Gebhard F.B. Schumacher

Radial diffusion in gel for micro determination of enzymes: II. Plasminogen activator, elastase, and nonspecific proteases☆

Abstract The principle of radial diffusion in substrate containing agar gel has been applied for quantitation of proteolytic activity in small volumes (2 μl). Urokinase, elastase, α-chymotrypsin, trypsin, and other proteases have been investigated. Clearing zones in the agar gel indicate the presence of the enzyme. A linear relationship was found between the logarithm of the proteinase concentrations and the diameters of the clearing zones for urokinase, elastin, and α-chymotrypsin. The trypsin standard curve represents a curved line in the semilogarithmic system. The reproducibility of these micro methods is satisfactory. They are suitable for the analysis of biological fluids, especially viscous secretions, and for screening of large numbers of chromatography fractions. The micro radial diffusion of enzymes in gel is a simple and time-saving method that does not require expensive equipment.

Radial diffusion in gel for micro determination of enzymes

Abstract The principle of radial diffusion in substrate containing agar gel has been applied for the quantitative assessment of several enzymes. Muramidase, alpha-amylase, DNase I, RNase A, acid phosphatase, and alkaline phosphatase have been investigated. Clearing zones in the opalescent agar, staining of the substrate incorporated in the agar, or a colored insoluble hydrolysis product indicate the diffusion zone of the enzyme. A linear relationship was found between the logarithm of the enzyme concentration and the corresponding diameters of the diffusion zones over a wide range. Standard dilutions of the different enzymes are used as reference. The methods have been elaborated for the quantitative assessment of these enzymes in biological material on a micro scale. The wells in the gel hold a volume of 2 μl under standard conditions. The wells can function as volumetric vessels, especially in case of investigations on very viscous mucoid material, which cannot be delivered accurately by a micropipet. The diffusible enzyme of small tissue pieces can also be estimated as well as enzymes in micro homogenates. The method has been found very helpful in screening large numbers of chromatography fractions. The necessary equipment is simple and inexpensive.

Immune Response after Vaginal Application of Antigens in the Rhesus Monkey

The immune response after vaginal application of antigens was investigated in six sexually mature female rhesus monkeys. Two model antigens, i.e., lipopolysaccharide (LPS) OF Salmonella typhosa and abortive type T-4 coliphages were applied with or without adjuvant. A plastic sponge used as the antigen carrier was introduced into the upper vagina and placed against the ectocervix. For primary immunization, each monkey received 18 vaginal antigen applications and 10 applications for each booster course. For comparison, three other female rhesus monkeys were immunized systemically. Alum or LPS was used as adjuvant. Blood was obtained two times and cervical mucus three times weekly from each monkey. Antibodies were only barely detectable in cervical mucus after the primary vaginal immunization. However, booster treatments resulted in definite antibody responses. Specific antibodies were also detected in the circulating blood after vaginal booster immunization. The antibody level in cervical secretion in three of four cases was higher than that in circulatin blood. Systemic immunization resulted in high levels of circulating antibodies, but less than 10% appeared in cervical secretions. A characteristic decrease in antibody levels in cervical mucus was usually observed at midcycle after local immunization as well as after systemic immunization. More than 90% of T-4 coliphages applied vaginally were absorbed within 48 hours. Although alum appeared to retard the absorption of antigens, it seemed to enhance the local response. More than 90% of the antibodies to the T-4 coliphages could be removed from the serum and cervial mucus by treatment with anti-immunoglobuin G antiserum. The lymphocyte response to antigens was studied by measuring the 3H-thymidine uptake by peripheral blood lymphocytes in culture. A positive response was observed in three of three systemically immunized and in only two of six locally immunized aminals. In general, the immune response was significantly weaker after local vaginal immunization than after systemic immunization.

Observation of two proacrosins in extracts of human spermatozoa

The observation of 2 proacrosins in extracts of human spermatozoa is described. When spermatozoa were extracted in the presence of .05 M benzamidine the resulting solutions reveled a time-dependent, sigmoidal increase of trypsinlike activity upon incubation at pH 8. Gel permeation chromatography of those extracts separated 2 species, P1 and P2, with apparent molecular weights of 75000 and 42000, respectively. Both species are autoactivatable at pH 7-8 and the kinetic parameters of activated P1 and P2 are indistinguishable from those of human acrosin. That both are inactive precursors of human acrosin is shown by the fact that in the presence of benzamidine they are obtained instead of and in greater yield than acrosin. The features of the activation process show that P1 and P2 are zymogens.

Serum proteins in cervical mucus

Abstract The occurrence of serum proteins in human cervical mucus was investigated in 41 cases using immunoelectrophoresis. Albumin, transferrin, and gamma globulin were always found in specimens from normal women, and in more than half of the cases alpha 1 antitrypsin and prealbumin were present. There were no significant differences between specimens from the eighth to the thirteenth day, from the fifteenth to the eighteenth day, or from the twenty-first to the twenty-eighth day of the menstrual cycle. The pattern of the cervical mucus from women taking Enovid as an oral contraceptive showed only slight differences: One alpha 2 globulin line was observed addition-ally in a number of cases. There was no indication of a change in the immunoelectrophoretic pattern of the cervical mucus from a surgical castrate under hormonal treatment, whereas the cytologic patterns showed typical changes. Unlike the group of normal women and those under Enovid treatment, the benzidine reaction, indicating the presence of haptoglobin, was positive in most of the cases with local pathologic changes. In cases of cervical erosion, cervicitis, or pelvic inflammatory diseases a higher number of precipitin lines was disclosed in conjunction with a remarkable increase in leukocytes. The limits of the methods are thoroughly discussed together with the significance of the findings.

Structural aspects of human cervical mucus

Two types of fibrous structures can be demonstrated in midcycle cervical mucus: (1) long, thick fibers that vary in diameter from 0.5 to 5 mum and run parallel to each other, and (2) microfibrils that vary in diameter from 500 to 1,500 A and form bundles or networks. The spaces in such networks usually measure from 800 to 4,0000 A. The fibers are made up of the microfibrils and most likely represent the micelles that give spermatozoa their directional transport through the cervix.

Estrogen and progestin binding and changes in secretions by human cervical tissue during the ovarian cycle.

Functional events in the cervix were correlated with sex steroid receptor activity and serum concentrations of sex steroids by measuring cytoplasmic estrogen and progestin binding capacities in cervices collected at hysterectomies performed during the proliferative, midcycle, and secretory phases. Ovarian endocrine status was determined from morphologic criteria for the endometrium as well as from serum concentrations of estrogens and progesterone. Also measured as indices of ovarian endocrine status and cervical function were concentrations of lysozyme, albumin and immunoglobulin G (IgG) in cervical mucus. Early to midfollicur phase specimens had progestin binding in cervical cytosol of 2.3+ or -.6 fmol, and in luteal phase specimens this binding was less than .3 fmol. There were no differences in cytoplasmic estrogen-binding capacities among the 3 phases. Serum estradiol levels were higher (P .05) for luteal phase specimens than for early to midfollicular phase specimens, and the IG, albumin, and lysozyme concentrations of cervical mucus were similar for luteal and early to midfollicular phase specimens, surprisingly. These finding support an hypothesis that progestins reduce cellular responsiveness to the estrogen-receptor complex.

Primate model for the evaluation of vaginal contraceptives

A good animal model that simulates the human subject has not been available for the evaluation of the in vivo effectiveness of vaginal contraceptives. After careful consideration. The stumptailed macaque (Macaca arctoides) was studied for its applicability since it has a reproductive tract similar to that of the woman, is easy to handle, does not require tranquilization or anesthesia when the contraceptive is deposited, and breeds and conceives readily under caged conditions. The reported observations show the usefulness of this animal. Both postcoital sperm motility studies and breeding experiments were performed with the use of Delfen vaginal cream and K-Y jelly. K-Y jelly had no effect on the motility of vaginal spermatozoa or on the conception rate of the primates. Although Delfen vaginal cream consistently immobilized all spermatozoa in the postcoital test, half of the animals became pregnant within an average of 3.7 breeding cycles. These results illustrate the discrepancy between spermicidal tests and fertility measurements, and it is recommended that primate-breeding experiments be performed before a spermicide is evaluated in women as a contraceptive.

Specific antibodies and immunoglobulins in the oviductal fluid of the rhesus monkey.

Levels of specific antibodies against model antigens, immunoglobulins G and A and also albumin, in oviductal fluid were studied in the rhesus monkey during the periovulatory period. Animals were systemically or intravaginally immunized against T4 coliphages. Attempts to induce ovulation were made with human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) monitored by radioimmunoassay of serum estrogen and progesterone. Collection of tubal fluid over 6 to 13 days was accomplished by surgical cannulation using a refrigerated extracorporeal collection device for each side. The results indicated the following: (1) The levels of specific antibodies against T4 coliphage and immunoglobulins (IgG, IgA) in the oviduct fluid averaged approximately one tenth of the serum values and showed a characteristic decrease and subsequent increase by a factor of 4 to 5 during and following treatment with hMG/hCG. The nadir was observed on the first or second day after hCG injection. (2) This pattern was similar in both ovulatory or nonovulatory cycles; therefore, these changes seem to be associated with the changes in serum estrogen levels. (3) There was a striking difference in serum and tubal fluid antibody levels after systemic versus after vaginal immunization by a factor of 10(3) and 10(4); however, the patterns in tubal fluid under treatment with hMG/hCG were very similar. (4) Specific antibodies in oviductal fluid and serum were mainly of the IgG class. (5) A concomitant change of total protein and albumin in oviduct fluid was also observed. The presence of sperm agglutination antibody in oviductal fluid was demonstrated in two monkeys after systemic immunization with homologous spermatozoa. The sperm antibody titers showed a similar pattern of change after hMG/hCG treatment.

Inhibition of rabbit sperm acrosomal protease by human alpha1-antitrypsin and other protease inhibitors

Abstract Human alpha 1 -antitrypsin preparations inhibit rabbit sperm acrosomal protease using BAEE and gelatin as substrate. In BAEE tests, the alpha 1 -antitrypsin shows a progressive kinetics of inhibition in contrast to the trypsin kallikrein inhibitor from bovine lung that reacts immediately. Human inter-alpha-trypsin inhibitor also inhibits rabbit sperm acrosomal protease. The reaction is of the immediate type but less extensive in the BAEE test. Human alpha 1x -antichymotrypsin does not inhibit rabbit sperm acrosomal protease.