Appraisal of rabies vaccine potency by determination of in vitro, specific interleukin-2 production

The potency of different rabies vaccines was measured via cell mediated immunity (CMI) assessed by the production of interleukin-2 (IL-2) by CD4+CD8- lymphocytes. IL-2 production by splenocytes from mice immunized with various vaccines was measured following in vitro stimulation with antigens from different rabies and rabies-related strains. IL-2 production was specific, reproducible and correlated with the vaccine protective activity as determined by the pre-exposure NIH test. Our results suggest that measurement of IL-2 production could be used for the appraisal of rabies vaccine potency.     

Mechanisms of dimethylbenzanthracene-induced immunotoxicity

Traditional methods for toxicological assessment have implicated the immune system as a frequent target organ of toxic insult following chronic exposure to certain environmental chemicals, radiation or therapeutic drugs (xenobiotics). Immunotoxicity is expressed as autoimmunity, chemical hypersensitivity or immunosuppression. A tiered approach for characterizing chemical and drug-induced immunomodulation has been developed and validated in laboratory animals. Polycyclic aromatic hydrocarbons (PAH) have been studied because of their ubiquitous presence in the environment and carcinogenic potential. Since immunosuppression induced by PAH carcinogens has been implicated as an epigenetic mechanism in the outgrowth of initiated cells, this tiered approach was used to characterize the mechanism of PAH immunosuppressive capacity. Previously, studies in this laboratory have demonstrated that subchronic exposure of B6C3F1 mice to PAH carcinogens suppresses both humoral immunity (HI) and cell-mediated immunity (CMI), concurrently with decreased resistance to tumor challenge. The potent carcinogenic PAH, 7,12-dimethylbenz[a]anthracene (DMBA) was subchronically administered subcutaneously at 5, 50, or 100 micrograms/g of body weight. Natural killer (NK) cell tumor cytolysis, generation of cytotoxic T-cells (CTL), and lymphoproliferation to mitogens and allogeneic splenocytes in mixed leukocyte cultures (MLC) were quantitated 3-5 days after exposure to assess CMI. Mitogen and alloantigen-induced proliferation (MLC) of splenocytes was suppressed up to 90%. CTL and NK tumor cytolysis of radiolabelled target cells were similarly depressed up to 88 and 82%, respectively. Impairment of MLC or CTL responses correlated with increased susceptibility to challenge with PYB6 sarcoma cells. HI was measured by quantitating the number of antibody (IgM) plaque-forming cells (PFC) produced in response to T-cell dependent antigen challenge (sheep erythrocytes) and was similarly suppressed up to 95%. To understand the mechanism of PAH-induced immunotoxicity, splenocytes from DMBA-exposed mice were sensitized to alloantigens in the presence of interleukin-2 (IL-2) because there were indications that T-helper cell function was suppressed. In these preliminary studies, CTL suppression could be completely restored by the addition of the T-cell growth supporting lymphokine (IL-2) during the inductive phase of CTL generation, suggesting that DMBA exposure directly or indirectly induced deficits in T-helper cell function.     

Boron Induces Lymphocyte Proliferation and Modulates the Priming Effects of Lipopolysaccharide on Macrophages

Chemical mediators of inflammation (CMI) are important in host defense against infection. The reduced capacity of host to induce the secretion of these mediators following infection is one of the factors in host susceptibility to infection. Boron, which has been suggested for its role in infection, is reported in this study to increase lymphocyte proliferation and the secretion of CMI by the lipopolysaccharide (LPS)-stimulated peritoneal macrophages in BALB/c mice. Boron was administered to mice orally as borax at different doses for 10 consecutive days, followed by the stimulation of animals with ovalbumin and isolation of splenocytes for proliferation assay. The lymphocyte subsets were determined by flow cytometry in spleen cell suspension. The mediators of inflammation, TNF-α, IL-6, IL-1β and nitric oxide (NO), were measured in culture supernatant of LPS-primed macrophages isolated from borax treated mice. TNF and ILs were measured by ELISA. NO was determined by Griess test. The expression of inducible nitric oxide synthase (iNOS) in macrophages was studied by confocal microscopy. Results showed a significant increase in T and B cell populations, as indicated by an increase in CD4 and CD19, but not CD8, cells. Boron further stimulated the secretion of TNF-α, IL-6, IL-1β, NO and the expression of iNOS by the LPS-primed macrophages. The effect was dose dependent and most significant at a dose level of 4.6 mg/kg b. wt. Taken together, the study concludes that boron at physiological concentration induces lymphocyte proliferation and increases the synthesis and secretion of pro-inflammatory mediators by the LPS-primed macrophages, more specifically the M1 macrophages, possibly acting through Toll-like receptor. The study implicates boron as a regulator of the immune and inflammatory reactions and macrophage polarization, thus playing an important role in augmenting host defense against infection, with possible role in cancer and other diseases.     

Phenotypic and functional analysis of immune CD8+ T cell responses induced by a single injection of a HIV DNA vaccine in mice

HIV DNA vaccines are potent inducers of cell-mediated immune (CMI) response in mice but elicit poor HIV-specific IFN-gamma-producing T cells in monkeys and humans. In this study, we performed kinetic analyses on splenocytes of BALB/c mice that were immunized by a single injection with a unique DNA vaccine. Using IFN-gamma-ELISPOT and multiparametric FACS analysis, we characterized the induced CMI response. We found that the response was detectable for at least 63 wk. ELISPOT detection of IFN-gamma-producing T cells showed a profile with two waves separated by a long period of minimal response. Multiparametric FACS analysis showed two populations of CD3(+)CD8(+) T cells that were specific for all HIV Ags. These cells had similar robust proliferation abilities and contained granzyme B. However, only a few produced IFN-gamma. Both IFN-gamma-producing and non-IFN-gamma-producing HIV-specific CD8(+) T cells were detected in the early stage (week (W)1 and W2 postimmunization (PI)), in the prolonged intermediate period of minimal response (W4-W26 PI), and in the final late phase of increased response (W30-W63 PI). Our longitudinal characterization showed that both subsets of cells underwent expansion, contraction, and memory generation/maintenance phases throughout the lifespan of the animal. Altogether, these findings bring insight to the heterogeneity of the immune T cell response induced by a single immunization with this DNA and strengthen the concept that used of the IFN-gamma-ELISPOT assay alone may be insufficient to detect critical T cell responses to candidate HIV vaccines.     

Inhibition of specific immune responses by feeding protein antigens. IV. Evidence for tolerance and specific active suppression of cell-mediated immune responses to ovalbumin.

We studied the effect of a single intragastric administration of ovalbumin (OVA) on the subsequent development of OVA-specific cell-mediated immune (CMI) responses in BDF1 mice. In animals fed OVA 7 days before subcutaneous sensitization with OVA-CFA, we observed a concomitant dose-dependent decrease in both the humoral and CMI responses specific for OVA. The CMI tolerance was found to be antigen-specific when assayed in vivo by ear swelling or in vitro by an antigen-induced T cell proliferation assay because OVA-fed mice responded normally to sensitization with horse gamma-globulin. It was also shown that either spleen or lymph node cells, but not serum, from OVA-fed donors transferred suppression to normal recipients. The transfer was mediated by antigen-specific suppressor T cells (Ts) that appeared to inhibit the induction phase (afferent limb) of the CMI response, since the Ts were only effective when transferred before or shortly after the onset of sensitization.     

Central thyrotropin-releasing hormone increases hepatic cyclic AMP through vagal-cholinergic and prostaglandin-dependent pathways in rats

Central neuropeptides play roles in many physiologic regulations through the autonomic nervous system. We have demonstrated that central thyrotropin-releasing hormone (TRH), one of neuropeptides, induces a stimulation of hepatic proliferation through vagal-cholinergic pathways. Since cAMP is known to play an important role in the hepatic proliferation, effect of central TRH on hepatic cAMP was investigated. Rats were intracisternally injected with either a TRH analog, RX-77368 (1-100 ng), or saline. The liver was removed 2-72 h after the TRH analog and hepatic cAMP content was determined by radioimmunoassay. In some experiments, pretreatment with hepatic vagotomy, atropine methyl nitrate, or 6-hydroxydopamine (6-OHDA) was performed. Hepatic cAMP was dose-dependently increased by intracisternal TRH analog (5-100 ng) with a peak response occurring 12 h postinjection. The central TRH-induced increase in hepatic cAMP was abolished by vagotomy, atropine and indomethacin, but not by 6-OHDA. Intravenous injection of the TRH analog (10 ng) did not affect hepatic cAMP. These results demonstrate that TRH acts in the brain to increase hepatic cAMP through vagal-cholinergic and prostaglandin-dependent pathways, suggesting that central TRH modulates hepatic functions through cAMP-mediated signaling pathways.     

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.     

Mac1 positive cells are required for enhancement of splenocytes proliferation caused by bisphenol a

We examined the effects of bisphenol A (BPA) on immune cells and it was shown that BPA upregulated the proliferation of murine splenocytes stimulated with Concanavalin A (ConA). The upregulating effects of BPA were removed with depleting Mac1+ cells from the splenocytes. This study provides evidence for the first time that Mac1+ cells were required for enhancement of splenocytes proliferation caused by bisphenol A.     

Effect of thyrotropin-releasing hormone on immune functions of peripheral blood mononuclear cells

The tripeptide thyrotropin-releasing hormone (TRH) works as a hypothalamic hormone, but is found also outside the brain in intrinsic nerve fibers of the gastrointestinal tract. There is evidence that TRH modulates the activity of immunocompetent cells, although there are only very few data on TRH-mediated immune effector functions. Since we could recently show that TRH inhibits monocyte activities we were also interested in other possible TRH modulated immune functions. Peripheral blood mononuclear cells (PBMC) from ten healthy subjects were cultured for 7 days and pulsed with 0.125 and 0.250 microgram/ml Pokeweed mitogen (PWM). 10(-12) to 10(-6) M TRH was added simultaneously with PWM. Lymphocyte proliferation [(3H]thymidine incorporation), interferon-gamma (IFN-gamma) activity (RIA) and immunoglobulin activities (IgG, IgM, IgA; ELISA) were determined in the supernatants. We could demonstrate a TRH-dependent decrease in PWM-pulsed IgG activity with significant (alpha = 0.05) values at 10(-8) and 10(-10) M (-29 +/- 6%/-16 +/- 3% for PWM 0.125 microgram/ml and -17 +/- 9%/-11 +/- 9% for PWM 0.250 microgram/ml). This inhibitory effect could be abolished by an anti-TRH antiserum. There was no TRH effect on IgM and IgA activities, IFN-gamma activity and lymphocyte proliferation compared with the PWM stimulated values alone. The described TRH effect on the polyclonal IgG response by PBMC gives further evidence for a functional link between the immune system and the endocrine system, although its underlying mechanism is not yet clear.     

Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhbition of cell proliferation

GH(4)C(1) cells are a clonal strain of rat pituitary cells that synthesize and secrete prolactin and growth hormone. Chronic treatment (longer than 24 h) of GH(4)C(1) cells with epidermal growth factor (EGF) (10(-8) M) decreased by 30-40 percent both the rate of cell proliferation and the plateau density reached by cultures. Inhibition of cell proliferation was accompanied by a change in cellular morphology from a spherical appearance to an elongated flattened shape and by a 40-60 percent increase in cell volume. These actions of EGF were qualitatively similar to those of the hypothalamic tripeptide thyrotropin-releasing hormone (TRH) (10(-7) M) which decreased the rate of cell proliferation by 10-20 percent and caused a 15 percent increase in cell volume. The presence of supramaximal concentrations of both EGF (10(-8)M) and TRH (10(-7)M) resulted in greater effects on cell volume and cell multiplication than either peptide alone. EGF also altered hormone production by GH(4)C(1) cells in the same manner as TRH. Treatment of cultures with 10(-8) M EGF for 2-6 d increased prolactin synthesis five- to ninefold compared to a two- to threefold stimulation by 10(-7) M TRH. Growth hormone production by the same cultures was inhibited 40 percent by EGF and 15 percent by TRH. The half- maximal effect of EGF to increase prolactin synthesis, decrease growth hormone production, and inhibit cell proliferation occurred at a concentration of 5 x 10 (-11) M. Insulin and multiplication stimulating activity, two other growth factors tested, did not alter cell proliferation, cell morphology, or hormone production by GH(4)C(1) cells, indicating the specificity of the EGF effect. Fibroblast growth factor, however, had effects similar to those of EGF and TRH. Of five pituitary cell strains tested, all but one responded to chronic EGF treatment with specifically altered hormone production. Acute chronic EGF treatment with specifically altered hormone production. Acute treatment (30 min) of GH(4)C(1) cells with 10(-8) M EGF caused a 30 percent enhancement of prolactin release compared to a greater than twofold increase caused by 10(-7) M TRH. Therefore, although EGF and TRH have qualitatively similar effects on GH(4)C(1) cells, their powers to affect hormone release acutely or hormone synthesis and cell proliferation chronically are distinct.     

Unsuitability of the assay for cell-mediated lympholysis in inbred mice for H-Y antigen determination of human cells

Summary This study examined the H-Y-specific in vitro restimulation of splenocytes from in vivo intraperitoneally (i.p.) primed C57B1/6 (B6) female mice. In vivo priming was carried out with human male or female fibroblasts or peripheral blood lymphocytes, respectively. It was attempted to measure the in vitro H-Y-specific activity by cell-mediated lympholysis and by cell proliferation. ³[H]Thymidine incorporation was determined in mixed lymphocyte cultures (MLCs) of xenogenetic primed female splenocytes (responder cells) and of syngeneic lethally irradiated male splenocytes (stimulator cells). The xenogenic H-Y presentation by in vivo sensitization did not induce in the in vitro restimulation system an H-Y-specific cell proliferation or in the effector phase the generation of H-Y-specific killer cells. The assay for celimediated lympholysis and lymphocyte proliferation after xenogeneic priming and syngeneic in vitro restimulation is, thus, not suitable for H-Y testing of human cells.     

Assessing labile organic carbon in soils undergoing land use change in Brazil: A comparison of approaches

The labile organic C (LC) and C management index (CMI) may be useful indicators of alterations of soil organic matter (SOM) in areas undergoing land use change (LUC) for biofuels production. However, there is no consensus on which methodology is best suited for quantifying LC and CMI. The main goal of this study was to assess alterations on LC contents and CMI values in sites undergoing the LUC native vegetation (NV)-pasture (PA)-sugarcane (SC) in south-central Brazil, and evaluated the sensitivity of different methods commonly used to assess LC and CMI, in order to select a best-suited method to quantify these indicators in tropical regions. The conversion NV-PA decreased the LC and CMI, whilst the conversion of PA-SC tended to increase the CMI. Accordingly, cropping sugarcane in areas previously used as pastures, as currently has been observed in Brazil, enhance SOM quality. The methodology used to quantify the LC and the CMI is critical to infer about LUC effects. Both methods proposed by Blair et al. (1995) and Diekow et al. (2005) were highly sensitive to the conversions evaluated in this research. However, Diekow et al. (2005) is the most suitable method to estimate the LC and CMI in sites undergoing LUC in Brazil, since the approach of Blair et al. (1995) notably overestimates these SOM quality indicators. We reiterate that the SOM changes are well expressed by the total soil organic C in areas undergoing LUC and, integrated approaches, such as the CMI, are quite suitable to evaluate the effects of LUC on SOM.     

Characterization of protective immunity induced against Schistosoma mansoni via DNA priming with the large subunit of calpain (Sm-p80) in the presence of genetic adjuvants

Despite advances in control via snail eradication and large-scale chemotherapy using praziquental, schistosomiasis continues to spread to new geographic areas particularly in sub-Saharan Africa. Presently, there is no vaccine for controlling this disease. We have concentrated on a functionally important schistosome antigen Sm-p80 as a possible vaccine candidate for schistosomiasis. Here we report the proliferation of spleen cells in response to the recombinant Sm-p80 protein and cytokine (IFN-gamma and IL-4) production by the splenocytes. These spleen cells were obtained from groups of mice that were vaccinated with a DNA vaccine formulation containing Sm-p80 and one of the Th-1 (IL-2 or IL-12) or Th-2 (GM-CSF, IL-4) enhancer cytokines. The splenocytes from the groups of mice vaccinated with Sm-p80 DNA in the presence of Th-2 enhancer cytokines showed moderate but detectable proliferation. The splenocytes obtained from mice vaccinated with Sm-p80 DNA with Th-1 enhancer cytokines IL-2 and IL-12 provided the highest proliferation. The IFN-gamma production by splenocytes was found to follow the similar pattern [(Sm-p80) < (Sm-p80 + IL-4) < (Sm-p80 + GMCSF) < (Sm-p80 + IL-12) < (Sm-p80 + IL-2)], as has been observed for the proliferation and protection data. However, the elevated IL-4 production was inversely correlated to Sm-p80-induced splenocyte proliferation or the protection. These results show again that protective immune response induced by Sm-p80 is of Th-1 type.     

Clomipramine actions on firing rate in septal nuclei of the rat are not related to anaesthesia (urethane).

An increased firing rate in lateral septal nuclei (LSN) appears in urethane-anesthetized rats after several acute drug and non-drug human antidepressant treatments. A still more pronounced increase in firing rate is produced in LSN after clomipramine (CMI) long-term treatment. In spite of urethane is a widely used anesthetic for single unit extracellular recordings, it modifies evoked potentials wave-form. Therefore, present study discards urethane interaction with CMI in LSN single unit extracellular recordings. CMI was acutely injected (1.25 mg/kg: IP) either to urethane-anesthetized, or non-anesthetized encephale-isolé rats. The CMI treated groups showed higher rates of firing in LSN regardless of the use of general anesthesia during recordings. Another group of urethane-anesthetized rats received intracerebroventricular (ICV) microinjections of CMI (100 micrograms/10 microliters/1 min). An amount of 42.8% of LSN-recorded neurons responded with a long-lasting increased firing rate. Results discard urethane and CMI interactions. Additionally, systemic actions of CMI on firing rate of LSN are reproduced by ICV/route microinjections.     

Selenium promotes T-cell response to TCR-stimulation and ConA, but not PHA in primary porcine splenocytes

There is controversy in the literature over whether the selenium (Se) influences cellular immune responses, and the mechanisms possibly underlying these effects are unclear. In this study, the effects of Se on T-cell proliferation and IL-2 production were studied in primary porcine splenocytes. Splenocytes were treated with different mitogens in the presence of 0.5-4 μmol/L sodium selenite. Se significantly promoted T-cell receptor (TCR) or concanavalin A (ConA)-induced T-cell proliferation and IL-2 production but failed to regulate T-cell response to phytohemagglutinin (PHA). In addition, Se significantly increased the levels of cytosolic glutathione peroxidase (GPx1) and thioredoxin reductase 1 (TR1) mRNA, the activity of GPx1 and the concentration of reduced glutathione (GSH) in the unstimulated, or activated splenocytes. These results indicated that Se improved the redox status in all splenocytes, including unstimulated, TCR, ConA and PHA -stimulated, but only TCR and ConA-induced T-cell activation was affected by the redox status. N-acetylcysteine (NAC), a pharmacological antioxidant, increased T-cell proliferation and IL-2 production by TCR and ConA stimulated splenocytes but had no effect on the response to PHA in primary porcine splenocytes confirming that PHA-induced T-cell activation is insensitive to the redox status. We conclude that Se promotes GPx1 and TR1 expression and increases antioxidative capacity in porcine splenocytes, which enhances TCR or ConA -induced T-cell activation but not PHA-induced T-cell activation. The different susceptibilities to Se between the TCR, ConA and PHA -induced T-cell activation may help to explain the controversy in the literature over whether or not Se boosts immune responses.     

Inhibition of lymphocyte proliferation by prenylated flavones: artelastin as a potent inhibitor

Eight natural prenylated flavones, previously isolated from Artocarpus elasticus, were evaluated for their effect on the mitogenic response of human lymphocytes to PHA. They all exhibited a dose-dependent suppression effect. An interesting relationship was observed between their antiproliferative activity and their chemical structure. Indeed, the most potent flavones possessed a 3,3-dymethylallyl group (prenyl) at C-8, such as artelastin, which exhibited the highest antiproliferative activity. Studies of the mechanism underlying its effect revealed that artelastin had an irreversible inhibitory effect on the PHA-induced lymphocyte proliferation and could affect the course of the ongoing mitogenic response either at the initial induction phase or at the late phase of proliferation. This prenylated flavone was also shown to be a potent inhibitor of both T- and B-lymphocyte mitogen induced proliferation although B-mitogenic response was the more sensitive one. Artelastin did not affect either the basal levels of the early marker of activation CD69 on non-stimulated splenocytes or its expression on ConA- or LPS-stimulated splenocytes. However, it decreased the production of IFN-gamma, IL-2, IL-4 and IL-10 in ConA-stimulated splenocytes. Furthermore, artelastin had no effect on apoptosis of splenocytes.     

Lack of species specificity in the action of immunosuppressive factors of tumor cells and absence of competition between them and recombinant interleukin-2 and phytohemagglutinin for lymphocyte membrane receptors

We studied some possible mechanisms of action of immunosuppressor factors (ISF) produced by tumor cells on lymphocyte proliferation. ISF of murine tumor cell lines inhibited the mitogen induced proliferation of murine splenocytes as well as human mononuclear blood cells. Normal human mononuclear blood cells or concanavalin A-activated murine spleen cells preincubated with phytohemagglutinin (PHA) or interleukin 2 (IL-2) respectively, were strongly suppressed by ISF in response to these activators. When preincubated with splenocytes or blood cells for 2 h at 4 degrees C following washing, ISF suppressed the lymphocyte proliferation as effectively as when being with cells during all period of cultivation. ISF inhibited mitogen-induced lymphocyte proliferation at low dilutions. There was no competition for lymphocyte membrane receptors between these functionally heterogenic kinds of ISF. Collectively, these results show that ISF acted when being attached to some lymphocyte membrane receptors.     

Image-guided analyses reveal that non-CD4 splenocytes contribute to CD4+ T cell-mediated inflammation leading to islet destruction by altering their local function and not systemic trafficking patterns

Recruitment of CD4(+) T cells into islets is a critical component of islet inflammation (insulitis) leading to type 1 diabetes; therefore, determining if conditions used to treat diabetes change their trafficking patterns is relevant to the outcome. Cotransfer of CD4(+)BDC2.5 (BDC) cells with non-CD4 splenocytes obtained from newly diabetic NOD mice, but not when they are transferred alone, induces accelerated diabetes. It is unclear whether these splenocytes affect diabetes development by altering the systemic and/or local trafficking and proliferation patterns of BDC cells in target and nontarget tissues. To address these questions, we developed an animal model to visualize BDC cell trafficking and proliferation using whole-body in vivo bioluminescence imaging and used the images to direct tissue sampling for further analyses of the cell distribution within tissues. The whole-body, or macroscopic, trafficking patterns were not dramatically altered in both groups of recipient mice. However, the local patterns of cell distribution were distinct, which led to invasive insulitis only in cotransferred mice with an increased number of islet-infiltrating CD11b(+) and CD11c(+) cells. Taken together, the non-CD4 splenocytes act locally by promoting invasive insulitis without altering the systemic trafficking patterns or proliferation of BDC cells and thus contributing to diabetes by altering the localization within the tissue.     

Rate of thymidine incorporation and incidence of parenchymal cell division in adult rat hypophyseal cell cultures. Effect of thyroliberin and somatostatin

Cell suspensions derived from adult rat anterior pituitary glands were cultured for up to eight days. Prolactin immunoreactivity and/or tritiated thymidine incorporation into DNA of cell nuclei were demonstrated in cells with and without thyroliberin (TRH) and somatostatin (SRIF) treatment. It has been established that (a) TRH, which is effective in releasing both thyrotropin and prolactin, may stimulate cell proliferation in other than its target cells; that (b) SRIF has no effect on lactotropic cell proliferation and augments thymidine incorporation into DNA of unidentified cells; that (c) immunoreactive lactotropic cells with tritium-labelled nuclei are present in each culture, independent of hypothalamic hormone treatments.     

Soil carbon pool and fertility under natural evergreen broadleaved forest and its artificial regeneration forests in southern Sichuan Province, China

The measurement of total soil organic matter (SOM) is not sensitive enough to detect short and medium term changes, and thus meaningful fractions of SOM should be measured. The carbon management index (CMI) was shown to be a useful technique for describing soil fertility. Soil samples were collected from natural evergreen broadleaved forest and its artificial regeneration forests of Sassafras tzumu, Cryptomeria fortunei and Metasequoia glyptostroboides in southern Sichuan Province, China, to determine soil carbon fractions, available nutrients, enzyme activity and CMI. Regression analysis was used to determine the relationship between soil carbon fractions, CMI and fertility. The results showed that the contents of soil organic carbon, water-soluble carbon, microbial biomass carbon, labile carbon, non-labile carbon, hydrolysis-N, available-P and available-K, the activity of invertase, phosphatase and catalase, and CMI were ranked with different seasons and followed the order: natural evergreen broadleaved forest > Sassafras tzumu plantation > Metasequoia glyptostroboides plantation > Cryptomeria fortunei plantation. The soil carbon fractions and CMI were significantly positively (P < 0.05) correlated with available nutrients and enzyme activity. The results indicate that soil carbon fractions and CMI could be used to evaluate the soil fertility for natural evergreen broadleaved forest and its artificial regeneration forests.