Ageing of Neurospora crassa. VIII. Lethality and mutagenicity of ferrous ions, ascorbic acid, and malondialdehyde.

Ferrous ions were highly lethal and mutagenic to germinated conidia of Neurospora crassa. At comparable survival, treatment with 0.2 mM ferrous ions was 14- and 50-fold more mutagenic than ultra-violet irradiation or X-rays, respectively, in the reversion of an inositol auxotroph. Ascorbic acid alone (2 mM) was not reproducibly lethal and inhibited both the lethality and mutagenicity of ferrous ions. Bovine superoxide dismutase (SOD) completely inhibited the residual lethality of ferrous ascorbate. Protection by ascorbic acid and SOD indicates that superoxide radicals, generated by oxidation of Fe(II), are directly or indirectly mutagenic and lethal. Malondialdehyde (MDA) was lethal and appeared to be mutagenic; however, its action is probably different from that of superoxide. Therefore, superoxide-mediated production of endogenous MDA by way of peroxidation of polyunsaturated fatty acids is probably not an alternate mutagenic pathway, at least in the reversion of the allele of the inl locus examined. These results and the demonstration of superoxide-mediated decrease in the synthetic fidelity of DNA polymerase in vitro (Rana and Munkres, in preparation) warrant additional exploration of the hypothesis that endogenous cellular free radicals, generated by pre- and post-senescent metabolism, may enter into lethal and mutagenic reactions.     

A morphological and genetic analysis of conidiophore development in Neurospora crassa

The filamentous fungus Neurospora crassa responds to nutrient deprivation and dessication by producing asexual spores, or conidia. These conidia are derived from differentiated aerial structures called conidiophores. The process of conidiation was analyzed in wild-type and morphological mutants using scanning electron microscopy (SEM) and specific fluorescent probes. The first discernible morphological step of conidiation is the transition from growth by hyphal tip elongation to growth by repeated apical budding, resulting in the formation of chains of proconidia that resemble beads on a string. The initial proconidial chains are morphologically distinct from those that form later and are capable of reverting to hyphal growth, whereas the later chains are committed to conidiation. As the proconidial chains are formed, nuclei migrate into the conidiophore, and cross-walls arise between adjoining proconidia in a series of steps that have been defined by staining with Calcofluor, a fluorescent chitin-binding probe. The chains ultimately disarticulate in several discrete stages into free, morphologically mature conidia. Different conidiation-defective mutants were shown to be blocked at distinct stages in conidiation. Our observations permit us to derive a developmental timeline of conidiation relating the occurrence of morphological changes and the stage blocked in specific mutants.     

Ageing of Neurospora crassa. VII. Accumulation of fluorescent pigment (lipofuscin) and inhibition of the accumulation by nordihydroguairetic acid.

Continuous administration of the antioxidant nordihydroguairetic acid to clones of the natural-death nutant or wild-type Neurospora crassa growing on 5-fluorotryptophan not only alleviates the time-dependent deterioration of growth rate (senescence), but also inhibits the accumulation of a fluorescent pigment (lipofuscin), an end-product of lipid peroxidation. In the three model system of ageing in N. crassa that we have examined, it is now clear that massive accumulation of lipofuscin begins shortly before or concident with the period of senescence. By this and other criteria, the youngest cells in time of origin appear to be most senescent. Thus the degree of senscence appears to be a function of physiological rather than chronological age and, more specifically, is probably a function of the number of nuclear mitotic divisions. Normal, non-senescent, wild-type, clones also accumulate lipofuscin; but, unlike clones undergoing senscence, accumulate very low concentrations which are independent of subclonal age. Apparently, clones of wild type can tolerate a low level of lipid peroxidation without undergoing senescence; but even a low level is inimical since culture with nordihydroguaiaretic acid not only enhances their growth rate, but also inhibits the accumulation of lipofuscin.     

Expression of human mucin genes during normal and abnormal renal development

Human mucin genes encode large O-glycoproteins, which are expressed in various epithelial tissues. The proteins are the main components of mucus, but also might be involved in morphogenesis of or carcinogenesis in many organs. We studied the expression of human mucin genes during fetal kidney development and in malformed cystic renal diseases in 10 normal fetal kidneys and 12 malformed kidneys by in situ hybridization and immunohistochemical analysis. MUC1, MUC3, and MUC6 were expressed in normal fetal kidney. MUC1 was expressed from 7.5 weeks of gestation in the metanephric blastema and throughout fetal life in the ureteric buds, distal convoluted tubules, and collecting ducts. MUC3 was expressed weakly in immature tubules from 8 weeks of gestation, after which it was expressed weakly and focally in the proximal convoluted tubules. MUC6 was expressed at 9.5 weeks of gestation in the tips of the ureteric buds and later in the collecting ducts. In malformative cystic diseases, only MUC1 expression was retained; no expression of MUC6 and MUC3 was observed. These results implicate human mucin genes (MUC1, MUC3, and MUC6) in renal morphogenesis processes.     

Genetic organization and structural features of maranhar,a senescence-inducing linear mitochondrial plasmid of Neurospora crassa

Summary The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.     

Molecular cloning and physical mapping of the Neurospora crassa 74-OR23-1A mitochondrial genome

Summary To provide for thorough sampling of the Neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the EcoRI digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped HincII sites. Data used to locate new restriction sites were obtained from digestions of whole mitochondrial DNA, digestions of the cloned EcoRI mitochondrial DNA fragments and hybridizations between new restriction fragments and the cloned fragments. Length measurements of the total genome and of EcoRI fragment 1 are larger than commonly reported.     

Different respiratory-defective phenotypes of Neurospora crassa and Saccharomyces cerevisiae after inactivation of the gene encoding the mitochondrial acyl carrier protein

The nuclear genes (acp-1, ACP 1) encoding the mitochondrial acyl carrier protein were disrupted in Neurospora crassa and Saccharomyces cerevisiae. In N. crassa acp-1 is a peripheral subunit of the respiratory NADH: ubiquinone oxidoreductase (complex I). S. cerevisiae lacks complex I and its ACP1 appears to be located in the mitochondrial matrix. The loss of acp-1 in N. crassa causes two biochemical lesions. Firstly, the peripheral part of complex I is not assembled, and the membrane part is not properly assembled. The respiratory ubiquinol: cytochrome c oxidose (complex IV) are made in normal amounts. Secondly, the lysophospholipid content of mitochondrial membranes is increased four-fold. In S. cerevisiae, the loss of aCP1 leads to a pleiotropic respiratory deficient phenotype.     

A relational view of causality in normal and abnormal development

An understanding of developmental phenomena demands a relational or coactive concept of causality, as opposed to a conceptualization that assumes that singular causes can act in isolation. In this article we present a developmental psychobiological systems view of relational (bidirectional, coactional) causality, in which it is proposed that developmental outcomes are a consequence of at least two specific components of coaction from the same or different levels of a developmental system. The levels are genetic, neural, behavioral, and environmental; the latter level includes the cultural, social, and physical aspects of an organism's environment. We show the applicability of this view to the understanding of the development of normal and abnormal behavioral and psychological phenotypes through illustrations from the existing animal and human literature. Finally, we discuss future possibilities and potential stumbling blocks in the implementation of a more fully realized bidirectional, coactional perspective in developmental psychopathological research.     

Elimination of an abnormal cell line from blood lymphocytes during post-natal growth

A cell line carrying an additional metacentric chromosome of unknown origin was present in PHA-stimulated blood lymphocytes from a newborn female. The child had no marked physical or developmental abnormality but was small. The abnormal cell line, present in 20 % of blood cells at birth, progressively decreased and was absent by 16 months. Loss of the cell line appeared to involve a process of elimination rather than a chance event.     

Biochemical, genetic and ultrastructural defects in a mitochondrial mutant (ER-3) of Neurospora crassa with senescence phenotype

The structural and functional abnormalities in a new respiratory deficient, mitochondrial senescence mutant ER-3 of Neurospora crassa are described. The mitochondrial mutant, which grows at a rate of only 10% of that of the wild type, was found deficient in all three cytochromes, and completely lacking in cytochromes aa3. Cytochrome oxidase activity in the mutant mitochondria was only about 5% of the wild type mitochondria. However, the total whole cell respiration rate of the mutant was 33% greater than that of the wild type, while the cyanide-resistant respiration rates were equal. The results of inhibitor studies clearly demonstrate that the mutant possesses a defect in one or more components of the terminal oxidase. Electron microscopic examination of whole cell sections and subsequent morphometric analysis revealed a significant (33%) reduction in membrane surface density of mitochondrial cristae in the mutant as compared with the wild type. Results of genetic and heterokaryon analyses indicate the location of mutation (ER-3) in the mitochondrial DNA. It is concluded that the senescence mutant ER-3 possesses a defect in the terminal portion of the mitochondrial respiratory apparatus. These results are consistent with previous analyses of mitochondrial DNA populations, and support the notion that obligately aerobic eukaryotic cells deficient in mitochondrial respiration necessarily exist as a result of stable heteroplasmosis and that defects in mitochondria lead to senescence in Neurospora mutant ER-3.     

Cortical plasticity in normal and abnormal cognitive development: evidence and working hypotheses.

In this paper I review evidence and viewpoints on developmental plasticity in the cerebral cortex. Although there is some degree of plasticity in the cortex during early postnatal life in the human infant, this plasticity is constrained by various factors. Three working hypotheses about postnatal cortical specialization of function are advanced, and some specific predictions about the limits and extent of plasticity are assessed through both empirical evidence from infants and simulations on simple cortical network models.     

Inactivation of the Neurospora crassa mitochondrial outer membrane protein TOM70 by repeat-induced point mutation (RIP) causes defects in mitochondrial protein import and morphology

Mitochondrial biogenesis requires the efficient import of hundreds of different cytosolically translated preproteins into existing organelles. Recognition and translocation of preproteins at the mitochondrial outer membrane is achieved by the TOM complex (translocase of the outer mitochondrial membrane). The largest component of this complex is TOM70, an integral outer membrane protein with a large cytosolic domain thought to serve as a receptor for a specific group of preproteins. To investigate the functional role of TOM70 in Neurospora crassa the tom70 gene was inactivated using the natural phenomenon of repeat-induced point mutation (RIP). Mutant strains were identified that harbored RIPed tom70 alleles and contained no immunologically detectable TOM70. Strains that lack TOM70 grow more slowly than wild-type strains, conidiate poorly, and contain enlarged mitochondria. In vitro preprotein import studies using TOM70-deficient mitochondria revealed a defect in the uptake of the ADP/ATP carrier. Other preproteins tested were imported at wild-type rates with the exception of the precursor of the mitochondrial-processing peptidase (MPP) which was imported more efficiently by TOM70-deficient mitochondria. These data support the view that TOM70 plays a role as a specific receptor for carrier proteins in mitochondrial-preprotein import. The presence of tetratricopeptide repeats (TPRs) in the TOM70 sequence and the enlarged shape of mitochondria lacking TOM70 raise the possibility that the protein also plays a role in the maintenance of mitochondrial morphology. Received: 25 March 1999 / 18 May 1999     

The PAS/LOV protein VIVID controls temperature compensation of circadian clock phase and development in Neurospora crassa

Circadian clocks are cellular timekeepers that regulate aspects of temporal organization on daily and seasonal time scales. To allow accurate time measurement, the period lengths of clocks are conserved in a range of temperatures--a phenomenon known as temperature compensation. Temperature compensation of circadian clock period aids in maintaining a stable "target time" or phase of clock-controlled events. Here we show that the Neurospora protein VIVID (VVD) buffers the circadian system against temperature fluctuations. In vvd-null mutants, the circadian period of clock-controlled events such as asexual sporulation (conidiation) is temperature compensated, but the phase of this clock time marker is not. Consistent with delayed conidiation at lower temperatures in vvd(KO) strains, the levels of vvd gene products in the wild type increase with decreasing temperatures. Moreover, vvd(C108A) mutants that lack the light function of VVD maintain a dark activity that transiently influences the phase of conidiation, indicating that VVD influences the time of conidiation downstream from the clock. FREQUENCY (FRQ) phosphorylation is altered in a vvd(KO) strain, suggesting a mechanism by which VVD can influence the timing of clock-controlled processes in the dark. Thus, temperature compensation of clock-controlled output is a key factor in maintaining temperature compensation of the entire circadian system.     

Morphogenetic concepts of normal and abnormal growth in the human prostate

 Benign prostatic hyperplasia (BPH) and prostate cancer are multifactorial disease processes, involving a growing number of biochemical, genetic and epigenetic factors. Their pathogenesis, however, remains poorly understood. The present review examines current morphogenetic concepts of normal and abnormal growth in the human prostate. This includes the role of basal cells in organogenesis and cancerogenesis, the impact of cell–matrix interactions, and the importance of cellular heterogeneity in tumour progression and hormone-insensitive growth. Knowledge of morphogenesis and morphology is required in any scientific approach to BPH and prostate cancer. Received: 21 March 1998 / Accepted: 25 May 1998