CHANGES IN THE HEAT-RESISTANCE OF ASCOSPORES OF NEUROSPORA UPON GERMINATION

Lingappa , Yamuna , and A. S. Sussman . (U. Michigan, Ann Arbor.) Changes in the heat-resistance of ascospores of Neurospora upon germination. Amer. Jour. Bot. 46 (9): 671–678. Illus. 1959.—A rapid loss in heat-resistance accompanies activation of ascospores of Neurospora tetrasperma after incubation at 27°C. When activated spores are given a 5-min. “heat-flash” at 65°C. after only 5 min. at 27°C., fully % fail to germinate. Such treatment, if administered 25 min. after activation, results in the complete destruction of the spores. By contrast, when incubation at 27°C. is not interposed, more than ½ of the spores will germinate, even when they have been exposed to 65°C. for 30 min. Similar results were obtained with “heat-flashes” at 50 and 60°C., although exposures of longer duration were required to affect the spores. Conidia respond very differently to “heat-flashes” in that germination is stimulated if they are provided after an incubation period at 27°C. On the other hand, conidia are killed by short exposures to 60°C., so that they are far more susceptible to such treatment than are ascospores. A study of the cardinal temperatures of germination revealed that the maximum is about 44°C. for both conidia and ascospores. The maximum for the growth of two strains of N. tetrasperma and for one of N. crassa is between 40–45°C.; however, another strain of the latter species grows at 45°C. Dry heat was shown to be less effective than wet in activating ascospores. Removal of the exospore of ascospores results in the loss of considerable heat-resistance. In addition, the requirement for heat-activation is considerably mitigated in such spores, suggesting that the exospore, or an associated layer is the locus of the ascospore's heat-resistance.     

Location of a dicyclohexylcarbodiimide-reactive glutamate residue in the Neurospora crassa plasma membrane H+-ATPase

The proton pump (H+-ATPase) found in the plasma membrane of the fungus Neurospora crassa is inactivated by dicyclohexylcarbodiimide (DCCD). Kinetic and labeling experiments have suggested that inactivation at 0 degrees C results from the covalent attachment of DCCD to a single site in the Mr = 100,000 catalytic subunit (Sussman, M. R., and Slayman, C. W. (1983) J. Biol. Chem. 258, 1839-1843). In the present study, when [14C]DCCD-labeled enzyme was treated with the cleavage reagent, N-bromosuccinimide, a single major radioactive peptide fragment migrating at about Mr = 5,300 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was produced. The fragment was coupled to glass beads and partially sequenced by automated solid-phase Edman degradation at the amino terminus and at an internal tryptic cleavage site. By comparison to the DNA-derived amino acid sequence for the entire Mr = 100,000 polypeptide (Hager, K., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697), the fragment has been identified as arising by cleavage at tyrosine 100 and tryptophan 141. Covalently incorporated [14C]DCCD was released at a position corresponding to glutamate 129. The DCCD-reactive glutamate is located in the middle of the first of eight predicted transmembrane sequences. When the sequence surrounding the DCCD site is compared to that surrounding the DCCD-reactive residue of two other proton pumps, the F0F1-ATPase and cytochrome c oxidase, no homology is apparent apart from an abundance of hydrophobic amino acids.     

Individual- and Neighborhood-Level Predictors of Mortality in Florida Colorectal Cancer Patients

Purpose

We examined individual-level and neighborhood-level predictors of mortality in CRC patients diagnosed in Florida to identify high-risk groups for targeted interventions.

Methods

Demographic and clinical data from the Florida Cancer Data System registry (2007–2011) were linked with Agency for Health Care Administration and US Census data (n = 47,872). Cox hazard regression models were fitted with candidate predictors of CRC survival and stratified by age group (18–49, 50–64, 65+).

Results

Stratified by age group, higher mortality risk per comorbidity was found among youngest (21%), followed by middle (19%), and then oldest (14%) age groups. The two younger age groups had higher mortality risk with proximal compared to those with distal cancer. Compared with private insurance, those in the middle age group were at higher death risk if not insured (HR = 1.35), or received healthcare through Medicare (HR = 1.44), Medicaid (HR = 1.53), or the Veteran’s Administration (HR = 1.26). Only Medicaid in the youngest (52% higher risk) and those not insured in the oldest group (24% lower risk) were significantly different from their privately insured counterparts. Among 18–49 and 50–64 age groups there was a higher mortality risk among the lowest SES (1.17- and 1.23-fold higher in the middle age and 1.12- and 1.17-fold higher in the older age group, respectively) compared to highest SES. Married patients were significantly better off than divorced/separated (HR = 1.22), single (HR = 1.29), or widowed (HR = 1.19) patients.

Conclusion

Factors associated with increased risk for mortality among individuals with CRC included being older, uninsured, unmarried, more comorbidities, living in lower SES neighborhoods, and diagnosed at later disease stage. Higher risk among younger patients was attributed to proximal cancer site, Medicaid, and distant disease; however, lower SES and being unmarried were not risk factors in this age group. Targeted interventions to improve survivorship and greater social support while considering age classification may assist these high-risk groups.     

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.