Screening of anti-mycobacterial compounds in a naturally infected zebrafish larvae model.

OBJECTIVES: Mycobacterium tuberculosis is a deadly human pathogen that causes the lung disease TB. M. tuberculosis latently infects a third of the world's population, resulting in ∼1.5 million deaths per year. Due to the difficulties and expense of carrying out animal drug trials using M. tuberculosis and rodents, infections of the zebrafish Danio rerio with Mycobacterium marinum have become a useful surrogate. However, the infection methods described to date require specialized equipment and a high level of operator expertise. METHODS: We investigated whether zebrafish larvae could be naturally infected with bioluminescently labelled M. marinum by immersion, and whether infected larvae could be used for rapid screening of anti-mycobacterial compounds using bioluminescence. We used rifampicin and a variety of nitroimidazole-based next-generation and experimental anti-mycobacterial drugs, selected for their wide range of potencies against M. tuberculosis, to validate this model for anti-mycobacterial drug discovery. RESULTS: We observed that five of the six treatments (rifampicin, pretomanid, delamanid, SN30488 and SN30527) significantly reduced the bioluminescent signal from M. marinum within naturally infected zebrafish larvae. Importantly, these same five treatments also retarded the growth of M. tuberculosis in vitro. In contrast, only three of the six treatments tested (rifampicin, delamanid and SN30527) retarded the growth of M. marinum in vitro. CONCLUSIONS: We have demonstrated that zebrafish larvae naturally infected with bioluminescent M. marinum M can be used for the rapid screening of anti-mycobacterial compounds with readily available equipment and limited expertise. The result is an assay that can be carried out by a wide variety of laboratories for minimal cost and without high levels of zebrafish expertise.

Phytoestrogen metabolites are much more active than phytoestrogens themselves in increasing prostaglandin F(2alpha) synthesis via prostaglanin F(2alpha) synthase-like 2 stimulation in bovine endometrium.

Phytoestrogens have recently been suggested to be the cause of infertility by stimulating luteolytic prostaglandin (PG) F(2alpha) secretion from endometrium in cattle. The purpose of this study was to examine the enzymatic and molecular mechanisms involved in the preferential induction of PGF(2alpha) synthesis by phytoestrogens, and whether phytoestrogens influence endometrial cell viability. Cultured bovine endometrial epithelial and stromal cells were exposed to phytoestrogens (daidzein and genistein) and their metabolites (equol and p-ethyl phenol) for 24h. Prostaglandin F(2alpha) and PGE2 were stimulated by phytoestrogens in both stromal and epithelial cells, with a preference for PGF(2alpha) synthesis in epithelial cells (P<0.001). Although RT-PCR and Western Blot analyses did not reveal the influence of phytoestrogens on either gene expression or protein level of cyclooxygenase-2 (COX-2) and PGE2 synthase (PGES) in stromal and epithelial cells (P>0.05), the stimulative effects of equol and p-ethyl phenol on PGF(2alpha) synthase-like 2 (PGFSL2) gene expression and protein level were observed only in epithelial cells (P<0.05). The same compounds did not affect PGFSL2 gene expression and protein in stromal cells (P>0.05). Exposure to phytoestrogens and their metabolites decreased cell viability in both stromal and epithelial cells. Stromal cell viability decreased to 50% of the control and was more evident than that in epithelial cells (P<0.001). The overall results suggest that infertility in cattle, caused by phytoestrogen-dependent preferential stimulation of luteolytic PGF(2alpha) synthesis, is caused by increasing PGFSL2 in epithelial cells, and by decreasing stromal cell viability, which are the main source of luteotropic PGE2 production.

A comparison of the antibacterial efficacies of essential oils against oral pathogens.

Cariogenic bacteria and periodontopathic bacteria are present in dental plaque as biofilms. In this study, we investigated the antibacterial effects of essential oils on the following oral bacteria: Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Streptococcus mutans, and Streptococcus sobrinus. We tested manuka oil, tea tree oil, eucalyptus oil, lavandula oil, and romarinus oil and determined their minimum inhibitory concentration and minimum bactericidal concentration. The essential oils inhibited the growth of the bacteria tested, manuka oil being the most effective. Minimum bactericidal concentration values showed that lavandula oil acts bacteriostatically, and the remaining oils, bactericidally. Periodontopathic bacterial strains tested were killed completely by exposure for 30 s to 0.2% manuka oil, tea tree oil or eucalyptus oil. Tea tree oil and manuka oil showed significant adhesion-inhibiting activity against P. gingivalis. All the essential oils tested inhibited the adhesion of S. mutans. This study showed that, among the essential oils tested, manuka oil and tea tree oil in particular had strong antibacterial activity against periodontopathic and cariogenic bacteria. From the viewpoint of safety, we also examined the effects of these essential oils on cultured human umbilical vein endothelial cells and found that, at a concentration of 0.2%, they had little effect on cultured cells.

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses.

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.

ARG tyrosine kinase activity is inhibited by STI571.

The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces hematologic remission in most patients with chronic myeloid leukemia. In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF)beta receptor, and c-KIT, but it does not inhibit SRC family kinases, c-FMS, FLT3, the epidermal growth factor receptor, or multiple other tyrosine kinases. ARG is a widely expressed tyrosine kinase that shares substantial sequence identity with c-ABL in the kinase domain and cooperates with ABL to regulate neurulation in the developing mouse embryo. As described here, ARG has recently been implicated in the pathogenesis of leukemia as a fusion partner of TEL. A TEL/ARG fusion was constructed to determine whether ARG can be inhibited by STI571. When expressed in the factor-dependent murine hematopoietic cell line Ba/F3, the TEL/ARG protein was heavily phosphorylated on tyrosine, increased tyrosine phosphorylation of multiple cellular proteins, and induced factor-independent proliferation. The effects of STI571 on Ba/F3 cells transformed with BCR/ABL, TEL/ABL, TEL/PDGFbetaR, or TEL/ARG were then compared. STI571 inhibited tyrosine phosphorylation and cell growth of Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/PDGFbetaR, and TEL/ARG with an IC(50) of approximately 0.5 microM in each case, but it had no effect on untransformed Ba/F3 cells growing in IL-3 or on Ba/F3 cells transformed by TEL/JAK2. Culture of TEL/ARG-transfected Ba/F3 cells with IL-3 completely prevented STI571-induced apoptosis in these cells, similar to what has been observed with BCR/ABL- or TEL/ABL-transformed cells. These results indicate that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571.

Altered vasoreactivity to angiotensin II in experimental diabetic neuropathy: role of nitric oxide.

We evaluated the effects of angiotensin II and an angiotensin-converting enzyme inhibitor (cilazapril) on nerve blood flow (NBF) and electrophysiology in control and diabetic rats. When applied locally to the sciatic nerve, the dose-response curve of angiotensin II was more potent in experimental diabetic neuropathy (EDN) than control rats. No difference existed in plasma angiotensin II levels between EDN and controls. The rats were given typical rat pellets or pellets treated with 10 mg/kg per day cilazapril for 4 weeks. Diabetes caused a significant reduction in NBF, nerve conduction velocity, and compound muscle action potential (CMAP) amplitudes. NBF was significantly increased in diabetic rats supplemented with cilazapril diet, and nerve conduction velocity and amplitudes of the CMAP were also improved after 4 weeks on this diet. Direct application 10(-3) mol/L cilazapril on sciatic nerve did not increase NBF in normal and EDN rats. We topically applied the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine, on sciatic nerve and observed reduced inhibition of NBF in EDN, which was correctable with a cilazapril diet. These results suggest that diabetic neuropathy may have an increasing vasopressor action with angiotensin II and this is likely to be the mechanism of NOS inhibition. Angiotensin II-converting enzyme inhibitors may have potential in the treatment of diabetic neuropathy.

The efficacy of antimicrobial mouth rinses in oral health care.

There is growing public recognition of the importance of oral health, as symbolized by the theme. "Oral Health for a Healthy Life" proposed for the 1994 World Health Day. In this report, the efficacy of antimicrobial mouth rinses, mainly Listerine, was reviewed by three investigators who are working as a microbiologist, a microbiologist, a dentist, and a dental hygienist participating in oral health care. Listerine, an antimicrobial mouth rinse, completely killed microorganisms in 10 to 30 seconds; the microbes includes methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Helicobacter pylori, Candida albicans, Streptococcus mutans, Actinomyces viscosus, Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. Listerine was also weakly effective in inactivating human immunodeficiency viruses. Bacteria in samples collected from human dental plaque and saliva were completely killed within 30 seconds when exposed to Listerine. When saliva samples were collected from subjects who had rinsed their mouths with 20 ml of Listerine or 1:50 diluted povidone-iodine, levels of viable anaerobic bacteria in the samples were reduced to 1%. When Listerine was used for oral surgery such as tooth extraction and periodontal surgery, the agent was effective in relieving toothache. This was probably due to a decrease in oral bacteria by the antimicrobial action of Listerine, leading to lowering the inflammatory response of the host. The use of antimicrobial mouth rinse during dental treatments such as endodontic treatment proved effective for more reliable infection control. In Japan, there are an increasing number of elderly and medically compromised hosts who are potentially at risk for developing pneumonia due to silent aspiration of microbes in the oral cavity and throat. For the aged with such potential risk, using of antimicrobial mouth rinse may be effective in preventing dental plaque accumulation when used in addition to the mechanical control of plaque, since they tend to have difficulty in brushing teeth by themselves. Indeed, the use of antimicrobial mouth rinse in these elderly people proved useful not only in preventing bacterial pneumonia, but also in improving their quality of life by preserving their oral health.

[Continuous arterial infusion of low-dose cisplatin, 5-fluorouracil as adjuvant therapy in cytoreductive surgery for advanced hepatocellular carcinoma].

We investigated the effectiveness of continuous arterial infusion of low-dose CDDP, 5-FU for residual cancer after cytoreductive surgery for advanced hepatocellular carcinoma. Thirty-one patients with unresectable advanced hepatocellular carcinoma were classified into two groups by adjuvant therapy after reduction surgery: 1) Low-dose FP Group: 17 patients; continuous arterial infusion of low-dose CDDP, 5-FU via implanted port system; 2) Conventional group: 8 patients; Lipiodolization (6 cases) and transcatheter arterial embolization (2 cases). The five-year survival rate in the low-dose FP group was 34.8%, the efficacy was 64.7%, CR: 6 (35.3%); PR: 5; NC: 4; PD: 2. Thus, continuous arterial infusion of low-dose CDDP, 5-FU was effective as adjuvant therapy in cytoreductive surgery for advanced hepatocellular carcinoma.

Case report: right-sided periadrenal metastasis supplied by the hepatic artery. Clue to the genesis of pedunculated hepatocellular carcinoma.

The adrenal is the second most common site of haematogenous metastasis of hepatocellular carcinoma (HCC). The right adrenal is much more frequently affected than the left, but no reason has been offered for this difference. An aetiological connection has never been suggested between adrenal metastasis and pedunculated HCC. Hepatocellular carcinoma was resected in two patients who subsequently developed right-sided adrenal metastasis diagnosed by imaging. The adrenal mass was enhanced by hepatic arteriography and took up lipiodol injected into the hepatic artery. Reoperation was performed to remove the adrenal mass, which was abutting on the liver but was readily separable. Histopathologically, the adrenal gland was compressed by a metastatic HCC that developed in the immediate periadrenal tissue or adrenal capsule. By conventional imaging, the adrenal gland could not be recognized and the mass was thought to have arisen within the adrenal gland. In conclusion, periadrenal growth of HCC is a hitherto unrecognized type of metastasis and must have been mistaken either for an adrenal metastasis or a pedunculated HCC in the past. If left unresected, it would have fused with the liver and grown into a pedunculated HCC. Cancer cell invasion through an adrenohepatic fusion is the most likely mode of periadrenal metastasis; it explains the arterial communication between the mass and the liver.

Functional oxytocin receptors in bovine granulosa cells.

The aim of this study was to identify the presence of functional oxytocin (OT) receptors on bovine granulosa cells. Freshly prepared bovine granulosa cells from small (3-5 mm in diameter) or preovulatory (mature) follicles were examined for OT receptors by a radioreceptor assay. Scatchard analysis revealed that both binding capacity and affinity in granulosa cells from small follicles were significantly higher than those in granulosa cells from mature follicles (p < 0.01). With use of a reverse transcriptase polymerase chain reaction analysis, expression of OT receptor mRNA was detected in granulosa cells obtained from both small and mature follicles. When the granulosa cells obtained from small follicles were cultured in Dulbecco's Modified Eagle's Medium and Ham's F-12 medium (1:1 [v:v]) with 10% calf serum up to 72 h, as the period of culture was prolonged, the concentration of OT receptor decreased with increases of progesterone and OT release in the medium. However, the binding affinity was not changed during culture for 72 h. When bovine follicular oocytes with cumulus oophorus were cultured for 24 h in tissue culture Medium-199 with 10% fetal calf serum and OT (0-10 nM), the percentages of oocytes reaching maximum cumulus expansion were significantly increased at 0.5, 1, and 10 nM OT, although nuclear maturation in oocytes surrounded by compact cumulus cells was not affected by the addition of OT. Coexposure with OT antagonist blocked the stimulatory effect of OT on cumulus expansion, confirming the specificity of the effect. Furthermore, anti-OT rabbit serum inhibited the percentages of oocytes with expanded cumulus compared to those supplemented with normal rabbit serum (p <0.05). The overall results indicate the presence of functional OT receptors in bovine granulosa cells and support the hypothesis that OT plays a role (or roles) in regulating the function of granulosa cells as an autocrine factor during follicular growth.

An expression cloning method to identify monomeric GTP-binding proteins by GTP overlay.

We have developed a method for identifying monomeric GTP-binding proteins that is based on probing plasmid expression libraries with [alpha-32P]GTP. The method involves the production of nitrocellulose replica filter lifts from a plasmid cDNA expression library and treatment of the filters with chloroform vapor to lyse the Escherichia coli and to denature and inactivate endogenous E. coli GTP-binding proteins, thus allowing the direct identification of cDNA clones which encode Ras-like small GTP-binding proteins by ligand blotting. Using this procedure we have cloned a series of small Ras-like GTP-binding proteins from human retina. The method relies on a functional test, ligand specificity of the expressed proteins, to identify candidate molecules. This results in the isolation of predominantly full-length cDNA clones without relying on DNA sequence similarity. Thus, this method may be particularly useful for the cloning of novel Ras-related GTP-binding proteins which share limited sequence similarity with previously identified members of the Ras superfamily.

Effects of intracerebroventricular administration of 6-hydroxydopamine on ouabain-like immunoreactivity in plasma and the hypothalamo-pituitary axis in rats.

OBJECTIVE: To examine the role of central mechanisms on the production and release of an ouabain-like factor, the effects of intracerebroventricular injections of 6-hydroxydopamine on the tissue content and on the plasma level of the ouabain-like factor were determined in rats. METHODS: The vehicle (0.1% ascorbic acid in 0.9% saline) and 6- hydroxydopamine (250 micrograms/rat) were injected into the left lateral ventricle in ether-anaesthetized Wistar rats. Hypothalamus, pituitary, adrenal and venous blood was sampled 24h and 7 days later. The procedure was repeated using another rat group 7 days later. Characteristics of immunoreactive ouabain-like factor were determined by a combination of high-performance liquid chromatography and a highly sensitive enzyme-linked immunosorbent assay for ouabain. The level of the ouabain-like factor in these tissues and in plasma extracts measured by the enzyme-linked immunosorbent assay was compared between the two groups receiving 6-hydroxydopamine and the vehicle. RESULTS: Twenty-four hours after the intracerebroventricular injections of 6-hydroxydopamine, the ouabain-like factor level in the pituitary, hypothalamus and plasma had decreased significantly, whereas the ouabain-like factor level in the adrenal had not changed. The content of noradrenaline in the hypothalamus was also decreased markedly 7 days later and the content of ouabain-like factor in the pituitary remained low. On liquid chromatography the elution pattern of the ouabain-like factor in plasma and in tissue extracts coincided with that of authentic ouabain. CONCLUSIONS: Intracerebroventricular treatments with 6-hydroxydopamine elicited decreases in ouabain-like factor contents in the pituitary, the hypothalamus and the plasma. These results suggest that the production and release of ouabain-like factor are closely associated with the brain, particularly the hypothalamus-pituitary axis, and that noradrenergic or dopaminergic neurons, or both, play a key role in this mechanism.

Effects of osmolarity and amino acids in a chemically defined medium on development of rat one-cell embryos.

Rat one-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (R1ECM) under different experimental conditions. When the osmolarity of the medium with a reduced concentration (63.8 mmol l-1) of NaCl was varied by adding different amounts of D-sorbitol, more (79-91%) of the one-cell embryos developed to the four-cell stage at 212-278 mosmol than at 306 mosmol (13%). The greatest proportions of morulae (74%) and blastocysts (60%) were obtained at 246 mosmol. When the medium was supplemented with amino acids in various combinations and the osmolarity adjusted to about 246 mosmol, more (80-98%) of the embryos developed to the morula stage. More blastocysts were obtained in medium supplemented with glutamine (Gln: 80%), minimal essential medium (MEM) essential amino acids (EAA) (90%), Gln+EAA (83%), EAA+MEM nonessential amino acids (NEAA) (83%) or EAA+Gln+NEAA (90%) than in medium without amino acids (59%). Few (3-10%) hatching or hatched blastocysts were observed 120 h after the start of culture in the medium with EAA plus Gln or NEAA. The mean number of cells in blastocysts developed in the medium with EAA+Gln+NEAA was 46.7 +/- 7.2. When a total of 82 morulae or early blastocysts that had developed in culture were transferred to eight pseudopregnant rats on day 4, six recipients into which 62 embryos were transferred maintained their pregnancies beyond day 23, although no deliveries had occurred by day 25 or 26. When the rats were killed, 42 (68%) implantation sites and eight (13%) full-term fetuses with no gross abnormality were observed in the uterine horns.

An investigation of diterpenes from the leaves of Rabdosia trichocarpa and their antibacterial activity against oral microorganisms.

The ethanolic extract of Rabdosia trichocarpa leaves showed antibacterial activity against cariogenic mutans streptococci and periodontopathic Porphyromonas gingivalis. Chromatographic separation and purification of the extract afforded ten diterpenes, including one novel ent-kauren type compound named trichoranin. Some of these compounds possess potent antibacterial activity against these oral micro-organisms, indicating that these diterpenes may be useful natural substances for the maintenance of oral health.

Development of rat one-cell embryos in a chemically defined medium: effects of glucose, phosphate and osmolarity.

Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In the presence of 0.4 mmol phosphate l-1 (NaH2PO4), no embryos developed beyond the two-cell stage, regardless of the presence of 5.0 mmol glucose l-1. This inhibition was dose dependent at very low concentrations of phosphate in the medium supplemented with 7.5 mmol glucose l-1 and osmolarity adjusted to 244 mosmol; development to the blastocyst stage was not inhibited at 0.001-0.01 mumol phosphate l-1, but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 mumol phosphate l-1. In the medium without phosphate, glucose did not inhibit or promote development to the morula stage, but adequate concentrations of glucose were necessary for the development of morulae to the blastocyst stage; the percentage of one-cell embryos that developed to the blastocyst stage at 7.5 mmol glucose l-1 (67%) and 10.0 mmol glucose l-1 (60%) were not statistically different from the percentage at 5.0 mmol glucose l-1 (46%), but was significantly greater than the percentage at 2.5 mmol l-1 (33%). When osmolarity of the medium with 5.0 mmol glucose l-1 was varied by adjusting the amount of NaCl added, more (82-98%) of the one-cell embryos developed to the four-cell stage at 212-276 mosmol, but development was greatly inhibited at 304 mosmol. Development to the blastocyst stage was largely dependent on osmolarities; at 244 mosmol, 61% of embryos developed to the blastocyst stage, although this percentage was not significantly different from the percentage (43%) at 264 mosmol.

Cloning and expression of cDNA of human delta 4-3-oxosteroid 5 beta-reductase and substrate specificity of the expressed enzyme.

The enzyme delta 4-3-oxosteroid 5 beta-reductase (3-oxo-5 beta-steroid: NADP+ oxidoreductase and 4,5 beta-dihydrocortisone: NADP+ delta 4-oxidoreductase) catalyzes the reduction of the delta 4 double bond of bile acid intermediates and steroid hormones carrying the delta 4-3-one structure in the A/B cis configuration. Human delta 4-3-oxosteroid 5 beta-reductase cDNA was isolated from a liver cDNA library by cross hybridization with a previously cloned rat cDNA, which was used as a probe [Onishi, Y. Noshiro, M., Shimosato, T. & Okuda, K.-I. (1991) FEBS Lett. 283, 215-218]. DNA sequence analysis of a hybridization-positive clone predicted the human delta 4-3-oxosteroid 5 beta-reductase to contain 326 amino acids. The amino acid sequence of the human delta 4-3-oxosteroid 5 beta-reductase had 79% overall identity to the rat enzyme sequence. It also showed 54% and 50% overall identity with rat 3 alpha-hydroxysteroid dehydrogenase and human aldose reductase, respectively. RNA blotting analysis demonstrated the existence of a single delta 4-3-oxosteroid 5 beta-reductase mRNA of approximately 2.7 kb in human liver. Transfection of the cDNA into COS cells resulted in the expression of an active enzyme with a high activity toward the bile acid intermediates 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one and 7 alpha-hydroxy-4-cholesten-3-one. In addition, the expressed enzyme showed a small but significant 5 beta-reduction activity toward 11 beta,17 alpha,21-trihydroxy-delta 4-pregnene-3,20-dione (cortisol) and 17 beta-hydroxy-delta 4-androsten-3-one (testosterone) whereas no activity was observed toward delta 4-pregnene-3,20-dione (progesterone) or delta 4-androstene-3-17-dione (androstenedione). The substrate specificity of the human enzyme is considerably narrower than that of the rat enzyme, and the enzyme seems to be more important for bile acid biosynthesis than for metabolism of steroid hormones.

Strong immunogenicity of a multicomponent peptide vaccine developed with the branched lysine oligopeptide method for human immunodeficiency virus infection.

We synthesized one V3 peptide each from HTLV-IIIB, Thai A and Thai B, conjugating them to the T cell epitope of the env region, and we also synthesized a p17 protein peptide of the gag region (HGP-30). These peptides were then coupled to 8-lysine copolymers using N-succinimidyl maleimido carboxylate (M(r) = ca 60,000). We designated this the branched lysine oligopeptide method. The large peptide complexes constructed from these four macromolecular peptides were used with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay showed high titres of anti-peptide antibodies to each V3 loop peptide and the HGP-30 peptide. Strong inhibition of CD4+ dependent cell fusion was obtained with these antisera when IIIB, Thai A and Thai B strains of the human immunodeficiency virus (HIV) were used. Strong anti-fusion inhibition was also observed with two other HIV strains. In addition, an increase of the anti-HIV effect was observed when we used sera obtained by multicomponent vaccine immunization. The same kind of inhibition was also observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in mice immunized with this vaccine. These results suggest that immunization with macromolecular peptide complexes can result in strong immunogenicity towards HIV-1.

Maturation-promoting factor (MPF) is responsible for the transformation of sperm nuclei to metaphase chromosomes in maturing bovine oocytes in vitro.

Bovine oocytes at the germinal vesicle stage were inseminated in Brackett and Oliphant's medium in the presence of BSA (10 mg ml-1), caffeine (5 mmol l-1) and heparin (10 micrograms ml-1). When oocytes were transferred into tissue culture medium (TCM)-199 containing 10% fetal calf serum (FCS) with or without 6-dimethylaminopurine (6-DMAP; 2 mmol l-1) 8 h after insemination and cultured for 15-40 h at 39 degrees C in 5% CO2 in air, 74-83% of oocytes were penetrated and polyspermy (67-80%) was common. At 40 h after culture in 6-DMAP-free medium, 65% and 63% of unpenetrated and penetrated oocytes, respectively, reached metaphase II or beyond. A few (6%) oocytes were activated and contained both male and female pronuclei. Sperm metaphase chromosomes were observed in 90% of the penetrated oocytes. Penetration by more than four spermatozoa greatly retarded the meiotic maturation of the oocyte. However, sperm chromosomes were never observed in oocytes cultured in 6-DMAP supplemented medium and oocyte maturation did not proceed beyond the stage of prometaphase I. These results demonstrate the possible participation of maturation-promoting factor in metaphase chromosome formation in spermatozoa.