Ligand/receptor internalization: a kinetic, flow cytometric analysis of the internalization of N-formyl peptides by human neutrophils

Fluorescence flow cytometry was used to measure the internalization of the fluorescent ligand N-formyl-nle-leu-phe-nle-tyr-lys-fluorescein by human neutrophils. The internalization process was monitored by the accessibility of the receptor-bound fluorescent ligand to quenching following a change in the pH of the extracellular medium from 7.4 to 3.0. In such a pH change, extracellular ligand or fluorescein are quenched immediately (excitation 488 nm). In contrast, intracellular fluorescein (derived from fluorescein diacetate) or intracellular ligand are quenched with half-times of approximately 20 or approximately 40 sec, respectively, at 37 degrees C. The fraction of internalized ligand is calculated by resolving the fast and slow components of the quenching process. Temporal resolution of the internalization process in this system depends upon two factors. We have previously shown that it is possible to examine essentially continuously the kinetics of ligand binding in the nM concentration range without removing the free ligand (Sklar LA, Finney DA, Cytometry 3:161, 1982). We have now modified a Becton Dickinson FACS IV sample head assembly to permit direct addition of reagents into the cell suspension while on-line. This enables us to change the suspension pH and evaluate internalization with a time resolution of a few seconds. We observe that internalized ligand can be detected within 1 min and that the rate is proportional to the number of receptors occupied. The rate is essentially linear over the first few minutes and approximately 60% of the receptor-bound ligand is internalized after 3 min.     

Hydration-induced conformational and flexibility changes in lysozyme at low water content

Using laser Raman spectroscopy, we are able to study conformational changes that occur as previously-dried hen egg-white lysozyme is sequentially rehydrated. Parallel n.m.r. exchangeability studies enable us to monitor flexibility changes also during this rehdyration. The results are consistent with a general loosening up of the protein at a water content of ～0.08 g water/g protein, followed by (probably small) local conformational changes. The enzyme regains its activity only after both these processes have gone to completion; thus these solvent-related changes may be necessary before activity can recommence.     

Myth, Experiment, and the Reinvention of Polynesian Voyaging

Experimental canoe voyages between Hawai'i, Tahiti, and Aotearoa (New Zealand) indicate that proponents of the "mythical" view of voyaging traditions cannot casually dismiss their historical basis because they believe it impossible for canoes navigated without instruments to have been intentionally sailed over such long distances. Furthermore, the key role of Hawaiians in this reinvention of Polynesian voyaging, and in particular their efforts to extend the sailing and navigational trials far beyond the original experimental plan, point toward a more Polynesian interpretation of voyaging traditions than one developed solely through Western analytical approaches.     

Cyclic AMP inhibits dedifferentiation in the cellular slime mold Dictyostelium discoideum

When aggregating amoebas of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, the amoebas will reaggregate in less than $\text{1}\text{10}\text{th}$ the original time. When aggregating amoebas are disaggregated and resuspended either in full nutrient medium or in buffered salts solution containing dextrose, they retain this developmentally acquired capacity to rapidly reaggregate for approximately 1 hr and then lose it completely in a synchronous and discrete step which we have referred to as the “erasure event.” In this report, it is demonstrated that micromolar concentrations of cAMP completely block this transition from the developmental to vegetative state, and that other cyclic nucleotides also inhibit it, but they do so at 20-fold higher concentrations. Neither the hydrolysis products of cAMP nor the vegetative chemoattractant folic acid inhibit dedifferentiation at concentrations as high as 10^?3M, demonstrating a specificity for cyclic nucleotides and cAMP in particular. The addition of cAMP at any time during the lag period preceding the erasure event inhibits it and addition immediately after the erasure event reverses it. Since cAMP may inhibit the transition from the developmental to vegetative state intracellularly or extracellularly, we have also examined the intracellular concentration of cAMP and the levels of cAMP binding sites on the cell surface during the erasure process. Evidence is presented that the majority of cAMP binding sites on the cell surface are not necessary for the inhibition of erasure by cAMP. The results of these latter studies are discussed in terms of alternative models for the involvement of cAMP in the transition from the developing to vegetative state.     

Effects of Holocene Alnus Expansion on Aquatic Productivity, Nitrogen Cycling, and Soil Development in Southwestern Alaska

Numerous pollen records provide evidence for the widespread range expansion of Alnus throughout Alaska and adjacent Canada during the middle Holocene. Because Alnus can fix atmospheric N₂, this vegetational change probably had a profound effect on N availability and cycling. To assess this effect, we analyzed a sediment core from Grandfather Lake in southwestern Alaska for a suite of geochemical indicators, including elemental composition, biogenic silica (BSi) content, and carbon (C) and nitrogen (N) isotopes of organic matter. These data, in conjunction with a pollen record from the same site, are used to infer biogeochemical processes associated with the mid-Holocene Alnus expansion. The increase in Alnus pollen percentages from 10% to 70% circa 8000-7000 BP (¹⁴C years before present) suggests the rapid spread of Alnus shrub thickets on mountain slopes and riparian zones in the Grandfather Lake region. Coincident with this vegetational change, the mean value of the sediment BSi content increases from 20.4 to 106.2 mg/g, reflecting increased diatom productivity within the lake as a result of Alnus N₂ fixation in the watershed soils and the associated N flux to the lake. Elevated aquatic productivity at this time is also supported by increased percentages of organic C and N, decreased C:N ratios, and decreased values of δ ¹³C. Furthermore, the δ ¹⁵N values of sediments increase substantially with the establishment of Alnus shrub thickets, suggesting enhanced N availability and accelerated N cycling within the lake and its watershed. Superimposed on a general trend of soil acidification throughout the postglacial period, soil acidity probably increased as a result of the Alnus expansion, as can be inferred from decreasing ratios of authigenic base cations to allogenic silica (Si) and increasing ratios of authigenic aluminum (Al) to allogenic Si. The ultimate cause of these mid-Holocene ecosystem changes was an increase in effective moisture in the region. Received 21 July 2000; accepted 3 January 2001.     

Identifying metalloproteins through X-ray fluorescence mapping and mass spectrometry

Metals are critical and dynamic components of biochemistry. To understand their roles, we greatly need tools to identify the ligands that bind them within the complexity of natural systems. This work describes the development of methods that not only detect and distinguish metals, but also characterize the proteins that bind them. We describe an approach to expose, identify and quantify metalloproteins in complex mixtures by sequential non-denaturing 2D-gel electrophoresis (2D GE)/X-ray Fluorescence (XRF) and tandem mass spectrometry (MS/MS) in the same spot of a sample. We first apply the development of 2D GE/XRF to Shewanella oneidensis MR-1, a well-studied system, and verify our electrophoretic approach. Then, we identified a novel periplasmic zinc protein in Pseudomonas aeruginosa PAO1 through 2D GE/XRF followed by MS/MS. The identity and function of this protein was verified through a gene mutation experiment.     

Surveillance of Infection Severity: A Registry Study of Laboratory Diagnosed Clostridium difficile

Background

Changing clinical impact, as virulent clones replace less virulent ones, is a feature of many pathogenic bacterial species and can be difficult to detect. Consequently, innovative techniques monitoring infection severity are of potential clinical value.

Methods and Findings

We studied 5,551 toxin-positive and 20,098 persistently toxin-negative patients tested for Clostridium difficile infection between February 1998 and July 2009 in a group of hospitals based in Oxford, UK, and investigated 28-day mortality and biomarkers of inflammation (blood neutrophil count, urea, and creatinine concentrations) collected at diagnosis using iterative sequential regression (ISR), a novel joinpoint-based regression technique suitable for serial monitoring of continuous or dichotomous outcomes. Among C. difficile toxin-positive patients in the Oxford hospitals, mean neutrophil counts on diagnosis increased from 2003, peaked in 2006–2007, and then declined; 28-day mortality increased from early 2006, peaked in late 2006–2007, and then declined. Molecular typing confirmed these changes were likely due to the ingress of the globally distributed severe C. difficile strain, ST1. We assessed the generalizability of ISR-based severity monitoring in three ways. First, we assessed and found strong (p<0.0001) associations between isolation of the ST1 severe strain and higher neutrophil counts at diagnosis in two unrelated large multi-centre studies, suggesting the technique described might be useful elsewhere. Second, we assessed and found similar trends in a second group of hospitals in Birmingham, UK, from which 5,399 cases were analysed. Third, we used simulation to assess the performance of this surveillance system given the ingress of future severe strains under a variety of assumptions. ISR-based severity monitoring allowed the detection of the severity change years earlier than mortality monitoring.

Conclusions

Automated electronic systems providing early warning of the changing severity of infectious conditions can be established using routinely collected laboratory hospital data. In the settings studied here these systems have higher performance than those monitoring mortality, at least in C. difficile infection. Such systems could have wider applicability for monitoring infections presenting in hospital.