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Biomedical subjects

S Zhang

Publications and source records attributed to S Zhang.

At least 19 recordsLinked to original sources

Effects of neonatal transection of the infraorbital nerve upon the structural and functional organization of the ventral posteromedial nucleus in the rat.

The present study examined the way in which an indirect partial deafferentation of the medial portion of the ventrobasal complex (VPM/VPL) induced by neonatal transection of the infraorbital nerve (ION) altered the structural and functional properties of its constituent neurons. This manipulation significantly reduced the volume of the contralateral VPM/VPL. In addition, cell counts in Nissl-stained material revealed a significant reduction of the number of VPM/VPL neurons contralateral to neonatal ION transection. We also analyzed the effect of neonatal ION transection on the soma-dendritic morphology of individual neurons in the ventral posteromedial nucleus of the thalamus (VPM) by intracellular injection of horseradish peroxidase (HRP) in vivo and Lucifer yellow in fixed slices. Neonatal transection of the ION resulted in increased dendritic length, area, and volume of VPM neurons in both preparations; however only the changes observed in fixed slices reached statistical significance. Alterations in the functional characteristics of VPM neurons were also observed following neonatal nerve damage. There was a significant decrease in the percentage of vibrissae-sensitive neurons and a corresponding increase in the percentages of neurons responsive to guard hair deflection or that were unresponsive to peripheral stimulation. Neonatal nerve damage also resulted in significantly longer latencies of VPM cells after stimulation of either trigeminal nucleus principalis or subnucleus interpolaris. The present results indicate that the development of normal response properties and soma-dendritic morphology of VPM neurons is dependent upon intact afferent input during development. Indirect partial deafferentation of VPM/VPL by neonatal transection of the ION results in reduced neuron number, which may result in decreased competition among the dendrites of these neurons. This proposal is consistent with observations of increased dendritic dimensions of VPM neurons contralateral to neonatal ION damage.

Animals

Single-stranded DNA binding protein from calf thymus. Purification, properties, and stimulation of the homologous DNA-polymerase-alpha-primase complex.

A binding protein for single-stranded DNA (ssDNA) was purified from calf thymus to near homogeneity by chromatography on DEAE-cellulose, blue-Sepharose, ssDNA-cellulose and FPLC Mono Q. The most purified fraction consisted of four polypeptides with molecular masses of 70, 55, 30, and 11 kDa. The polypeptide with the molecular mass of 55 kDa is most likely a degraded form of the largest polypeptide. The complex migrated as a whole on both glycerol gradient ultracentrifugation (s = 5.1 S) and gel filtration (Stokes' radius approximately 5.1 nm). Combining these data indicates a native molecular mass of about 110 kDa, which is in accord with a 1:1:1 stoichiometry for the 70 + 55/30/11-kDa complex. The ssDNA binding protein (SSB) covered approximately 20-25 nucleotides on M13mp8 ssDNA, as revealed from both band shift experiments and DNase I digestion studies. The homologous DNA-polymerase-alpha-primase complex was stimulated by the ssDNA binding protein 1.2-fold on poly(dA).(dT)14 and 10-13-fold on singly primed M13mp8 DNA. Stimulation was mainly due to facilitated DNA synthesis through stable secondary structures, as demonstrated by the vanishing of many, but not all, pausing sites. Processivity of polymerase-primase was not affected on poly(dA).(dT)14; with poly(dT).(rA)10 an approximately twofold increase in product lengths was observed when SSB was present. The increase was attributed to a facilitated rebinding of polymerase alpha to an already finished DNA fragment rather than to an enhancement of the intrinsic processivity of the polymerase. Similarly, products 300-600 nucleotides long were formed on singly primed M13 DNA in the presence of SSB, in contrast to 20-120 nucleotides when SSB was absent. DNA-primase-initiated DNA replication on M13 DNA was inhibited by SSB in a concentration-dependent manner. However, with less sites available to begin with RNA priming, more homogeneous products were formed.

Animals

DNA junctions, antijunctions, and mesojunctions.

Antijunctions and mesojunctions are new classes of multistranded DNA complexes. They represent a generalization of DNA branched junction complexes, such as the Holliday recombination intermediate. Each strand of a conventional branched junction participates in two different double helices, and this is also true for mesojunctions and antijunctions. The helix axes of conventional branched junction complexes may be drawn to converge at a point, but this convergence occurs for lines drawn perpendicular to the helix axes of antijunctions. Mesojunctions are complexes that mix these features of junctions and antijunctions. Antijunction complexes require an even number of strands. We have synthesized the mesojunction containing three strands, the two mesojunctions containing four strands, and the antijunction containing four strands; we compare them with branched junctions containing three and four strands, derived by permutations of the same sequences. Each double helix is designed to contain 1.5 turns of DNA. A tendency to oligomerize makes it difficult to capture antijunctions and mesojunctions in stable discrete complexes, in contrast to conventional branched junctions. For both three-strand and four-strand complexes, Tm is highest for conventional branched junctions. Ferguson analysis indicates similarities in the occluded surface area of junctions, antijunctions, and one four-strand mesojunction, but the other four-strand mesojunction has a much lower apparent surface area. Hydroxyl radical cleavage patterns suggest that the four-strand antijunction and the low-surface-area four-strand mesojunction form stacking domains, analogous to the behavior of conventional branched junctions. These new structures are related to replicational and recombinational intermediates and to single-stranded nucleic acid knots.

Base Sequence

A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase alpha on double-stranded DNA.

A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB), also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase alpha to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200-600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase alpha-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.

Adenosine Triphosphate

Characteristics of the beta-galactosidase-carboxypeptidase complex in GM1-gangliosidosis and beta-galactosialidosis fibroblasts.

Lysosomal beta-galactosidase (beta-Gal) occurs either alone in monomeric and dimeric forms, or in a high-M(r) complex with at least two additional proteins. One is neuraminidase and the second is the protective protein, which has also been shown to possess carboxypeptidase activity. beta-Gal activity is deficient in GM1-gangliosidosis as a primary defect, and is secondarily affected in galactosialidosis (GS), where the primary defect is the absence of protective protein activity. Fibroblasts from three patients with GM1-gangliosidosis, type 1, showed markedly reduced amounts of beta-Gal cross-reacting material (CRM), and a fourth appeared to have normal levels. A patient with type 2 GM1-gangliosidosis was also found to be CRM-normal. These findings demonstrate that patients with GM1-gangliosidosis type 1 are heterogeneous with respect to the level of residual beta-Gal protein. Fibroblasts from four patients with GS were strongly CRM-positive with an anti-beta-Gal antibody, as was a sample of brain from one of these patients, suggesting that the loss of beta-Gal activity is linked to a subtler change in the primary structure of the enzyme than has been previously thought. While three GS cell lines displayed reduced carboxypeptidase activity (to 32-42% of the control), one cell line was completely devoid of activity, demonstrating that while carboxypeptidase activity is a property of the protective protein this action is distinct and separate from its protective role. On direct immunoprecipitation with anti-beta-Gal antibody, a portion of the total carboxypeptidase activity co-precipitated with beta-Gal from extracts of normal and GM1-gangliosidosis cells, consistent with the presence of the complex in these cells. However, no carboxypeptidase activity was precipitable with this antibody from GS fibroblasts, suggesting the absence of complex from these cells. To examine this further, the various forms of beta-Gal were resolved by h.p.l.c. molecular-sieve chromatography. Three forms of beta-Gal activity were resolved in normal cells: a complex, a dimer and a monomer. Residual beta-Gal activity of GS cells resolved into two of these forms, the complex and the monomer. In normal and GM1-gangliosidosis cells a portion of the total carboxypeptidase activity co-chromatographed with the complex while the bulk of the activity occurred in a single 36,000-M(r) peak. Only the low-M(r) carboxypeptidase activity was detected in GS cells. This confirms our results on immunoprecipitation indicating that portions of the beta-Gal and the carboxypeptidase activities exist outside the complex in normal, GM1-gangliosidosis and GS cells. In summary, the loss of protective protein function from GS cells results in disproportionate loss of the dimeric and monomeric forms of beta-Gal activity, but does not result in the complete degradation of the protein.

Carbohydrate Metabolism, Inborn Errors

Occurrence of butyltin species in sewage and sludge in Canada.

Samples of sewage treatment influent, effluent and sludges collected monthly from five Canadian cities over the period from July 1990 to January 1991 were analysed for butyltin and octyltin species. Monobutyltin was found in all influent samples, but dibutyltin and tributyltin were found only infrequently, and octyltin species were not found at all. In the case of monobutyltin, there was significant reduction in its concentration by degradation and adsorption to sludge during passage through the sewage treatment plant. The average reduction was 40%. The monobutyltin found in the effluent likely came from its use as a poly (vinyl chloride) stabilizer, and from the degradation of tributyltin, which is used as a slimicide. No butyltin or octyltin species was found in five landfill leachate samples in southern Ontario during the same period.

Canada

Visualization of the plasmin receptor on carcinoma cells.

In order to visualize the receptor for plasmin and plasminogen present on human carcinoma cells, SW1116 and MCF7-MF cells were incubated with biotinylated plasminogen or plasmin and fluoresceinated streptavidin, and counterstained with propidium iodide. It was first demonstrated that biotinylation did not alter the binding properties of plasminogen and plasmin, provided that the biotinylation rate was around 2. Specific staining of tumor cells was obtained using these reagents. Images with green fluorescence were clearly visible as grains or contours at the surface of tumor cells. The localization of fluorescent grains was analyzed more precisely using confocal microscopy. The percentage of stained cells varied from one experiment to another between 10 and 60%. In no experiment were all the cells observed to be positive. Mitotic cells were more frequently stained than non-mitotic cells, suggesting a relationship between the presence of plasmin receptors and cell proliferation. This was confirmed by the use of Ki67 monoclonal antibody (MAb), as B-Pg-binding tumor cells generally had their nucleus stained by this antibody. Other images indicated staining of the extracellular matrix. Finally, 2 rat tumor-cell sub-lines of colonic origin (DHD K12 Pro-b and Reg-b) were shown to bind human biotinylated plasminogen, confirming the strong interspecies reactivity of plasmin receptors.

Animals

Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae.

A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

Amino Acid Sequence

Liposomes and conjugate vaccines for antigen delivery and induction of mucosal immune responses.

In this brief review, emphasis was placed on the effectiveness of liposomes as carriers/vehicles of soluble antigens and as adjuvants for use in oral vaccine development. Evidence was provided that oral administration of antigen in liposomes resulted in a mucosal response which was higher than that obtained when the oral vaccine consisted of antigen alone. Specific mucosal responses were enhanced by incorporating lipophilic MDP into the antigen/liposome vaccines. Antigens shown to be effective in inducing a protective mucosal response when given in an oral liposome vaccine were anti-idiotypic antibodies, purified S. mutans GTF, CHO and Ag I/II. Evidence is also provided that CTB may be an effective oral adjuvant when coupled to proteins or peptides by either chemical or genetic methods. Further studies, however, will be required to characterize the effectiveness and safety of CTB in conjugate vaccines for inducing specific mucosal responses and to develop practical means to prepare oral liposome vaccines for use in humans.

Animals

Study of the relationships between weather conditions and the marathon race, and of meteorotropic effects on distance runners.

The relationships between the results of Beijing International Marathon Races and the corresponding weather conditions are analysed quantitatively. There are obvious interrelationships between the marathon results and weather factors such as air temperature, wet bulb temperature and human biometeorological indices. For example, the correlation coefficient between the average times of the top ten finishers and temperature is r = 0.8910. The meteorological conditions are classified into three categories, suitable, moderate and unsuitable for running a marathon race, and the optimum meteorological index is given. Also the concept of a meteorological result, i.e. the part of the actual performances fluctuating with the changes in weather conditions, is presented. This plays an important role in some kinds of sports such as marathon racing. Finally, the results of physiological tests are given, which illustrate the physiological reactions of long- and middle-distance runners to the surrounding temperature.

Exercise

Isolation and characterization of a DNA helicase from cytosolic extracts of calf thymus.

A DNA helicase has been isolated from calf thymus tissue. The enzyme was enriched from crude cytosolic extracts by batchwise chromatography on phosphocellulose, followed by 35% ammonium sulfate precipitation, and subsequent chromatography on phenyl-Sepharose, single-stranded DNA cellulose, and AcA 44 gel filtration. The DNA helicase had a Stokes' radius of about 45 A and a sedimentation coefficient of 4.3 S. The most purified fractions contained three polypeptides with apparent molecular weights of 110, 65, and 34 kDa. UV crosslinking with radioactive dATP stained all three major polypeptides. The helicase catalyzed the unwinding of a DNA primer from a single-stranded DNA template in an ATP- or dATP-dependent manner. DNA unwinding was also observed with CTP or dCTP, but with reduced efficiency. The helicase translocated from 3' to 5' on the single-stranded template it was bound to. Relationships between this DNA helicase and other calf thymus helicases will be discussed.

Adenosine Triphosphate

Boosted mucosal immune responsiveness in the rat intestine by actively transported hexose.

Anaphylaxis-mediated intestinal fluid secretion was measured in Trichinella spiralis- or ovalbumin-immunized rats challenged intraduodenally with T. spiralis somatic antigen (1 mg protein/0.5 mL saline) or ovalbumin (1 mg/0.5 mL saline), respectively. Intestinal fluid volume was measured 30 minutes after challenge as an index of net secretion. Challenge with the antigenic bolus containing 40 mmol/L D-glucose induced twice the fluid secretion as that induced by either antigen alone. L-Glucose was an ineffective substitute for D-glucose. The enhancement of secretion by D-glucose was dependent on prior immunologic sensitization, was diminished in the presence of phlorizin, and was mimicked by beta-methyl glucoside. Results indicate that the active transport of D-glucose augments the antigen-mediated fluid secretion, possibly by enhancing permeation of the intestinal epithelium to antigen, thereby providing greater access to the mucosal immune system.

Anaphylaxis

Adenovirus type 12 early region 1B proteins and metabolism of early viral mRNAs.

Early region 1B (E1B) of human adenoviruses encodes two major proteins. The 19-kDa polypeptide appears to prevent E1A-induced cytolysis and DNA degradation. The larger E1B product of approximately 55 kDa, which is essential for viral replication, plays a role in the accumulation and stability of viral mRNAs and the late shutoff of host metabolism. For serotype 12 (Ad12), this 482-residue (482R) protein is essential for viral DNA replication. In the present report we have used a series of mutants to examine the roles of Ad12 482R and the 19-kDa, 163R protein in the metabolism of early viral mRNAs. No specific effects on the accumulation of early (or late) mRNAs were detected with any of the mutants affecting 163R. With mutant dl42, which encodes an altered 482R product that lacks residues 114-155, both viral DNA replication and late viral protein synthesis were defective. Accumulation of E1A transcripts in the nucleus and cytoplasm resembled wt. The levels of mRNAs from early regions E1B, E2A and E3 at later times during infection were somewhat lower than those of wt, but this decrease may have been due to the absence of progeny viral DNA in dl42-infected cells. However, the accumulation of both E2B and E4 mRNAs at all times was severely reduced. These data suggested that the requirement of 482R for Ad12 DNA replication may be related to its specific role in the metabolism of E2B and E4 mRNAs that encode products necessary for viral DNA synthesis.

Adenovirus E1B Proteins

An immunosuppressant compound, FK-506, prevents the progression of autoimmune myocarditis in rats.

A new immunosuppressive compound, FK-506, is a macrolide produced by Streptomyces tsukubaensis. It is reported that FK-506 prolongs the viability of allogenic grafts of the heart and kidney in vivo and inhibits the development of autoimmune diseases. Furthermore, immunosuppressive therapy of myocarditis in humans has been given special attention by various observers; however, it is controversial. This study investigates the effects of FK-506 on experimental autoimmune myocarditis in rats. We performed two experiments. In Experiment 1, FK-506 was given intramuscularly on Days 11-20 after the first immunization. The rats were immunized twice (on Day 0 and Day 7). They were injected subcutaneously in the footpads with 1.0 mg of human cardiac myosin in equal volumes of complete Freund's adjuvant supplemented with Mycobacterium tuberculosis. They were divided into four groups: Control (six rats, saline), group 1 (six rats, FK-506: 0.1 mg/kg/day), group 2 (seven rats, FK-506: 0.32 mg/kg/day), and group 3 (six rats, FK-506: 1.0 mg/kg/day). To investigate the histologic extent of myocarditis, we formulated a histologic score (0-3). Histologic scores were: Control, 1.90 +/- 0.14; group 1, 0.97 +/- 0.46; group 2, 0.03 +/- 0.05; and group 3, 0 +/- 0. The indices of heart weight/body weight were: Control, 0.74 +/- 0.10%; group 1, 0.45 +/- 0.05%; group 2, 0.35 +/- 0.03%; and group 3, 0.35 +/- 0.03%. In Experiment 2, FK-506 was given on Days 1-10 after the first immunization, earlier than in Experiment 1. The rats were similarly divided into four groups. Each group was given the same dose of FK-506 as in Experiment 1. Histologic scores were: Control 1.49 +/- 0.24; group 1, 1.60 +/- 0.22; group 2, 0.29 +/- 0.41; and group 3, 0.03 +/- 0.03. The indices of heart weight/body weight were: Control, 0.69 +/- 0.15%; group 1, 0.76 +/- 0.09%; group 2, 0.42 +/- 0.08%; and group 3, 0.37 +/- 0.03%. Accordingly, in Experiments 1 and 2, the effects of FK-506 on autoimmune myocarditis were dose-dependent. On the other hand, in Experiments 1 and 2, not only in the control group but also in all treated groups, the titers of anti-myosin IgG were high. In conclusion, even if it is administered just before the onset of myocarditis, FK-506 is extremely effective at suppressing autoimmune myocarditis, despite a high titer of anti-myosin IgG.

Animals

Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactors.

Foam formation and the subsequent cell damage/losses in the foam layer were found to be the major problems affecting cell growth and monoclonal antibody (MAb) production in stirred and sparged bioreactors for both serum-supplemented and serum-free media. Surfactants in the culture media had a profound effect on cell growth by changing both the properties of bubbles and the qualities of foam formed. Comparable cell growth and MAb production in sparged bioreactors and in stirred and surface-aerated control cultures were observed only in Pluronic F-68 containing culture media. In media devoid of Pluronic F-68, cells became more sensitive to direct bubble aeration in the presence of antifoam agent which was used to suppress foam formation. Compared with serum-supplemented medium, more severe cell damage effects were observed in serum-free medium. In addition, serum-free medium devoid of cells was partially degraded under continuous air sparging. The mechanism of this damage effect was not clear. Pluronic F-68 provided protective effect to cells but not to the medium. A theoretical model based on the surface active properties of Pluronic F-68 was proposed to account for its protective effect on cell growth. Optimum media surfactant composition in terms of maximum cell growth and minimum foam formation was proposed for stirred and sparged animal cell bioreactor.

Animals

Developmental and age-related changes in the D2 dopamine receptor mRNA subtypes in rat brain.

The influence of ontogeny and aging on the D2 dopamine receptor mRNA in rat brain were examined using in situ hybridization histochemistry and Northern analysis utilizing oligonucleotide probes complementary to the different D2 mRNA subtypes. At birth, there was a high level of D2 dopamine receptor mRNA in corpus striatum relative to that found in the cerebral cortex and other brain areas. The hybridization signal of striatum (using a probe that hybridizes to both the D2A and D2B mRNA) increased during the first two postnatal weeks, reached a peak at day 16, then declined slightly. The D2A mRNA showed a similar distribution and developmental pattern. Intracisternal injection of 6-hydroxydopamine into neonates did not significantly alter the increase of the D2 dopamine receptor mRNAs, suggesting that neuronal input does not influence the ontogenetic development of this mRNA. In striatum, olfactory tubercule and inferior colliculus, the D2A mRNA declined between 3 and 24 months of age. By contrast, there was an age-related increase in the D2A mRNA in the anterior and intermediate lobes of the pituitary. The mRNA for the D2B dopamine receptor showed very low but nevertheless detectable levels in striatum, olfactory tubercule and pituitary. Like with the D2A mRNA, in 24-month-old rats the D2B mRNA declined in striatum and olfactory tubercule and increased in pituitary. These results show that there are differential tissue-related changes in the mRNAs for the D2 dopamine receptor during both development and aging.

Aging

Determination of butyltin species in sewage and sludge by gas chromatography-atomic absorption spectrometry.

A method for the extraction and determination of butyltin compounds in sewage and sludge is reported. Sewage and sludge samples are acidified and shaken for 2 h. The various butyltin species are extracted quantitatively by tropolone (cycloheptatrienone) in toluene, followed by ethylation to their tetraalkyl-substituted forms, BuSnEt3, Bu2SnEt2, Bu3SnEt and Et4Sn, all of which can be separated and determined by a gas chromatographic-atomic absorption spectrometric technique. The non-pesticidal octyltin species and acid-leachable SnIV species can also be determined by this method. Detection limits expressed as Sn are 40 ng dm-3 and 2 ng g-1 dry mass for sewage and sludge, respectively. Analyses of some samples from Canadian treatment plants are given.

Chromatography, Gas

Inhibitory effect of tetrandrine on lens proteins-induced ocular inflammation in rabbits.

Ocular inflammation was induced by 25 microliters of lens proteins (62.5mg/ml) injected into anterior chamber of the rabbit eye. Tetrandrine (Tet) (50mg/kg ip) and Indomethacin (Ind) (20 mg/kg ip) showed marked inhibition on this ocular inflammation. Maximum inhibition rate of Tet and Ind was 65% and 66% and their anti-inflammation action lasted 5 and 4 h, respectively. In the early phase of ocular inflammation (at 2 h) the total content of prostaglandin E(PGES) in the iris was reduced by Tet and Ind. Ind showed a greater effect than Tet on PGES. Tet also inhibited leukocyte chemotaxis significantly at late phase of inflammation. No significant effect was observed on the IOP recovery following reduction by 20% NaCl iv. Topical instillation of 50 microliters of 2% Tet did not display any inhibition of ocular inflammation. These results indicate that Tet is an effective ocular antiinflammatory agent without producing ocular hypertension. The antiinflammatory mechanism of Tet in early phase (at 2 h) was related to inhibition of PGES synthesis. The relationship between ocular anti-inflammation and calcium antagonism of Tet was discussed.

Alkaloids