STUDY OF BULL SPERMATOZOA : Quantitative Electron Microscopy

The electron microscope has been used to determine the characteristic dimensions and the distribution of the dry mass in bull spermatozoa. The most important result is that all characteristic data are occurring in logarithmic distributions. Furthermore, no correlation between such parameters as head weight and tail weight or head length and tail length was found. The occurrence in logarithmic distributions and the non-correlation of parts in the assembly of a spermatozoon are considered to reflect significant biologic principles. Methodologically, a new procedure is added to quantitative electron microscopy permitting the recording of the mass cross-section (total mass per unit length) of an object. This approach makes possible determinations of the distribution and the total mass of very long and narrow structures.     

The distribution of nuclear mass, volume and dimensions

Our own measurements and a review of measurements presented in the literature showed that the mass, volume and dimensions of cell nuclei are distributed with a skew towards higher values, a distribution that can best be described as lognormal. The practical consequences of this finding suggest that the measurements of mass and of size should be plotted on a logarithmic scale while DNA values are appropriately presented on a linear scale. The distribution of DNA values of normal cells represents errors introduced by random disturbances in preparations and measurements.     

Proliferative potential of human fibroblasts: an inverse dependence on cell size

Human foreskin fibroblast-like cells were separated on the basis of DNA content and cell size by fluorescence-activated cell sorting. Subpopulations of "large" or "small" cells with the same (G1) DNA content were clonally expanded and found to contain predominantly nondividing or highly proliferative cells, respectively. From the rate of clonal growth, we deduce that small cells divide faster than large cells. Intermediate-sized cells were found to yield primarily smaller ("attenuated") clones. The clonal data can be incorporated into a previously reported kinetic model of clonal attenuation. This version of the model postulates that small "stem" cells yield larger daughters which have only a limited proliferative potential. We also postulate that a progressive increase in cell size can account for the decreasing concentration of DNA polymerase alpha, which has been reported in older cultures.     

Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry

Sphingolipids are a highly diverse category of compounds that serve not only as components of biologic structures but also as regulators of numerous cell functions. Because so many of the sphingolipids in a biological system are bioactive and are often closely related structurally and metabolically (for example, complex sphingolipids<-->ceramide<-->sphingosine<-->sphingosine 1-phosphate), to understand the role(s) of sphingolipids in a given context one must conduct a "sphingolipidomic" analysis-i.e., a structure-specific and quantitative measurement of all of these compounds, or at least all members of a critical subset. Liquid chromatography tandem mass spectrometry (LC MS/MS) is currently the only technology with the requisite structural specificity, sensitivity, quantitative precision, and relatively high-throughput capabilities for such analyses in small samples ( approximately 10(6) cells). This review describes a series of protocols that have been developed for the relatively rapid analysis of all of the molecular species from 3-ketosphinganines through sphingomyelins and some glycosphingolipids (including all the compounds that are presently regarded as sphingolipid "second messengers") using normal- and reverse-phase LC to separate isometric and isobaric species (such as glucosylceramides and galactosylceramides) in combination with triple quadrupole (for MS/MS) and hybrid quadrupole-ion trap (for MS3) mass spectrometry. Also discussed are some of the issues remaining to be resolved in the analysis of the full sphingolipidome.     

Direct mass spectrometry approaches to characterize polyphenol composition of complex samples

Lower molecular weight polyphenols including proanthocyanidin oligomers can be analyzed after HPLC separation on either reversed-phase or normal phase columns. However, these techniques are time consuming and can have poor resolution as polymer chain length and structural diversity increase. The detection of higher molecular weight compounds, as well as the determination of molecular weight distributions, remain major challenges in polyphenol analysis.Approaches based on direct mass spectrometry (MS) analysis that are proposed to help overcome these problems are reviewed. Thus, direct flow injection electrospray ionization mass spectrometry analysis can be used to establish polyphenol fingerprints of complex extracts such as in wine. This technique enabled discrimination of samples on the basis of their phenolic (i.e. anthocyanin, phenolic acid and flavan-3-ol) compositions, but larger oligomers and polymers were poorly detectable. Detection of higher molecular weight proanthocyanidins was also restricted with matrix-assisted laser desorption ionization (MALDI) MS, suggesting that they are difficult to desorb as gas-phase ions. The mass distribution of polymeric fractions could, however, be determined by analyzing the mass distributions of bovine serum albumin/proanthocyanidin complexes using MALDI-TOF-MS.     

Physical and functional heterogeneity in TYMV RNA: evidence for the existence of an independent messenger coding for coat protein.

Turnip yellow mosaic virus RNA can be separated into two distinct components of 2 times 10(6) and 300 000 daltons molecular weight after moderate heat treatment in the presence of SDS or EDTA. The two species cannot have arisen by accidental in vitro degradation of a larger RNA, as they both possess capped 5' ends. Analysis of the newly synthesized proteins resulting from translation of each RNA by a wheat germ extract shows that the 300 000 molecular weight RNA can be translated very efficiently into coat protein. When translated in vitro the longer RNA gave a series of high molecular weight polypeptides but only very small amounts of a polypeptide having about the same mass as the coat protein. Thus our results suggest that the small RNA is the functional messenger for coat protein synthesis in infected cells.     

THE LIKELIHOOD OF SPERM COMPETITION IN MANATEES-EXPLAINING AN APPARENT PARADOX

Florida manatees ( Trichechus manatus latirostris ) are promiscuous, with multiple males mating with individual females. This suggests manatees are sperm competitors. Surprisingly, manatee testes are not relatively large. For adult males in non-winter, testicular size is approximately twice what is expected, based on allometry, for "typical" ( i. e. , non-sperm competitor) male mammals of similar size; for these manatees, combined testicular weight represents 0.19% of the body weight ( n = 27 manatees). Testicular weight was generally largest in manatees older than 7 yr in non-winter. Testicular size of some sperm competitors is as much as an order of magnitude (or more) larger than expected in a "typical" species. Perhaps in compensation for the testes not being remarkably large, the seminal vesicles of mature manatees may be larger than the testes. Production of notably large volumes of seminal fluid characterizes sperm competitor primate species and may have positive energy consequences for species such as the manatee that have extremely low metabolic rates. Another possible explanation for the observed relationship between testicular mass and body mass in manatees is that selection for a greatly expanded hindgut and extremely dense, heavy integument could make the body mass of manatees "artificially" high, and the relative testicular mass "artificially" low.     

Fractionation of vanadium complexes in serum, packed cells and tissues of Wistar rats by means of gel filtration and anion-exchange chromatography

Male Wistar rats were intraperitoneally injected with [(48)V]vanadium tracer to (1) investigate the distribution of vanadium over different tissues and (2) study the distribution of vanadium over the proteins and peptides in serum, packed cells and homogenates of tissues by means of liquid chromatography experiments (size exclusion, ion exchange). Target organs were primarily kidney, bone, spleen and liver. In serum we found that vanadium was mainly bound to transferrin; however, a small amount was also bound to albumin. Besides these two complexes, a significant part of vanadium occurred as readily exchangeable ("free") vanadium. In packed cells, vanadium is mainly bound to hemoglobin and to two abundant low molecular mass complexes. The chromatograms of tissues (kidney, liver, testes, spleen and lung) show similar high molecular mass complexes (vanadium co-elutes with ferritin, transferrin and hemoglobin). Between the low molecular mass complexes there are similar peaks for spleen, testes and kidneys on the one hand, and liver and lung on the other hand, albeit the differences are small. In the case of lung, there is an additional low molecular mass peak.     

A small-scale five-hour procedure for isolating multiple samples of CsCl-purified DNA: application to isolations from mammalian, insect, higher plant, algal, yeast, and bacterial sources

A rapid and simple procedure is described for obtaining CsCl-purified DNA from multiple small samples of cells or tissue. The DNA is recovered in a high-molecular-weight form (greater than or equal to 50 kb) that is readily cleaved with restriction enzymes. Sufficient quantities of DNA (10-50 micrograms) are recovered to allow multiple analyses by Southern blotting and most cloning procedures. The isolation procedure involves addition of intact cells or powders of frozen tissues directly to a simple lysis buffer containing detergent (sodium dodecyl sulfate or sodium sarcosinate) and high concentrations of EDTA. Ultra-high-speed centrifugation of CsCl gradients allows the isolation of DNA from 10 different samples in as little as 5 h. Applications are described for mammalian cells (HeLa cells), insect tissues (Drosophila melanogaster adults and pupa, Manduca sexta pupa, and Musca domestica pupa), higher plant tissues (Vicia faba leaves and meristems), algal cells (walled and wall-less Chlamydomonas reinhardi), yeast cells (Saccharomyces cerevisiae), and bacterial cells (Escherichia coli spheroplasts for preparation of both chromosomal and plasmid DNA). The procedure can be scaled up with larger sample sizes and longer centrifugation times to provide bulk quantities of DNA.     

Generation of anaphylatoxins by human beta-tryptase from C3, C4, and C5

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.     

Dynamic strain similarity in vertebrates; an alternative to allometric limb bone scaling

Galileo (1638) observed that "nature cannot grow a tree nor construct an animal beyond a certain size, while retaining the proportions which suffice in the case of a smaller structure". However, subsequent measurement has shown that limb bone dimensions are scaled geometrically with body size (Alexander et al., 1979a), and that the material properties of their constituent bone tissue are similar in animals over a wide range of body weight (Sedlin & Hirsch, 1966; Yamada, 1970; Burstein et al., 1972; Biewener, 1982). If, as suggested in previous scaling arguments (McMahon, 1973; Biewener, 1982), vigorous locomotion involved the same proportional forces over a wide range of animal size, this would create a paradox since large animals would be in far greater danger of skeletal failure than small ones. However, in vivo strain gauge implantations have shown that, during high speed running, axial force as a proportion of body weight (G) in the limb bones of animals decreases as a function of body size from 6.9 G in a 7 kg turkey to 2.8 G in a small (130 kg) horse. Estimates of axial force in larger animals suggest that this is further reduced to 0.8 G in a 2500 kg elephant. Nevertheless, it appears that, regardless of animal size or locomotory style, the peak stresses in the bones of these animals are remarkably similar. Therefore, throughout the range of animals considered (350 times differences in mass), we suggest that similar safety factors to failure are maintained, not by allometrically scaling bone dimensions, but rather by allometrically scaling the magnitude of the peak forces applied to them during vigorous locomotion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of the eukaryotic ARP2/3 activators of the WASP family: WASP, WAVE, WASH, and WHAMM, and the proposed new family members WAWH and WAML

Background

The sizes of proteins are relevant to their biochemical structure and for their biological function. The statistical distribution of protein lengths across a diverse set of taxa can provide hints about the evolution of proteomes.

Results

Using the full genomic sequences of over 1,302 prokaryotic and 140 eukaryotic species two datasets containing 1.2 and 6.1 million proteins were generated and analyzed statistically. The lengthwise distribution of proteins can be roughly described with a gamma type or log-normal model, depending on the species. However the shape parameter of the gamma model has not a fixed value of 2, as previously suggested, but varies between 1.5 and 3 in different species. A gamma model with unrestricted shape parameter described best the distributions in ~48% of the species, whereas the log-normal distribution described better the observed protein sizes in 42% of the species. The gamma restricted function and the sum of exponentials distribution had a better fitting in only ~5% of the species. Eukaryotic proteins have an average size of 472 aa, whereas bacterial (320 aa) and archaeal (283 aa) proteins are significantly smaller (33-40% on average). Average protein sizes in different phylogenetic groups were: Alveolata (628 aa), Amoebozoa (533 aa), Fornicata (543 aa), Placozoa (453 aa), Eumetazoa (486 aa), Fungi (487 aa), Stramenopila (486 aa), Viridiplantae (392 aa). Amino acid composition is biased according to protein size. Protein length correlated negatively with %C, %M, %K, %F, %R, %W, %Y and positively with %D, %E, %Q, %S and %T. Prokaryotic proteins had a different protein size bias for %E, %G, %K and %M as compared to eukaryotes.

Conclusions

Mathematical modeling of protein length empirical distributions can be used to asses the quality of small ORFs annotation in genomic releases (detection of too many false positive small ORFs). There is a negative correlation between average protein size and total number of proteins among eukaryotes but not in prokaryotes. The %GC content is positively correlated to total protein number and protein size in prokaryotes but not in eukaryotes. Small proteins have a different amino acid bias than larger proteins. Compared to prokaryotic species, the evolution of eukaryotic proteomes was characterized by increased protein number (massive gene duplication) and substantial changes of protein size (domain addition/subtraction).     

A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells

In vitro assays involving primary cells are used routinely to evaluate organ-specific toxic effects, for instance, the use of primary hepatocytes to evaluate hepatotoxicity. A major drawback of an in vitro system is the lack of multiple organ interactions as observed in a whole organism. A novel cell culture system, the integrated discrete multiorgan cell culture system (IdMOC), is described here. The IdMOC is based on the "wells within a well" concept, consisting of a cell culture plate with larger, containing wells, within each of which are multiple smaller wells. Cells from multiple organs can be cultured initially in the small wells (one organ per well, each in its specialized medium). On the day of toxicity testing, a volume of drug-containing medium is added to the containing well to flood all inner wells, thereby interconnecting all the small wells. After testing, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate endpoints. We report here the application of IdMOC in the evaluation of the cytotoxicity of tamoxifen, an anticancer agent with known human toxicity, on primary cells from multiple human organs: liver (hepatocytes), kidney (kidney cortical cells), lung (small airway epithelial cells), central nervous system (astrocytes), blood vessels (aortic endothelial cells) as well as the MCF-7 human breast adenocarcinoma cells. IdMOC produced results that can be used for the quantitative evaluation of its anticancer effects (i.e., cytotoxicity towards MCF-7 cells) versus its toxicity toward normal organs (i.e., liver, kidney, lung, CNS, blood vessels).     

Rapid discrimination of Bacillus anthracis from other members of the B. cereus group by mass and sequence of "intact" small acid soluble proteins (SASPs) using mass spectrometry

The intentional contamination of buildings, e.g. anthrax in the bioterrorism attacks of 2001, demonstrated that the population can be affected rapidly and lethally if the appropriate treatment is not provided at the right time. Molecular approaches, primarily involving PCR, have proved useful in characterizing "white powders" used in these attacks as well as isolated organisms. However there is a need for a simpler approach, which does not involve temperamental reagents (e.g. enzymes and primers) which could potentially be used by first responders. It is demonstrated here that small acid-soluble proteins (SASPs), located in the core region of Bacillus spores, are reliable biomarkers for identification. The general strategy used in this study was to measure the molecular weight (MW) of an intact SASP by electrospray ionization mass spectrometry (ESI MS) followed by generation of sequence-specific information by ESI MS/MS (tandem mass spectrometry). A prominent SASP of mass 6679 was present in all B. anthracis strains. For B. cereus and B. thuringiensis strains the SASP had a mass of 6712. This represents a two amino acid substitution (serine to alanine; phenylalanine to tyrosine). The only SASP present in the B. anthracis genome consistent with this sequence is encoded by the gene ssB. This protein has a predicted mass of 6810, presumably post-translational processing leads to loss of methionine (mass 131) generating a SASP of mass 6679. This study showed that intact SASPs can be used as a biomarker for identification of B. anthracis; the protocol is simple and rapid. Extrapolation of this approach might prove important for real-time biodetection.     

Characterization of the cellular basis for the inhibition of cytolytic effector cells by murine placenta

Direct suppression of cytolytic effector cell function by cells of the placenta may represent one mechanism that protects the "fetal allograft" from rejection by maternal transplantation immunity. Collagenase disaggregated murine placental cells block target cell lysis by natural killer, lymphokine-activated killer, and (CTL)-type killer cells. This inhibition is reversible and noncompetitive, similar to a previously described inhibitor of CTL found in spleens of mice undergoing an acute graft vs host (GVH) response. Velocity sedimentation separation of placental cells shows that the inhibitory activity is primarily associated with cells that cosediment with nucleated fetal erythrocytes. When these erythrocytes were lysed, an increased number of non-erythrocytic cells could be separated and under this circumstance, inhibitory activity was seen in association with either small white cells or fetal erythrocytes and with large white cells. There may be several cell populations in murine placenta that can inhibit cytolytic effector cells. The possible relevance of direct placental inhibition of cytolytic effectors to protection of the "fetal allograft" is discussed.     

Body Mass Estimates in Extinct Mammals from Limb Bone Dimensions: the Case of North American Hyaenodontids

The body mass estimation of several limb bone dimensions (shaft cross-sectional properties, articular sizes, and bone lengths) were examined using bivariate linear regression analyses. The sample included taxonomically and behaviourally diverse small to medium-sized Recent carnivorans and carnivorous marsupials. All examined limb bone dimensions indicated low errors (percentage standard error of estimate, 8–13) for the body mass estimations. Among them, humeral and femoral shaft properties correlated best with body weight, while limb bone lengths gave larger errors. Both humeral and femoral head dimensions have relatively large individual variations, and distal humeral articular dimensions seem to be influenced more by phylogenetic differences. The regressions based on each locomotor group gave slightly lower errors than those based on the total pooled sample. The results were then applied to hyaenodontid creodonts from the Eocene–Oligocene of North America. The estimated body masses (kg) are: Arfia , 5.4–9.5; Prototomus , <6.0; Pyrocyon , 2.6; Sinopa , 1.3–1.4; Tritemnodon , 7.6–13; Prolimnocyon , 1.6; Thinocyon , 0.7–2.5; Machaeroides , 12; Limnocyon , 7.8– 16; Hyaenodon , 9.1–43. The various limb bone dimensions give different body mass values, but the variation in estimates is smaller compared to those derived from dental or cranial measurements.     

Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics

We have shown that sample fractionation is an effective method for increasing the detection coverage of the proteome of complex samples, such as organs, by mass-spectrometric techniques. Further fractionating a sample based on subcellular compartments can generate molecular information on the state of a tissue and the distribution of its protein components. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs or tissues for subsequent analysis by mass spectrometry. It uses differential centrifugation in density gradients to isolate nuclear, cytosolic, mitochondrial and mixed microsomal (Golgi, endoplasmic reticulum, other vesicles and plasma membrane) fractions. Once the fractions are isolated, they are extracted for protein and the samples can then be frozen for processing and analysis at a later date. The procedure can typically be completed in 5 h.     

Seasonal variation in size spectra of phytoplankton assemblages in Loch Leven,Scotland

Greatest axial dimensions (GALD) of phytoplankton cells, colonies and filaments etc, are used to describe the size structure of whole assemblages of species in the shallow eutrophic Loch Leven (S.E. Scotland). Two-weekly samples over the period 1979–1982 have been analysed to determine whether variation in size spectra show seasonal trends. Size frequency distributions are displayed using — for the first time in studies of phytoplankton assemblages — the graphical method based on rankits. The paper describes how individuals to be measured were chosen without bias towards any particular type. Seasonal variation in temperature and nutrient concentrations are discussed in relation to algal size structure.In spite of irregular shifts in species composition and abundance, phytoplankton assemblage size spectra (PASS) exhibit seasonal patterns. Early in the year, when temperatures are low and herbivorous zooplankton sparse, small algae ( 15 µm) predominate. The winter-early spring assemblages often exhibit a normal size frequency distribution. Later in the year larger algae occasionally become relatively more numerous, and skewed or polymodal frequency distributions are recorded. Increases in large algae are usually associated with Daphnia population maxima; the inter-relationship is clearly demonstrated in time-series plots of GALD isopleths and Daphnia numbers.The potential of the PASS method to further knowledge on ecological controls of phytoplankton is discussed. The investigator is compelled to include all species in a sample. The rankit-dimension graphs retain all the data, so the position of each algal measurement and its influence on the size distribution can be observed. This approach may help to identify size ranges of algae removed by a wide variety of grazing zooplankton.     

Human SERPINB12 Is an Abundant Intracellular Serpin Expressed in Most Surface and Glandular Epithelia

The intracellular serine protease inhibitors (serpins) are an important family of proteins that protect cells form proteinase-mediated injury. Understanding the tissue and cellular expression pattern of this protein family can provide important insights into their physiologic roles. For example, high expression in epithelial tissues, such as lung, may suggest a biologic function in cellular defense, secretion, or selective absorption. Although the expression pattern of many of the intracellular serpins has been well described, one member of this class, SERPINB12, has not been carefully examined. We generated a mouse monoclonal antibody directed against human SERPINB12 and delineated its specificity and tissue and cell type distribution pattern through immunoblotting and immunohistochemistry, respectively. This monoclonal antibody was human specific and did not cross-react with other human intracellular serpins or mouse Serpinb12. SERPINB12 was found in nearly all the tissues investigated. In addition, this serpin was found in multiple cell types within individual tissues but primarily the epithelium. These data suggest that SERPINB12, like some other intracellular serpins, may play a vital role in barrier function by providing protection of epithelial cells.