Development of coding single nucleotide polymorphic markers in the pearl oyster Pinctada fucata based on next-generation sequencing and high-resolution melting analysis.

The pearl oyster Pinctada fucata is an important commercial marine shellfish that is cultured for producing saltwater pearls. In this study, 468 single nucleotide polymorphisms (SNPs) were screened from P. fucata transcriptome data, and 119 polymorphic SNPs were successfully isolated by a two-step small-amplicon high-resolution melting assay. Of these, 88 were annotated with BLAST in the Nr database and 90 were in the open reading frame, including 16 non-synonymous SNPs and 74 synonymous SNPs; 12 SNPs were in the 3'-untranslated region (UTR) and 1 was in the 5'-UTR. Twenty-five SNPs were randomly chosen to test the genetic diversity of 40 wild individuals from Liusha Bay, China. All of the loci had two alleles. The observed and expected heterozygosities ranged from 0.0417 to 0.6042 and from 0.2945 to 0.5053, respectively. Minor allele frequencies ranged from 0.1771 to 0.5000, and the polymorphism information content ranged from 0.2516 to 0.3750. These novel SNP markers can contribute to P. fucata genetics and breeding studies.

Identification of 15 novel polymorphic microsatellite loci in pearl oyster (Pinctada fucata).

The pearl oyster Pinctada fucata is a commercially important marine shellfish. As a result, genetic improvement and selective-breeding program have been conducted for this species. Polymorphic microsatellites are effective molecular markers to investigate molecular marker-assisted selection and genetic variance. In this study, microsatellite DNAs were screened and characterized based on the partial genome sequence of P. fucata. We identified 111 microsatellite DNA motifs through mining the published draft genome sequence of P. fucata. Forty-two loci were screened with 8 P. fucata individuals, and 15 were found to be polymorphic and were therefore further evaluated using 40 wild individuals from the Daya Bay, Shenzhen City, Guangdong Province, China. The number of alleles per locus ranged from 3 to 8, with an average of 5.2667 for the 15 polymorphic loci. Observed and expected heterozygosities ranged from 0.1154 to 0.6216 (0.3321 on average) and 0.4950 to 0.8491 (0.6768 on average), respectively. Of the 15 polymorphic loci, 12 loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0033). Polymorphism information content ranged from 0.44 to 0.83 with a mean value of 0.63. The results suggest that the markers isolated in this study can be used for research on molecular marker-assisted selection and genetic variance of P. fucata.

[Obesity and the central nervous system regulation].

There are increasing evidences to show that central nervous system is involved in the regulation of energy homeostasis. Energy intake is usually matched to energy expenditure over a period of time. Obesity occurs when the amount of energy intake (or food intake) is more than the energy expenditure. Because of the enormous tolls on human health taken by obesity and related disorders, an improved understanding of the control of food intake is an important priority. The aim of this article is to briefly review the advances in recent years on long-term maintenance of energy homeostasis and the role of central nervous system. In the present review, the following contents are included: (1) satiety and its production, (2) adiposity signals and the regulation of food intake, (3) nuclei in central nervous system involved in food intake, (4) the first- and the second-order neuronal signaling in hypothalamus on control of food intake and (5) clinic implications.

Naltrexone suppresses the rejection of cardiac tissue transplantation.

The present study demonstrates the following: 1. Transplantation of cardiac tissue induces an inflammatory response that ultimately leads to the rejection of the tissue by the host within 9 days; 2. Treatment with the opiate antagonist, naltrexone, significantly increased the survival of the transplanted cardiac tissue to 13 days, suggesting the involvement of opioid signaling molecules in tissue rejection; 3. In further experiments it was demonstrated that in mixed lymphocyte populations from different mice, the DNA synthesis inhibitor, mitomycin C, reduced the lymphocyte proliferative response as did naltrexone; 4. Mice injected with naltrexone for 10 days and given concanavalin A exhibited a suppressed spleen lymphocyte proliferative response compared to controls. Taken together, these data suggest that endogenous opioid signals not only activate immunocytes, but also stimulate DNA synthesis.

A suppressive protein generated in peripheral lymph tissue induced by restraint stress.

The results discussed here indicate that under the conditions of restraint stress and under the control of CNS, a suppressive protein (NIP) was generated in peripheral lymph tissue and released into the blood stream, which acts as a immune suppressor. It is potentially a very important molecule that could be very important to our understanding of the interaction between CNS and immune function.

[Building up of an animal model of conditioned immunosuppression and analysis of its possible mechanism].

In the present study, camphor odor and intraperitoneal (ip) injection of cyclophosphamide (CY) were used as conditional and unconditional stimulus, respectively, in mice. Mice were exposed to camphor odor for 1 h in their cage in a closed area followed by an ip injection of CY (75 mg.kg-1). This association trial session was repeated once on the next day. Delayed type hypersensitivity response (DTH) was induced as follows: six days after the second association trial session the mice were sensitized by smearing dinitrochlorobenezene (DNCB) on their abdominal skin. The mice were challenged by smearing DNCB on the left ear 5 days after the antigen sensitization. The left and right ears were removed 24 h after the challenge and weighed, the weight ratio of left/right ears was calculated for identification of the response. The ratio was 1.30 +/- 0.113 (+/- s, P < 0.001), indicating that the challenged ear was heavier than the other and DTH was induced. In the unconditioned response (UCR) group, CY (75 mg.kg-1) was given 24 h prior to the challenge and the ratio was 1.09 +/- 0.024 (P < 0.001) indicating that DTH was suppressed by unconditional stimulus (CY). In the conditioned response (CR) group mice were reexposed to camphor odor 24 h prior to the challenge and normal saline was injected instead of CY. The ratio was 1.13 +/- 0.074 (P < 0.001), indicating that DTH was also suppressed by conditional stimulus (camphor odor). These results show that a conditioned immunosuppressive response was induced. In the experiment, many other groups, including unconditioned response group, CYE group and camphor control group, were described in more details in the text. In order to further analyse the mechanisms of the conditioned response, the blood from the mice in CR group was obtained 6 h after reexposure to camphor odor and the serum was injected to normal mice 6 h prior to the challenge. DTH was found to be suppressed significantly when compared with the mice injected with normal serum. The conditioned serum was dialyzed against a membrane with a 10,000 molecular weight cut off. The suppressive activity of the conditioned serum disappeared, suggesting that the molecular weight of the suppressive element in the serum was probably less than 10,000 kDa.

[Intracerebroventricular injection of interleukin 1 receptor antagonist antagonises on the generation of immuno-suppressive factor in restraint mice].

Our previous work showed that a lymphocyte proliferation suppressing factor could be found in the serum of restraint mice. In the present work, it was found that intracerebroventricular (icv) injection of IL-1 receptor antagonist (IL-1Ra) was found capable of suppressing the production of such a serum protein under restraint stress. Nearly complete suppression could be achieved by 5.0 micrograms IL-1Ra. Intracerebroventricular injection of IL-1 beta (1 pg), however, increased the generation of the supressive protein. Neither intraperitoneal (ip) injection of IL-1Ra or IL-1 beta had any effect on the generation of the protein. The fact that icv. injection of a very small dose of IL-1 beta (0.06 fmol) was effective on the generation of the supressive protein led us to suggest that IL-1 in brain might act as an important mediator between CNS and the immune system.

A factor in lymph node and spleen induced by restraint stress in mice and rats suppresses lymphocyte proliferation.

Extracts from lymph node and spleen in mice and rats subjected to restraint stress significantly suppressed lymphocyte proliferation, but extracts from brain, skeletal muscle, and thymus gland had no effect on lymphocyte proliferation, suggesting that a suppressive factor for lymphocyte proliferation might selectively be induced in lymph node and spleen. Further experiments showed that biochemical properties, molecular weight, correlation between suppressive factors in serum and in extract of the lymph tissue from stressed animals, and control of the generation, all indicated that under the conditions of restraint stress and under the control of central nervous system a suppressive factor was generated in peripheral lymph tissue and then released into the blood-stream, which acted as a strong suppressor of lymphocyte proliferation.

Brain interleukin-1 is involved in generation of the serum suppressive factor induced by restraint stress in mice.

Our previous work showed that a factor (a protein with a high molecular weight) in serum was induced by restraint stress in mice and rats, and suppressed lymphocyte proliferation induced by concanavalin A. It was also found that the generation of the serum suppressive factor was under the control of the central nervous system. The present work was designed to investigate the role of interleukin-1 (IL-1) in the brain in the serum suppressive factor. IL-1 receptor antagonist (IL-1ra) was injected intracerebroventricularly in mice and the generation of the serum suppressive factor was found to be significantly decreased in a dose-dependent manner. When the dose of IL-1ra reached 5 micrograms, the generation of the suppressive factor was almost totally abolished. Intracerebroventricular injection of IL-1 beta (1.0 pg) enhanced the generation of the suppressive factor. Taken together, these results indicate the involvement of IL-1 in the brain in mediating generation of the suppressive factor.

[Effect of intracerebroventricular injection of IL-1 beta on generation of immuno-suppressive factor in lymphocytes in stressed mice].

Our previous work showed that a suppressive factor (a protein with large molecular weight) in serum was induced by restraint stress in mice and rats, which suppressed Con A induced lymphocyte proliferation. It was also found that the generation of serum suppressive factor was under control of the central nervous system. Our further study showed that intracerebroventricular (icv) injection of interleukin 1 receptor antagonist (IL-1Ra) antagonised the generation of serum suppressive factor induced by restraint stress and icv injection of interleukin-1 beta (IL-1 beta) increased the generation of the suppressive factor. Our experiment also showed that the serum suppressive factor induced by restraint stress was first made in lymph tissue and then released into blood. The present work was designed to investigate the role of IL-1 in the brain in generation of the suppressive factor in lymph node in mice. Icv injection of IL-1 beta (1 pg/mouse) was shown to significantly increase the generation of the suppressive factor in lymph node. Icv injection of IL-1Ra, however, antagonised generation of the suppressive factor. In mice without restraint stress, both the suppressive factor in serum and in lymph node were found to be induced in dose-dependent manner by icv injection of IL-1 beta. Taken together, these results suggest that IL-1 beta in brain played a very important role in generation of the suppressive factor in lymph node. The positive correlation between the suppressive action of lymph node and of serum added to the evidence that lymph tissue is probably the source of the serum suppressive factor.

[A study on the location of immuno-suppressive factor(s) in restraint rats and mice].

A serum lymphocyte-proliferation suppressive factor(s) induced by restraint stress over 10 h was found in previous studies in both rats and mice. The present study was undertaken to investigate the sites of its production. The results show that large doses of irradiation and cyclophosphamide (CY) decreased the total number of splenic nucleated cells, but the production of the suppressive factor was inhibited only by irradiation. This indicates that the drop in total number of lymphocytes does not play any key role in the production of the serum suppressive factor. Cell classification showed that the ratio of T to B cell was decreased by radiation but increased by CY, suggesting that this ratio may be relevant to the production of the factor. Inhibition of the production was also observed in nude mouse (an animal showing a lack of T cell activity), again supporting that T cells are involved in the production of the inhibitory factor.

[A study on serum suppressive factor(s) on lymphocyte proliferation in rats under restraint stress].

In order to study the effect of stress on lymphocyte proliferation, SD rats were restrained with four limbs tied on a frame in supine position at room temperature (20 degrees C) for 20 h, and control animals were not disturbed in home cage. The blood was then collected from the heart under light ether anesthesia. The peripheral blood lymphocytes were separated from heparinized whole blood by density gradient (d 1.077) centrifugation, or the serum was obtained after the blood coagulated at 4 degrees C for about 6h. It was found that the blood lymphocyte proliferation induced by Con A was significantly inhibited in the stressed group as compared with the control (P less than 0.01, n = 8, ANOVA). The result was in accordance with our earlier study in which the animals were stressed with electric shock. In the present study, it was also found that the serum of the stressed animals was capable of suppressing Con A-induced lymphocyte proliferation of normal mice (P less than 0.01, n = 8, ANOVA) to a significant extent. Thus the present experiment suggests that there is some substance with suppressive activity on lymphocyte proliferation in the serum of the stressed rats. The serum lost its suppressive activity when it was heated to 100 degrees C (3 min), treated with 60% methanol or incubated with trypsin (64 micrograms/ml), thus suggesting that the suppressive factor(s) most likely is a kind of protein.

[Possible mechanisms of the action of lymphocyte proliferation-inhibitory factor(s) in rat serum receiving electroacupuncture stimulation].

Previous reports showed that EA stimulation (3V, 2Hz, 30 min/d, 5 d) induced the production of one or more lymphocyte proliferation-inhibitory factor(s) in the rat serum. In this paper, the mechanisms of the action for the inhibitory factor(s) to suppress lymphocyte proliferation were studied. (1) the lymphocytes from different immune organs of the mice were prepared and cultured with the rat serum stimulated by EA. The results show that the serum not only inhibited the mouse lymph node T cell proliferation induced by Con A, but also inhibited the mouse thymocyte and spleen T cell proliferation induced by Con A. When B cells were stimulated by LPS, the proliferative effect can also be inhibited significantly by the rat serum stimulated by EA. This implies that the effect of the lymphocyte proliferation-inhibitory factor(s) has no specificity. (2) Incubation of the mouse lymph node cell with serum for one hour is enough to cause an inhibitory effect on Con A stimulated lymphocyte proliferation. However, no inhibitory effect was observed if the mouse lymph node cells were incubated with Con A for 15 min or 30 min before the addition of rat serum. The results demonstrate that the lymphocyte proliferation-inhibitory factor(s) act on the early events of T lymphocyte activation induced by Con A. (3) Protein kinase C (PKC) is a key link in the activation of T and B lymphocyte proliferation by Con A and LPS respectively. So it would be interesting to learn whether the inhibitory effect of the lymphocyte proliferation-inhibitory factor(s) is caused by the inhibition of PKC activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholecystokinin-8 suppressed 3H-etorphine binding to rat brain opiate receptors.

Radio receptor assay (RRA) was adopted to analyse the influence of CCK-8 on 3H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 (1pM to 1 microM) suppressed the binding of 3H-etorphine. This effect was completely reversed by proglumide at 1 microM. Rosenthal analysis for saturation revealed two populations of 3H-etorphine binding sites. CCK-8 (1pM to 1 microM) inhibited 3H-etorphine binding to the high affinity sites by an increase in Kd (up to +235%) and decrease in Bmax (up to -80%) without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 (10nM) was also completely reversed by proglumide at 1 microM. Unsulfated CCK-8 (100pM to 1 microM) produced only a slight increase in Kd of the high affinity sites (+64%) without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.

The protein kinase C inhibitor H-7 inhibits concanavalin A induced T-lymphocyte activation.

1. 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, was found to inhibit con A stimulated [3H]thymidine incorporation and cytosolic protein kinase C (PKC) activation in T-lymphocytes of mouse spleen. 2. The inhibitory effect of H-7 was both concentration and time-dependent. 3. H-7 exerted no inhibition when T-lymphocytes have been preincubated with con A for 10 hours or longer. 4. These results support the notion that PKC is an important element of the con A mitogenic signal transduction mechanism and the PKC signal is completed within the first 10 hr of con A incubation.

Reversal of tolerance to morphine but no potentiation of morphine-induced analgesia by antiserum against cholecystokinin octapeptide.

Tolerance to morphine analgesia was induced in rats by chronic treatment with morphine (5-30 mg/kg, t.i.d. for 6 days). Intracerebroventricular (i.c.v.) injection of antiserum against cholecystokinin octapeptide (CCK-8) reversed tolerance to morphine by 50% (P less than 0.001). Intrathecal (ith) injection of the CCK-8 antiserum produced a similar, although less marked, reversal of tolerance to morphine. Rats made tolerant to analgesia induced by morphine developed a cross tolerance to electroacupuncture-induced analgesia. This cross tolerance was also reversed by the CCK-8 antiserum by more than 50% (P less than 0.001). Intracerebroventricular or intrathecal injection of the CCK-8 antiserum per se produced no significant changes in the basal level of the latency of the tail flick response, nor did it affect the analgesia induced by morphine in naive rats. The results suggest that prolonged activation of opioid receptors may trigger the CCK-8 system in the central nervous system to exert a negative feedback control, which may constitute one of the mechanisms for the development of tolerance to opioids.

Cholecystokinin octapeptide (CCK-8): antagonism to electroacupuncture analgesia and a possible role in electroacupuncture tolerance.

The analgesic effect produced by electroacupuncture (EA) stimulation in the rat was dose-dependently antagonized by cholecystokinin octapeptide (CCK-8) administered intracerebroventricularly (i.c.v.) or intrathecally (i.th) at a dose range of 0.25-4 ng. This effect had an immediate onset and lasted for at least 4 h. CCK-8 per se, however, did not affect baseline tail flick latency. Rats subjected to prolonged EA stimulation developed EA tolerance as well as cross-tolerance to morphine. These tolerances could be postponed or reversed by i.c.v. or i.th injection of antiserum against CCK-8. While CCK-8 antagonized opioid analgesia, it did not affect analgesia induced by 5-hydroxytryptamine (5-HT) or norepinephrine (NE). Moreover, CCK-8 antiserum did not alter the basic level of nociception, nor did it potentiate EA analgesia in naive rats. It is concluded that prolonged EA stimulation results in a profound release of opioids which may trigger the release of CCK-8 in the central nervous system to counteract the opioid component of EA analgesia. This mechanism may account, at least in part, for the development of EA tolerance.