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Developmental maps of acetylcholinesterase and G4-antigen of the early chicken brain: long-distance tracts originate from AChE-producing cell bodies.

After approaching the outer surface of the neuroepithelium, postmitotic cell bodies abruptly start to synthesize acetylcholinesterase (AChE). Their easy histochemical detection allows us to trace sensitively spatiotemporal patterns of differentiation processes of the chicken nervous system. To investigate the relationship between postmitotic AChE production and the first formation of neurites, AChE histochemistry is combined here with immunohistochemistry using the neurite-specific G4-antibody. Spatial computer reconstructions from double-stained serial sections of whole brains of H.H. stages 10-20 demonstrate that G4-neurite expression spatio-temporally follows the expression of AChE in its complex polycentric pattern closely, the details of which have been described earlier. By comparing both differentiative steps at the single cell level reveals that a great majority (if not all) of the G4-positive neurites originate from AChE-positive cell bodies. Based on both the computer reconstructions as well as single cell analysis, including [3H]-thymidine pulse-experiments followed by autoradiography, we conclude, that AChE expression precedes formation of G4-neurites by about 15 h. In addition, the reconstructions provide the first detailed maps of G4-fiber tract formation and shows that G4-neurites form fascicles, most of which travel over long distances to targets within or without the central nervous system (CNS). This is the first demonstration for the entire young chicken brain which verifies that AChE-expressing cells, generally, are those that will establish efferents to distant targets.

Acetylcholinesterase↗

Pregnancy after preimplantation genetic diagnosis for Sanjad-Sakati syndrome.

Sanjad-Sakati syndrome (SSS) is an autosomal recessive disorder characterized by congenital hypoparathyroidism, growth and mental retardation. In Saudi Arabia, the disease is caused by a deletion of 12 bp (155-166nt) in the tubulin-specific chaperone E gene. In a family with two affected siblings with SSS, preimplantation genetic diagnosis (PGD) was performed. Fluorescent PCR (F-PCR) was utilized to check the heterozygosity and the homozygosity status of the parents and the affected children, respectively. F-PCR was then optimized for single-cell analysis by using peripheral blood lymphocytes. The patient underwent a cycle with intra-cytoplasmic sperm injection. A total of 11 embryos were obtained and biopsied. There were five heterozygous, three homozygous affected and three normal embryos. One heterozygous and one normal embryo were transferred because of their very good quality (morula). A singleton pregnancy was obtained, and amniosynthesis confirmed the presence of the heterozygous fetus. These results show for the first time, the feasibility of PGD for SSS.

DNA↗

Functional protein nanoarrays for biomarker profiling.

The use of microarrays for parallel screening of nucleic acid profiles has become an industry standard. Similar efforts for screening protein-protein interactions are gaining momentum, however, they remain limited by the requirement for relatively large sample volumes. One strategy for overcoming this problem is to significantly decrease the size and consequently the sample volume of the protein interaction assay. We report here on our progress over the last two years in the construction of ultraminiaturized, functional protein capture assays. Each one micron spot in these array-based assays covers less than 1/1000(th) of the surface area of a conventional microarray spot while still maintaining enough antibodies to provide a useful dynamic range. These nanoarray assays can be read by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy. The size reduction realized by functional protein nanoarrays also creates opportunities for novel applications including highly multiplexed single cell analysis and integration with microfluidics and other "lab-on-a-chip" technologies.

Biomarkers↗

Degradation of IkappaBalpha is limited by a postphosphorylation/ubiquitination event.

Regulation of IkappaBalpha during activation was examined using EGFP. Single cell analysis showed that both localisation- and cytokine-induced degradation of IkappaBalpha are dependent on expression levels. Cells expressing higher levels of the inhibitor demonstrated an increase in nuclear IkappaBalphaEGFP with a pronounced enhancement in the nuclear/cytoplasmic ratio. Enhancing the levels of the endogenous IkappaBalpha by relA transfection caused significant reduction in IL-1-mediated degradation of the fusion protein. Similarly, IkappaBalphaEGFP-transfected cells showed an inverse correlation between the level of the fusion protein and IL-1-mediateddegradation. Comparing absolute levels demonstrated a biphasic response, with reduction in cells expressing over 15-fold that of endogenous levels. Further experiments using Western analysis showed a positive correlation between both phosphorylation and ubiquitination of IkappaBalphaEGFP, and the level the inhibitor. In contrast, and in agreement with the singlecell analysis, while IL-1 stimulation caused the expected degradation at lower levels of the fusion protein,breakdown of IkappaBalphaEGFP was totally inhibited at the higher transfection levels. The data show that turnover of IkappaBalpha is saturable and suggest that limitation of the pathway by enhanced inhibitor expression is regulated through a post phosphorylation/ubiquitination event, at the level of degradation.

Blotting, Western↗

Mechanism of norepinephrine-mediated inhibition of human NK cytotoxic functions: inhibition of cytokine secretion, target binding, and programming for cytotoxicity.

Norepinephrine (NE) has been shown to inhibit human peripheral blood-derived natural-killer (NK) cell cytotoxicity (NKCC) in vitro. We demonstrate in this study that NE not only inhibits IL-2-activated NKCC but antibody-dependent cellular cytotoxicity (ADCC) as well. NK cytotoxicity by purified NK cells against K562 (NKCC) and against Raji cells (ADCC) were inhibited by NE (1-100 microM) by more than 50% in a 4-h (51)Cr release assay. The mechanism underlying the inhibition has been examined. NK cytotoxicity is dependent on target recognition and formation of NK-target conjugates, and activation by IL-2 is dependent on the secretion of cytokines (such as TNF-alpha) by NK cells. We hypothesized that the inhibition of NK functions by NE may be due to disruption of NK-target conjugation, blocking programming for lysis, and/or inhibition of cytokine secretion. Pretreatment of human peripheral blood mononuclear cells (PBMC) with NE for 15 min significantly reduced the binding to K562 cells by CD16(+) NK lymphocytes. In the presence of K562 cells, NE down-regulated the expression of CD16 (FcgammaRIII) by human PBMC, an NK cell receptor responsible and necessary for ADCC and cytokine secretion. We also demonstrate that NE inhibited the IL-2-mediated up-regulation of the activation marker CD69. At concentrations of 10(-6) to 10(-5) M, NE inhibited TNF-alpha, IFN-gamma, and GM-CSF secretion by NK cells, which are essential for IL-2-driven NK maturation and functions. In addition, using single-cell analysis, NE pretreatment of lymphocytes reduced the frequency of killer cells in the NK-K562 conjugate population in a concentration-dependent manner, indicating an inhibition of the programming for lysis by NK cells. In summary, these data demonstrate that NE-induced inhibition of NK cytotoxicity is manifested at multiple levels, including a modification of NK cell receptor ligation to target cells, blockade of NK cytokine secretion necessary for NK maturation and differentiation, and inhibition of the target-induced activation of the cytotoxic mechanism(s) in NK cells. Thus, sympathetic activation, as often induced experimentally, may profoundly impair natural cellular immunity through varied measurable pathways.

Antigens, CD↗

Differentiation of resting human peripheral blood gamma delta T cells toward Th1- or Th2-phenotype.

Depending on the microenvironment, murine gamma delta T cells differentiate into either Th1 (IFN-gamma-producing) or Th2 (IL-4-producing) cells. It is unclear, however, whether circulating human peripheral blood gamma delta T cells can be driven into Th1 or Th2 cells by modulation of the priming cytokine milieu. In this study, peripheral blood gamma delta T cells were stimulated by phosphoantigen (isopentenyl pyrophosphate) or Daudi lymphoma cells in the presence of Th1-priming (rIL-12, anti-IL-4 Ab) or Th2-priming (rIL-4, anti-IL-12 Ab) conditions. Single-cell analysis of cytokine secretion (IFN-gamma and IL-4) was performed by flow cytometry after 18 h and after restimulation of expanded gamma delta T cells. The early activation of resting gamma delta T cells was characterized by the induction of IFN-gamma. Priming under Th1 conditions induced a Th1 profile characterized by increased secretion of IFN-gamma and TNF-alpha, while Th2 conditions caused increased production of IL-4 (Th2 profile) by the gamma delta T cells. These results indicate that the major subset of human gamma delta T cells can be polarized into either Th1 or Th2 cytokine pattern depending on the cytokine milieu in which contact with antigen occurs.

Antibodies, Monoclonal↗

Cell proliferation is not required for productive HIV-1 infection of macrophages.

Lentiviruses, including HIV-1, are considered a rare example of retroviruses which do not require cell proliferation for their replication. However, this notion was questioned in several publications where productive HIV-1 infection was found to be restricted to a small fraction of macrophages with proliferative capacity. Since the mode of HIV-1 replication in macrophages is of great clinical relevance, we performed a single-cell analysis of HIV-1 replication and [3H]thymidine incorporation. Our results indicate that while 17% macrophages were detected as HIV-1 DNA-positive 12 hr after infection, only 2% of those cells had incorporated tritium, about the same percentage as in the uninfected cell population. Forty-eight hours after infection, 38% macrophages were HIV-1 DNA-positive and 47% of those had incorporated tritium, while the percentage of tritium-positive uninfected cells did not change (1%). These results demonstrate directly that HIV-1 DNA does not colocalize with [3H]thymidine and support the notion that cell proliferation is not required for HIV-1 infection of macrophages.

Cell Division↗

Polymerase chain reaction amplification specificity: incidence of allele dropout using different DNA preparation methods for heterozygous single cells.

PURPOSE: The purpose was to evaluate methods of DNA preparation in a single cell to determine the ability to amplify and correctly diagnose a targeted gene. METHODS: One- or two-cell lymphoblasts (n = 100/group), heterozygous for the normal and 4-base pair insertion on exon 11 of the beta-hexosaminidase A gene, were collected and prepared under the following conditions: (1) freeze-thaw liquid nitrogen, then boiling (LN2); (2) potassium hydroxide/dithiothreitol, heated to 65 degrees C, followed by acid neutralization (KOH); (3) boiling only (Bl); and (4) water only (H2O). Cells were analyzed by polymerase chain reaction using nested primers. RESULTS: The total number of cells amplifying [in brackets] and the cells with amplification for both alleles (heterozygous), the normal allele, or the mutant allele were as follows, respectively: LN2 [38], 11, 16, 11; KOH [97], 91, 5, 1; Bl [41], 17, 13, 11; and H2O [85], 41, 16, 28. With two cells per reaction tube the results were as follows: LN2 [85], 53, 14, 18; and KOH [97], 96, 1, 0. CONCLUSIONS: KOH lysis was significantly greater than with all other methods (P < 0.006) and should be used for single cells. This study also demonstrates the importance of using heterozygous cells to determine the ability to amplify both alleles as a method of quality control for single-cell analysis.

Alleles↗

Reduction in HLA-DR antigen density on alveolar macrophages of smokers.

The expression of HLA class II genes is essential to the antigen-presenting function of monocytes and macrophages. With a new cytofluorometric method of single-cell analysis we investigated the density of HLA-DR antigen expression on alveolar macrophages (AM) of smokers. Although all AM of nonsmokers (100 +/- 1%) expressed HLA-DR antigens, only 79 +/- 12% of AM from smokers showed positive findings (p less than 0.001). Furthermore, the antigen density of those cells expressing this antigen was significantly reduced in smokers (p less than 0.001). The defective expression of class II genes on AM of smokers might contribute to the impaired antigen-presenting function found by some previous investigators.

Antibodies, Monoclonal↗

Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry.

Quorum sensing enables bacteria to regulate expression of certain genes according to population density. N-acyl homoserine lactone (AHL)-based quorum sensing is known to be widespread among gram-negative bacteria. Several bacterial whole-cell biosensors for AHL detection have been developed and some were used in in situ studies of AHL production. From these studies our knowledge of the significance of quorum sensing in various environments has been improved. However, very little is known about production of AHLs in soil environments. In the present study, an approach for detecting AHL production in bulk soil was developed. A whole-cell biosensor based on the regulatory region of the lux-operon from Vibrio fischeri fused to gfp was constructed, resulting in a luxR-PluxI-gfpmut3*-fusion in the high copy plasmid, pAHL-GFP. Escherichia coli MC4100 harboring pAHL-GFP responded to the AHL-compound N-octanoyl homoserine lactone (OHL) by expressing green fluorescence. In situ application of E. coli MC4100/pAHL-GFP was tested by adding OHL in different concentrations to sterile soil microcosms. E. coli MC4100/pAHL-GFP were incubated in the soil microcosms and extracted by an improved Nycodenz-extraction method optimized for flow cytometry. The presence of induced cells was then verified by single-cell analysis by flow cytometry. OHL concentrations between 0.5 and 50 nmol per g soil were detected. When introducing the AHL-producing Serratia liquefaciens to soil microcosms, expression of green fluorescent protein was induced in E. coli MC4100/pAHL-GFP. Thereby, the ability of this strain to detect excretion of AHLs by S. liquefaciens in sterile soil was shown. The use of an improved extraction method and a whole-cell biosensor combined with flow cytometry analysis proved to be promising tools in future studies of AHL production by microbial populations in soil environments.

4-Butyrolactone↗

Juvenile hormone-dependent motor activation in the adult locust Locusta migratoria.

Abdominal motoneurones of the locust Locusta migratoria were investigated in immature, mature and allatectomised females to compare their response characteristics during reproductive development. These motoneurones were chosen because they control muscles which are involved in extreme lengthening during egg-laying behaviour. The study focused on changes in motoneurone firing activity and its possible regulation by juvenile hormone. In isolated nerve-muscle preparations, increased resting motor activity was found in mature (>14 days) but not in immature females (<5 days). Removing the corpora allata, the gland producing juvenile hormone in insects, prevented increased motor activity. Stimulus evoked activation of the motor system led to a characteristic burst of action potentials which lasted for a few seconds. The time-course and amount of activation changed significantly during reproductive development. Mature females displayed longer lasting and higher activity than immature or allatectomised females, but only those segments involved in egg-laying were found to express the altered firing properties. Single cell analysis of motoneurone dendritic morphology or membrane properties revealed no evidence that could be causative for the activity changes seen during reproductive development. The results suggest that altered motoneurone activity serves to adapt females to the neuromuscular requirements of egg-laying behaviour.

Action Potentials↗

Progress in Raman spectroscopy in the fields of tissue engineering, diagnostics and toxicological testing.

This review summarises progress in Raman spectroscopy and its application in diagnostics, toxicological testing and tissue engineering. Applications of Raman spectroscopy in cell biology are in the early stages of development, however, recent publications have demonstrated its utilisation as a diagnostic and development tool with the key advantage that investigations of living cells can be performed non-invasively.Some of the research highlighted here demonstrates the ability of Raman spectroscopy to accurately characterise cancer cells and distinguish between similar cell types. Many groups have used Raman spectroscopy to study tissues, but recently increased effort has gone into single cell analysis of cell lines; the advantages being that cell lines offer ease of handling and increased reproducibility over tissue studies and primary cells. The main goals of bio-Raman spectroscopy at this stage are twofold. Firstly, the aim is to further develop the diagnostic ability of Raman spectroscopy so it can be implemented in a clinical environment, producing accurate and rapid diagnoses. Secondly, the aim is to optimise the technique as a research tool for the non-invasive real time investigation of cell/material interactions in the fields of tissue engineering and toxicology testing.

Animals↗

Inhibition of neutrophil oxidative burst and phagocytosis by meconium.

OBJECTIVE: Meconium in amniotic fluid has been associated with an increased prevalence of chorioamnionitis. In an effort to delineate the mechanism of this association, we determined the effect of meconium on the neutrophil's capacity for phagocytosis and microbial killing by oxidative burst in vitro. STUDY DESIGN: Sterile meconium samples were obtained from four fetuses at the time of breech delivery and were then pooled and lyophilized. Neutrophils were purified from whole blood of each of 13 pregnant nonlaboring patients. Phagocytosis and the oxidative burst of neutrophils in the presence and absence of meconium were assessed by single-cell analysis with flow cytometry. Phagocytosis was measured as the mean fluorescence intensity produced after 30 minutes of incubation with fluorescein-labeled Escherichia coli. Oxidative burst was measured as the mean fluorescence intensity resulting from the oxidation of internalized reduced dichlorodihydrofluorescein after 15 minutes of stimulation with phorbol myristate acetate. Oxidative burst was expressed as the neutrophil oxidative index and the net fluorescence intensity. Neutrophil oxidative index was equivalent to the quotient of the mean fluorescence intensity for phorbol myristate acetate-stimulated and unstimulated cells. Net fluorescence intensity was equivalent to the absolute difference between stimulated and unstimulated cells. RESULTS: Exposure of neutrophils to light and very light meconium each resulted in significantly lower mean neutrophil oxidative index compared with unexposed controls (3.2 +/- 4.9 and 4.2 +/- 5.9 vs 16.2 +/- 7.5, p = 0.00002 and p = 0.0007, respectively) and significantly lower mean net fluorescence intensity than that of control cells (112 +/- 220 and 188 +/- 294 vs 613 +/- 328, p = 0.0001 and p = 0.005, respectively). Phagocytosis was significantly impaired in the presence of moderate meconium compared with control cells (2239 +/- 393 vs 4645 +/- 2071, p = 0.0001). Light meconium did not significantly affect phagocytosis. CONCLUSION: Meconium has significant effects on neutrophil function in vitro. Both light and very light meconium inhibit the oxidative burst. Moderate meconium inhibits phagocytosis.

Chorioamnionitis↗

Flow cytometric evaluation of phagocytosis by peripheral blood polymorphonuclear leucocytes in human periodontal diseases.

The complement-dependent phagocytic functions of polymorphonuclear leucocytes (PMNL) in peripheral blood from 15 patients with localized juvenile periodontitis (LJP), 13 with generalized juvenile periodontitis (GJP) and 52 with adult periodontitis (AP), and from 30 normal subjects as controls were measured by flow cytometry. Heparinized blood was collected and incubated with fluorescent microspheres, and erythrocytes were removed. By means of single-cell analysis the percentage of phagocytosing cells (% phagocytosis) and the mean number of microspheres phagocytosed by one PMNL (degree of phagocytosis; d-phagocytosis) were measured. Some but not all patients with LJP (53%) and GJP (46%) showed consistently low % phagocytosis and d-phagocytosis. On the other hand, only 6% of AP patients and no healthy subjects showed a reduction of PMNL phagocytosis. Phagocytosis was unchanged after initial periodontal treatment in all subjects, suggesting the depression of PMNL phagocytosis may not be a transient phenomenon associated with periodontal status. Furthermore, PMNLs from the LJP patients that showed depressed phagocytic function exhibited depressed phagocytic responses with either autologous or normal plasma, while control PMNLs with either normal or the patients' plasma showed normal responses. These results suggested that the depressed phagocytic responses in LJP patients could be due to cell-associated defect(s) on the PMNL.

Adolescent↗

The antidromic compound action potential of the hypothalamo-neurohypophysial tract, a tool for assessing posterior pituitary activity in vivo.

A sensitive technique was developed for measuring continuously the summated neural activity of the rat hypothalamo-neurohypophysial tract. Large compound potentials of 4-9 mV peak amplitude were recorded from the fibre tract in response to electrical stimulation of the neurohypophysis. It was estimated that all fibres in the tract were electrically activated and contributed to the compound potential. It was shown that a decrease of peak amplitude could be used as a measure of changes in endogenous neural activity. The decrease in peak amplitude during an increase of endogenous neural activity was due to fatigue and temporal spread of the compound potential, and also to collision of antidromic and orthodromic action potentials. Decrease of the compound potentials was related to the intensity of vaginal distension, haemorrhage and plasma hyperosmolality, and it correlated with neurohypophysial hormone release. This method may usefully complement hormone assays and single cell analysis.

Action Potentials↗

Cytofluorometry as a diagnosis of protoporphyria.

Protoporphyria is a disorder that usually has cutaneous symptoms. Only in a few cases liver disease develops, invariably progressing to cirrhosis and liver failure. A case of a patient who suffered from photosensitivity as a result of protoporphyria since the age of 2 is described. At age 33, she first presented with cholestatic hepatitis. At age 40, liver failure developed requiring liver transplantation. Quantitative cytofluorometry proved to be a quick and simple method in the follow-up of her erythrocyte protoporphyrin levels before, during, and after transplantation. Fluorescence correlated well with the protoporphyrin levels obtained by high-performance liquid chromatography (r = 0.98), a comparably cumbersome and complicated method for the determination of protoporphyrin. In addition, single cell analysis enabled us to follow the effects of transfusion therapy on protoporphyrin levels of the patient's own erythrocytes, which has not been possible by previous methods. Thus, cytofluorometry might prove to be elegant and useful for screening and future studies on therapy and pathophysiology of protoporphyria.

Adult↗

Finite element modeling of damage accumulation in trabecular bone under cyclic loading.

A two-dimensional finite element model of an idealized trabecular bone specimen was developed to study trabecular bone damage accumulation during cyclic compressive loading. The specimen was modeled as a two-dimensional honeycomb-like structure made up of an array of hexagonal cells. Each trabecula was modeled as a linearly elastic beam element with the same material properties as cortical bone. Initial microcracks were assumed to exist within the oblique trabeculae and to grow according to the Paris law. Forces and moments were computed in each trabecula and the microcracks were allowed to propagate until fracture occurred. Between cycles, fractured trabeculae were removed from the finite element mesh, and force and moment distributions were calculated for the next cycle. This iterative process was continued until the simulated trabecular bone specimen showed a 10% reduction in modulus. Creep failure was also studied using a single cell analysis, in which a closed-form solution was obtained after prescribing the creep properties of the trabeculae. The results of the crack propagation analysis showed that fractures of only a small number of individual trabeculae can cause a substantial reduction in the modulus of the trabecular bone specimen model. Statistical tests were performed to compare the slopes and intercepts of the S-N curves of our model predictions to those of experimentally derived S-N curves for bovine trabecular bone. There was no significant difference (p > 0.2 for both slope and intercept) between our model predictions and the experimentally derived S-N curves for the low-stress, high-cycle range. For the high-stress, low-cycle range, the crack propagation model overestimated the fatigue life for a given stress level (for slope, p < 0.001), while the creep analysis agreed well with the experimental data (for slope, p > 0.2). These findings suggest that the primary failure mechanism for low-stress, high-cycle fatigue of trabecular bone is crack growth and propagation, while the primary failure mechanism for high-stress, low-cycle fatigue is creep deformation and fracture. Furthermore, our results suggest that the modulus of trabecular bone at the specimen level may be highly sensitive to fractures of individual trabeculae.

Algorithms↗

Reprogramming cell differentiation in the absence of DNA synthesis.

We examined whether the activation of muscle gene expression in nonmuscle cells required DNA synthesis. Human fibroblasts from amniotic fluid and fetal lung were fused with differentiated mouse muscle cells in the presence or absence of the DNA synthesis inhibitor, cytosine arabinoside. In the stable heterokaryons formed, the human contractile enzyme, MM-creatine kinase (CK), and the cell surface antigen, 5.1H11, were detected in comparable amounts regardless of whether DNA synthesis had occurred. A single cell analysis revealed that the efficiency of gene activation was high and that DNA synthetic activity was not affected by the ratio of muscle to nonmuscle nuclei in the heterokaryons. In addition, muscle gene expression was not restricted to the G1 phase of the cell cycle. We conclude that cell differentiation can be reprogrammed in heterokaryons regardless of cell cycle phase and in the absence of detectable DNA synthesis.

Animals↗