Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

An agarose gel method for the determination of DNA interstrand crosslinking applicable to the measurement of the rate of total and "second-arm" crosslink reactions

A simple agarose gel electrophoresis method for the determination of DNA interstrand crosslinks is described. Following complete denaturation of 32P-end-labeled DNA the presence of an interstrand crosslink results in renaturation to double-stranded DNA. The single- and double-stranded bands separated on an agarose gel can be accurately quantitated by densitometry of the autoradiograph produced from the dried gel. The technique is particularly applicable to detailed time-course experiments of both total crosslink formation and, following removal of free drug, the "second-arm" of the crosslink reaction. The method is illustrated for a number of nitrogen mustard antitumor agents, showing how the moiety attached to a bifunctional reactive group can influence the extent and rate of crosslink formation and, in particular, the conversion of monoadducts to crosslinks. It is sensitive enough to follow the formation of crosslinks by slow and inefficient cross-linking agents such as busulfan which have not previously been measured by physical procedures.     

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C.     

Hospital work load produced by breast-cancer screening programme run by trained non-medical staff.

In a feasibility study of mass population screening for breast cancer by annual clinical examination and mammography the findings of non-medical staff (nurses and radiographers) were used to estimate the hospital work load generated by such a programme. Among 2490 women who attended for the first time by invitation the rate of referral for a surgical opinion based on the findings of the non-medical staff was 7.9% and the biopsy rate 2.5%. In the second and third years referral rates fell to 4.3% and 2.7% respectively and the biopsy rates to 1.1% and 1.4%. The rates of referral and biopsy among 1203 women who referred themselves for screening were higher, but many self-referred women were symptomatic; those without symptoms had rates of referral and biopsy similar to those of the invited women. Extrapolation of these findings to a population of 200,000 in a typical health district showed that the hospital work load would be high in the first year of screening with 44 outpatient referrals per week and 14 biopsies. By the third year, however, only seven referrals and four biopsies a week could be expected. The work load would be reduced by a third if screening were confined to women over the age of 50.     

Screening for breast cancer.

The feasibility of mass population screening for breast cancer by clinical examination and x-ray mammography was studied. The results indicate that such a programme could be conducted effectively by non-medical staff and be safe from the dangers of irradiation. The response rate of women invited for screening suggests that such a service is acceptable to the general public. The additional work load produced by screening would not overburden the existing surgical services.     

Aldosterone dynamics during graded exercise at sea level and high altitude.

Hormonal responses to graded exercise of eight low altitude residents were examined at sea level (SL) and after 1 (acute) and 11 (chronic) days at 4,300 m (HA). Caloric, water, and electrolyte intakes were controlled, as were temperature and humidity. Blood was sampled at rest and during light and moderate upright bicycle exercise (20 min at 40% and 75% of maximal O2 uptake, respectively). Mean VO2 max at HA was 27% lower than at SL. Resting plasma levels of aldosterone (Aldo), renin, and angiotensin II (A II) were significantly lower (P smaller than 0.05) on day 1 at HA compared to SL, but returned to SL values by day 11. Plasma cortisol values at rest were similar at SL and HA and were not significantly altered by light or moderate exercise. Renin, A II, and Aldo rose progressively with increasing workload in each environment. With acute HA, renin and Aldo were lower than at either SL or chronic HA. The chronic HA levels tended to approximate SL findings, implying adaptation. The data suggest that aldosterone is predominantly under the control of the renin-angiotensin system during graded exercise at sea level and that the response of this system is altered on acute high-altitude exposure.     

Ataxin1L Is a Regulator of HSC Function Highlighting the Utility of Cross-Tissue Comparisons for Gene Discovery

Hematopoietic stem cells (HSCs) are rare quiescent cells that continuously replenish the cellular components of the peripheral blood. Observing that the ataxia-associated gene Ataxin-1-like (Atxn1L) was highly expressed in HSCs, we examined its role in HSC function through in vitro and in vivo assays. Mice lacking Atxn1L had greater numbers of HSCs that regenerated the blood more quickly than their wild-type counterparts. Molecular analyses indicated Atxn1L null HSCs had gene expression changes that regulate a program consistent with their higher level of proliferation, suggesting that Atxn1L is a novel regulator of HSC quiescence. To determine if additional brain-associated genes were candidates for hematologic regulation, we examined genes encoding proteins from autism- and ataxia-associated protein–protein interaction networks for their representation in hematopoietic cell populations. The interactomes were found to be highly enriched for proteins encoded by genes specifically expressed in HSCs relative to their differentiated progeny. Our data suggest a heretofore unappreciated similarity between regulatory modules in the brain and HSCs, offering a new strategy for novel gene discovery in both systems.     

Data-Driven Modeling to Assess Receptivity for Rift Valley Fever Virus

Rift Valley Fever virus (RVFV) is an enzootic virus that causes extensive morbidity and mortality in domestic ruminants in Africa, and it has shown the potential to invade other areas such as the Arabian Peninsula. Here, we develop methods for linking mathematical models to real-world data that could be used for continent-scale risk assessment given adequate data on local host and vector populations. We have applied the methods to a well-studied agricultural region of California with 1 million dairy cattle, abundant and competent mosquito vectors, and a permissive climate that has enabled consistent transmission of West Nile virus and historically other arboviruses. Our results suggest that RVFV outbreaks could occur from February–November, but would progress slowly during winter–early spring or early fall and be limited spatially to areas with early increases in vector abundance. Risk was greatest in summer, when the areas at risk broadened to include most of the dairy farms in the study region, indicating the potential for considerable economic losses if an introduction were to occur. To assess the threat that RVFV poses to North America, including what-if scenarios for introduction and control strategies, models such as this one should be an integral part of the process; however, modeling must be paralleled by efforts to address the numerous remaining gaps in data and knowledge for this system.