Characterization and immunohistochemical localization of rat salivary cobalamin-binding protein and comparison with human salivary haptocorrin.

Rat saliva contains a cobalamin-binding protein that binds cobalamin as well as cobinamide. The protein binds cobalamin with an affinity constant of 8 X 10(10) l X mol-1, and it binds cobalamin over a more narrow pH range (pH 7.5-10) than does human haptocorrin. It has a Stokes radius of 2.45 nm as compared to the Stokes radius of 4.50 nm for human haptocorrin. Upon isoelectricfocusing it dissociates into four strong bands with pI between 7 and 8, while human haptocorrin dissociates into acid isoproteins. Since human haptocorrin binds to concanavalin A while rat haptocorrin does not, we suggest that rat haptocorrin lacks carbohydrate. The substance concentration of rat saliva haptocorrin is 0.04-12.9 nmol X l-1 (median 7.5 nmol X l-1, n = 9) for control animals. After stimulation with isoproterenol, a beta-adrenergic agent, the substance concentration is 46.4-96.6 nmol X l-1 (median 69.7 nmol X l-1, n = 8). Immunohistochemical studies show haptocorrin in the secretory acini of the submandibular and parotid glands of the rat. In the human submandibular gland, the protein is detected both in the mucous secretory acini and in the intercalated ducts.

Excess antibody immunoassay for rat glandular kallikrein. Monosized polymer particles as the preferred solid phase material.

The development of an excess antibody assay for rat glandular kallikrein is described. This assay permits immunological determination of kallikrein as well as a simultaneous specific measurement of kallikrein enzymatic activity. The assay is based on coupling of immunopurified anti-kallikrein immunoglobulin to a solid phase. In a first incubation step, kallikrein was bound to the immobilized antibody. Determination of kallikrein was subsequently done in a second incubation step; immunologically by addition of iodinated anti-kallikrein antibody, or enzymatically by a kallikrein substrate. Enzymatic quantification could also be followed by immunological measurements on the same sample. Comparison of Sepharose, cellulose, and acrylate based polymer particles proved the latter to be the best matrix in this assay. The main advantage of the polymer particles was the low non-specific binding of labelled antibody.

Analysis of S-35 labeled WR-2721 and its metabolites in biological fluids.

Studies with WR-2721 and related compounds have been hindered by the lack of a suitable assay for the drug and its major metabolites. We have developed a chromatographic method which requires no derivatization for the separation and detection of WR-2721, the free thiol, its symmetrical disulfide and other mixed disulfides. Our procedure involves ion-pairing for separation of ionizable compounds by causing polar molecules to become more lipophilic and hence separable using reverse phase HPLC. Detection is based upon liquid scintillation counting of S-35 incorporated during the synthesis of the parent compound. This method requires no pre-column preparation of samples and, by detecting the S-35 label, eliminates the chance that a coeluting species could interfere with detection, as might occur with post-column derivatization. Chromatography was done using a 10 micron C8RP column and 35% MeOH/65% 0.0113M NaH2PO4, 0.005 M hexanesulfonate, pH 5.9, flowing at 1 ml/min. Half-minute fractions were collected into scintillation vials for counting. Retention volumes for the various compounds were: column breakthrough (3.5 ml), WR-2721 (4.5 ml), WR-1065 (9 ml), and WR-33278 (24 ml). This analytical technique employing radiotracers can be used to study radioprotective mechanisms by time dependent measurements of the tissue distribution and chemical form of labeled drug. Such chemical information can then be correlated with biological measures of radiation protection.

The isolation, purification and some properties of pheromaxein, the pheromonal steroid-binding protein, in porcine submaxillary glands and saliva.

Pheromaxein, the 16-androstene steroid-binding protein with a relative molecular mass of 15,000 was isolated in sub-milligram quantities from the submaxillary gland and saliva of the Gottingen miniature boar, after a fourfold purification involving the following methods: ultrafiltration for submaxillary gland cytosols and ethanol precipitation for saliva, Concanavalin-A-Sepharose affinity chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, 'Extractigel-D' affinity chromatography (to remove sodium dodecyl sulphate) and fast protein-liquid chromatography. Yields of purified pheromaxein obtained after fast protein-liquid chromatography represented 10-20% of total protein present in an ultrafiltrate of a submaxillary gland cytosol. Fast protein-liquid chromatography separated the alpha- and beta-charge isomers of pheromaxein which were shown to have isoelectric points of 4.78 and 5.35 respectively on flat-bed isoelectric focusing. Some data are provided for the variable occurrence of the isomeric forms of pheromaxein in relation to different breeds of pig. Five 16-unsaturated steroids showed the highest binding to pheromaxein. Other steroids of the 5 alpha- and 5 beta-androstane series also showed some binding to pheromaxein, i.e. 17 beta-hydroxy-5 alpha-androstan-3-one (19.2%), with 5 alpha-androstan-3-one, which has a similar urinous odour to 5 alpha-androst-16-en-3-one, showing the greatest binding (42.6%) relative to 5 alpha-androst-16-en-3-one (100%).

Submaxillary secretion in rats treated with guanethidine.

The composition of final submaxillary saliva and of whole gland homogenates were compared in rats treated with 14 daily doses of guanethidine (20 mg/kg body weight) and in untreated controls after stimulation with pilocarpine (10 mg/kg) or carbamylcholine (50-100 mg/kg). A 44% reduction in the volume of saliva and elevations in the salivary concentrations of K+, Ca2+ and protein were found in the treated animals after pilocarpine stimulation. Similarly, a 25% reduction in salivary volume and elevations in salivary Ca2+ and protein concentrations were observed following stimulation with carbamylcholine. A less significant elevation in salivary K+ was seen after this secretagogue. The Na+ and protein contents, but not the K+ and Ca2+ contents, were found to be elevated in the glands of the treated animals in the resting (unstimulated) state. After stimulation with the two secretagogues, however, similar changes in glandular Na+ and K+ contents were found in the glands of control and treated animals. The glandular water content was also similar in both types of glands in the resting and stimulated states. It is concluded that a reduced salivary secretion, rather than supersensitivity, is observed in the rat submaxillary gland following chronic guanethidine administration. The drug treatment does not impair the glandular electrolyte changes that occur upon stimulation, but most likely impairs the release of protein from the gland and also transductal K+ transport. Both these effects may result from the depletion of sympathetic neurotransmitter caused by the guanethidine administration.

Immunochemical identification and determination of proline-rich proteins in salivary secretions, enamel pellicle, and glandular tissue specimens.

Acidic proline-rich proteins were localized histologically by means of the indirect immunoperoxidase technique in the acinar cells of parotid and submandibular gland sections of both man and Macaca fascicularis. These proteins were also identified as constituents of the long term in vivo-formed acquired pellicle on human tooth surfaces employing the same immunoperoxidase technique. Immunological quantitation of proline-rich proteins in whole and parotid saliva of 42 subjects indicates the presence of high concentrations in parotid secretions varying between 19-80 mg%, while their levels in whole saliva are significantly lower, ranging between 0-18 mg%. The concentrations of these proteins in whole saliva exhibited a negative correlation with plaque accumulation (r = -0.464) and gingival index scores (r = -0.576).

The effects of various secretagogues on the mucin content of pure submandibular salivas.

The concentrations of a specific mucin and total protein were compared in pure submandibular saliva samples elicited by pilocarpine (PILO), epinephrine (EPI), and isoproterenol (IPR) in mice. At the dosages employed, IPR-stimulated samples contained, on the average, the highest concentrations of protein and mucin. EPI samples contained intermediate levels, and PILO the lowest concentrations. The average mucin-to-protein ratio in the PILO saliva samples was intermediate between the IPR and EPI samples. Non-stimulated saliva samples showed a broader range of mucin to total protein than did the stimulated saliva sets. Within the PILO saliva samples, there was a very strong positive correlation between the mucin content of saliva and the mucin content in the pre-secretory gland prior to stimulation. The mucin content of EPI and IPR samples also showed strong positive relationships with the content of mucin in the gland. The mucin and protein in EPI saliva samples were significantly correlated in at least one of the collections, whereas in the IPR samples, there was a much weaker relationship. With the exception of the third sequential collection of EPI saliva, all nine of the other EPI, IPR, and PILO collection sets showed only a weakly negative or no correlation between flow rate and saliva mucin or protein content. This is in sharp contrast with the strongly negative correlation which was noted between saliva protein or mucin contents and flow rate when all of the data were combined. These observations suggest that the nature of the stimulant is a very important determinant of overall flow rates, saliva protein, and mucin concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Methotrexate-induced changes in rat parotid and submandibular gland function.

Methotrexate (MTX) was injected intraperitoneally at a dose of 15 mg/kg into adult rats daily for three consecutive days. When compared with saline-injected, ad libitum-fed rats or pair-fed saline-injected rats, MTX decreased unstimulated parotid gland weight, RNA content, and amylase content. RNA content was also decreased in submandibular glands. The parotid and submandibular gland outputs of sodium and chloride, but not that of potassium, were decreased following MTX injection. Ductal perfusion demonstrated decreased net efflux of sodium and chloride from the excretory duct of the submandibular gland. The effects of MTX on unstimulated parotid gland weight, RNA content, and amylase content are consistent with its ability to inhibit protein synthesis through depletion of folate co-factors. However, the mechanism by which the alterations in ion transport occurred is not clear at this time.

Lead distribution in the saliva and blood fractions of rats after intraperitoneal injections.

Sprague-Dawley rats were treated with 1, 2 or 3 i.p. injections of lead acetate (100 mg/kg) and sacrificed 24 h, 3 days, 7 days, 14 days and 21 days after the last injection. Lead concentration was determined by flameless AAS technique in whole blood, plasma, plasma filtrate, saliva and submaxillary gland tissue. The concentration of lead in saliva was about 5% of whole blood lead concentration and around 61% of plasma filtrate lead level. Saliva lead concentration was significantly related both to whole blood lead concentration and plasma filtrate lead concentration (r = 0.78, P less than 0.001; r = 0.80, P = 0.001 respectively). Lead was present in the submaxillary gland tissue; the amount of lead increased with increasing amounts administered.

The parotid gland is the main source of human salivary epidermal growth factor.

To clarify the production of human epidermal growth factor (EGF) by different salivary glands, we measured its concentration by radioimmunoassay separately in whole saliva, in parotid gland (PG) saliva and in mixed submandibular (SMG) and sublingual gland (SLG) saliva. Also, we studied the presence of EGF in PG and SMG by immunohistochemistry. The mean (geometric) concentrations of EGF in PG saliva (2704 pg/ml, +/- SEM interval 2393-3056 pg/ml, n = 20) was higher (p less than 0.001) than in whole saliva (864 pg/ml, +/- 733-1019 pg/ml, n = 29), which in turn was higher (p less than 0.001) than in mixed SMG + SLG saliva (357 pg/ml, +/- 296-430 pg/ml, n = 16). No sex difference existed in any salivary gland EGF. Immunohistochemistry revealed EGF in the acinar cells of both PG and SMG, but only in PG there were prominent EGF deposits in luminal spaces. Our data suggest that EGF is produced by both PG and SMG, but that more of it is secreted from the PG. This result is new and challenges the general view that human salivary EGF is mainly from SMG. In mouse almost all salivary EGF comes from SMG and its amount is androgen dependent. Thus there are great differences in sources and regulation of salivary EGF between man and mouse.

Two-dimensional electrophoresis in the analysis of a mixture of human sublingual and submandibular salivary proteins.

Complex protein mixtures of unstimulated human sublingual and submandibular saliva were fractionated by two-dimensional (2-D) gel electrophoresis, visualized by silver staining and then analysed by immunostaining. Specific proteins were identified by incubation with specific antibody and peroxidase-conjugated second antibody (Western blot). Electrophoresis and silver staining revealed over 50 protein components in 2 microliter of unconcentrated mixture. The Western-blot technique allowed detection of protein spots of plasma origin when an antibody against whole serum was used, but only the albumin spot could be found. Albumin, secretory IgA, acid phosphatase and alpha-amylase were identified with specific antibodies.

[Fundamental and clinical studies of cefaclor in oral surgery].

Fundamental and clinical studies on cefaclor (CCL) have been performed and the following results were obtained. CCL was orally administered to NZW rabbits at the dose of 20 mg/kg, and its concentrations in blood and various tissues of oral organs were determined. Pattern of change in its blood concentration after the administration was similar to that of change in its concentration in the tissues of oral organs. Its concentration in blood was the highest followed by gingiva, parotid gland, submandibular gland, cervical lymph node and tongue in descending order. Comparative studies of CCL against cephalexin (CEX) were conducted in 5 healthy volunteers with cross over method. The 5 volunteers were orally given 500 mg of CCL or CEX at 1 dose after meal. Peak blood levels of CCL and CEX were 14.8 micrograms/ml at 2 hours and 11.5 micrograms/ml at 3 hours, respectively. The dose of 750 or 1,500 mg/day of CCL in 3 divided doses was orally administered to 71 patients with acute purulent infections in oral tissues for 3 to 13 days. Evaluation of effect was determined by the criteria for evaluation of antimicrobial agents in oral surgery. Out of the 70 patients, excellent clinical response was observed in 18 patients, good in 40, and poor in 12. Effective rate was 83%. In vitro antibacterial activities (MIC) of CCL and CEX were studied in 74 out of 81 strains (41 from aerobes and 40 from anaerobes) isolated from 47 patients. CCL showed stronger antibacterial activities than CEX. MICs of CCL against 30 strains of Gram-positive anaerobes were distributed from 0.10 to 3.13 micrograms/ml with a peak of 0.78 micrograms/ml. As adverse reaction due to CCL, eruption was observed in only 1 patient. Laboratory tests in 61 patients who received CCL showed elevation of GOT in 1 patient and elevation of GOT and GPT in 1 patient. From the above fundamental and clinical results, CCL was considered to be a useful antibiotic for the treatment of acute purulent infections caused by aerobes and anaerobes in oral surgery field.