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Biomedical subjects

C He

Publications and source records attributed to C He.

At least 19 recordsLinked to original sources

Effect of a lipopolysaccharide from E. coli on the proliferation of fibroblasts and keratinocytes in vitro.

Studies previously conducted in our laboratory have shown that an extract from the leaves of Chromo-laena odorata is mitogenic for human skin fibroblasts and keratinocytes. However, lipopolysaccharides, sometimes present in plant extracts, can also play a role in cell growth and might have been responsible for or contributed to the mitogenic activity observed. The present study aimed to investigate whether a lipopolysaccharide would have any effect on the proliferation of human fibroblasts and keratinocytes. Cells were seeded in 96-well plates and concentrations from 0.0 to 5.0 microg/mL of lipopolysaccharide in basal or growth medium were added. Cell growth was determined over a period of 10 days using a colorimetric assay. Lipopolysaccharide at concentrations between 0.05 microg/mL and 0.5 microg/mL in the growth medium significantly stimulated fibroblast proliferation after incubation for more than 6 days. In basal medium, more than 8 days of incubation was needed for significant stimulation of growth. Lipopolysaccharide stimulation of keratinocytes was evident at 0.5 microg/mL by day 3 in basal medium and by day 5 in growth medium. Although the lipopolysaccharide did stimulate cell growth it did so only at higher concentrations than were present in our plant extracts and to a lesser degree.

Cell Division↗

Manipulating the direction of electron transfer in the bacterial reaction center by swapping Phe for Tyr near BChl(M) (L181) and Tyr for Phe near BChl(L) (M208).

We have investigated the primary charge separation processes in Rb. capsulatus reaction centers (RCs) bearing the mutations Phe(L181) --> Tyr, Tyr(M208) --> Phe, and Leu(M212) --> His. In the YFH mutant, decay of the excited primary electron donor P occurs with an 11 +/- 2 ps time constant and is trifurcated to give (1) internal conversion to the ground state ( approximately 10% yield), (2) charge separation to the L side of the RC ( approximately 60% yield), and (3) electron transfer to the M-side bacteriopheophytin BPh(M) ( approximately 30% yield). These results relate previous work in which the ionizable residues Lys (at L178) and Asp (at M201) have been used to facilitate charge separation to the M side of the RC, and the widely studied L181 and M208 mutants. One conclusion that comes from this work is that the Tyr (M208) --> Phe and Gly(M201) --> Asp mutations near the L-side bacteriochlorophyll (BChl(L)) raise the free energy of P(+)BChl(L)(-) by comparable amounts. The results also suggest that the free energy of P(+)BChl(M)(-) is lowered more substantially by a Tyr at L181 than a Lys at L178. The results on the YFH mutant further demonstrate that the free energy differences between the L- and M-side charge-separated states play a significant role in the directionality of charge separation in the wild-type RC, and place limits on the contributing role of differential electronic matrix elements on the two sides of the RC.

Electron Transport↗

Biphasic response of the regional lymphatics in the normal lymphocyte transfer reaction.

BACKGROUND: Initially developed for histocompatibility testing, the normal lymphocyte transfer (NLT) reaction involves the intradermal injection of allogeneic lymphocytes from one individual to another. Because of the unique kinetics of the immunological response to allogeneic lymphocytes, the NLT reaction has been considered an informative system for the analysis of transplant immunity. METHODS: In this study, we used bilateral efferent lymph duct cannulations in sheep to examine the regional lymphatic response to the NLT reaction. Our studies used monoclonal antibodies to define lymphocyte population dynamics and DNA flow cytometry to reflect lymphocyte proliferative responses. RESULTS: The results confirmed a biphasic NLT reaction. An unexpected finding was the marked differences between the early and late NLT responses. The early response was characterized by T-lymphocyte proliferation, as reflected by S-phase DNA, which was comparable in both the NLT-stimulated and contralateral control efferent lymphocytes. This bilateral proliferative response was observed in both CD4+ and CD8+ lymphocytes. In contrast, the late response was restricted to the efferent lymph from the NLT-stimulated lymph node. Dual-parameter flow cytometry demonstrated that the dominant component of this unilateral NLT response was CD8+ lymphocytes. CONCLUSIONS: These results suggest important functional distinctions between systemic and regional lymphatic responses to intradermal alloantigens.

Animals↗

Glial cell line-derived neurotrophic factor enhances axonal regeneration following sciatic nerve transection in adult rats.

Adult rat sciatic nerve was transected and sutured with an entubulation technique. The nerve interstump gap was filled with either collagen gel (COL) or collagen gel mixed with glial cell line-derived neurotrophic factor (COL/GDNF). Four weeks after nerve transection, horseradish peroxidase (HRP)-labelled spinal cord motoneurons and the myelinated distal stump axons were quantified. Compared with the COL group, the percentages of labeled spinal somas and axon number were significantly increased after topically applied glial cell line-derived neurotrophic factor (GDNF). The functional recovery of the transected nerve was improved in COL/GDNF group. GAP-43 expression was also significantly higher in COL/GDNF group 1 and 2 weeks after sciatic nerve axotomy vs. COL group. These data provide strong evidence that GDNF could promote axonal regeneration in adult rats, suggesting the potential use of GDNF in therapeutic approaches to peripheral nerve injury and neuropathies.

Animals↗

Cytolytic peptides induce biphasic permeability changes in mammalian cell membranes.

The cytolytic peptides melittin and gramicidin S are naturally occurring agents that provide a comparative model for studies of complement, immunotoxin and cell-mediated membrane permeability. Most attempts to characterize cytolytic peptides have used model membrane systems including phospholipid vesicles or erythrocytes. Membrane vesicles permit the use of self-quenching concentrations of fluorescent permeability markers, while erythrocytes release measurable hemoglobin. Attempts at measuring early membrane permeability changes in nucleated mammalian cells have been limited. To measure the kinetics of mammalian cell membrane permeability changes induced by cytolytic peptides, we developed a 96-well fluorescence cytolysis assay using the cytoplasmic fluorescent dye calcein as the membrane permeability marker. To facilitate rapid assessment of membrane permeability, trypan blue was added to the assay solution to quench (a) released fluorescence and (b) retained intracellular fluorescence. Trypan blue also provided a complementary visual assessment of cell viability. Using this assay, a detailed kinetic analysis demonstrated permeability of the cell membranes within seconds of exposure to the cytolytic peptides. The rapid permeabilization of the cell membranes was confirmed by flow cytometry using the calcium indicator dye fluo-3. The assay also demonstrated a second slower phase of marker release over the next several hours. The fluorescence cytolysis assay was able to reliably detect the biphasic permeability changes associated with the melittin and gramicidin S peptides suggesting the potential utility of this assay in the assessment of other cytolytic agents.

Animals↗

The expression of G-protein-gated inwardly rectifying K+ channels GIRK1 and GIRK2 mRNAs in the supraoptic nucleus of the rat and possible role involved.

The expression of G-protein-gated inwardly rectifying K channels subunits GIRK1 and GIRK2 mRNAs in the supraoptic nucleus (SON) was investigated in the rat by in situ hybridization with non-radioactive dig-labeled cRNA probes. Double-labeled methods were used to study the co-localization of GIRK1 and 2 and oxytocin (OT) and vasopressin (AVP) in the SON. The present study revealed wide and intense expression of GIRK1 and GIRK2 mRNAs with high overlapping in the SON, indicating the heterologous channel of GIRK1/GIRK2 was a major functional channel in the SON. Given that 100% of OT-positive and 95% of (AVP)-positive neurons in the SON expressed GIRK1/GIRK2 mRNAs, it is possible that GIRK1/GIRK2 channel, activated through G-protein coupled receptors, may be involved in the inhibitory regulation of the release of OT and AVP from the SON.

Animals↗

A novel chordin-like protein inhibitor for bone morphogenetic proteins expressed preferentially in mesenchymal cell lineages.

Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem cells in vitro and interacted directly with BMPs, similar to chordin. However, CHL also weakly bound to TGFbetas. In situ hybridization revealed that the mouse CHL gene, located on the X chromosome, was expressed predominantly in mesenchyme-derived cell types: (1) the dermatome and limb bud mesenchyme and, later, the subdermal mesenchyme and the chondrocytes of the developing skeleton during embryogenesis and (2) a layer of fibroblasts/connective tissue cells in the gastrointestinal tract, the thick straight segments of kidney tubules, and the marrow stromal cells in adults. An exception was expression in the neural cells of the olfactory bulb and cerebellum. Interestingly, the spatiotemporal expression patterns of CHL were distinct from those of chordin in many areas examined. Thus, CHL may serve as an important BMP regulator for differentiating mesenchymal cells, especially during skeletogenesis, and for developing specific neurons.

Alternative Splicing↗

Antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene and transferred them into malignant cells in vitro and tumors in vivo. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of activation of pro-caspase-8 and cleavage of poly(ADP-ribose) polymerase. The intratumoral delivery elicited tumor cell apoptosis and suppressed tumor growth. In comparison with Bax gene treatment, which is toxic to normal cells, TRAIL gene treatment caused no detectable toxicity in cultured normal fibroblasts nor in mouse hepatocytes after systemic gene delivery. Furthermore, coculture of cancer cells expressing TRAIL with those expressing green fluorescent protein (GFP) resulted in apoptosis of both cells, whereas coculture of Bax-expressing cells with GFP-expressing cells resulted in the cell death of the Bax-expressing cells only, which suggested that the transfer of the TRAIL gene resulted in bystander effects. Moreover, culture of cells with medium from TRAIL-expressing cells showed the proapoptotic activity and bystander effect of the TRAIL gene to be not transferable with medium. To further demonstrate the bystander effect of the TRAIL gene, we constructed plasmid vectors encoding GFP-TRAIL or GFP-Bik chimeric proteins. Transfection of the GFP-TRAIL gene into cancer cells resulted in the death of GFP-positive cells and their neighbors, whereas transfection of the GFP-Bik gene killed GFP-positive cells only. Finally, GFP-TRAIL genes, transfected into normal human fibroblasts or bronchial epithelial cells, did not kill such cells, whereas transfected GFP-Bik genes did. Thus, the direct transfer of the TRAIL gene led to selective killing of malignant cells with bystander effect, which suggests that the TRAIL gene could be valuable for treatment for cancers. Together, these results suggest that delivering the TRAIL gene to cancerous cells may be an alternative approach to cancer treatment.

Adenoviridae↗

Synthesis and electrochemical studies of diiron complexes of 1,8-naphthyridine-based dinucleating ligands to model features of the active sites of non-heme diiron enzymes.

A bis(mu-carboxylato)(mu-1,8-naphthyridine)diiron(II) complex, [Fe2(BPMAN)(mu-O2CPhCy)2](OTf)2 (1), was prepared by using the 1,8-naphthyridine-based dinucleating ligand BPMAN, where BPMAN = 2,7-bis[bis(2-pyridylmethyl)aminomethyl]-1,8-naphthyridine. The cyclic voltammogram (CV) of this complex in CH2Cl2 exhibited two reversible one-electron redox waves at +296 mV (DeltaE(p) = 80 mV) and +781 mV (DeltaE(p) = 74 mV) vs Cp2Fe+/Cp2Fe, corresponding to the FeIIIFeII/FeIIFeII and FeIIIFeIII/FeIIIFeII couples, respectively. This result is unprecedented for diiron complexes having no single atom bridge. Dinuclear complexes [Fe2(BPMAN)(mu-OH)(mu-O2CPhCy)](OTf)2 (2) and [Mn2(BPMAN)(mu-O2CPhCy)2](OTf)2 (3) were also synthesized and structurally characterized. The cyclic voltammogram of 2 in CH2Cl2 exhibited one reversible redox wave at -22 mV only when the potential was kept below +400 mV. The CV of 3 showed irreversible oxidation at potentials above +900 mV. Diiron(II) complexes [Fe2(BEAN)(mu-O2CPhCy)3](OTf) (4) and [Fe2(BBBAN)(mu-OAc)2(OTf)](OTf) (6) were also prepared and characterized, where BEAN = 2,7-bis(N,N-diethylaminomethyl)-1,8-naphthyridine and BBBAN = 2,7-bis[2-[2-(1-methyl)benzimidazolylethyl]-N-benzylaminomethyl]-1,8-naphthyridine. The cyclic voltammograms of these complexes were recorded. The Mössbauer properties of the diiron compounds were studied.

Binding Sites↗

[Mechanism of the activation of extracellular signal-regulated kinase (ERK) in prostate cancer cell lines with different metastatic potential].

OBJECTIVE: To investigate the mechanism of the activation of signal transduction of ERK induced by purinergic receptor agonist ATP in prostate cancer cells with different metastatic potential. METHODS: Cell counts and MTT method were used to detect the influence of ATP on the growth of 1E8 (metastatic) and 2B4 (non-metastatic) cells derived from human prostate cancer cell line PC3M. The activity of ERK1/2 was analyzed with phosphospecific antibodies directed against the dually phosphorylated, active forms of ERK1/2 (p44/p42) by Western Blot. RESULTS: ATP can significantly inhibit the growth of 1E8 and 2B4 cells in vitro (inhibition rate in the 6th and the 8th day were 54% and 59% for 1E8 and 67% and 39% for 2B4 respectively). ATP activated both ERK1 and ERK2 in 1E8 and 2B4 cells with a time and dose dependent pattern. The activation of ERK1/2 by ATP was blocked by the P2 purinoceptor antagonist, suramin with an inhibitory rate of 82% +/- 9% for 1E8 and an inhibitory rate of 81% +/- 6% for 2B4. The activation of ERK1/2 by ATP can be inhibited by the inhibitor of the upstream kinase MEK- PD098059 with an inhibitory rate of 94% +/- 4% for 1E8 and 91% +/- 4% for 2B4,which suggests a link between the G protein coupled P2 purinoceptor and activation of Ras MEK MAPK pathway. ATP-stimulated ERK activation was sensitive to treatment with G protein modulator pertussis toxin (PTX) with an inhibitory rate of 50% +/- 3% for 1E8 and 51% +/- 4% for 2B4. The activating potential of ATP to ERK1/2 in metastatic 1E8 cells is greater than that to nonmetastatic 2B4 cells, and the response of 1E8 cells to TPA was quite different from the response of 2B4 cells, thus implying a potential signaling mechanism in regulating metastasis phenotypes. CONCLUSION: The metastatic 1E8 and non-metastatic 2B4 cells show differential response to ATP-induced ERK activation. This may provide an instructive clue to cancer metastasis research.

Adenosine Triphosphate↗

Stochastic regulation of cell migration from the efferent lymph to oxazolone-stimulated skin.

The systemic immune response is a dynamic process involving the trafficking of lymphocytes from the Ag-stimulated lymph node to the peripheral tissue. Studies in sheep have demonstrated several phases of cell output in the efferent lymph after Ag stimulation. When skin contact sensitizers are used as Ag, the efferent lymph cell output peaks approximately 96 h after Ag stimulation and is temporally associated with the recruitment of cells into the skin. To investigate the relative contribution of this high-output phase of efferent lymphocytes to lymphocytic inflammation in the skin, we used a common contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (oxazolone) to stimulate the skin and draining prescapular lymph node of adult sheep. The efferent lymph ducts draining the Ag-stimulated and contralateral control lymph nodes were cannulated throughout the experimental period. The lymphocytes leaving the lymph nodes during the 72-h period before maximum infiltration were differentially labeled with fluorescent tracers, reinjected into the arterial circulation, and tracked to the site of Ag stimulation. Quantitative tissue cytometry of the skin at the conclusion of the injection period (96 h after Ag stimulation) demonstrated more migratory cells derived from the Ag-stimulated lymph node than the contralateral control (median 18.5 vs 15.5 per field; p < 0.05). However, when corrected for total cell output of the lymph node, the Ag-stimulated migratory cells were 3.8-fold more prevalent in the skin than the contralateral control cells. These results suggest that the in situ immune response generally mirrors the frequency of recruitable lymphocytes in the peripheral blood.

Adjuvants, Immunologic↗

Effects of histone acetylation on the solubility and folding of the chromatin fiber.

The folding ability of chromatin fractions containing approximately identical nucleosome numbers and the same linker histone composition, but with different extents of core histone acetylation, were analyzed by analytical ultracentrifugation. It was found that the acetylated fractions consistently exhibited a relatively small but significantly lower extent of compaction than that of their native nonacetylated counterparts. This was regardless of the extent of the size distribution heterogeneity of the fractions analyzed. Furthermore the acetylated chromatin fibers exhibited an enhanced solubility in both NaCl and MgCl(2), which is neither the result of a differential binding affinity of the linker histones to chromatin nor of an alteration in the relative amounts of the histone H1 variants.

Acetylation↗

Analysis of expressions of components in the plasminogen activator system in high- and low-metastatic human lung cancer cells.

PURPOSE: To determine the expressive patterns of the components of the plasminogen activator system in human large-cell lung carcinoma strains and to analyze the effects of the patterns on tumor invasion and metastasis. METHODS: The in vitro and in vivo invasive and metastatic potential of two human large-cell lung carcinoma strains with high (strain 95D) and low (strain 95C) metastatic potential was further confirmed by the Boyden chamber model and nude mice model. After this, the expressions of the components of the plasminogen activator system--including urokinase-type and tissue-type plasminogen activator (uPA and tPA), urokinase receptor (uPAR), and type-1 and type-2 plasminogen activator inhibitor (PAI-1 and PAI-2) in strain 95D and 95C cells--were determined by RT-PCR and immunohistochemical staining. The effects of monoclonal antibodies of uPA, uPAR, and PAI-1 on the invasive potential of strain 95D cell line were also evaluated. RESULTS: Strain 95D cells were found to have a stronger in vitro and in vivo invasive and metastatic potential than strain 95C cells. In the former, the average number of infiltrating cells in the in vitro model in one field of vision (40055) was 73.75 +/- 7.42, while in the latter, it was 56.33 +/- 6.28 (P < 0.001). Lung metastatic loci were observed in all six nude mice inoculated with 95D cells (6/6), but not in any of the nude mice inoculated with 95C cells (0/6). The high-metastatic strain 95D cells expressed higher uPA and uPAR and lower tPA and PAI-2 than the low-metastatic strain 95C cells. The PAI-1 expressions in both 95D and 95C cells were almost the same. Monoclonal antibodies of uPA and uPAR greatly reduced the invasive potential of strain 95D cells in vitro. CONCLUSIONS: These data suggest that the invasive and metastatic potential of human large-cell lung carcinoma cell lines is associated with differential expressions of the components of the plasminogen activator system and that the determination of these components may be used as a marker for judging clinically the possibility of tumor metastasis as well as the prognoses of patients.

Carcinoma, Large Cell↗

Oxytocin mediates the inhibitory action of acute lithium on the morphine dependence in rats.

The role of central oxytocin in inhibitory action of lithium on the development of morphine dependence was behavioral investigated in rats. Acute lithium could enhance the morphine-induced analgesia in rats with or without chronic morphine treatment; this effect could be inhibited by intraventricular injection of oxytocin antagonist d (CH(2))(5)-Tyr (Me)-[Orn(8)]-Vasotocin (OVT). Lithium could attenuate naloxone-precipitated withdrawal signs in morphine dependent rats. The reduction of the expression of naloxone-precipitated withdrawal signs by lithium was reversed by ICV of OVT. The lithium significantly inhibited the conditioned place preference (CPP) induced by morphine, which inhibitory action of lithium could also reverse by ICV injection of OVT. These results suggested that lithium might inhibit the physical dependence on morphine as well as psychological dependence in rats, and that this inhibitory effect of lithium on the development of morphine dependence might be associated with oxytocin systems in the central nervous system.

Analgesics, Opioid↗

Glial cell line-derived neurotrophic factor promotes survival and induces differentiation through the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathway respectively in PC12 cells.

PC12-GFRalpha1 cells, a clonal cell line engineered to express glial cell line-derived neurotrophic factor receptor alpha1 were constructed. Given glial cell line-derived neurotrophic factor could induce the differentiation and promote the survival of PC12-GFRalpha1 cells at low concentrations, the cells provide an unlimited source of monoclonal cells for studies on the signal transduction pathway of glial cell line-derived neurotrophic factor. To characterize the involvement of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in the biological effect of glial cell line-derived neurotrophic factor, we used the mitogen-activated protein kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor LY294002. PD98059 blocked glial cell line-derived neurotrophic factor-induced PC12-GFRalpha1 cells neurite formation in a dose-dependent manner, without significantly altering cell viability. LY294002 reversed the survival-promoting effect of glial cell line-derived neurotrophic factor on the PC12-GFRalpha1 cells in serum-deprived medium. The present study demonstrates that phosphatidylinositol 3-kinase pathway seems to mediate the survival-promoting effect of glial cell line-derived neurotrophic factor on PC12-GFRalpha1 cells, while the activation of mitogen-activated protein kinase pathway could be an important step in mediating PC12-GFRalpha1 cells differentiation induced by glial cell line-derived neurotrophic factor. Therefore, it is inferred that similar intracellular signaling components are used by distinct growth factors toward a common biological effect.

Animals↗

Prevalence of vaccine-induced escape mutants of hepatitis B virus in the adult population in China: a prospective study in 176 restaurant employees.

BACKGROUND AND AIM: Hepatitis B virus (HBV) variants with mutations in the S gene would pose a substantial risk to the community as current HBV vaccines are not effective in preventing infection with them. The majority of such vaccine escape mutants so far reported have been found while studying vertical transmission of HBV; the vaccine failure rate in connection with vaccine escape mutants in adults is not clear at the moment. The purpose of this study was to evaluate the efficacy of immunization against HBV in the adult population by analysis using polymerase chain reaction (PCR) to detect HBV-DNA, and also to elucidate the type of mutation encountered in vaccine failure cases. METHOD: A total of 176 adult restaurant employees in China, who had been vaccinated according to the food epidemic law, were enrolled in a standard vaccination program. Their serum HBV-DNA was determined before and 1 year after the completion of the vaccination program. In those infected with HBV, despite having received the HBV vaccine, direct sequencing within the S gene of the amplified samples was conducted. RESULTS: Although only two cases were found to be hepatitis B surface antigen (HBsAg) positive 1 year after the completion of the vaccination program, six subjects (3.4%) were found to be HBV-DNA positive assessed by a nested PCR. Four out of these six cases had a point mutation within the 'a' determinant; they were Gly-145-Ala, and Ile/Thr-126-Asn/Ser. CONCLUSION: The HBV vaccine failure rate assessed by using PCR analysis was 3.4% (six of 176) in the Chinese adult population undergoing the HBV vaccination program. Hepatitis B virus variants with missense mutation within the 'a' determinant were responsible in most cases.

Adolescent↗

Focal topographic changes in inflammatory microcirculation associated with lymphocyte slowing and transmigration.

Microcirculation is the primary mechanism for delivering lymphocytes to inflammatory tissues. Blood flow within microvessels ensures a supply of lymphocytes at the blood-endothelial interface. Whether the structure of the inflammatory microcirculation facilitates lymphocyte transmigration is less clear. To illuminate the microcirculatory changes associated with lymphocyte transmigration, we used intravital videomicroscopy to examine the dermal microcirculation after application of the epicutaneous antigen oxazolone. Intravascular injection of fluorescein-labeled dextran demonstrated focal topographic changes in the microcirculation. These focal changes had the appearance of loops or hairpin turns in the oxazolone-stimulated skin. Changes were maximal at 96 h and coincided with peak lymphocyte recruitment. To determine whether these changes were associated with lymphocyte transmigration, lymphocytes obtained from efferent lymph of draining lymph nodes at 96 h were fluorescently labeled and reinjected into inflammatory microcirculation. Epifuorescence intravital video microscopy demonstrated focal areas were associated with lymphocyte slowing and occasional transmigration. In contrast, focal loops and lymphocyte slowing were rarely observed in the contralateral control microcirculation. Results suggest that structural adaptations in inflammatory microcirculation represented by focal topographic changes may contribute to regulation of tissue entry by recirculating lymphocytes.

Adjuvants, Immunologic↗

Aldehyde fixation of thiol-reactive fluorescent cytoplasmic probes for tracking cell migration.

Tracking of cell migration plays an important role in the study of morphogenesis, inflammation, and metastasis. The recent development of probes that exist as intracellular peptide-fluorescence dye adducts has offered the possibility of aldehyde fixation of these dyes for detailed anatomic studies of lymphocyte trafficking. To define the conditions for fixation of these cytoplasmic fluorescent probes, we compared fixation conditions containing formaldehyde, glutaraldehyde, paraformaldehyde, zinc formaldehyde, and glyoxylate, as well as fixation by quick-freezing in liquid nitrogen-cooled methylbutane. The efficacy of aldehyde fixation of the cell fluorescence was assessed by quantitative tissue cytometry and flow cytometry. We studied cytoplasmic fluorescent dyes with discrete emissions in the green [5-chloromethylfluorescein diacetate (CMFDA); 492 ex, 516 em] and orange [5-(and-6)-(4-chloromethyl(benzoyl)amino) tetramethylrhodamine (CMTMR); 540 ex, 566 em] spectra. The results demonstrated that aldehyde fixation preserved cell fluorescence for more than 6 months. The primary difference between the aldehyde fixatives was variability in the difference between the yield of the cell fluorescence and the relevant background fluorescence. Formaldehyde and paraformaldehyde were superior to the other fixatives in preserving cell fluorescence while limiting background fluorescence. With these fixatives, both the CMFDA and CMTMR fluorescent dyes permitted sufficient anatomic resolution for reliable localization in long-term cell tracking studies.

Aldehydes↗