A spin-labeled abasic DNA substrate for AP endonuclease.

We report the first observation of a spin-labeled ds 23-mer oligonucleotide by high-field electron spin resonance (ESR) and demonstrate that it interacts with AP endonuclease, the key enzyme in DNA abasic site repair. The spin labeled 23-mer with a U at position 12 of the upper strand is processed by uracil DNA glycosylase to provide the abasic substrate. With a spin-label two nucleotides away from the abasic site, AP endo binds and cleaves when the label is 3' but not 5' to the abasic site. These results confirm that the disposition of the bases immediately upstream of the abasic site is particularly critical for cleavage by AP endo, and establish that DNA-protein interactions in this important enzyme can be examined using spin-labeled substrates.     

Calcium carbonate budgets for two coral reefs affected by different terrestrial runoff regimes, Rio Bueno, Jamaica

A process-based carbonate budget was used to compare carbonate framework production at two reef sites subject to varying degrees of fluvial influence in Rio Bueno, Jamaica. The turbid, central embayment was subjected to high rates of fluvial sediment input, framework accretion was restricted to ≤30 m, and net carbonate production was 1,887 g CaCO₃ m⁻² year⁻¹. Gross carbonate production (GCP) was dominated by scleractinians (97%), particularly by sediment-resistant species, e.g. Diploria strigosa on the reef flat (<2 m). Calcareous encrusters contributed very little carbonate. Total bioerosion removed 265 g CaCO₃ m⁻² year⁻¹ and was dominated by microborers. At the clear-water site, net carbonate production was 1,236 g CaCO₃ m⁻² year⁻¹; the most productive zone was on the fore-reef (10 m). Corals accounted for 82% of GCP, and encrusting organisms 16%. Bioerosion removed 126 g CaCO₃ m⁻² year⁻¹ and was dominated by macroborers. Total fish and urchin grazing was limited throughout (≤20 g CaCO₃ m⁻² year⁻¹). The study demonstrates that: (1) carbonate production and net reef accretion can occur where environmental conditions approach or exceed perceived threshold levels for coral survival; and (2) although live coral cover (and carbonate production rates) were reduced on reef-front sites along the North Jamaican coast, low population densities of grazing fish and echinoids to some extent offset this, thus maintaining positive carbonate budgets.     

ALFRED: An allele frequency database for anthropology

The deluge of data from the human genome project (HGP) presents new opportunities for molecular anthropologists to study human variation through the promise of vast numbers of new polymorphisms (e.g., single nucleotide polymorphisms or SNPs). Collecting the resulting data into a single, easily accessible resource will be important to facilitate this research. We created a prototype Web-accessible database named ALFRED (ALelle FREquency Database, http://alfred.med.yale.edu/alfred/) to store and make publicly available allele frequency data on diverse polymorphic sites for many populations. In constructing this database, we considered many different concerns relating to the types of information needed for anthropology, population genetics, molecular genetics, and statistics, as well as issues of data integrity and ease of access to data. We also developed links to other Web-based databases as well as procedures for others to make links to the data in ALFRED. Here we present an overview of the issues considered and provisional solutions, as well as an example of data already available. It is our hope that this database will be useful for research and teaching in a wide range of fields, and that colleagues from various fields will contribute to making ALFRED an important resource for many studies as yet unforeseen.     

Island‐wide aridity did not trigger recent megafaunal extinctions in Madagascar

Researchers are divided about the relative importance of people versus climate in triggering the Late Holocene extinctions of the endemic large‐bodied fauna on the island of Madagascar. Specifically, a dramatic and synchronous decline in arboreal pollen and increase in grass pollen ca 1000 yr ago has been alternatively interpreted as evidence for aridification, increased human activity, or both. As aridification and anthropogenic deforestation can have similar effects on vegetation, resolving which of these factors (if either) led to the demise of the megafauna on Madagascar has remained a challenge. We use stable nitrogen isotope (δ¹⁵N) values from radiocarbon‐dated subfossil vertebrates to disentangle the relative importance of natural and human‐induced changes. If increasing aridity were responsible for megafaunal decline, then we would expect an island‐wide increase in δ¹⁵N values culminating in the highest values at the time of proposed maximum drought at ca 1000 yr ago. Alternatively, if climate were relatively stable and anthropogenic habitat alteration explains the palynological signal, then we would anticipate little or no change in habitat moisture, and no systematic, directional change in δ¹⁵N values over time. After accounting for the confounding influences of diet, geographic region, and coastal proximity, we find no change in δ¹⁵N values over the past 10 000 yr, and no support for a period of marked, geographically widespread aridification culminating 900–950 yr ago. Instead, increases in grasses at around that time may signal a transition in human land use to a more dedicated agro‐pastoralist lifestyle, when megafaunal populations were already in decline. Land use changes ca 1000 yr ago would have simply accelerated the inevitable loss of Madagascar's megafauna.     

Distribution,levels, and activation of MEK1 in Alzheimer's disease

Extracellular-signal-regulated kinase (ERK) has been implicated in the pathogenesis of Alzheimer's disease (AD), but the upstream cascade leading to ERK activation has not been elucidated. In this study, we focused on one of the physiological activators of ERK, mitogen-activated protein kinase (MAPK)/ERK kinase 1 (MEK1). Although there was no significant difference in the level and distribution of total MEK1 between AD and age-matched control cases, increased levels of activated phospho-MEK1 were specifically localized to neuronal intracytoplasmic granular structures in severe AD (Braak stage V-VI). The considerable overlap between MEK1 and its downstream effector, phospho-ERK, suggests both a functional and mechanistic link. Nuclear localization of phospho-MEK1 was a prominent feature in both mild AD cases (Braak stage III-IV) and control cases with limited pathology (Braak stage I-II). Since MEK1 is normally cytoplasmic due to the active export from nucleus because of the presence of nuclear export signal in its amino-terminus, we suspect that the apparent nuclear accumulation of phospho-MEK1 in AD patients at early stages suggests that abnormal nuclear trafficking may contribute to the pathogenesis of AD. By immunoblot analyses, phospho-MEK1 was significantly increased in AD over control cases. Together, these findings lend further credence to the notion that the ERK pathway is dysregulated in AD and also indicate an active role for this pathway in disease pathogenesis.     

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating

Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.     

Biotechnology for phosphorus removal during wastewater treatment

Advanced biological wastewater treatment for the removal of phosphorus in excess of the normal metabolic requirements of activated sludge type processes has been developed as an alternative to chemical addition. Current laboratory and pilot plant investigations have confirmed that a preliminary anaerobic zone and plug-flow type configuration are necessary for good enhanced biological phosphorus removal. Nitrate in the anaerobic stage inhibits the process whereas acetate enhances phosphorus uptake. The bacteria probably responsible are of the Acinetobacter genus and the presence of stored polyphosphate within these bacteria has been demonstrated. It has also been shown that pure cultures of Acinetobacter do not necessarily take up soluble substrate as phosphate is released during the anaerobic phase, in contrast to the current proposed mechanism, and that in certain cases natural chemical precipitation could make a significant contribution towards overall phosphorus removal. Several studies of pilot and full-scale plants have been reported.     

Plasma Lipoprotein(a) Levels Are Associated with Mild Renal Impairment in Type 2 Diabetics Independent of Albuminuria

Background

CKD, an independent risk factor for CV disease, increases mortality in T2DM. Treating modifiable CV risk factors decreases mortality in diabetics with microalbuminuria, but the role of early CV prevention in diabetics with mild CKD by GFR criteria alone remains unclear. The purpose of this study was to probe whether T2DM patients with mild GFR impairment have atherogenic lipid profiles compared to diabetic counterparts with normal renal function.

Methods

In the Penn Diabetes Heart Study (PDHS), a single-center observational cohort of T2DM patients without clinical CVD, cross-sectional analyses were performed for directly measured lipid fractions in 1852 subjects with eGFR>60 mL/min/1.73 m² determined by the CKD-EPI equation (n = 1852). Unadjusted and multivariable analyses of eGFR association with log-transformed lipid parameters in incremental linear and logistic regression models (with eGFR 90 mL/min/1.73 m² as a cut-point) were performed.

Results

Mild GFR impairment (eGFR 60–90 mL/min/1.73 m², median urinary ACR 5.25 mg/g) was associated with higher log-transformed Lp(a) values (OR 1.17, p = 0.005) and with clinically atherogenic Lp(a) levels above 30 mg/dL (OR 1.35, p = 0.013) even after full adjustment for demographics, medications, metabolic parameters, and albuminuria. Logistic regression demonstrated a trend towards significance between worse kidney function and apoB (p = 0.17) as well as apoC-III (p = 0.067) in the fully adjusted model.

Conclusions

Elevated Lp(a) levels have a robust association with mild GFR impairment in type 2 diabetics independent of race, insulin resistance, and albuminuria.