Screening of in Vitro Inhibition of Lactoperoxidase Enzyme by Methyl Benzoate Derivatives with Molecular Docking Studies

Lactoperoxidase enzyme (LPO) is secreted from salivary, mammary, and other mucosal glands including the bronchi, lungs, and nose, which had functions as a natural and the first line of defense towards viruses and bacteria. In this study, methyl benzoates were examined in LPO enzyme activity. Methyl benzoates are used as precursors in the synthesis of aminobenzohydrazides used as LPO inhibitors. For this purpose, LPO was purified in a single step using sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography with a yield of 9.91 % from cow milk. Also, some inhibition parameters including the half maximal inhibitory concentration (IC₅₀) value and an inhibition constant (K_i) values of methyl benzoates were determined. These compounds inhibited LPO with K_i values ranging from 0.033±0.004 to 1540.011±460.020 μM. Compound 1 a (methyl 2-amino-3-bromobenzoate) showed the best inhibition (K_i=0.033±0.004 μM). The most potent inhibitor ( 1 a ) showed with a docking score of −3.36 kcal/mol and an MM-GBSA value of −25.05 kcal/mol, of these methyl benzoate derivatives ( 1 a – 16 a ) series are established H-bond within the binding cavity with residues Asp108 (distance of 1.79 Å), Ala114 (distance of 2.64 Å), and His351 (distance of 2.12 Å).     

Effect of 41-CRF antiserum on the secretion of ACTH, B-endorphin and alpha-MSH in the rat

In order to elucidate the physiological role of the 41 amino-acid residue corticotropin-releasing factor (41-CRF) on the secretion of ACTH, B-Endorphin and alpha-MSH, plasma levels of these peptides were measured by radioimmunoassay in intact and adrenalectomized rats, two hours after the injection of either 41-CRF antiserum (CRF-AS) or normal rabbit serum for controls. The administration of CRF-AS strikingly lowered the plasma ACTH levels in both intact and adrenalectomized rats. A statistically significant reduction of plasma levels of B-Endorphin was also observed in the same rats. However, the effect of CRF-AS on B-Endorphin release was less pronounced than the effect on ACTH release. No changes in plasma alpha-MSH levels were observed after passive immunization with CRF-AS. We conclude that, in the rat, 41-CRF plays a physiological role in the regulation of ACTH and B-Endorphin secretion, but is not involved in the regulation of alpha-MSH release from the pituitary gland.     

Distribution and localization of neurokinin A-like immunoreactivity and neurokinin B-like immunoreactivity in rat peripheral tissue

Using specific radioimmunoassays and immunocytochemistry for neurokinin A (NKA) and neurokinin B (NKB), distribution and localization of these peptides in rat peripheral tissues were studied. NKA-like immunoreactivity (NKA-LI) was present in highest levels of 15.7–23.9 pmol/g wet wt. and NKB-like immunoreactivity (NKB-LI) was in levels of 0.33–0.67 pmol/g wet wt., throughout the gastrointestinal tract involving stomach, duodenum, jejunum, ileum and colon. Immunocytochemical analysis of gastrointestinal tract revealed that NKA-LI and NKB-LI localized in ganglia of both the submucosal and myenteric plexuses as well as varicose neurons in the mucosa and the muscle layer of the small and large intestine. On the other hand, high levels of NKB-LI were observed in oesophagus (0.83 ± 0.08 pmol/g wet wt.), adrenal (1.02 ± 0.21), head of pancreas (0.73 ± 0.06) and kidney (0.98 ± 0.05).

The present study shows the difference of localization of NKA-LI and NKB-LI in peripheral tissues and suggests that NKB may have some physiological role differing from that of NKA in peripheral tissues.     

Plasmodium berghei: Use of free blood stage parasites to demonstrate protective humoral activity in the serum of recovered rats

Free infectious Plasmodium berghei parasites (FP) were used in a system suitable for measurement of protective antibody in the serum of rats recovered from malaria. By the fluorescent antibody technique it was demonstrated that the free parasites, but not parasites in erythrocytes, became coated with antibody after incubation in recovered rat serum. Because immune sera capable of coating free parasites did not protect mice against FP inocula, but partially or completely protected rats, it is probable that antibody coating alone is not sufficient to kill the parasites. It was further demonstrated in vitro, with one strain of P. berghei, that phagocytes more readily ingested parasites in the presence of immune serum than in the presence of normal serum. This observation suggests that phagocytosis of the antibody coated parasite probably was required to prevent infection.     

Characterization and tissue distribution of a novel human cytochrome P450-CYP2U1

A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and the corresponding gene is located on chromosome 4. The deduced 544 amino acid sequence displays up to 39% identity to other CYP2 family members, with closest resemblance to CYP2R1 and is highly conserved between species. CYP2U1 shows some structural differences compared to other CYP2 family members. The gene has only five exons and the enzyme harbors two insertions in the N-terminal region. Northern blot analysis revealed high mRNA expression in human thymus, with weaker expression in heart and brain, whereas in the rat similar mRNA levels were detected in thymus and brain. Western blot analysis revealed much higher CYP2U1 protein expression in rat brain than in thymus, particularly in limbic structures and in cortex. The physiological and toxicological role of this novel P450 is still unknown, but the selective tissue distribution suggests an important endogenous function.     

Production and Characterization of a New Specific Antiserum Against the Taurine Putative Biosynthetic Enzyme Cysteine Sulfinate Decarboxylase

Abstract: We have shown previously that cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme of taurine in the brain, is identical to the liver enzyme according to biochemical, kinetic, and immunochemical criteria. In the present work, CSD was purified in its native form from rat liver. The purification was performed in eight steps, which included conventional chromatography (diethylaminoethyl cellulose, hydroxylapatite), followed by HPLC (hydrophobic, adsorption, and ion-exchange HPLC). The purification factor was 11,000, and the final yield was around 2%. The procedure led to the enrichment of a protein, the molecular mass of which was 51,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final fraction was more than 90% homogeneous. By using this fraction as the antigen, an antiserum was raised in rabbit that (a) quantitatively immunoprecipitated CSD activity from liver and brain extract, and (b) immunolabeled one band (51,000 daltons) on immunoblots of partially purified fractions from liver. Enrichment of CSD specific activity and that of the protein immunolabeled by the antiserum for a given step, e.g., hydrophobic HPLC, were consistently parallel. The antiserum was used to carry out CSD immunocytochemistry in cerebellum. Numerous small cells were labeled in the Purkinje cell layer, the granular layer, and the white matter. In the molecular layer, Bergmann radial fibers were im munostained. The Purkinje and stellate cells were devoid of any labeling at the cell body and terminal levels. The antiserum appears to be specific for CSD and suitable for immunocytochemical visualization of CSD in the brain.     

Use of mucin and hemoglobin in experimental murine Gram-negative bacteremia enhances the immunoprotective action of antibodies reactive with the lipopolysaccharide core region

An antiserum with a high content of antibodies, binding to the Gram-negative lipopolysaccharide core region, was prepared by immunizing rabbits with the rough Escherichia coli mutant J5. This antiserum was capable of protecting mice against lethal challenge doses of E. coli 0 111: B4 in a mouse model where the animals were compromised by means of mucin plus hemoglobin (LD 50=10³ bacteria). However, no protection was observed in a non-compromised mouse model (LD 50=10⁷ bacteria). This observation might explain why in the past so many discrepant results have been obtained in mouse protection studies with cross-reactive antisera.Department of Medical Microbiology, Research Group for Commensal Infections     

Antiserum directed against cell surface antigens is lethal toLeishmania donovani promastigotes

The purified flagellar fraction ofLeishmania donovani promastigotes consists of 30–35 polypeptides. Antiserum raised against this fraction reacts with both flagella and pellicular membrane antigens as evident from immunoblot and immunofluorescence studies. Only 3 of these immunoreactive polypeptides are flagellum-specific. The antiserum agglutinates the cells and inhibits their growth in liquid culture medium. Moreover, glucose uptake and glucose-stimulated oxygen uptake of the promastigotes are significantly inhibited by the antiserum. The results indicate that the antiserum has a profound lethal effect on the invitro propagation of the parasite.     

A polyclonal antiserum against the rabbit progesterone receptor recognizes the human receptor: biochemical characterization

Polyclonal antiserum was generated in guinea pigs immunized with the 116,000 Mr rabbit uterine progesterone receptor (PR). The PR antigen was partially purified by DEAE-cellulose chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and the 116,000 Mr band excised and injected into guinea pigs. The antiserum recognized on protein blots rabbit uterine PR of Mr 116,000 and 81,000. The antiserum was judged to be specific for PR from normal and malignant human tissues as determined by sedimentation shift on sucrose gradients, immunoprecipitation studies, protein blotting, and fluorographic analysis using photolabelled samples. Comparison of protein blots probed with this polyclonal antiserum or with a recently obtained monoclonal antibody to human PR indicated that similar PR structures were recognized in rabbit and human samples by both antisera. Characterization of the polyclonal antiserum has demonstrated its suitability for investigating the immunolocalization or PR in normal and malignant human tissues as well as the receptor structure detected on protein blots.     

Interaction of L antibody with low potassium-type sheep red cells: Resolution of two separate functional antibodies

Summary Antibodies of two specificities in alloimmune sheep anti-L sera, anti-L _P and anti-L _l , were separated by a new technique and characterized. Absorption of anti-L serum with trypsinized LK (LL) sheep red cells left anti-L _P antibodies; the absorbed anti-L _l antibodies were then eluted. Anti-L _P was only weakly lytic in the presence of complement; it had no effect on passive K influx, but stimulated active K influx. The stimulation could be reversed by eluting the antibody in glycine buffer at low pH. Stimulatory activity in the eluted cells could be restored by resensitization with anti-L _P . Anti-L _l was more strongly lytic than anti-L _P in the presence of complement; it had no effect on active K influx, but inhibited passive K influx. Pig anti-ruminant IgG conjugated to hemocyanin was used to visualize by electron microscopy the number of L _P and L _l antigen sites onLL sheep red cells sensitized with anti-L _P and anti-L _l . The values obtained were 590 L _P sites/cell and 847 L _l sites/cell.