观察和生化试验特性,确定为一例由小型无绿藻（Ｐｒｏｔｏｔｈｅｃａｗｉｃｋｅｒｈａｍｉｉ     

In vitro acitivity of ribostamycin against Prototheca sp.

The in vitro activity of ribostamycin against algae of the genus Prototheca was evaluated by minimum inhibitory concentration (MIC) studies on solid media. Concentrations of 4 mcg/ml were required to inhibit 100% of the P. zopfii strains; 16 mcg/ml inhibited 100% of the P. stagnora strains and 95% of the P. wickerhamii strains. These values are inferior to plasma concentrations obtained after injection of ribostamycin. It is likely that this antibiotic could be effective in the treatment of protothecosis in man.     

Human Cutaneous Protothecosis: A Case Report and Review of Cases from Mainland China,Hong Kong,and Taiwan

We present a case of cutaneous protothecosis caused by Prototheca wickerhamii infection. The patient was a 72-year-old man with hypoalbuminemia. He responded well to fluconazole treatment. We reviewed this case along with 17 other cases of cutaneous protothecosis reported from mainland China, Hong Kong, and Taiwan. Of the 18 cases, 7 each occurred in mainland China and Taiwan, and 4 occurred in Hong Kong. Thirteen cases were caused by P. wickerhamii (72.2%), and three were caused by P. zopfii (16.7%); in two cases, the species was not identified (11.1%). In all, 9 (50%) patients were immunocompromised, and 10 (55.5%) patients denied having a history of trauma. All patients presented with polymorphic skin lesions, and erythematous papules, plaques, or nodules was the most common presentation (15/18, 83.3%). Genotyping was performed in five cases, mostly by means of small subunit ribosomal DNA amplification (four cases). Susceptibility tests (6 patients) showed that P. wickerhamii was sensitive to amphotericin B and voriconazole but resistant to fluconazole or itraconazole. Treatment succeeded in 15 (83.3%) patients and failed in 3 (16.7%). Our data indicate that the number of cutaneous protothecosis cases is underestimated in China, and the skin lesions have some diagnostic value.     

Cloning and characterization of a gene encoding a cell-bound, extracellular beta-glucosidase in the yeast Candida wickerhamii.

The ability of yeasts to ferment cellodextrins is rare. Candida wickerhamii is able to use these sugars for alcohol production because of a cell-bound, extracellular, beta-glucosidase that is unusual by not being inhibited by glucose. A cDNA expression library in lambda phage was prepared with mRNA isolated from cellobiose-grown C. wickerhamii. Immunological screening of the library with polyclonal antibodies against purified C. wickerhamii cell-bound, extracellular beta-glucosidase yielded 12 positive clones. Restriction endonuclease analysis and sequence data revealed that the clones could be divided into two groups, bglA and bglB, which were shown to be genetically distinct by Southern hybridization analyses. Efforts were directed at the study of bglB since it appeared to code for the cell-bound beta-glucosidase. Sequence data from both cDNA and genomic clones showed the absence of introns in bglB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of cell lysates from Escherichia coli bglB clones confirmed the presence of an expressed protein with an apparent molecular mass of 72 kDa, which is consistent with that expected for an unglycosylated form of the enzyme. Amino acid comparisons of BglB with other beta-glucosidase sequences suggest that it is a member of family 1 glycosyl hydrolases but is unusual in that it contains an additional 100 to 130 amino acids at the N terminus. This sequence did not have homologies to other known protein sequences and may impart unique properties to this beta-glucosidase.     

Ribosomal Internal Transcribed Spacer of Prototheca wickerhamii Has Characteristic Structure Useful for Identification and Genotyping

Prototheca species are achlorophyllous algae ubiquitous in nature and known to cause localized and systemic infection both in humans and animals. Although identification of the Prototheca species in clinical specimens is a challenge, there are an increasing number of cases in which molecular techniques have successfully been used for diagnosis of protothecosis. In this study, we characterized nuclear ribosomal DNA (rDNA) of a strain of Prototheca (FL11-0001) isolated from a dermatitis patient in Japan for its species identification. When nuclear rDNA of FL11-0001 and that of various other Prototheca strains were compared by polymerase chain reaction (PCR), the results indicated that the sizes of ribosomal internal transcribed spacer (ITS) were different in a species-dependent manner, suggesting that the variation might be useful for differentiation of Prototheca spp. Especially, ITS of P. wickerhamii, the most common cause of human protothecosis, was distinctively larger than that of other Prototheca spp. FL11-0001, whose ITS was comparably large, could easily be identified as P. wickerhamii. The usefulness of the PCR analysis of ITS was also demonstrated by the discovery that one of the clinical isolates that had previously been designated as P. wickerhamii was likely a novel species. Furthermore, our data demonstrated that nucleotide sequences of P. wickerhamii ITS are heterogenous between different rDNA copies in each strain and also polymorphic between strains. Phylogenetic analysis suggested that the ITS sequences could be classified to four clades, based on which P. wickerhamii strains might be grouped into at least two genotypes. Comprehensive characterization of Prototheca rDNA may provide valuable insights into diagnosis and epidemiology of protothecosis, as well as evolution and taxonomy of Prototheca and related organisms.     

Phenoptosis as genetically determined aging influenced by signals from the environment

Aging is a complex and not well understood process. Two opposite concepts try to explain its causes and mechanisms — programmed aging and aging of “wear and tear” (stochastic aging). To date, much evidence has been obtained that contradicts the theories of aging as being due to accumulation of various damages. For example, creation of adequate conditions for the functioning of the organism’s components (appropriate microenvironment, humoral background, etc.) has been shown to cause partial or complete reversibility of signs of its aging. Programmed aging and death of an organism can be termed phenoptosis by analogy to the term apoptosis for programmed cell death (this term was first suggested by V. P. Skulachev). The necessity of this phenomenon, since A. Weismann, has been justified by the need for population renewal according to ecological and evolutionary requirements. Species-specific lifespan, age-dependent changes in expression pattern of genes, etc. are compatible with the concept of phenoptosis. However, the intraspecific rate of aging was shown to vary over of a wide range depending on living conditions. This means that the “aging program” is not set rigidly; it sensitively adjusts an individual to the specific realities of its habitat. Moreover, there are indications that in rather severe conditions of natural habitat the aging program can be completely cancelled, as the need for it disappears because of the raised mortality from external causes (high extrinsic mortality), providing fast turnover of the population.     

Cutaneous Protothecosis in Patient with Diabetes Mellitus and Review of Published Case Reports

Protothecosis is an opportunistic infection caused by Prototheca, usually called as saprophytes, and is frequently found in natural and living surroundings with low virulence, but may cause chronic infection in immunocompromised individuals. We report a case of cutaneous protothecosis with zopfii var. portoricensis infection in a 66-year-old diabetic woman following hand surgery on middle right finger. Mycology study showed that smooth, creamy white, yeast-like colonies grown after necrotic tissue was cultured on Sabouraud dextrose agar at both 37 and 25°C. The organism was then identified as Prototheca zopfii var. portoricensis by molecular identification and also found from histopathology of the lesion. The lesion got improved with intravenous amphotericin B and itraconazole.     

Isolation of Pittsburgh pneumonia agent from a hospital shower.

Tatlockia (Legionella) micdadei, the Pittsburgh pneumonia agent, was isolated from a hospital shower. Although it was not possible, at the current time, to establish an epidemiological link to disease acquisition, this information may be significant because it provides further evidence that a water-associated reservoir of this organism exists within the hospital.     

Cytogerontology at the beginning of the third millenium: from "correlative" to "gist" models

For the most part, research in the area of cytogerontology, i.e., investigation of the mechanisms of aging in the experiments on cultured cells, is carried out using the "Hayflick's model". More than forty years have passed since the appearance of that model, and during this period of time, very much data were obtained on its basis. These data contributed significantly to our knowledge of the behavior of both animal and human cultured cells. Specifically, we already know of the mechanisms underlying the aging in vitro. On the other hand, in my opinion, little has changed in our knowledge of the aging of the whole organism. In all likelihood, this can be explained by that the Hayflick's model is, like many others used in the experimental gerontology, correlative, i.e. based on a number of detected correlations. In the case of Hayflick's model, these are correlations between the mitotic potential of cells (cell population doubling potential) and some "gerontological" parameters and indices: species life-span, donor age, evidence of progeroid syndromes, etc., as well as various changes of normal (diploid) cells during long-term cultivation and during aging of the organism. It is, however, well known that very frequently a good correlation has nothing to do with the essence (gist) of the phenomenon. For example, we do know that the amount of gray hair correlates quite well with the age of an individual but is in no way related to the mechanisms of his/her aging and probability of death. In this case, the absence of cause-effect relationships is evident, which are, at the same time, indispensable for the development of gist models. These models, as distinct from the correlative ones, are based on a certain concept of aging. In the case of Hayflick's model, such a concept is absent: we cannot explain, using the "Hayflick's limit", why our organism ages. This conclusion was convincingly confirmed by the discovery of telomere mechanism which determines the aging of cells in vitro. That discovery initiated the appearance of theories attempting to explain the process of aging in vivo also on its basis. However, it has become clear that the mechanisms of aging of the entire organism, located, apparently, in its postmitotic cells, such as neurons or cardiomyocytes, cannot be explained in the framework of this approach. Hence, we believe that it is essential to develop "gist" models of aging using cultured cells. The mechanisms of cell aging in such models should be similar to the mechanisms of cell aging in the entire organism. Our "stationary phase aging" model could be one of such models, which is based on the assumption of the leading role of cell proliferation restriction in the processes of aging. We assume that the accumulation of "senile" damage is caused by the restriction of cell proliferation either due to the formation of differentiated cell populations during development (in vivo) or to the existence of saturation density phenomenon (in vitro). Cell proliferation changes themselves do not induce aging, they only lead to the accumulation of macromolecular defects, which, in turn, lead to the deterioration of tissues, organs, and, eventually, of the entire organism, increasing the probability of its death. Within the framework of our model, we define cell aging as the accumulation in a cell population of various types of damage identical to the damage arising in senescing multicellular organism. And, finally, it is essential to determine how the cell is dying and what the death of the cell is. These definitions will help to draw real parallels between the "genuine" aging of cells (i.e., increasing probability of their death with "age") and the aging of multicellular organisms.     

Experimental cultivation of the invasive freshwater bryozoan <Emphasis Type="Italic">Pectinatella magnifica</Emphasis>

The freshwater bryozoan Pectinatella magnifica (Leidy, 1851) is an invasive species in many countries all over the world. Although native to North America, it has been found in many countries of Europe and Asia. Pectinatella magnifica forms the largest colonial masses from all recently known bryozoan species. Culturing this organism in an aquarium has never been achieved for more than few days so far. Colonies from laboratory culture are important for various studies on its biology and life cycle of this species in experimental conditions. Young colonies successfully hatched from germinating statoblasts of P. magnifica in the laboratory and were maintained over eight weeks. Moreover, this was the first time when the compound colonies of this species were carried from its natural habitat to the laboratory, into a special aquarium system, and kept alive for more than three weeks. In both experiments the physicochemical parameters of the water (temperature, concentration of dissolved oxygen, electrolytic conductivity and pH) and changes in weight of compound colonies of P. magnifica in laboratory conditions were checked. The results found in this study are essential for understanding the invasiveness of this species and identifying methods for elimination of its ecological risks because these are closely resembling those of other invasive species.     

<Emphasis Type="Italic">Phragmites</Emphasis><Emphasis Type="Italic">australis</Emphasis> as a model organism for studying plant invasions

The cosmopolitan reed grass Phragmites australis (Poaceae) is an intensively studied species globally with a substantial focus in the last two decades on its invasive populations. Here we argue that P. australis meets the criteria to serve as a model organism for studying plant invasions. First, as a dominant species in globally important wetland habitats, it has generated significant pre-existing research, demonstrating a high potential for funding. Second, this plant is easy to grow and use in experiments. Third, it grows abundantly in a wide range of ecological systems and plant communities, allowing a broad range of research questions to be addressed. We formalize the designation of P. australis as a model organism for plant invasions in order to encourage and standardize collaborative research on multiple spatial scales that will help to integrate studies on the ecology and evolution of P. australis invasive populations, their response to global environmental change, and implications for biological security. Such an integrative framework can serve as guidance for studying invasive plant species at the population level and global spatial scale.     

A Case of <Emphasis Type="Italic">Prototheca zopfii</Emphasis> Genotype 1 Infection in a Dog (<Emphasis Type="Italic">Canis lupus familiaris</Emphasis>)

Protothecosis is a rare disease caused by environmental algae of the genus Prototheca. These are saprophytic, non-photosynthetic, aerobic, colorless algae that belong to the Chlorellaceae family. Seven different species have been described. Prototheca zopfii genotype 2 and P. wickerhamii are most commonly involved in pathogenic infections in humans and animals. The objective of this work is to describe, for the first time, a case of protothecosis caused by P. zopfii genotype 1 in a dog. The dog, a 4-year-old mix bred male, was presented to a veterinary clinic in Montevideo, Uruguay, with multiple skin nodules, one of which was excised by surgical biopsy. The sample was examined histologically and processed by PCR, DNA sequencing, and restriction fragments length polymorphisms for the detection and genotyping of P. zopfii. In addition, transmission electron microscopy and scanning electron microscopy were performed. Histology showed severe ulcerative granulomatous dermatitis and panniculitis with myriads of pleomorphic algae. Algal cells were 4–17 µm in size, with an amphophilic, 2–4-µm-thick wall frequently surrounded by a clear halo, contained flocculant material and a deeply basophilic nucleus, and internal septae with daughter cells (endospores) consistent with endosporulation. Ultrastructurally, algal cells/endospores at different stages of development were found within parasitophorous vacuoles in macrophages. Prototheca zopfii genotype 1 was identified by molecular testing, confirming the etiologic diagnosis of protothecosis.     

Complete genome sequence of the aerobic, heterotroph Marinithermus hydrothermalis type strain (T1(T)) from a deep-sea hydrothermal vent chimney

Marinithermus hydrothermalis Sako et al. 2003 is the type species of the monotypic genus Marinithermus. M. hydrothermalis T1(T) was the first isolate within the phylum "Thermus-Deinococcus" to exhibit optimal growth under a salinity equivalent to that of sea water and to have an absolute requirement for NaCl for growth. M. hydrothermalis T1(T) is of interest because it may provide a new insight into the ecological significance of the aerobic, thermophilic decomposers in the circulation of organic compounds in deep-sea hydrothermal vent ecosystems. This is the first completed genome sequence of a member of the genus Marinithermus and the seventh sequence from the family Thermaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,269,167 bp long genome with its 2,251 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Cloning and characterization of an ftsZ homologue from a bacterial symbiont of Drosophila melanogaster

A 1194 by open reading frame that codes for a 398 amino acid peptide was cloned from a gt11 library of Drosophila melanogaster genomic DNA. The predicted peptide sequence is very similar to three previously characterized protein sequences that are encoded by the ftsZ genes in Escherichia coli, Bacillus subtilis and Rhizobium meliloti. The FtsZ protein has a major role in the initiation of cell division in prokaryotic cells. Using a tetracycline treatment that eradicates bacterial parasites from insects, the ftsZ homologue has been found to be derived from a bacterium that lives within the strain. However, polymerase chain reaction (PCR) amplification of the gene from treated embryos suggests that it is not derived from a gut bacterium. Nevertheless, by amplifying and characterizing part of the 16S rRNA from this bacterium we have been able to demonstrate that it is a member of the genus Wolbachia, a parasitic organism that infects, and disturbs the sexual cycle of various strains of Drosophila simulans. We suggest that this ftsZ homologue is implicated in the cell division of Wolbachia, an organism that fails to grow outside the host organism. Sequence and alignment analysis of this ftsZ homologue show the presence of a potential GTP-binding motif indicating that it may function as a GTPase. The consequences of this function particularly with respect to its role in cell division are discussed.     

Fermentation of cellodextrins to ethanol using mixed-culture fermentations

The potential for enhancing ethanol production from cellodextrins by employing mixed-culture (Candida wickerhamii-Saccharomyces cerevisiae) fermentations was investigated. Initially, ethanol production was monitored in fermentation medium containing 50 g/L glucose plus 45 g/L cellobiose. Inoculum levels and times of inoculum addition were varied. Of the conditions tested, the most rapid rates of ethanol formation occurred in fermentations in which either C. wickerhamii and S. cerevisiae were coinoculated at a ratio of 57 : 1 cell/mL or in fermentations in which a 10-fold-greater S. cerevisiae inoculum was added to a pure culture C. wickerhamii fermentation after 1 day incubation. These conditions were used to attempt to enhance fermentations in which cellodextrins produced by trifluoroacetic acid hydrolysis of cellulose served as the sole carbon source. Cellodextrins that were not further purified after cellulose hydrolysis contained compounds that were slightly inhibitory to C. wickerhamii. In this case the mixed-culture fermentations produced 12-45% more ethanol than a pure culture C. wickerhamii fermentation. However, if the substrate was treated with Darco G-60 charcoal, the toxic materials were apparently removed and the pure culture C. wickerhamii fermentations performed as well as the mixed-culture fermentations.     

Characterization of a new methylotrophic strain,Methylomonas clara

Summary The organism examined was identified as an obligate methylotroph and as an organism of type I, because it has some characteristics of this group: methanol is utilized via the hexulose phosphate pathway, the enzymes glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase (both NAD- and NADP-specific) could be detected, whereas the citric acid cycle is incomplete (in particular -ketoglutarate dehydrogenase could not be found). Furthermore, the organism is able to fix nitrogen. In contrast, there are some different characteristics: carbon dioxide can be fixed in connection with methanol. There is no formate dehydrogenase, and formaldehyde dehydrogenase can be induced by different culture conditions. Moreover, the GC content (49.2%) is distinctly lower than normal, and the hexulose phosphate synthase shows a higher activity with NAD as cosubstrate than with NADP. Carbon is not metabolized via the Embden-Meyerhoff-Parnaß pathway, but via the Entner-Doudoroff pathway. In the disk test, the organism is resistant to some antibiotics: chloramphenicol, fusidic acid, methicillin and lincomycin. A partial resistance can be observed with penicillin, neomycin, novobiocin, and cloxacillin.     

Identification and Characterization of Prokaryotic Dipeptidyl-peptidase 5 from Porphyromonas gingivalis

Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN_0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated K_m and k_cat/K_m values for Lys-Ala-MCA of 688 μm and 11.02 μm⁻¹ s⁻¹, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea.