Homologous expression of a mutated beta-tubulin gene does not confer benomyl resistance on Trichoderma virens.

AIMS: To clone the beta-tubulins and to induce resistance to benzimidazoles in the biocontrol fungus Trichoderma virens through site-directed mutagenesis. METHODS AND RESULTS: Two beta-tubulin genes have been cloned using PCR amplification followed by the screening of a T. virens cDNA library. The full-length cDNA clones, coding for 445 and 446 amino acids, have been designated as T. virens tub1 and T. virens tub2. A sequence alignment of these two tubulins with tubulins from other filamentous fungi has shown the presence of some unique amino acid sequences not found in those positions in other beta-tubulins. Constitutive expression of the tub2 gene with a histidine to tyrosine substitution at position 6 (known to impart benomyl/methyl benzimadazol-2-yl carbamate resistance in other fungi), under the Pgpd promoter of Aspergillus nidulans, did not impart resistance to benomyl. CONCLUSIONS: The homologous expression of tub2 gene with a histidine to tyrosine mutation at position +6, which is known to impart benomyl tolerance in other fungi, does not impart resistance in T. virens. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike other Trichoderma spp., T. virens, has been difficult to mutate for benomyl tolerance. The present study, through site-directed mutagenesis, shows that a mutation known to impart benomyl tolerance in T. viride and other fungi does not impart resistance in this fungus. Understanding the mechanisms of this phenomenon will have a profound impact in plant-disease management, as many plant pathogenic fungi develop resistance to this group of fungicides forcing its withdrawal after a short period of use.

Serotonergic dorsal raphe neurons from obese zucker rats are hyperexcitable.

Release of serotonin (5-HT) from dorsal raphe nucleus (DRN) neurons projecting to the ventromedial hypothalamus (VMH) has a modulatory effect on the neural pathway involved in feeding, hunger, and satiety. The obese Zucker rat, an animal model of genetic obesity, exhibits differences in serotonin signaling as well as a mutated leptin receptor. To evaluate possible mechanisms underlying this difference in serotonin signaling, we have compared electrophysiological responses of DRN neurons from 14- to 25-day-old male lean (Fa/Fa) and obese (fa/fa) Zucker rats using the whole-cell patch clamp technique on cells in brain slices from these animals. We found that the resting properties of these neurons are not different, but the DRN neurons from obese rats are hyperexcitable in response to current injection. This hyperexcitability is not accompanied by an increase in the depolarization caused by current injection or by changes in the threshold for spiking. However, the hyperexcitability is accompanied by reduction in the size and time course of the afterhyperpolarization (AHP) following an action potential. DRN neurons of obese rats recover from the AHP faster due to a smaller amplitude AHP and a faster time constant (tau) of decay of the AHP. These deficits are not due to changes in the spike waveform, as the spike amplitude and duration do not differ between lean and obese animals. In summary, we provide evidence that serotonergic DRN neurons from obese Zucker rats are intrinsically hyperexcitable compared with those from lean rats. These results suggest a potential mechanism for the reported increase in 5-HT release at the VMH of obese rats during feeding, and provide the first direct evidence of changes in the intrinsic activity of serotonergic neurons, which are crucial regulators of feeding behavior, in a genetic model of obesity.

Meal-induced changes in extracellular 5-HT in medial hypothalamus of lean (Fa/Fa) and obese (fa/fa) Zucker rats.

Hypothalamic serotonin (5-HT) is involved in appetite regulation and sympathetic stimulation of thermogenesis. This study tested the hypothesis that the enhanced energetic efficiency of obese Zucker rats involves blunted serotonergic release within the medial hypothalamus (MH). We used microdialysis and HPLC-EC to measure dynamic changes in extracellular 5-HT levels in the MH of 10-13-week-old male lean (Fa/Fa) and obese (fa/fa) Zucker rats before and after a meal. No differences were noted in basal levels of 5-HT between lean and obese rats. Consistent with the suggestion that hypothalamic 5-HT plays a physiological role in feeding, extracellular 5-HT levels increased significantly in both lean and obese rats given a meal. This increase was observed in the 20 min interval in which they ate the 8.1 kcal meal and remained for an additional 60 min. The net release of 5-HT during the meal interval was comparable in the lean (1.46+/-0.38 fmol/microl) and obese (1.21+/-0.82 fmol/microl) rats. However, the 5-HT levels of the leans (1.80+/-0.29 fmol/microl) plateaued in the next 20 min interval, whereas they continued rising (2.74+/-0.53 fmol/microl) in obese rats and were significantly higher than those in the leans during the 40 and 60 min intervals after the meal was presented. This resulted in a total net release during the meal plus the next three 20 min intervals that was significantly higher in obese (9.83+/-1.16 fmol/microl) than in lean (5.59+/-0.85 fmol/microl) rats. Thus, the enhanced energetic efficiency of the obese Zucker rats may not be associated with attenuated serotonin release in response to a meal. Rather their enhanced release of 5-HT in the MH may reflect compensatory mechanisms for the elevated orexigen NPY, the reduction in meal-induced CCK release, and/or a functional resistance to 5-HT.

Reduced feeding response to neuropeptide Y in senescent Fischer 344 rats.

The anorexia of aging syndrome in humans is characterized by spontaneous body weight loss reflecting diminished food intake. We reported previously that old rats undergoing a similar phenomenon of progressive weight loss (i.e., senescent rats) also display altered feeding behavior, including reduced meal size and duration. Here, we tested the hypothesis that blunted responsiveness to neuropeptide Y (NPY), a feeding stimulant, occurs concurrently with senescence-associated anorexia/hypophagia. Young (8 mo old, n = 9) and old (24-30 mo old, n = 11) male Fischer 344 rats received intracerebroventricular NPY or artificial cerbrospinal fluid injections. In response to a maximum effective NPY dose (10 microg), the net increase in size of the first meal after injection was similar in old weight-stable (presenescent) and young rats (10.85 +/- 1.73 and 12.63 +/- 2.52 g/kg body wt (0.67), respectively). In contrast, senescent rats that had spontaneously lost approximately 10% of body weight had significantly lower net increases at their first post-NPY meal (1.33 +/- 0.33 g/kg body wt (0.67)) than before they began losing weight. Thus altered feeding responses to NPY occur in aging rats concomitantly with spontaneous decrements in food intake and body weight near the end of life.

Effects of 2 G on adiposity, leptin, lipoprotein lipase, and uncoupling protein-1 in lean and obese Zucker rats.

Male Zucker rats were exposed to 2 G for 8 wk to test the hypothesis that the leptin regulatory pathway contributes to recovery from effects of 2 G on feeding, growth, and nutrient partitioning. After initial hypophagia, body mass-independent food intake of the lean rats exposed to 2 G surpassed that of the lean rats maintained at 1 G, but food intake of the obese rats exposed to 2 G remained low. After 8 wk at 2 G, body mass and carcass fat were less in both genotypes. Leptin and percent fat were lower in lean rats exposed to 2 G vs. 1 G but did not differ in obese rats exposed to 2 G vs. 1 G. Although exposure to 2 G did not alter uncoupling protein-1 levels, it did elicit white fat pad-specific changes in lipoprotein lipase activity in obese but not lean rats. We conclude that 2 G affects both genotypes but that the lean Zucker rats recover their food intake and growth rate and retain "normal" lipoprotein lipase activity to a greater degree than do the obese rats, emphasizing the importance of a functional leptin regulatory pathway in this acclimation.

Nucleotide effects on liver and muscle mitochondrial non-phosphorylating respiration and membrane potential.

Uncoupling protein-1 homologs are hypothesized to mediate mitochondrial proton leak. To test this hypothesis, we determined the effects of ATP and other nucleotides on liver and skeletal muscle mitochondrial non-phosphorylating respiration (VO(2)), membrane potential, FCCP-stimulated respiratory control ratios, and swelling. Neither ATP nor CTP affected liver or muscle proton leak, but both inhibited the respiratory chain. Unexpectedly, CMP stimulated liver proton leak (EC(50) approximately 4.4+/-0.5 mM). Using CMP chromatography, we identified two proteins (M(r)=31.2 and 32.6 kDa) from liver mitochondria that are similar in size to members of the mitochondrial carrier protein family. We conclude (a) liver and muscle mitochondrial proton leak is insensitive to ATP and CTP, and (b) CMP activates a leak in liver mitochondria. The CMP-inducible leak may be mediated by a 30-32 kDa protein. Based on the high concentrations required, CMP is unlikely to be a physiologically important leak regulator. Nonetheless, our results show that tissues other than brown fat have inducible leaks that may be protein-mediated.

A convenient fluorometric method for the detection of extracellular N-acetylglucosaminidase production by filamentous fungi.

A rapid and convenient method for the detection of chitinases accumulating in filamentous fungal cultures was developed. The assay is performed on cultures growing in microtiter plates, with a fluorogenic substrate: 4-methylumbelliferyl-N-acetyl-D-glucosaminide (4-MeUNAG). The fluorescence of the product, 4-methylumbelliferone, was detected. This method was successfully used to follow induction and repression of extracellular exochitinase activity in the biocontrol fungus Trichoderma harzianum.

Helping undergraduates repair faulty mental models in the student laboratory.

Over half of the undergraduate students entering physiology hold a misconception concerning how breathing pattern changes when minute ventilation increases. Repair of this misconception was used as a measure to compare the impact of three student laboratory protocols on learning by 696 undergraduate students at 5 institutions. Students were tested for the presence of the misconception before and after performing a laboratory activity in which they measured the effect of exercise on tidal volume and breathing frequency. The first protocol followed a traditional written "observe and record" ("cookbook") format. In the second treatment group, a written protocol asked students to complete a prediction table before running the experiment ("predictor" protocol). Students in the third treatment group were given the written "predictor" protocol but were also required to verbalize their predictions before running the experiment ("instructor intervention" protocol). In each of the three groups, the number of students whose performance improved on the posttest was greater than the number of students who performed less well on the posttest (P < 0.001). Thus the laboratory protocols helped students correct the misconception. However, the remediation rate for students in the "instructor intervention" group was more than twice that observed for the other treatment groups (P < 0.001). The results indicate that laboratory instruction is more effective when students verbalize predictions from their mental models than when they only "discover" the outcome of the experiment.

Extracellular hypothalamic serotonin levels after dorsal raphe nuclei stimulation of lean (Fa/Fa) and obese (fa/fa) Zucker rats.

Serotonin (5-HT), acting in the medial hypothalamus (MH), is involved in appetite/satiety and sympathetic stimulation of thermogenesis. This study tested the hypothesis that the enhanced energetic efficiency of obese Zucker rats is associated with a reduced capacity of activated dorsal raphe (DR) neurons to release 5-HT in the MH. We used microdialysis and HPLC-EC to measure dynamic changes in extracellular 5-HT levels in the MH of urethane-anesthetized, 10-14 week old male lean and obese Zucker rats. These concentrations did not differ significantly between the two genotypes prior to stimulation (mean+/-S.E.M.=3.8+/-0.5 fmol/microl, lean; 3.6+/-1.0 fmol/microl, obese) or following DR stimulation at 25 Hz (200 microA). The latter elicited initial net increases of 0.54+/-0.15 fmol/microl in lean and 0.58+/-0.20 fmol/microl in obese rats; and 20 min post-stimulus, 5-HT values were still elevated and comparable in the two genotypes. Although a 50-Hz (200 microA) stimulus evoked initial increases that were similar in lean (1.37+/-0.23 fmol/microl) and obese (0.95+/-0.24 fmol/microl,) rats, the net increase in 5-HT concentration during the next 20-40 min period was higher in the lean (2.03+/-0.55 fmol/microl vs. 1.18+/-0.24 fmol/microl in the obese animals). Also, in the lean, but not obese rats, extracellular 5-HT levels were significantly greater at 50 vs. 25 Hz. These results support the hypothesis that the capacity of midbrain serotonergic neurons to release 5-HT at the MH is reduced in obese Zucker rats, consistent with their blunted responsiveness to dietary stimuli and greater energetic efficiency.

Characterization of blue-light and developmental regulation of the photolyase gene phr1 in Trichoderma harzianum.

Blue light and development regulate the expression of the phr1 gene of the filamentous fungus Trichoderma harzianum. The predicted product of phr1, the DNA repair enzyme photolyase, is likely to help protect Trichoderma, which grows in the soil as a mycoparasite or saprophyte, from damage upon emergence and exposure to ultraviolet-c. phr1 is transiently expressed in mycelium and conidiophores after illumination. phr1 mRNA also accumulates in conidiophores during development and spore maturation. As no other genes displaying rapid, direct light regulation have been described previously in this organism, we have characterized the fluence and time dependence of phr1 induction, and its relation to sporulation and photoreactivation. Induction is transient following a pulse, and, with slower decay, in continuous light. This implies that the photoreceptor, transducers or response are capable of adaptation. About two-fold more light is required to induce phr1 than conidiation, but this difference is modest, so both responses could use the same or similar chromophore. Adenosine 3':5'-cyclic monophosphate bypasses the requirement for light for sporulation, while atropine prevents sporulation even after photoinduction. Light regulation of phr1, however, is indifferent to both these effectors. Induction of photolyase expression behaves as a direct, rapid response to light, independent of the induction of sporulation. Indeed, illumination of mature spores increases their capacity for photoreactivation. Blue light seems to warn the organism against the harmful effects of short wave-lengths, inducing phr1 expression and sporulation by pathways that are, at least in part, distinct.

Effects of 2G exposure on lean and genetically obese Zucker rats.

Changes in the ambient force environment alter the regulation of adiposity, food intake and energy expenditure (i.e., energy balance). Lean (Fa/Fa) and obese (fa/fa) male Zucker rats were exposed to 2G (twice Earth's normal gravity) for eight weeks via centrifugation to test the hypothesis that the Fa/Fa rats recover to a greater degree from the effects of an increased ambient force environment on body mass and food intake, than do the fa/fa rats which have a dysfunctional leptin regulatory system. The rats (lean and obese exposed to either 1G or 2G) were individually housed in standard vivarium cages with food and water provided ad libitum. The acute response to 2G included a transient hypophagia accompanied by decreased body mass, followed by recovery of feeding to new steady-states. In the lean rats, body mass-independent food intake had returned to 1G control levels six weeks after the onset of centrifugation, and body mass increased towards that of the 1G rats. In contrast, food intake and body mass of the 2G obese rats plateaued at a level lower than that of the 1G controls. Although percent carcass fat was reduced more in the 2G leans vs. 2G obese rats, the latter lost significantly more grams of fat than did the leans. Our data suggest that with respect to food intake and body mass, the lean rats recover from the initial effects of 2G exposure to a greater degree than do the fatty rats, a difference that likely reflects the functionality of the leptin regulatory system in the leans.

Brief periods of hyperphagia cause renal injury in the obese Zucker rat.

BACKGROUND: Female obese (fa/fa) Zucker rats are maximally hyperphagic from the beginning of access to solid food until 20 weeks of age and die primarily from renal failure. We documented that urinary albumin excretion (UAE) rises early in obese rats during this time of greatest hyperphagia. This study was conducted to examine if this early surge of hyperphagia is critical to the initiation of glomerular damage. METHODS: Three groups of six-week-old rats were used: (a) obese females fed ad libitum (AL-obese), (b) obese females pair fed to lean controls until 21 weeks and then allowed to eat ad libitum until 57 weeks (PF. AL-obese), (c) lean (Fa/Fa) Zucker rats fed ad libitum (AL-lean). Cohorts of AL-obese and PF.AL-obese rats were allowed to continue to death or 57 weeks of age, and the rest were terminated at 21 weeks for renal histology. RESULTS: At 21 weeks, neither PF.AL-obese nor AL-lean rats had elevated UAE or glomerular histopathology. In contrast, glomerular injury was severe in AL-obese rats. UAE increased by 10 and 29 weeks in AL- and PF.AL-obese rats, respectively. Plasma triglycerides increased prior to UAE in both PF. AL- and AL-obese rats. CONCLUSIONS: In obese rats fed ad libitum, hyperphagia is followed within a few weeks by hypertriglyceridemia and then by glomerular injury regardless of when ad libitum feeding is initiated. These events do not occur in lean rats or in obese rats pair fed to lean rats. Protective effects of pair feeding did not extend into the period of ad libitum feeding for PF.AL-obese rats. Hyperphagia quickly initiates glomerular injury in obese female Zucker rats.

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: diverse roles for mitogen-activated protein kinase homologs in foliar pathogens.

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Deltachk1 mutants is attempted. The ability of Deltachk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

T(3) stimulates resting metabolism and UCP-2 and UCP-3 mRNA but not nonphosphorylating mitochondrial respiration in mice.

The molecular basis for variations in resting metabolic rate (RMR) within a species is unknown. One possibility is that variations in RMR occur because of variations in uncoupling protein 2 (UCP-2) and uncoupling protein 3 (UCP-3) expression, resulting in mitochondrial proton leak differences. We tested the hypothesis that UCP-2 and -3 mRNAs positively correlate with RMR and proton leak. We treated thyroidectomized and sham-operated mice with triiodothyronine (T(3)) or vehicle and measured RMR, liver, and skeletal muscle mitochondrial nonphosphorylating respiration and UCP-2 and -3 mRNAs. T(3) stimulated RMR and liver UCP-2 and gastrocnemius UCP-2 and -3 expression. Mitochondrial respiration was not affected by T(3) and did not correlate with UCP-2 and -3 mRNAs. Gastrocnemius UCP-2 and -3 expression did correlate with RMR. We conclude 1) T(3) did not influence intrinsic mitochondrial properties such as membrane structure and composition, and 2) variations in UCP-2 and -3 expression may partly explain variations in RMR. One possible explanation for these data is that T(3) stimulates the leak in vivo but not in vitro because a posttranslational regulator of UCP-2 and -3 is not retained in the mitochondrial fraction.

Developmental regulation of cmp1, a gene encoding a multidomain conidiospore surface protein of Trichoderma.

A gene encoding a developmentally regulated polypeptide of Trichoderma (strain ATCC 32173) was isolated, with the help of an antibody against a 62-kDa protein whose abundance strongly increases during photoinduced sporulation. The amino acid sequence deduced from this gene, cmp1 (conidial multidomain protein), is a 135-kDa polypeptide consisting of several domains. Although reminiscent of known structural modules, two of the domains may define novel families. The protein is apparently processed to give the 62-kDa species. Immunogold labeling electron microscopy localized the antigen to the membrane or inner wall layers. The mRNA is strongly up-regulated during sporulation. At least part of this regulation is likely to be conferred by several elements identified in the upstream region, with homology to elements recognized by fungal transcription factors for regulation by conidiation, light, and nitrogen stress. The developmental regulation, cell surface location, and modular structure suggest a function in cell-cell interactions, detection of the wall by the cell, or anchoring of the plasma membrane to the wall.

Neurochemical alterations during age-related anorexia.

Unexplained weight loss during the latter stages of aging is commonly preceded by a spontaneous diminution in food intake. Multiple etiologies of age-related anorexia in humans, ranging from social isolation to impaired gastrointestinal function, have been proposed. The observation of this phenomenon in older laboratory animals suggests that physiological changes play a significant causal role. A continually expanding body of information on the neurochemical control of food intake supports a contribution of altered neurochemistry to dysregulated feeding behavior. This review provides an update on the relationship between declining food intake during advanced age and physiological (specifically neurochemical) function. The complexity of the control of food intake as well as the variety of investigative methods used in this field of study render the identification of definitive causes difficult. Evidence presented here is evaluated and possible etiologic factors are suggested.

Rapid blue light regulation of a Trichoderma harzianum photolyase gene.

Photolyases and blue light receptors belong to a superfamily of flavoproteins that make use of blue and UVA light either to catalyze DNA repair or to control development. We have isolated a DNA photolyase gene (phr1) from Trichoderma harzianum, a common soil fungus that is of interest as a biocontrol agent against soil-borne plant pathogens and as a model for the study of light-dependent development. The sequence of phr1 is similar to other Class I Type I eukaryotic photolyase genes. Low fluences of blue light rapidly induced phr1 expression both in vegetative mycelia, which lack photoprotective pigments, and, to a greater extent, in conidiophores. Thus, visible light induces the development of pigmented, resistant spores as well as the expression of phr1, perhaps announcing in this way the imminent exposure to the more damaging short wavelengths of sunlight. Light induction of phr1 in non-sporulating mutants shows that a complete sporulation pathway is not required for photoregulation. The light requirements for photoinduction of phr1 were not altered in dimY photoperception mutants. This suggests that photoinduction of sporulation and of photolyase expression is distinct in their photoreceptor system or in the transduction of the blue light signal.

Alterations in endogenous circadian rhythm of core temperature in senescent Fischer 344 rats.

We assessed whether alterations in endogenous circadian rhythm of core temperature (CRT) in aging rats are associated with chronological time or with a biological marker of senescence, i.e., spontaneous rapid body weight loss. CRT was measured in male Fischer 344 (F344) rats beginning at age 689 days and then continuously until death. Young rats were also monitored. The rats were housed under constant dim red light at 24-26 degrees C, and core temperature was recorded every 10 min via biotelemetry. The CRT amplitude of the body weight-stable (presenescent) old rats was significantly less than that of young rats at all analysis periods. At the onset of spontaneous rapid weight loss (senescence), all measures of endogenous CRT differed significantly from those in the presenescent period. The suprachiasmatic nucleus (a circadian pacemaker) of the senescent rats maintained its light responsiveness as determined by an increase in c-fos expression after a brief light exposure. These data demonstrate that some characteristics of the CRT are altered slowly with chronological aging, whereas others occur rapidly with the onset of senescence.

A G protein alpha subunit from Cochliobolus heterostrophus involved in mating and appressorium formation.

A Galpha subunit-encoding gene (CGA1) was cloned from Cochliobolus heterostrophus, a heterothallic foliar pathogen of corn. The deduced amino acid sequence showed similarity to Galpha proteins from other filamentous fungi and suggested that CGA1 is a member of the Galphai class. cga1 mutants had reduced ability to form appressoria on glass surfaces and on corn leaves; mutants nevertheless caused lesions on corn plants like those of wild type. cga1 mutants were female sterile; sexual development was completely abolished when the mutant allele was homozygous in a cross. Ascospores produced in crosses heterozygous at Cga1 were all wild type. The signal transduction pathway represented by CGA1 appears to be involved in developmental pathways leading to either appressorium formation or mating; in sexual development CGA1 is required for both fertility and ascospore viability.