Mutations in proteins of the Conserved Oligomeric Golgi Complex affect polarity, cell wall structure, and glycosylation in the filamentous fungus Aspergillus nidulans.

We have described two Aspergillus nidulans gene mutations, designated podB1 (polarity defective) and swoP1 (swollen cell), which cause temperature-sensitive defects during polarization. Mutant strains also displayed unevenness and abnormal thickness of cell walls. Un-polarized or poorly-polarized mutant cells were capable of establishing normal polarity after a shift to a permissive temperature, and mutant hyphae shifted from permissive to restrictive temperature show wall and polarity abnormalities in subsequent growth. The mutated genes (podB=AN8226.3; swoP=AN7462.3) were identified as homologues of COG2 and COG4, respectively, each predicted to encode a subunit of the multi-protein COG (Conserved Oligomeric Golgi) Complex involved in retrograde vesicle trafficking in the Golgi apparatus. Down-regulation of COG2 or COG4 resulted in abnormal polarization and cell wall staining. The GFP-tagged COG2 and COG4 homologues displayed punctate, Golgi-like localization. Lectin-blotting indicated that protein glycosylation was altered in the mutant strains compared to the wild type. A multicopy expression experiment showed evidence for functional interactions between the homologues COG2 and COG4 as well as between COG2 and COG3. To date, this work is the first regarding a functional role of the COG proteins in the development of a filamentous fungus.

In vitro induction of infection-related hyphal structures in plant pathogenic fungi.

In recent years, a voluminous amount of genomic data has been generated for several plant pathogenic fungi. Multiple studies have utilized these genomic data to advance our knowledge about the molecular mechanisms of plant pathogenesis. However, not all plant pathogenic fungi share the same infection strategies, and several genes have been identified that are crucial for plant pathogenesis in one fungus, but dispensable in others. In order for data on biological relevance to keep pace with accumulating genomic data, new biological assays need to be developed for several pathogenic fungi. Accordingly, we have developed an in vitro assay that allows us to monitor morphological changes in hyphal development as the head blight pathogen Fusarium graminearum infects wheat. Using previously frozen detached wheat glumes, we are able to monitor both subcuticular and intercellular hyphal development of F. graminearum. The method described takes only 3-5 days from inoculation to microscopic observation (depending on time point) and does not require any elaborate laboratory equipment or supplies. This method could be adapted for different necrotrophic or hemi-biotrophic pathogens, on their host tissue types, in order to characterize their hyphal differentiation in vitro.

An antibiotic complex from Lysobacter enzymogenes strain C3: antimicrobial activity and role in plant disease control.

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 mug/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 mug/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and DeltaNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.

Septum formation in Aspergillus nidulans.

Filamentous fungi form multicellular hyphae that are partitioned by septa. In A. nidulans, septum formation requires the assembly of a septal band following the completion of mitosis. Recent observations show that this band is a dynamic structure composed of actin, a septin and a formin. In addition, assembly is dependent upon a conserved protein kinase cascade that regulates mitotic exit and septation in yeast. Hyphal differentiation may reflect the regulation of this cascade by cyclin-dependent kinase activity. In this review, the dynamics and regulation underlying the assembly of the septal band are discussed.

The Aspergillus nidulans snt genes are required for the regulation of septum formation and cell cycle checkpoints.

In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before forming a septum. Previous genetic results suggest that the ability to separate nuclear division and septum formation depends upon a threshold level of activity of the cyclin-dependent kinase NIMX(cdk1). Mutations in nimX and nimT, the gene encoding the NIMX(cdk1)-activating phosphatase, have revealed that Tyr-15 phosphorylation is important for determining the timing of the formation of the first septum. Here, we describe a screen for suppressors of nimT23 (snt), designed to identify additional components of the pathway regulating septum formation. We show that a subset of the snt mutants are defective in the temporal regulation of septum formation and in cell cycle checkpoint responses. Molecular characterization of sntA shows that it is allelic to the previously described ankA gene, which encodes the NIMX(cdk1) Tyr-15 kinase. Additional experiments described in this study show that nutritional conditions modulate the timing of septum formation and alter the phenotypes displayed by the snt mutants. A model that suggests that the timing of septum formation is influenced by DNA damage and glucose availability via the sntA and sntB gene products is proposed.

Cognitive-behavioral stress management intervention decreases the prevalence of depression and enhances benefit finding among women under treatment for early-stage breast cancer.

The authors tested effects of a 10-week group cognitive-behavioral stress management intervention among 100 women newly treated for Stage 0-II breast cancer. The intervention reduced prevalence of moderate depression (which remained relatively stable in the control condition) but did not affect other measures of emotional distress. The intervention also increased participants' reports that having breast cancer had made positive contributions to their lives, and it increased generalized optimism. Both remained significantly elevated at a 3-month follow-up of the intervention. Further analysis revealed that the intervention had its greatest impact on these 2 variables among women who were lowest in optimism at baseline. Discussion centers on the importance of examining positive responses to traumatic events--growth, appreciation of life, shift in priorities, and positive affect-as well as negative responses.

The Aspergillus nidulans musN gene encodes a RecQ helicase that interacts with the PI-3K-related kinase UVSB.

In Aspergillus nidulans, the uvsB gene encodes a member of the PI-3K-related kinase family of proteins. We have recently shown that UVSB is required for multiple aspects of the DNA damage response. Since the musN227 mutation is capable of partially suppressing defects caused by uvsB mutations, we sought to understand the mechanism underlying the suppression by cloning the musN gene. Here, we report that musN encodes a RecQ helicase with homology to S. pombe rqh1, S. cerevisiae sgs1, and human BLM and WRN. Phenotypic characterization of musN mutant alleles reveals that MUSN participates in the response to a variety of genotoxic agents. The slow growth and genotoxin sensitivity of a musN null mutant can be partially suppressed by a defect in homologous recombination caused by the uvsC114 mutation. In addition, we present evidence suggesting that MUSN may promote recovery from the DNA damage response. We suggest that a block to recovery caused by the musN227 mutation, coupled with the modest accumulation of recombination intermediates, can suppress defects caused by uvsB mutations. Finally, we report that another RecQ helicase, ORQA, performs a function that partially overlaps that of MUSN.

Improved FSH sensitisation and aromatase assay in human granulosa-lutein cells.

Granulosa-lutein (GL) cells from follicular aspirates from women undergoing in-vitro fertilization (IVF) treatment are usually refractory to follicle stimulating hormone (FSH) regarding the induction and/or maintenance of aromatase activity which converts androgens (e.g. testosterone) to oestrogens. The normal method of assaying FSH-stimulated aromatase activity in GL cell cultures is to add exogenous testosterone throughout the cell culture period and measure the secreted oestradiol. Thus under the conditions usually employed for studying FSH-stimulated oestradiol secretion in GL cells, the 'total' FSH effect is dependent both on the decay of the aromatase concentration in culture relative to its induction/maintenance by FSH and on changes in its activity in the face of a declining substrate concentration as the exogenous testosterone is converted over several days to oestradiol. We have therefore used a technique for challenging the cells with testosterone (10 micromol/l) for just 2 h at the end of the normal longer-term culture period such that its concentration was essentially unchanged, thus ensuring that there was no depletion of the aromatase substrate and that the FSH stimulation phase could be performed independently of exogenous testosterone. Consequently, GL cells were incubated for 0, 24 and 48 h prior to stimulation with FSH (100 IU/l) for 24 h after which they were washed and challenged with testosterone for 2 h and the secreted oestradiol was assayed. Freshly isolated GL cells from women undergoing IVF were refractory to FSH but after preincubation were responsive such that there was a 3-14-fold increase over basal activity depending on the cell preparation. In conclusion, we have developed a simple 48 h procedure for sensitizing GL cells to FSH and established the conditions for optimizing the assay of aromatase activity independently from the effect of FSH on its induction.

The Aspergillus nidulans uvsB gene encodes an ATM-related kinase required for multiple facets of the DNA damage response.

In Aspergillus nidulans, uvsB and uvsD belong to the same epistasis group of DNA repair mutants. Recent observations suggest that these genes are likely to control cell cycle checkpoint responses to DNA damage and incomplete replication. Consistent with this notion, we show here that UVSB is a member of the conserved family of ATM-related kinases. Phenotypic characterization of uvsB mutants shows that they possess defects in additional aspects of the DNA damage response besides checkpoint control, including inhibition of septum formation, regulation of gene expression, and induced mutagenesis. The musN227 mutation partially suppresses the poor growth and DNA damage sensitivity of uvsB mutants. Although musN227 partially suppresses several uvsB defects, it does not restore checkpoint function to uvsB mutants. Notably, the failure of uvsB mutants to restrain septum formation in the presence of DNA damage is suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DNA damage response, whereas MUSN promotes recovery by modulating a subset of the response.

Morphogenesis is coordinated with nuclear division in germinating Aspergillus nidulans conidiospores.

Germinating Aspergillus nidulans conidiospores switch to polarized apical growth following an initial period of isotropic expansion. At the same time, they re-enter the nuclear division cycle. The relationship between spore polarization and nuclear division was investigated by testing the effect of cell cycle inhibitors and temperature-sensitive cell cycle mutations on spore morphogenesis. On rich media, it was found that spore polarization is delayed if completion of the first mitosis is blocked. The observed delay may be dependent upon the activity of the mitosis-promoting NIMA kinase. An additional mechanism appears to prevent polarization as the spore progresses through its first S phase. In contrast, on poor media, spore polarization does not require completion of the first mitosis. These observations suggest that spore morphogenesis is influenced by cell cycle signals in a growth-dependent manner.

Identification and characterization of genes required for hyphal morphogenesis in the filamentous fungus Aspergillus nidulans.

In the filamentous fungus Aspergillus nidulans, germination of an asexual conidiospore results in the formation of a hyphal cell. A key feature of spore germination is the switch from isotropic spore expansion to polarized apical growth. Here, temperature-sensitive mutations are used to characterize the roles of five genes (sepA, hypA, podB-podD) in the establishment and maintenance of hyphal polarity. Evidence that suggests that the hypA, podB, and sepA genes are required for multiple aspects of hyphal morphogenesis is presented. Notably, podB and sepA are needed for organization of the cytoskeleton at sites of polarized growth. In contrast, podC and podD encode proteins that appear to be specifically required for the establishment of hyphal polarity during spore germination. The role of sepA and the pod genes in controlling the spatial pattern of polarized morphogenesis in germinating spores is also described. Results obtained from these experiments indicate that the normal pattern of germ-tube emergence is dependent upon the integrity of the actin cytoskeleton.

Concern about aspects of body image and adjustment to early stage breast cancer.

OBJECTIVE: Several authors have suggested that patients adjust more poorly to breast cancer if they are heavily invested in body image as a source of their sense of self-worth. This prospective study examined this possibility, looking at two aspects of concern about body image as predictors of several indices of adjustment over the first postoperative year. METHODS: At diagnosis (and again a year later) 66 women with early stage breast cancer reported how much they valued a) a sense of body integrity (or intactness) and b) a good physical appearance. The day before surgery, a week afterward, and at 3-month, 6-month, and 12-month follow ups, they reported on their mood. At presurgery and at follow ups they also rated their attractiveness and sexual desirability and reported on frequency of sexual interaction. At follow-ups they also indicated how much their illness and treatment were interfering with social and recreational activities. RESULTS: Initial investment in appearance was related to distress across the postsurgical year. In contrast, investment in appearance made women more resilient against deterioration in their perceptions of attractiveness. Concern about body integrity did not strongly predict emotional distress, but it related to adverse impact on social and recreational activities in the follow-up period, to deterioration in feelings of sexual desirability, and to feelings of alienation from the self (feeling "not like yourself anymore"). CONCLUSIONS: Body image is often thought of in terms of physical appearance, but there is also a body image pertaining to integrity, wholeness, and normal functioning. People who are greatly concerned about either aspect of their body image are vulnerable to poorer psychosocial adjustment when confronting treatment for breast cancer. The poorer adjustment takes a different form, however, depending on the nature of the patient's body-image concern.

Regulation of septum formation in Aspergillus nidulans by a DNA damage checkpoint pathway.

In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34nimX activity to control the timing of septum formation.

The duplication cycle in Aspergillus nidulans.

The duplication cycle encompasses the spectrum of events required for the growth and division of individual cells within a fungal hyphae. Recent advances in understanding the mechanisms which underlie nuclear division and cellular morphogenesis in the filamentous fungus Aspergillus nidulans have shown that in many respects, the duplication cycle differs significantly from the cell cycles of both budding and fission yeast. The purpose of this review is to summarize these advances and to highlight the fundamental differences between the duplication cycle and the yeast cell cycles. In addition, it is argued that the duplication cycle is controlled by cellular regulatory networks which integrate the processes of nuclear division, cellular morphogenesis, and cell growth with each other. Functional dissection of these networks should help to reveal features that are unique to the hyphal mode of growth.

The Aspergillus nidulans sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity.

Cytokinesis (septation) in the fungus Aspergillus nidulans occurs through the formation of a transient actin ring at the incipient division site. Temperature-sensitive mutations in the sepA gene prevent septation and cause defects in the maintenance of cellular polarity, without affecting growth and nuclear division. The sepA gene encodes a member of the growing family of FH1/2 proteins, which appear to have roles in morphogenesis and cytokinesis in organisms such as yeast and Drosophila. Results from temperature shift and immunofluorescence microscopy experiments strongly suggest that sepA function requires a preceding mitosis and that sepA acts prior to actin ring formation. Deletion mutants of sepA exhibit temperature-sensitive growth and severe delays in septation at the permissive temperature, indicating that expression of another gene may compensate for the loss of sepA. Conidiophores formed by sepA mutants exhibit abnormal branching of the stalk and vesicle. These results suggest that sepA interacts with the actin cytoskeleton to promote formation of the actin ring during cytokinesis and that sepA is also required for maintenance of cellular polarity during hyphal growth and asexual morphogenesis.

Internal carbohydrate complexity of the oligosaccharide chains of recombinant human follicle stimulating hormone (Puregon, Org 32489): a comparison with Metrodin and Metrodin-HP.

Glycoforms of recombinant human follicle stimulating hormone (rhFSH) (Org 32489, Puregon) were characterized using concanavalin A lectin affinity chromatography to reveal information about the internal carbohydrate complexity (extent of carbohydrate side-chain branching) of the preparations. The rhFSH glycoforms were measured by radioimmunoassay and a two-site immunoradiometric assay and compared with those in two urinary preparations (Metrodin and Metrodin-HP) used in assisted reproduction programmes and a urinary FSH international standard 70/45 (uFSH IS 70/45). Similar data were obtained with both assays; rhFSH had 6% complex internal carbohydrate structures compared with 22-27% for Metrodin, Metrodin-HP and uFSH. The proportion of simple carbohydrate structures was also different, with rhFSH having 18.5 compared with 4.5-9.3% for Metrodin, Metrodin-HP and uFSH. A linear relationship was observed between the percentage glycoforms with an isoelectric point (pl) < 4 and the log percentage simple forms (logarithmic regression; r = 0.93) indicating a direct relationship between carbohydrate complexity and charge heterogeneity. In summary, rhFSH contains fewer complex forms and an increased proportion of simple carbohydrate structures in comparison with Metrodin, Metrodin-HP and IS 70/45.

Cytokinesis in Aspergillus nidulans is controlled by cell size, nuclear positioning and mitosis.

The mycelium of Aspergillus nidulans is composed of multinucleate cellular compartments delimited by crosswalls called septa. Septum formation is dependent on mitosis and requires the recruitment of actin to the site of septum formation. Employing a collection of temperature sensitive nuclear distribution (nudA2, nudC3 and nudF7), nuclear division (nimA5, hfaB3), and septation (sepD5, sepG1) mutants, we have investigated the interdependency among nuclear positioning, mitosis, and cell growth in structuring the cellular compartments of A. nidulans. The cellular compartments of nud+ strains were highly uniform with regard to nuclear distribution and averaged 38 microns in length. Incubation of nud mutants at semi-restrictive temperature resulted in aberrant nuclear distribution that appeared to direct the formation of variable-sized cellular compartments, ranging from 5 microns to greater than 81 microns. In germinating spores, the first septum forms at the basal end of the germ tube following the third round of nuclear division. Germlings must undergo mitosis in order to form a septum. Temperature-sensitive mitotic mutants were used to show that a single nuclear division is sufficient to activate septum formation, provided a critical cell size has been attained. In mitotic mutants and wild-type cells, delays in nuclear division resulted in the misplacement of the first septum. These results strongly support the role of mitotic nuclei in determining septal placement, and suggest that cell size control is post-mitotic in A. nidulans.

Spontaneous cerebrospinal fluid leaks originating from multiple skull base defects.

Spontaneous cerebrospinal fluid (CSF) leaks of temporal bone origin are more prevalent than once believed. Twenty-eight of the 61 cases documented in the world literature have been reported since 1992. All but four of these cases involved unilateral defects. The authors have previously reported experiences with 12 cases, with the vast majority of defects localized to the tegmen tympani. These patients also had demonstrated a single area of bone and dural dehiscence. We report two additional cases of spontaneous CSF leak originating from multiple/distant skull base defects. As in previously reported multisite cases, one of our patients demonstrated an elevated opening pressure on lumbar puncture. Significant time intervals existed between leak site presentations, which emphasizes the importance of careful follow-up for treated patients. Potential etiologies and associated factors are also discussed. This patient subset contributes another dimension to the evolving natural history of spontaneous CSF leakage.

sepB: an Aspergillus nidulans gene involved in chromosome segregation and the initiation of cytokinesis.

In Aspergillus nidulans conidia, cytokinesis (septation) is delayed until three rounds of nuclear division have been completed. This has permitted the identification of essential genes that are involved in the coordination of cytokinesis with nuclear division. Conditional mutations in the sepB gene block septation but allow germinating spores to complete the first three rounds of nuclear division at restrictive temperature. sepB3 mutants demonstrate transient delays in M-phase, accumulate aneuploid nuclei and show defects in chromosome segregation. Molecular analysis of the sepB gene reveals that it is essential and possesses limited similarity to the CTF4 gene of Saccharomyces cerevisiae. Using temperature-shift analysis we show that sepB is required after the first nuclear division but before the onset of cytokinesis. A failure to execute the sepB function results in a block to nuclear division and leads to cell death at a time when wild-type cells would be undergoing cytokinesis. Finally, we demonstrate that sepB is also required for the uninucleate cell divisions of developing conidiophores. Our results suggest that sepB3 mutants accumulate specific nuclear defects that do not arrest mitosis, but block the initiation of septum formation. Thus, proper chromosome segregation and a functional sepB gene are required to initiate cytokinesis.

Ostrich slaughter and fabrication: 1. Slaughter yields of carcasses and effects of electrical stimulation on post-mortem pH.

A commercial ostrich slaughter protocol was developed. Ostriches (n = 7 males and n = 7 females) averaged 95.54 kg live weight and yielded 55.91-kg carcasses. By-product yields were measured. The most significant by-products by weight were full viscera (8.29 kg), hide (6.71 kg), full gizzard and crop (5.80 kg), and abdominal fat (4.11 kg). Sex had no effect on slaughter yields. Post-mortem temperature declines were measured on five separate muscles and showed that chilling for 24 h was sufficient to adequately chill the deep muscle temperature to under 4 C. The effect of electrical stimulation on post-mortem pH decline also was investigated and had no effect.