Immune surveillance against a solid tumor fails because of immunological ignorance

Many peripheral solid tumors such as sarcomas and carcinomas express tumor-specific antigens that can serve as targets for immune effector T cells. Nevertheless, overall immune surveillance against such tumors seems relatively inefficient. We studied immune surveillance against a s.c. sarcoma expressing a characterized viral tumor antigen. Surprisingly, the tumor cells were capable of inducing a protective cytotoxic T cell response if transferred as a single-cell suspension. However, if they were transplanted as small tumor pieces, tumors readily grew. Tumor growth correlated strictly with (i) failure of tumor cells to reach the draining lymph nodes and (ii) absence of primed cytotoxic T cells. Cytotoxic T cells were not tolerant or deleted because a tumor antigen-specific cytotoxic T cell response was readily induced in lymphoid tissue by immunization with virus or with tumor cells even in the presence of large tumors. Established tumors were rejected by vaccine-induced effector T cells if effector T cells were maintained by prolonged or repetitive vaccination, but not by single-dose vaccination. Thus, in addition to several other tumor-promoting parameters, some antigenic peripheral sarcomas—and probably carcinomas—may grow not because they anergize or tolerize tumor-specific T cells, but because such tumors are immunologically dealt with as if they were in a so-called immunologically privileged site and are ignored for too long.

Molecular mechanisms underlying differential odor responses of a mouse olfactory receptor

The prevailing paradigm for G protein-coupled receptors is that each receptor is narrowly tuned to its ligand and closely related agonists. An outstanding problem is whether this paradigm applies to olfactory receptor (ORs), which is the largest gene family in the genome, in which each of 1,000 different G protein-coupled receptors is believed to interact with a range of different odor molecules from the many thousands that comprise “odor space.” Insights into how these interactions occur are essential for understanding the sense of smell. Key questions are: (i) Is there a binding pocket? (ii) Which amino acid residues in the binding pocket contribute to peak affinities? (iii) How do affinities change with changes in agonist structure? To approach these questions, we have combined single-cell PCR results [Malnic, B., Hirono, J., Sato, T. & Buck, L. B. (1999) Cell 96, 713–723] and well-established molecular dynamics methods to model the structure of a specific OR (OR S25) and its interactions with 24 odor compounds. This receptor structure not only points to a likely odor-binding site but also independently predicts the two compounds that experimentally best activate OR S25. The results provide a mechanistic model for olfactory transduction at the molecular level and show how the basic G protein-coupled receptor template is adapted for encoding the enormous odor space. This combined approach can significantly enhance the identification of ligands for the many members of the OR family and also may shed light on other protein families that exhibit broad specificities, such as chemokine receptors and P450 oxidases.

Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution

A remarkable instability at simple repeated sequences characterizes gastrointestinal cancer of the microsatellite mutator phenotype (MMP). Mutations in the DNA mismatch repair gene family underlie the MMP, a landmark for hereditary nonpolyposis colorectal cancer. These tumors define a distinctive pathway for carcinogenesis because they display a particular spectrum of mutated cancer genes containing target repeats for mismatch repair deficiency. One such gene is BAX, a proapoptotic member of the Bcl-2 family of proteins, which plays a key role in programmed cell death. More than half of colon and gastric cancers of the MMP contain BAX frameshifts in a (G)8 mononucleotide tract. However, the functional significance of these mutations in tumor progression has not been established. Here we show that inactivation of the wild-type BAX allele by de novo frameshift mutations confers a strong advantage during tumor clonal evolution. Tumor subclones with only mutant alleles frequently appeared after inoculation into nude mice of single-cell clones of colon tumor cell lines with normal alleles. In contrast, no clones of BAX-expressing cells were found after inoculation of homozygous cell clones without wild-type BAX. These results support the interpretation that BAX inactivation contributes to tumor progression by providing a survival advantage. In this context, survival analyses show that BAX mutations are indicators of poor prognosis for both colon and gastric cancer of the MMP.

Connexin expression in electrically coupled postnatal rat brain neurons

Electrical coupling by gap junctions is an important form of cell-to-cell communication in early brain development. Whereas glial cells remain electrically coupled at postnatal stages, adult vertebrate neurons were thought to communicate mainly via chemical synapses. There is now accumulating evidence that in certain neuronal cell populations the capacity for electrical signaling by gap junction channels is still present in the adult. Here we identified electrically coupled pairs of neurons between postnatal days 12 and 18 in rat visual cortex, somatosensory cortex, and hippocampus. Notably, coupling was found both between pairs of inhibitory neurons and between inhibitory and excitatory neurons. Molecular analysis by single-cell reverse transcription–PCR revealed a differential expression pattern of connexins in these identified neurons.

Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic stem cells

Previous studies have demonstrated hematopoietic stem cell amplification in vitro after the activation of three cell-surface receptors: flt3/flk2, c-kit, and gp130. We now show flt3-ligand and Steel factor alone will stimulate >85% of c-kit+Sca-1+lin− adult mouse bone marrow cells to proliferate in single-cell serum-free cultures, but concomitant retention of their stem cell activity requires additional exposure to a ligand that will activate gp130. Moreover, this response is restricted to a narrow range of gp130-activating ligand concentrations, above and below which hematopoietic stem cell activity is lost. These findings indicate a unique contribution of gp130 signaling to the maintenance of hematopoietic stem cell function when these cells are stimulated to divide with additional differential effects dictated by the intensity of gp130 activation.

Synthesis of mitochondrial DNA in permeabilised human cultured cells

The mechanisms that underlie the maintenance of and increase in mutant mitochondrial DNA (mtDNA) are central to our understanding of mitochondrial disease. We have therefore developed a technique based on saponin permeabilisation that allows the study of mtDNA synthesis in intact cells. Permeabilisation of cells has been extensively used in an established method both for studying transcription and DNA replication in the nucleus and for measuring respiratory chain activities in mitochondria. We have quantitatively studied incorporation of radiolabelled DNA precursors into mtDNA in human cell lines derived from controls and from patients with mitochondrial DNA disease. Total cell DNA is extracted, restriction digested and Southern blotted, newly synthesised mtDNA being proportional to the label incorporated in each restriction band. A rate of synthesis can then be derived by estimating the relative steady-state mtDNA after probing with full-length mtDNA. Where co-existing mutant and wild-type mtDNA (heteroplasmy) can be distinguished using restriction digestion, their rates of synthesis can be compared within a single cell line. This will be particularly useful in elucidating the pathophysiology of mtDNA diseases in which the distribution of mutant and wild-type mtDNA in cell lines in patient tissues may evolve with time.

Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator

To investigate the dynamics of guanosine 3′,5′-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1–77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of ≈2 μM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr516 of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5–4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.

Localization and regulation of GLUTx1 glucose transporter in the hippocampus of streptozotocin diabetic rats

We describe the localization of the recently identified glucose transporter GLUTx1 and the regulation of GLUTx1 in the hippocampus of diabetic and control rats. GLUTx1 mRNA and protein exhibit a unique distribution when compared with other glucose transporter isoforms expressed in the rat hippocampus. In particular, GLUTx1 mRNA was detected in hippocampal pyramidal neurons and granule neurons of the dentate gyrus as well as in nonprincipal neurons. With immunohistochemistry, GLUTx1 protein expression is limited to neuronal cell bodies and the most proximal dendrites, unlike GLUT3 expression that is observed throughout the neuropil. Immunoblot analysis of hippocampal membrane fractions revealed that GLUTx1 protein expression is primarily localized to the intracellular compartment and exhibits limited association with the plasma membrane. In streptozotocin diabetic rats compared with vehicle-treated controls, quantitative autoradiography showed increased GLUTx1 mRNA levels in pyramidal neurons and granule neurons; up-regulation of GLUTx1 mRNA also was found in nonprincipal cells, as shown by single-cell emulsion autoradiography. In contrast, diabetic and control rats expressed similar levels of hippocampal GLUTx1 protein. These results indicate that GLUTx1 mRNA and protein have a unique expression pattern in rat hippocampus and suggest that streptozotocin diabetes increases steady-state mRNA levels in the absence of concomitant increases in GLUTx1 protein expression.

Appetite of an epiphyte: Quantitative monitoring of bacterial sugar consumption in the phyllosphere

We report here the construction, characterization, and application of a bacterial bioreporter for fructose and sucrose that was designed to monitor the availability of these sugars to microbial colonizers of the phyllosphere. Plasmid pPfruB-gfp[AAV] carries the Escherichia coli fruB promoter upstream from the gfp[AAV] allele that codes for an unstable variant of green fluorescent protein (GFP). In Erwinia herbicola, this plasmid brings about the accumulation of GFP fluorescence in response to both fructose and sucrose. Cells of E. herbicola (pPfruB-gfp[AAV]) were sprayed onto bean plants, recovered from leaves at various time intervals after inoculation, and analyzed individually for GFP content by quantitative analysis of digital microscope images. We observed a positive correlation between single-cell GFP accumulation and ribosomal content as determined by fluorescence in situ hybridization, indicating that foliar growth of E. herbicola occurred at the expense of fructose and/or sucrose. One hour after inoculation, nearly all bioreporter cells appeared to be actively engaged in fructose consumption. This fraction dropped to approximately 11% after 7 h and to ≈1% a day after inoculation. This pattern suggests a highly heterogeneous availability of fructose to individual E. herbicola cells as they colonize the phyllosphere. We estimated that individual cells were exposed to local initial fructose abundances ranging from less than 0.15 pg fructose to more than 4.6 pg.

Biomechanical activation of vascular endothelium as a determinant of its functional phenotype

One of the striking features of vascular endothelium, the single-cell-thick lining of the cardiovascular system, is its phenotypic plasticity. Various pathophysiologic factors, such as cytokines, growth factors, hormones, and metabolic products, can modulate its functional phenotype in health and disease. In addition to these humoral stimuli, endothelial cells respond to their biomechanical environment, although the functional implications of this biomechanical paradigm of activation have not been fully explored. Here we describe a high-throughput genomic analysis of modulation of gene expression observed in cultured human endothelial cells exposed to two well defined biomechanical stimuli—a steady laminar shear stress and a turbulent shear stress of equivalent spatial and temporal average intensity. Comparison of the transcriptional activity of 11,397 unique genes revealed distinctive patterns of up- and down-regulation associated with each type of stimulus. Cluster analyses of transcriptional profiling data were coupled with other molecular and cell biological techniques to examine whether these global patterns of biomechanical activation are translated into distinct functional phenotypes. Confocal immunofluorescence microscopy of structural and contractile proteins revealed the formation of a complex apical cytoskeleton in response to laminar shear stress. Cell cycle analysis documented different effects of laminar and turbulent shear stresses on cell proliferation. Thus, endothelial cells have the capacity to discriminate among specific biomechanical forces and to translate these input stimuli into distinctive phenotypes. The demonstration that hemodynamically derived stimuli can be strong modulators of endothelial gene expression has important implications for our understanding of the mechanisms of vascular homeostasis and atherogenesis.

Cellular Compartmentation of Zinc in Leaves of the Hyperaccumulator Thlaspi caerulescens1

Cellular compartmentation of Zn in the leaves of the hyperaccumulator Thlaspi caerulescens was investigated using energy-dispersive x-ray microanalysis and single-cell sap extraction. Energy-dispersive x-ray microanalysis of frozen, hydrated leaf tissues showed greatly enhanced Zn accumulation in the epidermis compared with the mesophyll cells. The relative Zn concentration in the epidermal cells correlated linearly with cell length in both young and mature leaves, suggesting that vacuolation of epidermal cells may promote the preferential Zn accumulation. The results from single-cell sap sampling showed that the Zn concentrations in the epidermal vacuolar sap were 5 to 6.5 times higher than those in the mesophyll sap and reached an average of 385 mm in plants with 20,000 μg Zn g−1 dry weight of shoots. Even when the growth medium contained no elevated Zn, preferential Zn accumulation in the epidermal vacuoles was still evident. The concentrations of K, Cl, P, and Ca in the epidermal sap generally decreased with increasing Zn. There was no evidence of association of Zn with either P or S. The present study demonstrates that Zn is sequestered in a soluble form predominantly in the epidermal vacuoles in T. caerulescens leaves and that mesophyll cells are able to tolerate up to at least 60 mm Zn in their sap.

Carbohydrates in Individual Cells of Epidermis, Mesophyll, and Bundle Sheath in Barley Leaves with Changed Export or Photosynthetic Rate1

Carbohydrate metabolism of barley (Hordeum vulgare) leaves induced to accumulate sucrose (Suc) and fructans was investigated at the single-cell level using single-cell sampling and analysis. Cooling of the root and shoot apical meristem of barley plants led to the accumulation of Suc and fructan in leaf tissue. Suc and fructan accumulated in both mesophyll and parenchymatous bundle-sheath (PBS) cells because of the reduced export of sugars from leaves under cooling and to increased photosynthesis under high photon fluence rates. The general trends of Suc and fructan accumulation were similar for mesophyll and PBS cells. The fructan-to-Suc ratio was higher for PBS cells than for mesophyll cells, suggesting that the threshold Suc concentration needed for the initiation of fructan synthesis was lower for PBS cells. Epidermal cells contained very low concentrations of sugar throughout the cooling experiment. The difference in Suc concentration between control and treated plants was much less if compared at the single-cell level rather than the whole-tissue level, suggesting that the vascular tissue contains a significant proportion of total leaf Suc. We discuss the importance of analyzing complex tissues at the resolution of individual cells to assign molecular mechanisms to phenomena observed at the whole-plant level.

Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.

Coordinated morphogenesis of epithelia during development of the Caenorhabditis elegans uterine-vulval connection.

Development of the nematode egg-laying system requires the formation of a connection between the uterine lumen and the developing vulval lumen, thus allowing a passage for eggs and sperm. This relatively simple process serves as a model for certain aspects of organogenesis. Such a connection demands that cells in both tissues become specialized to participate in the connection, and that the specialized cells are brought in register. A single cell, the anchor cell, acts to induce and to organize specialization of the epidermal and uterine epithelia, and registrates these tissues. The inductions act via evolutionarily conserved intercellular signaling pathways. The anchor cell induces the vulva from ventral epithelial cells via the LIN-3 growth factor and LET-23 transmembrane tyrosine kinase. It then induces surrounding uterine intermediate precursors via the receptor LIN-12, a founding member of the Notch family of receptors. Both signaling pathways are used multiple times during development of Caenorhabditis elegans. The outcome of the signaling is context-dependent. Both inductions are reciprocated. After the anchor cell has induced the vulva, it stretches toward the induced vulval cells. After the anchor cell has induced specialized uterine intermediate precursor cells, it fuses with a subset of their progeny.

Presence of mRNA for glutamic acid decarboxylase in both excitatory and inhibitory neurons.

Neurons in very low density hippocampal cultures that are physiologically identified as either GABAergic inhibitory or glutamatergic excitatory all contain mRNA for the gamma-aminobutyric acid (GABA) synthetic enzyme, glutamic acid decarboxylase (GAD), as detected by single cell mRNA amplification and PCR. However, consistent with the physiology, immunocytochemistry revealed that only a subset of the neurons stain for either GAD protein or GABA. A similar fraction hybridize with RNA probes for GAD65 and GAD67. Hippocampal CA1 pyramidal neurons in slice preparations, which are traditionally thought to be excitatory, also contain mRNA for GAD65 and GAD67. Hippocampal neurons in culture did not contain mRNA for two other neurotransmitter synthesizing enzymes, tyrosine hydroxylase, and choline acetyl transferase. These data suggest that in some neurons, presumably the excitatory neurons, GAD mRNA is selectively regulated at the level of translation. We propose that neurotransmitter phenotype may be posttranscriptionally regulated and neurons may exhibit transient phenotypic plasticity in response to environmental influences.