A comprehensive molecular phylogeny of the Mortierellales (Mortierellomycotina) based on nuclear ribosomal DNA.

The basal fungal order Mortierellales constitutes one of the largest orders in the basal lineages. This group consists of one family and six genera. Most species are saprobic soil inhabiting fungi with the ability of diverse biotransformations or the accumulation of unsaturated fatty acids, making them attractive for biotechnological applications. Only few studies exist aiming at the revelation of the evolutionary relationships of this interesting fungal group. This study includes the largest dataset of LSU and ITS sequences for more than 400 specimens containing 63 type or reference strains. Based on a LSU phylogram, fungal groups were defined and evaluated using ITS sequences and morphological features. Traditional morphology-based classification schemes were rejected, because the morphology of the Mortierellales seems to depend on culture conditions, a fact, which makes the identification of synapomorphic characters tedious. This study belongs to the most comprehensive molecular phylogenetic analyses for the Mortierellales up to date and reveals unresolved species and species complexes.

In vitro interactions of amantadine hydrochloride, R-(-)-deprenyl hydrochloride and valproic acid sodium salt with antifungal agents against filamentous fungal species causing central nervous system infection.

The mortality rates of fungal infections that affect the central nervous system are high in consequence of the absence of effective antifungal drugs with good penetration across the blood-brain barrier and the blood-cerebrospinal fluid barrier. In the present work in vitro antifungal activities of three good penetrating non-antifungal drugs (amantadine hydrochloride, R-(-)-deprenyl hydrochloride, valproic acid sodium salt) and their combinations with three antifungal agents (amphotericin B, itraconazole, terbinafine) were tested with broth microdilution method against eight fungal isolates belonging to Zygomycetes (Lichtheimia corymbifera, Rhizomucor miehei, Rhizopus microsporus var. rhizopodiformis, Saksenaeavasiformis) and Aspergillus genus (A. flavus, A. fumigatus, A. nidulans, A. terreus). These are known to be possible agents of central nervous fungal infections (CNFI). When used alone, the investigated nonantifungal drugs exerted slight antifungal effects. In their combinations with antifungal agents they acted antagonistically, additively and synergistically against zygomyceteous isolates. Primarily antagonistic interactions were revealed between the investigated drugs in case of Aspergilli, but additive and synergistic interactions were also observed. The additive and synergistic combinations allowed the usage of reduced concentrations of antifungal agents to inhibit the fungal growth in our study. These combinations would be a basis of an effective, less toxic therapy for treatment of CNFI.

Antifungal activity of statins and their interaction with amphotericin B against clinically important Zygomycetes.

The in vitro antifungal activity of different statins and the combinations of the two most effective ones (fluvastatin and rosuvastatin) with amphotericin B were investigated in this study on 6 fungal isolates representing 4 clinically important genera, namely Absidia, Rhizomucor, Rhizopus and Syncephalastrum . The antifungal effects of statins revealed substantial differences. The synthetic statins proved to be more effective than the fungal metabolites. All investigated strains proved to be sensitive to fluvastatin. Fluvastatin and rosuvastatin acted synergistically and additively with amphotericin B in inhibiting the fungal growth in clinically available concentration ranges. Results suggest that statins combined with amphotericin B have a therapeutic potential against fungal infections caused by Zygomycetes species.

Identification of acid- and thermotolerant extracellular beta-glucosidase activities in Zygomycetes fungi.

Extracellular beta-glucosidase activity of 94 strains, representing 24 species of the genera Gilbertella, Mucor, Rhizomucor , and Rhizopus was evaluated in submerged culture and under solid state fermentation on wheat bran. Gilbertella persicaria G1 isolate showed the highest activity (70.9 U ml -1 ) followed by other Gilbertella (58.6-59.0 U ml -1 ) and Rhizomucor miehei isolates (29.2-42.0 U ml -1 ). Optimum temperature for enzyme production was 25 degrees C for Gilbertella and Mucor , and 30 degrees C for Rhizomucor and Rhizopus strains. Enzymes of R. miehei strains proved to be thermotolerant preserving up to 92.8% residual activity after heating to 75 degrees C in the presence of cellobiose substrate. Enzymes of Mucor racemosus f. chibinensis, R. miehei and Rhizopus microsporus var. oligosporus strains were activated at acidic condition (pH 4). Glucose was a strong inhibitor for each fungal beta-glucosidase tested but some of them showed ethanol tolerance up to 20% (v/v). Ethanol also activated the enzyme in these strains suggesting glycosyl transferase activity.

High-affinity iron permease (FTR1) gene sequence-based molecular identification of clinically important Zygomycetes.

The clinical importance of zygomycosis, an emerging and frequently fatal mycotic disease, has increased during recent years. This report describes an identification method based on PCR amplification and sequencing of the high-affinity iron permease 1 gene (FTR1). Primers and amplification protocols were established and tested for the identification of Rhizopus oryzae, Rhizopus microsporus var. rhizopodiformis, R. microsporus var. oligosporus, Rhizopus schipperae, Rhizopus niveus and Rhizopus stolonifer. Rhizomucor and Syncephalastrum could be identified at the genus level. PCR-restriction fragment length polymorphism analysis of the amplified gene fragment using AluI digestion distinguished three subgroups among the R. oryzae isolates.

Differentiation between papillary and nonpapillary renal cell carcinomas by DNA analysis.

Recent studies have shown that the deletion of chromosome 3p or the loss of DNA sequences at 3p is generally associated with the development of renal cell carcinoma (RCC). However, chromosome analysis of some papillary RCCs suggested that this type of tumor differs genotypically from the most common nonpapillary RCCs. Therefore, by using cytogenetic and molecular genetic approaches, we examined human papillary and nonpapillary RCCs for the loss of heterozygosity or homozygosity at the short arm of chromosome 3. The constitutional heterozygosity for the DNF15S2 locus and for one allele of the c-erbA beta and the c-raf-1 proto-oncogenes was lost in nonpapillary RCCs, whereas both alleles were retained in each papillary RCC analyzed. We conclude that the loss of DNA sequences at the chromosome 3p region is a genomic change occurring consistently in nonpapillary RCCs, but never occurring in papillary RCCs.

Quality assurance challenges in X-ray emission based analyses, the advantage of digital signal processing.

There is a large scatter in the results of X-ray analysis with solid-state detectors suggesting methodological origin. In order to improve the methodology, detector response functions have been investigated by many researchers and analysts. This was necessary as the departure of the response function of some detector-signal processing electronics from the normally assumed Gaussian line shape can exceed 100% in area. Several detector models have been proposed to improve understanding and establish a firm basis for quantitative work. After reviewing some contradictory results, we describe a signal processor that offers quality assurance, by producing two spectra for each measurement. One is the normal spectrum of accepted events, while the second spectrum contains all of the rejected events. For each measurement, therefore, all X-ray events are recorded, enabling quality control. In addition to this improvement, the digital signal processor of Cambridge Scientific, Canada, delivers a high throughput rate, excellent resolution, decreased low energy tailing and a line shape justified by the physics of the detector. Comparative measurements are presented to demonstrate the improved rejection of background from gamma rays as well as a significant improvement in pile-up recognition. The rejected events spectrum gives insight into the origin of the response function, which suggests that the flat plateau of the frequently used Hypermet function, normally attributed to detector dead layers, originates from pile-up with the low energy noise events. A detailed analysis demonstrates how the relative intensities of the X-ray lines can change in a varying noise environment, thus potentially explaining the unacceptable large scatter in the experimental data currently found in the literature. The comparison of the accepted and rejected events adds the possibility of monitoring the electronic efficiency of signal recognition that has generally been ignored in this field.

Agrobacterium tumefaciens-mediated transformation of Mucor circinelloides.

The Agrobacterium tumefaciens-mediated transformation of the zygomycetous fungus Mucor circinelloides is described. A method was also developed for the hygromycin B-based selection of Mucor transformants. Transformation with the hygromycin B phosphotransferase gene of Escherichia coli controlled by the heterologous Aspergillus nidulans trpC promoter resulted in hygromycin B-resistant clones. The presence of the hygromycin resistance gene in the genome of the transformants was verified by polymerase chain reaction and Southern hybridization: the latter analyses revealed integrations in the host genome at different sites in different transformants. The stability of transformants remained questionable during the latter analyses.

Isolation of bovine adenovirus serotype 6 from a calf in the United Kingdom.

Two viruses, designated 99-8130(C) and 99-8130(I), were isolated in calf testis cells from the colon and ileum, respectively, of a suckled beef calf which had developed dysentery and died. Electron microscopy indicated that the mean (sd) size of the viral particles, 83 (2.5) nm, and their morphology were consistent with their being members of the family Adenoviridae. They were confirmed as adenoviruses by PCR when products of the expected size (608 bp) were amplified from both isolates by using a primer pair specific for members of the genus Atadenovirus. A comparison of the sequence of a 567 bp segment of the 99-8130(C) amplicon with that of other prototype bovine adenovirus (BAdV) strains of atadenoviruses identified the isolate as BAdV serotype 6 (BAdV-6), which had 99.3 per cent and 100 per cent identities at the nucleotide and amino acid levels, respectively, with the prototype BAdV-6 strain 671130. A virus neutralisation test was developed and indicated a high prevalence of antibody to BAdV-6 in Northern Irish cattle. There was no evidence of adenoviral inclusions in tissues from the affected calf and no antigen was detected when the tissues were stained by an immunoperoxidase technique, using a homologous antiserum raised in rabbits. The two viruses were the third reported isolation of BAdV-6, and the first from a clinically ill bovine animal.

Mapping of phenytoin-inducible cytochrome P450 immunoreactivity in the mouse central nervous system.

The distribution of phenytoin-inducible cytochrome P450 in non-treated mouse brain and spinal cord was analysed immunohistochemically using polyclonal antibodies against phenytoin-induced mouse cerebral microsomal P450. This P450 protein was proved in Ouchterlony [Volk B. et al. (1988) Neurosci. Lett. 84, 219-224], Western blot, and immunohistochemical analyses to be reactive to the specific antibodies and an IgG fraction raised against phenobarbital-induced rat liver microsomal P450IIB1. The phenytoin-induced P450 is designated P450IIB1* because immunologically it is comparable with P450IIB1; however, it has not yet been analysed for other characteristics of this enzyme. Immunocytochemistry was performed on acetone-fixed serial cryosections of the whole brain using the avidin-biotin-peroxidase detection system. Negative controls included incubations with preimmune serum of the immunized animal instead of the primary antibody and preabsorption of the antibody with the corresponding immunogen. The pattern of immunoreactive sites indicates that P450IIB1* is not distributed evenly throughout the CNS. It was found to be restricted to only some cellular populations. The most striking aspect of immunostaining was a predominant reactivity in the evolutionary old brain parts. Neuropil and neuronal staining was found in the spinal cord (motor neurons of the ventral horn), medulla oblongata (hypoglossal nuclei, magnocellular part of the lateral reticular nuclei), pons (trigeminal, facial, cochlear and pontine nuclei), cerebellum (granule cells), midbrain (dorsal raphe nucleus) and limbic lobe (hippocampal pyramidal cells). Neuropil reactivity alone appeared in cerebellar nuclei, midbrain, thalamus, basal ganglia, neopallium and olfactory brain. Generally, pia mater/arachnoid, ependyma, choroid plexus, vascular system and some astrocytic populations were found to be strongly P450IIB1* immunoreactive. In comparison with astroglia, which is characterized by glial fibrillary acidic protein-positiveness, the astrocytes, which are also P450IIB1* reactive, occurred only in subpial and subependymal layers, and in large fiber tracts of the spinal cord and brainstem, where they were attached to the vascular system. Otherwise, the glial fibrillary acidic protein-positive astrocytes were not P450IIB1* immunoreactive in the cerebellar molecular layer (fibers of Bergmann glia), in remaining neuropils and in white matter areas.

Detection of hyperdiploidy and chromosome breakage affecting the 1 (1cen-q12) region in lentigo malignant melanoma (LMM), superficial spreading melanoma (SSM) and congenital nevus (CN) cells in vitro by the multicolor FISH technique.

The centric/pericentric region of chromosome 1 (cen-q 2) of human melanoma cells of different stages of carcinogenicity (superficial spreading melanoma (SSM), lentigo malignant melanoma (LMM)) and premalignant precursor lesions (congenital nevus (CN)) were investigated by fluorescence in situ hybridization (FISH) with tandem DNA probes. The pericentric heterochromatin region 1(q12) is large and highly prone to breakage in contrast to the adjacent centromeric region which is much smaller and less prone to such events. All samples of melanoma cells were obtained from patients and cultivated in vitro. LMM cells showed the highest number of breakage events within the 1q12 region (90% of cells). The number of hyperdiploid cells was not increased in comparison to CN cells. In contrast to LMM cells, SSM cells showed a significant increased number of hyperdiploid cells which were mainly tetrasomic for chromosome 1 (P < or = 0.05). The number of chromosome breaks was not significantly increased in this type of melanoma cells. The spontaneous rates of chromosomal breakage and hyperdiploidy is relatively low in CN cells (1.5-2.5% and 3.2-5.8%, respectively) but these frequencies also differ between CN samples from different patients. These results show that the multicolor FISH technique represents a fast and reliable detection method, distinguishing structural and numerical chromosomal alterations in interphase nuclei. This technique is useful as a histological marker to differentiate between specific tumor subtypes and to investigate the relationship between genomic instability and clinopathological parameters (tumor grading and staging).

Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas.

Nineteen specimens from primary human malignant mesotheliomas obtained from 19 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 by combined RFLP-PCR/SSCP analysis. In addition, all tumours were screened for deletions and point mutations in the tumour suppressor genes p53, p16INK4a (CDKN2A) and p14ARF (exon-1beta) by combined multiplex-PCR/SSCP analysis. No mutations were found in N-ras, p53 and CDK4. Three tumours displayed homozygous deletion (co-deletion of exons 1, 2 and 3) of p16INK4a. One of them displayed additional homozygous deletion of p14ARF (exon-1beta). Two silent point mutations and 2 polymorphisms were found in p16INK4a in 3 tumours. Our preliminary data indicate that disarrangement of the Rb1 pathway may be involved in mesothelioma formation.

Lack of p53 mutations and loss of heterozygosity in non-cultured human melanocytic lesions.

In this study we analysed snap-frozen surgical resections of 16 superficial spreading melanomas, 13 nodular malignant melanomas, 2 lentigo maligna melanomas, 1 dysplastic nevus, 1 congenital nevus and 5 normal nevi from 38 patients for point mutations in the human p53 gene at exons 5-8 by polymerase chain reaction/single-strand conformation polymorphism as well as for loss of heterozygosity of p53 by restriction-fragment-length polymorphism/polymerase chain reaction in order to determine whether p53 aberrations are associated with melanoma subtypes. In addition, we analysed six melanoma cell lines for point mutations in p53. Our results revealed the absence of point mutations and loss of heterozygosity in all fresh resected lesions. However, a TAC (Tyr) to TGC (Cys) transition at codon 163 in exon 5 was found in one cell line.

Analysis of ras mutations in human melanocytic lesions: activation of the ras gene seems to be associated with the nodular type of human malignant melanoma.

We have analyzed the Ha-ras, Ki-ras and N-ras gene for point mutations at codons 12, 13 and 61 via restriction fragment length polymorphism/polymerase chain reaction analysis and subsequent direct sequencing in non-cultured fresh-frozen tissues of 16 superficial spreading melanomas (SSM), 13 nodular malignant melanomas (NMM), 2 lentigo malignant melanomas (LMM), 1 dysplastic nevus, 1 congenital nevus and 5 normal nevi from 38 patients. Mutations were found in 4 melanoma samples, all belonging to the nodular malignant type. Three of them were mutated in N-ras and one in the Ha-ras gene. Mutation in N-ras was also detected in the congenital nevus. All mutations were exclusively located at the first two base pairs of codon 61. No Ki-ras mutation was detected in any lesion. No mutation could be found in SSM and LMM in addition to dysplastic and normal nevi. The frequency of ras mutation in NMM was 31%, whereas in SSM it was 0%. Our study suggests (a) an association between ras mutations (mainly N-ras) and the NMM as a subgroup of human melanoma; (b) that activation of Ki-ras is not involved in the pathogenesis of melanoma. The role of UV radiation in point mutations of ras genes in human melanoma is discussed.

The mitochondrial genome of Mucor piriformis.

DNA was purified from the isolated mitochondria of a Mucor piriformis wild-type strain (NRRL 26211). A circular restriction map of the mitochondrial DNA was established on the basis of single and double digests with several restriction endonucleases. The average mitochondrial DNA size calculated from these data was 33.53 kbp; this is in good agreement with the contour length size (33.62 kbp) of the open circular molecules detected by electron microscopy. Heterologous hybridizations with cloned Aspergillus nidulans mitochondrial genes were used to locate some coding regions on the map.

Mutational analysis of the PTEN/MMAC1 tumour suppressor gene in primary human malignant mesotheliomas.

Eighteen primary human malignant mesotheliomas obtained from 18 patients were screened for point mutations and microdeletions/insertions in all exons of the tumour suppressor gene PTEN/MMAC1 by SSCP analysis. No mutation could be found. Our preliminary data indicate that disarrangements of PTEN/MMAC1 are at least not frequently involved in mesothelioma formation.

Cytogenetic changes in primary, immortalized and malignant mammalian cells.

Some chromosomes in transformed rat cells and somatic cell hybrids fail to display the presence of kinetochore proteins as detected by antikinetochore antibodies. Such chromosomes (K- chromosomes) may constitute a novel mechanism for the genesis of aneuploidy. We have analyzed primary, immortalized and malignant mammalian cells for the presence of kinetochore proteins and micronuclei. Our results suggest a correlation of the K- chromosome and micronucleus frequency with the variability in chromosome number. Upon in situ hybridization with the minor satellite and alpha satellite sequences some K- chromosomes showed a signal. This indicates that the observed lack of kinetochores is not necessarily due to a lack of centromeric DNA. We conclude that dislocated K- chromosomes may become incorporated into micronuclei which are prone to loss. Such events would be associated with the generation of aneuploidy.

Changes of the lumbar spinal canal proximal to spina bifida occulta. An archaeologic study with clinical significance.

STUDY DESIGN: This archaeologic study, based on four populations, examines the incidence of spina bifida occulta in the lumbar spine and the size of the vertebral canal proximal to the lesion. OBJECTIVES: To ascertain any significant change in the dimensions of the lumbar spinal canal of skeletons with spina bifida occulta. The incidence of the lesion also was compared in the separate genetic groups. METHODS: Central canals of 1760 lumbar vertebrae were examined. Silhouette, unmagnified pictures of the vertebral canals were measured by computerized image analysis. RESULTS: The mid-sagittal diameter at L4 and L5 and the cross-sectional area at L5 were found to be significantly larger proximal to the lesion compared with the unaffected spines. The overall incidence was 18%. CONCLUSIONS: The capacity of the lumbar canal is greater proximal to spina bifida occulta. Therefore, delayed closure of the neural arch at a single segment has morphologic significance to the more proximal spine.

The growth of the lumbar vertebral canal.

STUDY DESIGN: This study examines the growth and development of the lumbar spinal canal with emphasis on early life. OBJECTIVE: Changes in dimensions of the canal were investigated throughout life. SUMMARY OF BACKGROUND DATA: Seven hundred and fifteen lumbar vertebrae were examined from the Spitalfield Collection of Skeletons at the Natural History Museum, London. METHODS: Unmagnified silhouette pictures were taken of the canals with a specially designed photographic box. Computerized image analysis provided the accurate measurements. RESULTS: Regarding the midsagittal diameter and the cross-sectional area, the cranial four lumbar vertebrae were already fully matured in infants. At L5 there was significant increase up to 4 years of age when the midsagittal diameter was even larger than in the adult. The interpedicular diameter significantly increased at L1 until 10 years of age, at the other levels until adulthood, as did the perimeter at L4 and L5 until 14 years of age. The shape of the canal was assessed by measuring the circularity, the 'trefoilness' and the situation of the centroid. The first measurement significantly decreased with age, the trefoilness increased until adulthood, and the centroid of the canal approached the vertebral body. In spines with spina bifida occulta, the lumbar canal was significantly larger proximal to the lesion than in the unaffected spines. CONCLUSION: The lumbar spinal canal exhausts its growth potential by infancy as regards the midsagittal diameter and the cross-sectional area. Thus, in the case of delayed development, it is not capable of catch-up growth.

An in vitro study of the biomechanical effects of flexible stabilization on the lumbar spine.

STUDY DESIGN: Lumbar motion segments were tested in vitro to examine biomechanical changes after posterior fixation by a flexible device. OBJECTIVES: To assess changes in load distribution and conformation of vertebral structures after a flexible stabilization. This should provide the foundations for a scientific understanding of the immediate effects of this surgical procedure. METHODS: Hooks were placed over the proximal spinous process and the distal laminas of a motion segment and connected by a polyester braid. Tension applied to the braid then generated a compression of the posterior elements. The force between the articular facets, the displacement of the posterior anulus fibrosus of the intervertebral disc, and the change in the relative position of the adjacent vertebrae were measured as the applied tension was increased. RESULTS: Facet joint force, disc bulge, and vertebral angulation increased with applied tension until a position of "locking" was achieved, apparently when the bony margin of the superior half of the facet joint contacted the inferior pars interarticularis. A tension of between 50 to 100 N in the braid was required for this. Facet joint force was less than 40% of this, and disc bulge was only 0.15 mm. The extension of the motion segment was between 2 degrees and 8 degrees. CONCLUSIONS: The results suggest that if such a system is applied surgically, stabilization is produced by compaction of the bony margins of the facet joints. Only a relatively small proportion of the posteriorly applied load is carried by the facet joints themselves, and little angulatory change is expected with minimal disc bulge.