Genetic interactions between clock mutations in Neurospora crassa: can they help us to understand complexity?

Recent work on circadian clocks in Neurospora has primarily focused on the frequency (frq) and white-collar (wc) loci. However, a number of other genes are known that affect either the period or temperature compensation of the rhythm. These include the period (no relationship to the period gene of Drosophila) genes and a number of genes that affect cellular metabolism. How these other loci fit into the circadian system is not known, and metabolic effects on the clock are typically not considered in single-oscillator models. Recent evidence has pointed to multiple oscillators in Neurospora, at least one of which is predicted to incorporate metabolic processes. Here, the Neurospora clock-affecting mutations will be reviewed and their genetic interactions discussed in the context of a more complex clock model involving two coupled oscillators: a FRQ/WC-based oscillator and a 'frq-less' oscillator that may involve metabolic components.

Epistatic and synergistic interactions between circadian clock mutations in Neurospora crassa.

We identified a series of epistatic and synergistic interactions among the circadian clock mutations of Neurospora crassa that indicate possible physical interactions among the various clock components encoded by these genes. The period-6 (prd-6) mutation, a short-period temperature-sensitive clock mutation, is epistatic to both the prd-2 and prd-3 mutations. The prd-2 and prd-3 long-period mutations show a synergistic interaction in that the period length of the double mutant strain is considerably longer than predicted. In addition, the prd-2 prd-3 double mutant strain also exhibits overcompensation to changes in ambient temperature, suggesting a role in the temperature compensation machinery of the clock. The prd-2, prd-3, and prd-6 mutations also show significant interactions with the frq(7) long-period mutation. These results suggest that the gene products of prd-2, prd-3, and prd-6 play an important role in both the timing and temperature compensation mechanisms of the circadian clock and may interact with the FRQ protein.

Abnormal surfactant composition and activity in severe bronchiolitis.

A prospective study of infants under 1 y of age, ventilated for severe viral bronchiolitis, was carried out in four paediatric intensive care units in order to study surfactant activity and composition in this condition. Lung lavage fluid from 24 infants with bronchiolitis, 19 with bronchiolitis and sepsis or cardiac failure and 12 controls were analysed by the "click test" for surfactant activity and for phospholipids. Surfactant activity was present in all controls, but in only 2 of the 24 infants with bronchiolitis alone. The presence of phosphatidylglycerol correlated perfectly with the click test, suggesting that reduced activity is due to changes in surfactant lipid composition. In those with bronchiolitis plus coexisting disease, surfactant activity and phosphatidylglycerol were absent in only half. Surfactant activity and phosphatidylglycerol re-appeared by extubation. Severe viral bronchiolitis is associated with an absence of surfactant activity and PG, which resolves by clinical recovery. Infants with coexisting conditions are not always surfactant deficient. Surfactant administration is likely to be beneficial, but requires a selective approach.

Cobalamin deficiency associated with erythroblastic anemia and methylmalonic aciduria in a border collie.

Anemia due to cobalamin deficiency is a rare genetic disorder that has been recognized in dogs only recently. This report concerns a 14-month-old border collie that presented for chronic, nonregenerative anemia. Cytological examination of a peripheral blood smear showed the presence of erythroblasts. Serum cobalamin levels were below reference ranges reported for clinically normal dogs. A methylmalonic aciduria was found on urinalysis. These signs are consistent with the anemia in Imerslund-Graesbeck syndrome reported in humans. Anemia due to cobalamin deficiency responds to parenteral vitamin B12 therapy, and affected animals have a good prognosis for recovery.

Effects of in vitro hemolysis on serum biochemistry values of the bottlenose dolphin (Tursiops truncatus).

The effects of in vitro hemolysis on 23 biochemical analytes were assessed in sera from 14 clinically healthy Atlantic bottlenose dolphins (Tursiops truncatus). Each serum sample was divided into three portions for analysis: 1) nonhemolyzed control; 2) moderate hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 150 mg/dl Hb; and 3) severe hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 500 mg/dl Hb. Moderate hemolysis resulted in statistically significant increases in the mean values of iron, lactate dehydrogenase, potassium, and uric acid and a decrease in creatinine (P < 0.001). Severe hemolysis resulted in statistically significant changes in the mean values of the above analytes in addition to the following increases: alanine aminotransferase, calcium, and serum globulins (P < 0.001) and albumin and total protein (P < 0.01). Total bilirubin and gamma glutamyl transferase levels were lower in the severely hemolyzed sample (P < 0.001). Differences in mean values for alkaline phosphatase between nonhemolyzed and hemolyzed serum were not significant but did show a downward trend in the hemolyzed sera. The presence and severity of hemolysis must be considered in the interpretation of the serum chemistry values.

Isolation and characterization of a temperature-sensitive circadian clock mutant of Neurospora crassa.

A new circadian clock mutant has been isolated in Neurospora crassa. This new mutation, called period-6 (prd-6), has two features novel to known clock mutations. First, the mutation is temperature sensitive. At restrictive temperatures (above 21 degrees) the mutation shortens circadian period length from a wild-type value of 21.5 hr to 18 hr. At permissive temperatures (below 21 degrees) the mutant has a 20.5-hr period length close to that of the wild-type strain. Second, the prd-6 mutation is epistatic to the previously isolated clock mutation period-2 (prd-2). This epistasis is unusual in that the prd-2 prd-6 double mutant strain has an 18-hr period length at both the restrictive and permissive temperatures. That is, the temperature-sensitive aspect of the phenotype of the prd-6 strain is lost in the prd-2 prd-6 double mutant strain. This suggests that the gene products of the prd-2 and prd-6 loci may interact physically and that the presence of a normal prd-2+ protein is required for low temperature to "rescue" the prd-6 mutant phenotype. These results, combined with our recent finding that prd-2 and some alleles of the frq gene show genetic synergy, suggest that it may be possible to establish a more comprehensive model of the Neurospora circadian clock.

Analysis of frequency (frq) clock gene homologs: evidence for a helix-turn-helix transcription factor.

The frq gene plays a key role in the organization of the Neurospora crassa circadian clock. Our previous analysis of a C-terminal fragment of the putative FRQ protein suggested it is a nuclear transcription factor but did not identify a known DNA-binding domain. If the hypothesis is correct that FRQ is a transcription factor, sequences consistent with this function should be conserved in distantly related species. To investigate, we have cloned frq homologs from other filamentous fungi including Chromocrea spinulosa, and Leptosphaeria australiensis. Alignment of the Leptosphaeria and Chromocrea proteins with the published (complete) sequences for Neurospora crassa and Sordaria fimicola shows that they are respectively about 47%, and 43%, identical to both Neurospora and Sordaria. The alignment identifies several short regions of high conservation punctuated by regions showing near total divergence. Sequences consistent with FRQ being a transcription factor are generally conserved. Most importantly, we show that a highly conserved segment of the protein has strongly predicted helix-turn-helix (HTH) structure as supported by three independent methods. Further, this segment shows the defining sequence characteristics of known HTH DNA-binding domains. Amino acids at positions altered in frq mutant alleles are conserved in all species examined. Transformation of the Neurospora frq9 (conditionally arrhythmic) mutant with the Chromocrea homolog rescued the pigmentation and conidiation defects of the mutant but not the circadian defect; the Leptosphaeria homolog failed to rescue any defect. Together, these data provide the first testable hypotheses concerning several specific aspects of FRQ structure and function.

Metastatic disease first presenting as eyelid tumors: a report of two cases and review of the literature.

We present two unusual cases in which an eyelid tumor was the first sign of metastatic disease. The first involved a 53-year-old man with pulmonary carcinoma and the second a 71-year-old man with malignant lymphoma. Fifteen similar cases from the literature are reviewed. The most frequent primary lesion is breast carcinoma in women, which appears as a diffuse lesion of one or two eyelids. Metastatic lung carcinoma in men appears as solitary nodules, representing the second-most common type of lesion. The questions of left- or right-side predominance, age and sex of patients, types of tumors, and prognosis are discussed.

Informed consent in senile cataract extraction.

One day after surgery for senile cataract, we interviewed 50 patients to determine what percentage of the preoperative informed consent was retained. No patient felt less inclined to undergo surgery after hearing the informed consent and all patients believed the preoperative explanation had been adequate. This study found retention of relevant information was only 37% when assessed by ten standard questions. Only two (4%) of the patients remembered more than two of the five risks of surgery. Blindness, the most frequently recalled, was known by only 17 (34%). More than 80% failed to recall either hemorrhage (46/50), infection (47/50), failure of the procedure to improve visual acuity (42/50), or death (44/50) as other complications. Only 20% (10/50) would have remembered to protect the operated-on eye. Patients denied prior counseling for four of the ten questions (mean). Factors related to poor retention include advanced age and less than a high school education. Previous cataract surgery, level of anxiety prior to surgery, and the patient's sex did not appear to influence retention.

Epidemiologic aspects of senile cataract extraction stratified by visual acuity, age, and sex.

A review of records for 221 patients 50 years old and older undergoing first cataract extractions showed that women constituted 64% of this population (142 cases). The men had a mean age of 70.8 years and the women a mean age of 71.8 years. This difference was not significant. Men had a significantly (P less than .05) better mean best corrected visual acuity preoperatively than women (20/100 vs 20/400). In the two younger groups (those aged 50 to 59 years and those aged 60 to 69 years), men also had significantly (P less than .05) better preoperative visual acuities.