Direct Visualization of a Protein Nuclear Architecture

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca2+ signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca2+ concentration ([Ca2+]i), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca2+ spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca2+]i, induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca2+]i elevation, either by preincubation with the specific Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by removal of external Ca2+. CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca2+]i.

Increased liposome extravasation in selected tissues: effect of substance P.

We have used a pharmacologic mediator to open intercellular connections in selected vessels to allow liposomes to escape from the blood stream and to extravasate into tissues that have appropriate receptors. We have examined the effects of substance P (SP), a peptide known to increase vascular permeability in selected tissues, such as trachea, esophagus, and urinary bladder in rats. We used quantitative fluorescence analysis of tissues to measure two fluorescent markers, one attached to the lipid (rhodamine-phosphatidylethanolamine) and another, doxorubicin (an anti-tumor drug), encapsulated within the aqueous interior. We have also examined the deposition of liposomes microscopically by the use of encapsulated colloidal gold and silver enhancement. Analysis of the biochemical and morphological observations indicate the following: (i) Injection of SP produces a striking increase in both liposome labels, but only in tissues that possess receptors for SP in postcapillary venules; (ii) liposome material in these tissues has extravasated and is found extracellularly near a variety of cells beyond the endothelial layer over the first few hours; (iii) 24 h following injection of liposomes and SP, liposome material is found in these tissues, localized intracellularly in both endothelial cells and macrophages. We propose that appropriate application of tissue-specific mediators can result in liposome extravasation deep within tissues that normally do not take up significant amounts of liposomes from the blood. Such liposomes are able to carry a variety of pharmacological agents that can be released locally within selected target tissues for therapeutic purposes.

Modifications of RNA processing modulate the expression of hemoglobin genes.

The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC to analyze the mechanism of silencing of the embryonic epsilon-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state of their adult beta-globin genes. In hAEC, which express primarily the beta-globin gene, quantitative PCR analysis shows that beta-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the gamma-globin gene are low, with slight differences among the three exons. Although epsilon-globin is not expressed, epsilon-globin RNA transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than epsilon-exons II and III. As expected, in K562 cells that express high levels of epsilon- and gamma-globin, epsilon- and gamma-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells beta-mRNA levels are very low but beta-exon I levels are much higher than that of exons II or III. Moreover, all or most of the globin transcripts for the highly expressed globin genes in both cell types (gamma and beta in hAEC, epsilon and gamma in K562 cells) found in the cytoplasm or nucleus are correctly processed. The globin transcripts that are detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype.

Quantitative fine-structural analysis of olfactory cortical synapses

To determine the extent to which hippocampal synapses are typical of those found in other cortical regions, we have carried out a quantitative analysis of olfactory cortical excitatory synapses, reconstructed from serial electron micrograph sections of mouse brain, and have compared these new observations with previously obtained data from hippocampus. Both superficial and deep layer I olfactory cortical synapses were studied. Although individual synapses in each of the areas—CA1 hippocampus, olfactory cortical layer Ia, olfactory cortical area Ib—might plausibly have been found in any of the other areas, the average characteristics of the three synapse populations are distinct. Olfactory cortical synapses in both layers are, on average, about 2.5 times larger than their hippocampal counterparts. The layer Ia olfactory cortical synapses have fewer synaptic vesicles than do the layer Ib synapses, but the absolute number of vesicles docked to the active zone in the layer Ia olfactory cortical synapses is about equal to the docked vesicle number in the smaller hippocampal synapses. As would be predicted from studies on hippocampus that relate paired-pulse facilitation to the number of docked vesicles, the synapses in layer 1a exhibit facilitation, whereas the ones in layer 1b do not. Although hippocampal synapses provide as a good model system for central synapses in general, we conclude that significant differences in the average structure of synapses from one cortical region to another exist, and this means that generalizations based on a single synapse type must be made with caution.

On the reaction mechanism of l-lactate oxidase: Quantitative structure-activity analysis of the reaction with para-substituted l-mandelates

The rate constants for reduction of the flavoenzyme, l-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3–7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of α-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the α-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.

Quantitative analysis of the immunopotency of genetically transfected dendritic cells

Dendritic cells (DCs) instruct and activate a naive immune system to mount a response toward foreign proteins. Therefore, it has been hypothesized that an ideal vaccine strategy would be to directly introduce genes encoding antigens into DCs. To test this strategy quantitatively, we have compared the immune response elicited by a genetically transfected DC line to that induced by a fibroblast line, or standard genetic immunization. We observe that a single injection of 500–1,000 transfected DCs can produce a response comparable to that of standard genetic immunization, whereas fibroblasts, with up to 50-fold greater transfection efficiency, were less potent. We conclude that transfection of a small number of DCs is sufficient to initiate a wide variety of immune responses. These results indicate that targeting genes to DCs will be important for controlling and augmenting the immunological outcome in genetic immunization.

Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF2α—F2-iPs—in urine has reflected lipid peroxidation in humans. However, up to 64 F2-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F2-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F2-iPs, using liquid chromatography/tandem mass spectrometry (LC/MS/MS), in which sample preparation is minimized. Authentic standards of F2-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF2α-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F2-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F2-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC/MS and LC/MS/MS of iPF2α-VI in hypercholesterolemia and of 8,12-iso-iPF2α-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe’s average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 μg unspecific DNA without post-PCR probe manipulations could be achieved with different primer/probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Patterned library analysis: A method for the quantitative assessment of hypotheses concerning the determinants of protein structure

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we show that resin-splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455-member sample from a library wherein four solvent-exposed sites in an α-helix can contain any of nine different amino acids are highly correlated (P < 0.0001, R2 = 0.97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques.

The paradox of alloreactivity and self MHC restriction: Quantitative analysis and statistics

Although 1–24% of T cells are alloreactive, i.e., respond to MHC molecules encoded by a foreign haplotype, it is generally believed that T cells cannot recognize foreign peptides binding foreign MHC molecules. We show using a quantitative model that, if T cell selection and activation are affinity-driven, then an alloreactivity of 1–24% is incompatible with the textbook notion that self MHC restriction is absolute. If an average of 1% of clones are alloreactive, then according to our model, at most 20-fold more clones should, on average, be activated by antigens presented on self MHC than by antigens presented on foreign MHC. This ratio is at best 5 if alloreactivity is 5%. These results describe average properties of the murine immune system, but not the outcome of individual experiments. Using supercomputer technology, we simulated 100,000 MHC restriction experiments. Although the average restriction ratio was 7.1, restriction was absolute in 10% of the simulated experiments, greater than 100, although not absolute, in 29%, and below 6 in 24%. This extreme variability agrees with experimental estimates. Our analysis suggests that alloreactivity and average self MHC restriction both cannot be high, but that a low average restriction level is compatible with high levels in a significant number of experiments.

Quantitative analysis of mRNA amplification by in vitro transcription

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

Quantitative analysis of chromosomal CGH in human breast tumors associates copy number abnormalities with p53 status and patient survival

We present a general method for rigorously identifying correlations between variations in large-scale molecular profiles and outcomes and apply it to chromosomal comparative genomic hybridization data from a set of 52 breast tumors. We identify two loci where copy number abnormalities are correlated with poor survival outcome (gain at 8q24 and loss at 9q13). We also identify a relationship between abnormalities at two loci and the mutational status of p53. Gain at 8q24 and loss at 5q15-5q21 are linked with mutant p53. The 9q and 5q losses suggest the possibility of gene products involved in breast cancer progression. The analytical techniques are general and also are applicable to the analysis of array-based expression data.

Quantitative analysis of senile plaques in Alzheimer disease: observation of log-normal size distribution and molecular epidemiology of differences associated with apolipoprotein E genotype and trisomy 21 (Down syndrome).

The discovery that the epsilon 4 allele of the apolipoprotein E (apoE) gene is a putative risk factor for Alzheimer disease (AD) in the general population has highlighted the role of genetic influences in this extremely common and disabling illness. It has long been recognized that another genetic abnormality, trisomy 21 (Down syndrome), is associated with early and severe development of AD neuropathological lesions. It remains a challenge, however, to understand how these facts relate to the pathological changes in the brains of AD patients. We used computerized image analysis to examine the size distribution of one of the characteristic neuropathological lesions in AD, deposits of A beta peptide in senile plaques (SPs). Surprisingly, we find that a log-normal distribution fits the SP size distribution quite well, motivating a porous model of SP morphogenesis. We then analyzed SP size distribution curves in genotypically defined subgroups of AD patients. The data demonstrate that both apoE epsilon 4/AD and trisomy 21/AD lead to increased amyloid deposition, but by apparently different mechanisms. The size distribution curve is shifted toward larger plaques in trisomy 21/AD, probably reflecting increased A beta production. In apoE epsilon 4/AD, the size distribution is unchanged but the number of SP is increased compared to apoE epsilon 3, suggesting increased probability of SP initiation. These results demonstrate that subgroups of AD patients defined on the basis of molecular characteristics have quantitatively different neuropathological phenotypes.