Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian,halocynthia roretzi

The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl₂. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH₂Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH₂Cl, and tosyl-Phe-CH₂Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.     

Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

The use of volatile cues in recognition of kin eggs by predatory mites

1. Several animal species are known to distinguish between their own eggs and eggs of unrelated conspecifics. However, the cues involved in this discrimination are often unknown. These cues were studied using the predatory mite Gynaeseius liturivorus Ehara.
2. Adult females of these predatory mites oviposit in clusters and avoid oviposition close to eggs laid by other females, resulting in reduced cannibalism between offspring. Because predatory mites are blind, it was tested whether volatiles of eggs were used as a cue for egg recognition.
3. Adult female predatory mites were offered volatile cues of their own eggs and of unrelated conspecific eggs, and females were prevented from contacting the eggs. Predatory mites oviposited closer to their own eggs than to unrelated eggs. This preference was observed even when one own and one unrelated egg were offered as a volatile source.
4. These results suggest that adult female predatory mites can determine kinship using volatiles released from the eggs.

     

Induction of ovulation, mating, and conception in androgen-treated immature rats

Attempts were made to induce pregnancy in androgen-treated immature rats. Treatment with PMSG alone, which causes ovulation in normal immature rats, failed to cause ovulation in androgenized rats. However, treatment with PMSG plus LHRH was effective in causing ovulation. After ovulation, some of the normal and androgenized rats mated. Normal mated rats became pregnant but androgenized mated rats did not. However, when a pituitary gland was transplanted from a normal rat into the kidney capsule of an androgenized rat to maintain functional corpora lutea, implantation occurred in some of the mated animals. The positive decidual reaction in the uteri of such androgenized rats was similar to that observed in normal rats. These results suggest that the uterine sensitivity to blastocyst implantation of androgenized immature rats may be normal.     

Cytogenetic studies on 1,1-dichloroethylene and its two isomers in mammalian cells in vitro and in vivo

Chromosomal aberration and sister-chromatid exchange (SCE) tests in vitro on 1,1-dichloroethylene (1,1-DCE), its two isomers, cis- and trans-1,2-DCE, and two possible metabolites of 1,1-DCE, chloroacetyl chloride and chloroacetic acid, were carried out using a Chinese hamster cell line, CHL. 1,1-DCE induced chromosomal aberrations in the presence of S9 mix prepared from the rat liver, but not in the absence of S9 mix. SCEs were also slightly induced by 1,1-DCE only in the presence of S9 mix. On the other hand, two isomers and two metabolites of 1,1-DCE induced neither chromosomal aberrations nor SCEs with and without S9 mix. 1,1-DCE, however, was negative even at a sublethal dose in the micronucleus test using mouse bone marrow, fetal liver and blood.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

Uncoupling of Rat Cerebral Cortical α2-Adrenoceptors from GTP-Binding Proteins by N-Ethylmaleimide

Pretreatment of membranes from rat cerebral cortex with N-ethylmaleimide (NEM) decreased [3H]-clonidine binding in a concentration-dependent manner. The Bmax values of high-affinity sites for [3H]clonidine were reduced by 50 microM NEM treatment. Treatment with 500 microM NEM diminished the sum of Bmax of both high- and low-affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM-treated membranes. The addition of purified GTP-binding proteins caused an increase in the binding to the membranes pretreated with 50 microM NEM, but did not increase [3H]-clonidine binding in membranes treated with 500 microM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)-catalyzed ADP ribosylation of membrane-bound (41,000-dalton) and purified (39,000/41,000-dalton) GTP-binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, alpha 2-adrenoceptor, and GTP-binding protein. One is a highly sensitive site to NEM (a concentration range of 1-50 microM), which is probably a cysteine residue, IAP-catalyzed ADP-ribosylating site on the alpha-subunit of GTP-binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1-1 mM), and are the binding domain of agonist and/or the coupling domain of GTP-binding protein on the alpha 2-adrenoceptor. In addition, Ki-ras p21 protein may lack the capacity to couple with the alpha 2-adrenoceptor.     

Growth inhibitory and lethal effects of ethanol on Escherichia coli

The growth inhibitory and lethal effects of ethanol on Escherichia coli BB were investigated in batch cultures, by measuring total cell number, viable cell number, and cell mass concentration. Ethanol below ca. 50 g/L allowed exponential growth but depressed the specific growth rate. The effect of ethanol on the specific growth rate appeared to follow noncompetitive inhibition kinetics with apparently cooperative binding with a Hill coefficient of 2.5. The Hill coefficient and the inhibition constant were temperature independent over the range tested. Ethanol at 30 g/L decreased the growth yield. Ethanol enhanced the specific death rate in an experimental way. Stationary cell populations were more resistant than exponential ones but the degree of enhancement by ethanol was the same in both populations. Isopropanol and propanol also enhanced the specific death rate exponentially and the degree of enhancement was correlatedwith their membrane-buffer partition coefficients.     

Effects of multiple LH injections on the secretion of estrogens from polycystic ovaries of androgen-sterilized rats

Differences in the secretion of estrogens by follicular polycystic ovaries of androgen-sterilized rats and by normal follicular ovaries of early proestrous rats were compared. Some rats were injected i.v. with LH 30 min before bleeding. This injection of LH did not influence the secretion of estrogens by normal ovaries, but greatly increased that by polycystic ovaries, suggesting that there was abnormal steroidogenesis in cystic ovaries. In the ovaries of such androgen-sterilized rats, two types of enlarged abnormal follicles were seen. One of these was truly cystic with few or no granulosa cells (1st type). The other had a hyperplastic and infolded layer of granulosa cells with a papillary appearance (2nd type). Because it is known that the preovulatory LH surge is not found in androgen-sterilized rats, a classical approach was taken to circumvent the probable deficit in cyclic release of LH by giving an i.v. injection of LH every 4 days for 16 days, and ovarian venous blood was collected 4 days after the last injection. In consequence the 2nd type of abnormal follicle disappeared as did the abnormalities of estrogen production. These results suggest that the abnormalities of estrogen production by the polycystic ovaries of androgen-sterilized rats may be due to the 2nd type of abnormal follicle.