Determination of ribose and phosphorus contents in Haemophilus influenzae type b capsular polysaccharide by a quantitative NMR method using a single internal standard.

The ribose and phosphorus contents in Haemophilus influenzae type b (Hib) capsular polysaccharide (CPS) are two important chemical indexes for the development and quality control of Hib conjugate vaccine. A quantitative 1H- and 31P-NMR method using a single internal standard was developed for simultaneous determination of ribose and phosphorus contents in Hib CPS. Hexamethylphosphoramide (HMPA) was successfully utilized as an internal standard in quantitative 1H-NMR method for ribose content determination. The ribose and phosphorus contents were found to be affected by the concentration of polysaccharide solution. Thus, 15-20 mg·L-1 was the optimal concentration range of Hib CPS in D2O solution for determination of ribose and phosphorus contents by this method. The ribose and phosphorus contents obtained by the quantitative NMR were consistent with those obtained by traditional chemical methods. In conclusion, this quantitative 1H- and 31P-NMR method using a single internal standard shows good specificity, accuracy and precision, providing a valuable approach for the quality control of Hib glycoconjugate vaccines.

Immunogenicity and safety of a severe acute respiratory syndrome coronavirus 2 inactivated vaccine in healthy adults: randomized, double-blind, and placebo-controlled phase 1 and phase 2 clinical trials.

BACKGROUND: The significant morbidity and mortality resulted from the infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for urgent development of effective and safe vaccines. We report the immunogenicity and safety of an inactivated SARS-CoV-2 vaccine, KCONVAC, in healthy adults. METHODS: Phase 1 and phase 2 randomized, double-blind, and placebo-controlled trials of KCONVAC were conducted in healthy Chinese adults aged 18 to 59 years. The participants in the phase 1 trial were randomized to receive two doses, one each on Days 0 and 14, of either KCONVAC (5 or 10 µg/dose) or placebo. The participants in the phase 2 trial were randomized to receive either KCONVAC (at 5 or 10 µg/dose) or placebo on Days 0 and 14 (0/14 regimen) or Days 0 and 28 (0/28 regimen). In the phase 1 trial, the primary safety endpoint was the proportion of participants experiencing adverse reactions/events within 28 days following the administration of each dose. In the phase 2 trial, the primary immunogenicity endpoints were neutralization antibody seroconversion and titer and anti-receptor-binding domain immunoglobulin G seroconversion at 28 days after the second dose. RESULTS: In the phase 1 trial, 60 participants were enrolled and received at least one dose of 5-µg vaccine (n = 24), 10-µg vaccine (n = 24), or placebo (n = 12). In the phase 2 trial, 500 participants were enrolled and received at least one dose of 5-µg vaccine (n = 100 for 0/14 or 0/28 regimens), 10-µg vaccine (n = 100 for each regimen), or placebo (n = 50 for each regimen). In the phase 1 trial, 13 (54%), 11 (46%), and seven (7/12) participants reported at least one adverse event (AE) after receiving 5-, 10-µg vaccine, or placebo, respectively. In the phase 2 trial, 16 (16%), 19 (19%), and nine (18%) 0/14-regimen participants reported at least one AE after receiving 5-, 10-µg vaccine, or placebo, respectively. Similar AE incidences were observed in the three 0/28-regimen treatment groups. No AEs with an intensity of grade 3+ were reported, expect for one vaccine-unrelated serious AE (foot fracture) reported in the phase 1 trial. KCONVAC induced significant antibody responses; 0/28 regimen showed a higher immune responses than that did 0/14 regimen after receiving two vaccine doses. CONCLUSIONS: Both doses of KCONVAC are well tolerated and able to induce robust immune responses in healthy adults. These results support testing 5-µg vaccine in the 0/28 regimen in an upcoming phase 3 efficacy trial. TRIAL REGISTRATION: http://www.chictr.org.cn/index.aspx (No. ChiCTR2000038804, http://www.chictr.org.cn/showproj.aspx?proj=62350; No. ChiCTR2000039462, http://www.chictr.org.cn/showproj.aspx?proj=63353).

[Effects of purine nucleotide on the expressions of FSH and LH and the ultrastructure of endocrine cells in the pituitary gland of heroin-addicted male rats].

OBJECTIVE: To investigate the effects of purine nucleotide on the expressions of follicle-stimulating hormone (FSH) and luteotrophic hormone (LH) and the ultrastructures of the distal somatotrophic and gonadotrophic cells in the pituitary gland of heroin-addicted and -withdrawal rats. METHODS: Ninety-two male Wistar rats were randomly divided into a control group (ip saline for 14 d), a nucleotide group (ip AMP and GMP for 10 d), a heroin group (ip heroin for 10 d), a heroin + nucleotide group (ip AMP and GMP + heroin for 10 d), a 3 d withdrawal group (ip heroin for 10 d and killed at 14 d), a 9 d withdrawal group (ip heroin for 10 d and killed at 20 d), a 3 d nucleotide group (ip nucleotide for 3 d after 10 d heroin administration and killed at 14 d), and a 9 d nucleotide group (ip nucleotide for 9 d after 10 d heroin administration and killed at 20 d). Changes in the mRNA expressions of FSH and LH in the pituitary gland of the rats were analyzed by semi-quantitative RT-PCR, and alterations in the ultrastructures of the distal somatotrophic and gonadotrophic cells were observed under the microscope. RESULTS: The expression of FSH mRNA was significantly increased in the nucleotide, heroin + nucleotide, 3 d nucleotide and 9 d nucleotide groups (0.099 +/- 0.018, 0.177 +/- 0.046, 0.151 +/- 0.030 and 0.184 +/- 0.028) as compared with the control group (0.045 +/- 0.009) (P < 0.01); and so was that of LH mRNA in the heroin + nucleotide, 3 d nucleotide and 9 d nucleotide groups (0.950 +/- 0.169, 0.990 +/- 0.171 and 0.960 +/- 0.147) in comparison with the control group (0.700 +/- 0.099) (P < 0.01). In the heroin group, the nuclei of the distal somatotrophic and gonadotrophic cells exhibited morphological abnormality, unclear membrane, slightly pyknotic matrix, marginal and agglutinated heterochromatin, dilated rough endoplasmic reticula, swollen mitochondria, broken and vacuolated cristae in the cytoplasm, obviously decreased number of secretory granules, and myelin bodies in some cells. However, the heroin + nucleotide group showed no significant changes in the ultrastructures of somatotrophic and gonadotrophic cells compared with the control group. CONCLUSION: Short-term use of heroin does not obviously affect the expressions of FSH and LH mRNA in the pituitary gland of rats, while heroin + nucleotide, or nucleotide following heroin withdrawal can enhance their expressions significantly. Heroin damages the ultrastructures of the distal somatotrophic and gonadotrophic cells in the pituitary gland of male rats, and purine nucleotide can diminish or inhibit this damage.

Heroin affects purine nucleotides metabolism in rat brain.

OBJECTIVE: To explore the effects of heroin on purine nucleotides metabolism in rat brain. METHODS: Biochemical changes in association with heroin administration were compared between heroin-administered rats and non-heroin rats. HPLC method was used to detect the absolute content of purine nucleotides in brain tissues. Concentrations of uric acid (UA), blood urea nitrogen (BUN) and creatinine (Cre) in plasma were measured. Enzymatic activities of adenosine deaminase (ADA) and xanthine oxidase (XO) in brain tissue were analyzed. Real-time PCR was used to determine the relative level of transcripts of ADA, XO, adenine phosphoribosyl transferase (APRT), hypoxanthine-guaninephosphoribosyl transferase (HGPRT) and adenosine kinase (AK) in brain tissue. RESULTS: Compared with those in the saline group, the content of AMP and GTP of heroin group decreased significantly; the UA concentration in plasma, ADA and XO activities and the mRNA level of ADA and XO in brain tissues in heroin group increased significantly; the mRNA level of AK, APRT and HGPRT in brain tissues in heroin group decreased significantly (P<0.01). CONCLUSION: Heroin administration may enhance the catabolism and inhibit the anabolism of purine nucleotides in brain. There may be a deficiency of purine nucleotides, especially GTP and AMP in rat brain exposed to heroin. Our findings may provide a new potential approach to study the mechanism of heroin addiction.

Neanthes japonica (Iznka) fibrinolytic enzyme reduced cerebral infarction, cerebral edema and increased antioxidation in rat models of focal cerebral ischemia.

Thrombolytic agent is increasingly being used in treating acute ischemic stroke. A novel protease with strong thrombolytic activity, Neanthes japonica (Iznka) fibrinolytic enzyme (NJF) discovered in our laboratory has been reported with characteristics of direct hydrolyzing fibrin and fibrinogen. The neuroprotective effect of NJF and urokinase (UK) was tested in rat models of middle cerebral artery occlusion (MCAO). The model was successfully produced by introducing an intraluminal suture into the left middle cerebral artery (MCA). NJF (0.25, 0.5, 1mg/kg) was injected intravenously 1h after the onset of reperfusion. Compared with vehicle group, MCAO animals treated with NJF showed dose dependent reduction in cerebral infarction with improved neurological outcome. Meanwhile, ischemia induced cerebral edema was reduced in a dose dependent manner. Treatment with NJF at 0.5mg/kg was almost equivalent to UK at 15,000U/kg dosage in the reduction of cerebral infarction and cerebral edema. Biomedical assay showed that NJF treatment suppressed lipid peroxidation and restored superoxide dismutase (SOD) activities in brain tissue. These results suggest that NJF posses neuroprotective potential in rat MCAO and reperfusion model. Neuroprotection shown by NJF may be attributed to inhibition of lipid peroxidation, increase in endogenous antioxidant defense enzymes.

Morphine enhances purine nucleotide catabolism in vivo and in vitro.

AIM: To investigate the effect and mechanism of morphine on purine nucleotide catabolism. METHODS: The rat model of morphine dependence and withdrawal and rat C6 glioma cells in culture were used. Concentrations of uric acid in the plasma were measured by the uricase-rap method, adenosine deaminase (ADA) and xanthine oxidase (XO) in the plasma and tissues were measured by the ADA and XO test kit. RT-PCR and RT-PCR-Southern blotting were used to examine the relative amount of ADA and XO gene transcripts in tissues and C6 cells. RESULTS: (i) the concentration of plasma uric acid in the morphine-administered group was significantly higher (P<0.05) than the control group; (ii) during morphine administration and withdrawal periods, the ADA and XO concentrations in the plasma increased significantly (P<0.05); (iii) the amount of ADA and XO in the parietal lobe, liver, small intestine, and skeletal muscles of the morphine-administered groups increased, while the level of ADA and XO in those tissues of the withdrawal groups decreased; (iv) the transcripts of the ADA and XO genes in the parietal lobe, liver, small intestine, and skeletal muscles were higher in the morphine-administered group. The expression of the ADA and XO genes in those tissues returned to the control level during morphine withdrawal, with the exception of the skeletal muscles; and (v) the upregulation of the expression of the ADA and XO genes induced by morphine treatment could be reversed by naloxone. CONCLUSION: The effects of morphine on purine nucleotide metabolism might be an important, new biochemical pharmacological mechanism of morphine action.

[Effect of morphine on expression of gene of enzymes related to purine nucleotide metabolism in c6 glioma].

OBJECTIVE: To investigate the effect of morphine on catabolism and anabolism of purine nucleotide in c6 glioma cells. METHODS: C6 glioma cells were cultured and divided into 3 groups: 1) morphine group: morphine (10 micro g/ml culture) was added for 6 h, 12 h, 24 h, 48 h, and 72 h; 2) morphine + naloxone group: naloxone (1 micro mol/L) was added for 1 hour and then morphine (10 micro g/ml) was added for 24 hours; and 3) control group: normal saline was used for 6, 12, 24, 48, and 72 hours. The C6 glioma cells were centrifuged. RT-PCR was used to examine the gene transcripts of key enzymes of purine salvage way, hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) and adenylate kinase (AK). RT-PCR-Southern blotting was used to examine the gene transcripts of key enzymes of purine salvage way, xanthine dehydrogenase (XD)/xanthine oxidase (XO) mRNA. RESULTS: Compared with that in the control group, the transcript of AK mRNA was significantly lower in the C6 cells treated with morphine for 24 hours, and began to re-increase 48 hours after morphine treatment. The transcript of AK mRNA remained at a low level after treatment of naloxone for 1 hour and treatment of morphine for 24 hours. The levels of transcript of HGPRT mRNA were lower in the morphine group than in the control group at all time points after treatment. However, the level of transcript of HGPRT mRNA 72 hours after treatment was higher in the morphine group than in the control group. The level of transcript of HGPRT mRNA 24 hours after exposure to morphine in the naloxon2 + morphine group was still lower than in the control group. The levels of transcripts of XD/XO mRNA were significantly higher after exposure to morphine in comparison with those in the control group at all time points after treatment. However, the levels of XD/XO mRNA 24 hours after exposure to morphine in the naloxone + morphine group recovered to the normal level. CONCLUSION: The downregulation effect of morphine on the gene expression of AK and HGPRT may not be mediated by mu receptor. The upregulation effect of morphine on the gene expression of XD/XO may be mediated by mu receptor. Naloxone reverses the effect of morphine on enhancement of XD/XO gene expression and cannot reverse the inhibitory effect of morphine on HGPRT and AK gene expression.

Neurokinin-1 versus mu-opioid receptor binding in rat nucleus tractus solitarius after single and recurrent intermittent hypoxia.

G protein-coupled excitatory neurokinin-1 and inhibitory mu-opioid receptors exist in respiratory brainstem with their peptides and influence breathing. To assess their putative role in respiratory responses to hypoxia, neurokinin-1, and mu-opioid receptor binding was determined in the respiratory nucleus tractus solitarius of the mature rat after single and recurrent intermittent hypoxia versus normoxia. Hypoxia comprised six 5-min bouts of 8% O(2)-92% N(2) interceded by 5-min bouts in 21% O(2)-79% N(2) (normoxia), either on 6 consecutive days (recurrent intermittent hypoxia) or on the 6th day only (single intermittent hypoxia). Controls comprised six daily sessions in normoxia. To examine the plasticity in receptor response, brains were collected 5min, 2h, or 24h after the last gaseous exposure. Sections from each brainstem underwent quantitative film autoradiography with iodinated substance P and DAMGO for neurokinin-1 and mu-opioid receptors, respectively. Neurokinin-1 receptor binding decreased 5min after single and recurrent hypoxia and 2h after recurrent hypoxia, whereas mu-opioid binding remained unchanged. The binding of both receptors increased 24h after recurrent intermittent hypoxia. Neurokinin versus mu-opioid binding differences immediately posthypoxia might affect physiological responses to episodic hypoxia.