Capacitation,acrosome reaction,and egg penetration by canine spermatozoa in a simple defined medium

A defined medium (CCM) is described in which washed ejaculated canine spermatozoa can be induced to undergo capacitation and the acrosome reaction, and to penetrate corona-free eggs in vitro. The composition of the medium is similar to other Krebs-Ringer's bicarbonate buffered media except for the absence of magnesium and adjustments in the concentration of NaHCO₃, glucose, and albumin. The concentration of NaCI is adjusted to maintain the osmolality at approximately 300 mOsm, and the pH is 7.8 under 5% CO₂ in air. This pH was found more favorable for the occurrence of the acrosome reaction than the lower pH (7.4) of media with more usual bicarbonate concentrations. Calcium ions are essential not only for the occurrence of the acrosome reaction of canine spermatozoa, but also for their motility. Potassium ions are apparently necessary for the process of sperm-egg fusion following zona penetration. When CCM was compared with two other media, it allowed the best combination of good rates for acrosome reaction (46%), motility (83%), and zona penetration (71%). Sperm-egg fusion also occurred. The requirements for capacitation of canine spermatozoa are compared with those of some other species.     

Periovulatory time courses of plasma estradiol and progesterone in the Japanese monkey (Macaca fuscata)

Periovulatory time courses of plasma estradiol and progesterone were determined in 21 menstrual cycles of 20 Japanese monkeys. Both steroids were measured by radioimmunoassay. Ovulation was detected by serial laparoscopic observations of the ovaries. Three of the 21 cycles were anovulatory cycles. In the remaining 18 ovulatory cycles, a preovulatory estradiol peak occurred on day 12.2±1.4 (range 10–15) of the menstrual cycle. The estradiol concentration at the peak was 431±199 (range 210–930) pg/ml. The time interval between the estradiol peak and ovulation was within 48 hr; the shortest interval was 10–13 hr and the longest 32–48 hr. Although the progesterone levels began to increase slightly (0.6–1.4 ng/ml) before ovulation, they did not show a continuous increase but decreased once before ovulation. The increase in progesterone with development of the corpus luteum after ovulation was very gradual during the first 2 days after ovulation. Subsequently, in 13 of 18 ovulatory cycles the progesterone levels rose rapidly and reached a maximum, 4.0±1.2 (range 2.3–5.7) ng/ml, 4–8 days after ovulation. In 5 of the 18 cycles, the progesterone levels did not rise at all or did not exceed 2.0 ng/ml even if they showed more or less an increase. In the 5 cycles, the length of the luteal phase was 8.2±1.6 (range 6–10) days, which was significantly shorter than that of the former 13 cycles with 14.0±1.1 (range 13–16) days.     

Synthesis of glutamic oxaloacetic transaminase isozymes in rat liver cells

The synthesis of glutamic oxaloacetic transaminase isozymes in rat liver explants was studied using specific antisera against the cytosolic and mitochondrial isozymes. The pulse-labeled cytosolic isozyme was detected in the cytosolic fraction and remained there in pulse-chase experiments. On the other hand, the pulse-labeled mitochondrial isozyme was detected as a larger precursor in the cytosolic fraction. During chase, the amount of pulse-labeled precursor of the mitochondrial isozyme decreased and labeled mature mitochondrial isozyme appeared in the mitochondrial fraction.     

Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells

Erythropoietin (Epo), a glycoprotein that regulates the formation of erythrocytes in mammals, was produced in cultured tobacco BY2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) by introducing human Epo cDNA via Agrobacterium tumefaciens-mediated gene transfer. Epo was correctly processed and subsequently penetrated the plasma membrane of tobacco cells. However, it remained attached to the cell wall and was not released into the culture medium. Although Epo produced by tobacco cells was glycosylated with N-linked oligosaccharides, these carbohydrates were smaller than those of the recombinant Epo produced in mammalian cells. Epo produced in tobacco exhibited in vitro biological activities by inducing the differentiation and proliferation of erythroid cells. However, it had no in vivo biological activities. A lectin-binding assay indicated the lack of sialic acid residues in the N-linked oligosaccharides of Epo, suggesting that Epo was removed from the circulation before it reached erythropoietic tissues.     

Kinetic analyses of alcohol-induced potentiation of the response of GABA(A) receptors composed of alpha(1) and beta(1) subunits.

To investigate the kinetics of both the potentiation and desensitization of the response of ionotropic GABA receptors (GABA(A) receptors) in the presence of various compounds, we expressed receptors composed of alpha(1) and beta(1) subunits by injecting cells with the cRNAs synthesized from cloned bovine GABA(A) receptor cDNAs and measured the electrical responses of the cells electrophysiologically with or without the compounds. The potentiation of the GABA(A) receptor-mediated response was quantitatively analyzed using a simple model with the assumption that the receptors have two identical binding sites for GABA molecules with a dissociation constant of K(1), and one potentiation site for the compound with a dissociation constant of K(p), and that the binding of the compound to the potentiation site only increases the affinity of the GABA binding sites, changing K(1) to K(1p). The estimated K(p) and K(1p) were dependent on the functional groups and the chain length of the compounds. These results could be satisfactorily analyzed using this simple model. The potentiation of the GABA(A) receptor-mediated response by the components of essential oils used for aromatherapy was also examined. These compounds accelerated the decay of the response, possibly due to desensitization of the receptors, which was also analyzed on the basis of the model.     

Tumor immunotherapy using bone marrow-derived dendritic cells overexpressing Toll-like receptor adaptors

Myeloid dendritic cells (mDCs) play an important role in the initiation of immune responses to cancer and infectious diseases. Toll-like receptors (TLRs) expressed on mDCs recognize microbial products to elicit signals for mDC maturation, including cytokine production, antigen-presentation and induction of effector cells. TLR agonists work as adjuvants to modulate the function of mDCs. In TLR signaling, MyD88 and TRIF/TICAM-1 are major TLR adaptor molecules, which when overexpressed are able to transduce downstream signals without TLR stimuli. We successfully introduced the adaptors into mouse bone marrow-derived mDCs using lentiviral vectors. Introduction of MyD88 into mDCs in vitro led to the production of IL-6 and IL-12p40 while introduction of TICAM-1 stimulated interferon (IFN)-alpha production. Expression of TICAM-1, but not MyD88, in mDCs slightly induced the co-stimulatory molecule CD86, while significant upregulation of CD86 was observed in response to other TLR stimuli. Both MyD88 and TICAM-1 augmented allogeneic mixed lymphocyte reaction (MLR). Ex vivo mouse spleen cells pre-exposed to tumor antigen exhibited antitumor cytotoxicity when incubated with MyD88- or TICAM-1-expressing mDCs. Using mDC adoptive transfer and a syngeneic mouse tumor implant model, we established an antitumor immunotherapy whereby tumor growth is retarded by adaptor-manipulated mDCs.     

A BAC-based contig map of the cynomolgus macaque (Macaca fascicularis) major histocompatibility complex genomic region

The construction of a cynomolgus macaque (Macaca fascicularis, Mafa) BAC library for genomic comparison between rhesus and cynomolgus macaques is necessary to promote the cynomolgus macaque as one of the important experimental animals for future medical and biological research. In this paper, we constructed a cynomolgus macaque BAC library and a map of the MHC (Mafa) genomic region for comparison of the genomic organization and nucleotide similarities between the human, the chimpanzee, and the rhesus macaque. The BAC library consists of 221,184 clones with an average insert size of 83 kb, providing a sixfold coverage of the haploid genome. A total of 114 BAC clones and 54 PCR primer sets were used to construct a 4.3-Mb contig of the MHC region. Diversity analysis of genomic sequence from selected subregions of the MHC revealed that the cynomolgus sequence varied compared to rhesus macaque, human, and chimpanzee sequences by 0.48, 4.15, and 4.10%, respectively. From these findings, we conclude that the BAC library and Mafa genomic map are useful tools for genome analysis and will have important applications for comparative genomics and identifying regions of consequence in medical research.     

In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells

In the central nervous system, astrocytes produce erythropoietin (Epo) and neurons express its receptor. To examine whether or not the brain Epo protects the in vitro cultured neurons from glutamate-induced cell death, we established rat astrocyte cell lines containing the plasmid for production of recombinant rat Epo. Epo partially purified from the culture medium showed a neuroprotective effect similar to that of rat Epo produced by Chinese hamster ovary (CHO) cells. Comparison was made in some other properties between Epo produced by these astrocyte cell lines and that by CHO cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mutation patterns for two flaviviruses: hepatitis C virus and GB virus C/hepatitis G virus

We studied the mutation patterns of hepatitis C virus (HCV) and GB virus C/hepatitis G virus (HGV). Although the mutation patterns of the two viruses were similar to each other, they were quite different from that of HIV. In particular, the similarity of the patterns between HCV or HGV and human nuclear pseudogenes was statistically significant whereas there was no similarity between HIV and human nuclear pseudogenes. This finding suggests that the mutation patterns of HCV and HGV are similar to the patterns of spontaneous substitution mutations of human genes, implying that nucleotide analogues which are effective against HCV and HGV may have a side effect on the normal cells of humans.