Graham Bench

DNA AND TOTAL PROTAMINE MASSES IN INDIVIDUAL SPERM FROM FERTILE MAMMALIAN SUBJECTS

The total amount of phosphorus and sulfur inside the nuclei of individual bull, stallion, hamster, human, and mouse sperm from fertile subjects has been measured using Particle Induced X-ray Emission (PIXE). Using the sulfur masses, we determined the total protamine (protamine 1 plus protamine 2) mass within the sperm nuclei of each species. Using the phosphorus masses, we determined the DNA mass present within the sperm nuclei of each species. The results reveal that although the relative proportion of protamine 1 to protamine 2 varies among the species examined, the total protamine mass to DNA mass ratio is similar in bull, stallion, hamster, and mouse sperm nuclei. In contrast, mature human sperm nuclei were found to contain significantly less protamine. This observation is consistent with other studies, which suggest that as much as 15% of the DNA in human sperm remain packaged by histones. Using the data obtained for bull sperm, the length of DNA that could be covered by each protamine 1 molecule in bull sperm has been estimated. Making the assumption that the size of the protamine 1 binding site on DNA is similar in the sperm of these species, the length of DNA covered by a single protamine 2 molecule also has been estimated.

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH(3)CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.

Effects of Chlorophyll and Chlorophyllin on Low-Dose Aflatoxin B1 Pharmacokinetics in Human Volunteers

Chlorophyll (Chla) and chlorophyllin (CHL) were shown previously to reduce carcinogen bioavailability, biomarker damage, and tumorigenicity in trout and rats. These findings were partially extended to humans, where CHL reduced excretion of aflatoxin B1 (AFB1)-DNA repair products in Chinese unavoidably exposed to dietary AFB1. However, neither AFB1 pharmacokinetics nor Chla effects were examined. We conducted an unblinded crossover study to establish AFB1 pharmacokinetic parameters among four human volunteers, and to explore possible effects of CHL or Chla cotreatment in three of those volunteers. For protocol 1, fasted subjects received an Institutional Review Board–approved dose of 14C-AFB1 (30 ng, 5 nCi) by capsule with 100 mL water, followed by normal eating and drinking after 2 hours. Blood and cumulative urine samples were collected over 72 hours, and 14C- AFB1 equivalents were determined by accelerator mass spectrometry. Protocols 2 and 3 were similar except capsules also contained 150 mg of purified Chla or CHL, respectively. Protocols were repeated thrice for each volunteer. The study revealed rapid human AFB1 uptake (plasma ka, 5.05 ± 1.10 h−1; Tmax, 1.0 hour) and urinary elimination (95% complete by 24 hours) kinetics. Chla and CHL treatment each significantly impeded AFB1 absorption and reduced Cmax and AUCs (plasma and urine) in one or more subjects. These initial results provide AFB1 pharmacokinetic parameters previously unavailable for humans, and suggest that Chla or CHL co-consumption may limit the bioavailability of ingested aflatoxin in humans, as they do in animal models.

Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice

Abstract Disruption of the cutaneous permeability barrier induces metabolic responses in the epidermis which result in barrier recovery. Barrier disruption by either solvent treatment or tape stripping results in the loss of the epidermal calcium gradient. Previous studies in acetone treated hairless mice have shown that maintaining this calcium gradient inhibits barrier repair, suggesting that alterations in the epidermal calcium concentration may be an important signal for barrier homeostasis. In the present study, we show that in hairless mice disruption of the barrier by treatment with the detergent. SDS, also results in the loss of the calcium gradient, as demonstrated both semi‐quantitatively with ultrastructural cytochemical localization and quantitatively using proton induced X‐ray emission (PIXE). Additionally, immersion in calcium containing solutions delays barrier repair after either detergent (SDS treatment) or mechanical (tape stripping) disruption of the barrier, as reported previously for acetone treated skin. These results indicate that barrier disruption, regardless of the insult, induces changes in the epidermal calcium gradient which may play an important role in signaling the metabolic changes required for barrier homeostasis.

The Use of STIM and PESA to Measure Profiles of Aerosol Mass and Hydrogen Content, Respectively, across Mylar Rotating Drums Impactor Samples

A method has been developed for measuring profiles of aerosol mass on thin (480 w g/cm 2 ) Apiezon-L coated Mylar films employed in rotating drum aerosol impactor samplers using the ion beam analysis technique scanning transmission ion microscopy (STIM). The greased Mylar films are excellent impaction substrates and possess excellent uniformity in projected density, making them an ideal substrate for STIM analysis. The uniformity in projected density of a film enables STIM with a 3 MeV proton beam to produce profiles of aerosol mass with an accuracy of better than 90% and a mass sensitivity approaching 10 w g/cm 2 . Further, we have extended proton elastic scattering analysis (PESA) to the same films, achieving measurement of an organic surrogate. Although the films contain ∼ 20 w g/cm 2 hydrogen, the spatial uniformity in film hydrogen content enables PESA with a 3 MeV proton beam to produce profiles of hydrogen arising solely from the aerosols with an accuracy to within - 1 w g/cm 2 and a mass sensitivity of ∼ 1 w g/cm 2 . These measurements when combined with synchrotron-x-ray fluorescence (S-XRF) measurements on the same film allow mass closure, sum of species versus measured mass, a key quality assurance protocol, to be approached. All 3 techniques were applied to very fine and ultra-fine particles collected in Fresno, CA, November, 2000 by slotted DRUM samplers. Temporal resolution in the resulting profiles was h 6 h. The dramatic changes in composition versus size and time, and new types of elemental correlations unseen in PM 2.5 filters, will be major assets in correlating aerosols and health impacts, visibility degradation, and the effects of aerosols on climate.

The LLNL AMS facility

The AMS facility at Lawrence Livermore National Laboratory (LLNL) routinely measures the isotopes 3H, 7Be, 10Be, 14C, 26Al, 36Cl, 41Ca, and 129I. During the past two years, over 30000 research samples have been measured. Of these samples, approximately 30% were for 14C bioscience tracer studies, 45% were 14C samples for archaeology and the geosciences, and the other isotopes constitute the remaining 25%. During the past two years at LLNL, a significant amount of work has gone into the development of the Projectile X-ray AMS (PXAMS) technique. PXAMS uses induced characteristic X-rays to discriminate against competing atomic isobars. PXAMS has been most fully developed for 63Ni but shows promise for the measurement of several other long lived isotopes. During the past year LLNL has also conducted an 129I interlaboratory comparison exercise. Recent hardware changes at the LLNL AMS facility include the installation and testing of a new thermal emission ion source, a new multi-anode gas ionization detector for general AMS use, re-alignment of the vacuum tank of the first of the two magnets that make up the high energy spectrometer, and a new cryo-vacuum system for the AMS ion source. In addition, we have begun design studies and carried out tests for a new high-resolution injector and a new beamline for heavy element AMS.

Cadmium concentrations in the testes, sperm, and spermatids of mice subjected to long‐term cadmium chloride exposure

BACKGROUND Exposures to cadmium have been reported to reduce male fertility and there are several hypotheses that suggest how reduced male fertility may result from incorporation of cadmium into sperm chromatin. The purpose of this study was to determine whether mice subjected to long-term intraperitoneal cadmium exposure incorporated cadmium into their sperm chromatin. METHODS Male mice were exposed to 0.1 mg/kg body weight cadmium in the form of CdCl2 via intraperitoneal injection once per week for 4, 10, 26, and 52 weeks and then sacrificed. The cadmium contents of the liver, testes, pooled sperm, and pooled spermatids from dosed and control animals were determined by atomic absorption spectroscopy. Cadmium and zinc contents in individual sperm and spermatid heads were determined by particle-induced x-ray emission. RESULTS Atomic absorption spectroscopy revealed that although cadmium accumulated in the liver and testes, cadmium was not detected in pooled sperm or spermatid samples down to minimum detectable limits of 0.02 microg/g dry weight. Particle-induced x-ray emission analyses did not show the presence of cadmium in any sperm or spermatid head down to minimum detectable limits of 15 microg/g dry weight. Particle-induced x-ray emission analyses also demonstrated that phosphorus, sulfur, and zinc concentrations in individual sperm and spermatid heads were not altered by exposure to CdCl2. CONCLUSIONS Because cadmium was not incorporated into sperm chromatin at levels above 0.02 microg/g dry weight, the data cast doubt on hypotheses that suggest that reduced male fertility may result from incorporation of cadmium into sperm chromatin.

Biological sample preparation and 41Ca AMS measurement at LLNL

Abstract Calcium metabolism in biology may be better understood by the use of 41Ca tracer, although requiring detection by accelerator mass spectrometry (AMS). Methodologies for preparation of urine samples and subsequent AMS measurement were investigated. Novel attempts at preparing CaH2 were unsuccessful, but CaF2 of sufficient purity could be produced by precipitation of calcium from urine as oxalate, followed by separation of calcium by cation exchange chromatography and washing the CaF2 precipitate. The presence of some remaining impurities could be compensated for by selecting the appropriate accelerated ion charge state for AMS. The use of projectile X-rays for isobar discrimination was explored as an alternative to the conventional d E d x detector.

Cell-free expression for nanolipoprotein particles: Building a high-throughput membrane protein solubility platform

Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.