Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.     

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Background  

NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.     

Natal recruitment and adult retention in a population of nine-banded armadillos

Nine-banded armadillosDasypus novemcinctus Linnaeus, 1758 are interesting in part because (a) they give birth to litters of genetically identical quadruplets, and (b) the species’ range has expanded rapidly throughout the southern United States during the last 150 years, suggesting substantial dispersal of individuals. Using data from 7 field seasons between 1992 and 1999, we examined the extent of juvenile recruitment and retention of adults in a population of armadillos from northern Florida. There were no sex differences in the likelihood of recruitment or most attributes of male and female recruits at any age. In the few cases where more than one littermate was recruited into the population, siblings were significantly more widely dispersed as adults than they were as juveniles, thus limiting opportunities for interaction among clonal siblings. There was some evidence that recruits ranged more widely than other individuals, suggesting recruits may have been searching for suitable sites to establish themselves. Recruits were heavier than non-recruits as both juveniles and yearlings, which may have aided in establishing a home range, but recruits were lighter than other animals as adults. Overall, slightly less than 50% of armadillos first captured as adults were never seen in a subsequent year, suggesting these individuals may have been transients. However, some adults remained in the population for multiple years, moving very little from the area where they were first sighted. As with recruits, there were no sex differences in the likelihood of adults being retained in the population nor in the attributes of retained males and females. Retained animals exhibited more extensive anatomical damage and moved farther between successive sightings within years than did non-residents. Adults were more likely to be retained in the population than juveniles were to be recruited, and retained adults were older, heavier, and exhibited more extensive anatomical damage than did recruits. Our data seem to indicate a population characterized by limited recruitment of juveniles (particularly of clonemates) and an adult population exhibiting considerable turnover from year to year, but with a core of individuals who are long-term residents.     

A molecular genetic approach to the identification of isochromosomes of chromosome 21

Summary The largest class of de novo chromosomal rearrangements in Down syndrome are rea(21q21q). Classically, these rearrangements have been termed Robertsonian translocations, implying an attachment of two different chromosome 21 homologues. Additionally, a Robertsonian translocation between two chromosomes 21 cannot be distinguished from an isochromosome composed of genetically identical arms by cytogenetic analyses. Therefore, we have used molecular techniques to differentiate between true Robertsonian translocations and isochromosomes. Samples were obtained from 12 probands, ascertained for de novo rearrangements between homologous chromosomes 21 [11 rea(21q21q) and 1 rea (21;21)(q22;q22)], their parents (n = 24) and available siblings (n = 7). The parental origins of the de novo rearrangements were assigned using molecular and cytogenetic analyses. Although not statistically significant, there was a two-fold increase in the number of paternally derived de novo rearrangements (n = 8) as compared with maternally derived rearrangements (n = 4). To distinguish between rob(21q21q) and i(21q), we used restriction fragment length polymorphisms (RFLPs) spanning the length of chromosome 21. Using all informative and partially informative RFLPs, we used the method of maximum likelihood to assign the most likely rearrangement definition (i or rob) and parental origin in each family. The maximum likelihood estimates indicated that all rearrangements tested (n = 8) were isochromosomes. C-banding revealed two centromeres in three cases indicating that a U-type exchange occurred between sister chromatids in these rearrangements. Our results suggest that the majority of de novo rea(21q21q) are isochromosomes derived from a single parental chromosome 21.     

Cardiovascular responses to intrathecal administration of arginine vasopressin in rats

Arginine vasopressin (AVP) has been localized in numerous extrahypothalamic brain regions and in the spinal cord. The results of intracerebroventricular AVP injections and microinjection of AVP into the brain stem suggest that this peptide, acting centrally at higher levels, may influence cardiovascular function. No function for the AVP occurring at spinal levels has been reported. In this study we report that AVP, in picomole quantities, increased arterial blood pressure and integrated heart rate in a dose-dependent manner following intrathecal application to the thoracic region in the rat. This response was not blocked by intravenous administration of the AVP antagonist d(CH₂)₅-d-Tyr-VAVP. These results suggest that AVP, acting within the spinal cord, may alter neural outflow regulating blood pressure and heart rate.     

An approach to the collection and manipulation of time-based data using the IBM PC and BASICA

With the advent of increasingly integrated, powerful and inexpensivedigital electronics, relatively powerful computers have becomeavailable to the general public. Along with this technologicalboom there has been a concomitant increase in the availabilityof over-the-counter software packages which can be used by researchscientists for program development. In the past, the developmentof computer programs for the collection of large amounts oftime-based data was expensive and time consuming; however, theintroduction of the current generation of 16-bit microcomputersand associated hardware and software packages has enabled investigatorswith only a rudimentary knowledge of computers and interfacingto begin to design programs. The schemes and algorithms, developedusing BASICA on an IBM-Personal Computer, which are describedin this article can serve other investigators as models forthe assembly of their own programs for the collection, manipulationand plotting of time-based data. The incorporation of inexpensivecomputer graphics hardware and software, which provided a simplesolution to the problem of analysis and presentation of largeamounts of data, will also be discussed. Received on December 19, 1984; accepted on December 22, 1984     

Chromosome numbers of goldenrods,Euthamia and Solidago (Compositae: Astereae). II. Additional counts with comments on cytogeography

Chromosome number determinations were made from 407 wild or transplanted individuals and seedlings representing 65 taxa and hybrids inEuthamia andSolidago. The following are first reports:Euthamia remota, 2n=9II;Solidago leavenworthii, 2n=54;S. mollis, 2n=36;S. mollis var.angustata, 2n=36;S. rigida var.glabrata, 2n=9II;S. sempervirens var.azorica, 2n=9II; andS. sparsiflora, 2n=54. Most species have been sampled only a few times or are consistently of one cytotype. Sufficient counts have been made to indicate some general patterns of cytotype distribution in the following species complexes:S. gigantea, S. canadensis, S. flexicaulis, S. rugosa, andS. uliginosa.     

Three patterns of mitochondrial DNA nucleotide divergence in the meadow vole,Microtus pennsylvanicus

Summary The DNA sequence was determined for the cytochrome c oxidase II (COII), tRNA^Lys, and ATPase 8 genes from the mitochondrial genome of the meadow vole, Microtus pennsylvanicus. When compared to other rodents, three different patterns of evolutionary divergence were found. Nucleotide variation in tRNA^Lys is concentrated in the TC loop. Nucleotide variation in the COII gene in three genera of rodents (Microtus, Mus, Rattus) consists predominantly of transitions in the third base positions of codons. The predicted amino acid sequence in highly conserved (>92% similarity). Analysis of the ATPase 8 gene among four genera (Microtus, Cricetulus, Mus, Rattus) revealed more detectable transversions than transitions, many fixed first and second position mutations, and considerable amino acid divergence. The rate of nucleotide substitution at nonsynonymous sites in the ATPase 8 gene is 10 times the rate in the COII gene. In contrast, the estimated absolute mutation rate as determined by analysis of nucleotide substitutions at fourfold degenerate sites probably is the same for the two genes. The primary sequences of the ATPase 8 and COII peptides are constrained differently, but each peptide is conserved in terms of predicted secondary-level configuration.