Cation size effects in mixed-ion metaphosphate glasses: structural characterization by multinuclear solid state NMR spectroscopy.

Metaphosphate glasses with two monovalent species A(1-x)B(x)PO3 (0 ≤x≤ 1) show mixed-ion effects (MIE) in the dc conductivities and glass transition temperatures, which are strongly dependent on the cation size mismatch between the two mobile species. In the present contribution, mixed-ion metaphosphate glasses based on the cation combinations Cs-Li, Rb-Li, and Cs-Ag, exhibiting particularly large size mismatches, are analyzed by (31)P, (87)Rb, (109)Ag and (133)Cs NMR to determine possible correlations between this mismatch and some of the structural properties critical to the development of the MIE: the local environments around the mobile species and their spatial distribution relative to each other. The results are compared with those obtained in the Na-Ag metaphosphate series, which serves as a reference system, with minimized cation mismatch MIE. The local coordination environments of the Ag(+), Rb(+) and Cs(+) ions follow analogous compositional trends as previously observed in Na-based mixed-ion metaphosphate glasses: for a given cation species A, the average A-O distance shows an expansion/compression when this cation is replaced by a second species B with smaller/bigger ionic radius, respectively. This compositional differentiation of the structural sites for the mobile species may contribute to the MIE. Concerning the relative spatial distribution of the mobile ions, results from (7)Li-(133)Cs (SEDOR) experiments indicate a random mixture of Cs and Li in Cs-Li metaphosphate glasses. While this result is in agreement with one of the fundamental hypotheses of the models proposed to describe the MIE, it is at variance with the observation of various partial cation segregation phenomena observed in Na-based mixed alkali glasses. This result suggests that cation size mismatch is not the decisive parameter in determining segregation or non-statistical mixing of cations in the glass. In the Cs-Ag and Na-Ag glasses, (109)Ag spin-echo NMR reveals a progressive slowing down of the Ag(+) diffusion dynamics as this species is replaced by Cs(+) or Na(+). The substitution by the bigger Cs(+) ion causes a strong reduction in Ag(+) mobility suggesting the existence of separated diffusion pathways for these cations. In contrast, substitution by the similarly-sized Na(+) causes a much weaker mobility reduction consistent with the existence of Ag-Na cooperative hopping.

Identification and expression of the Xenopus homolog of mammalian p100-NFkappaB2.

We have molecularly cloned a cDNA encoding a new Rel-related protein in Xenopus laevis. The product is most homologous to mammalian p100-NFkappaB2. Furthermore, the putative protein kinase A-phosphorylation site (RRPS), which is found in most of the Rel family proteins and is replaced by KRKR in mammalian p100, is also replaced by KRKK in our clone, indicating that our cDNA most likely encodes the Xenopus p100 (Xp100). Like mammalian p52, a processed product of p100, Xp52 alone binds to the kappaB site but does not activate transcription, while the XRelB/Xp52 heterodimer activates transcription, which is inhibited by the carboxyl-terminal half of Xp100 (XIkappaBdelta). Xp100 transcripts are present at all stages of oocyte maturation and in all adult tissues examined. Xp100 transcripts decrease at the gastrula stage and resume their expression at the neurula stage, which is different from other Xenopus rel family. Xp100 is highly expressed in somitogenic mesoderm at the neurula stage, while in the gastrula and tailbud stages, Xp100 transcripts are not localized to restricted regions. These results suggest that Xp100 could be involved in the late-stage development of Xenopus laevis, especially in the maturation of somites.

Cloning and characterization of the human Calmegin gene encoding putative testis-specific chaperone.

The putative chaperone Calmegin is required for sperm fertility in mouse and the relevance of the gene to certain cases of human male infertility has been suggested. In the present paper, we have isolated and characterized the human homolog cDNA of the mouse germ cell-specific Calmegin. The entire coding region of the human cDNA showed 80% identity with the previously reported mouse Calmegin. The predicted amino acid sequence showed strong conservation of the two sets of internal repetitive sequences (Ca2+ binding motif), and the hydrophilic COOH terminus, which corresponds to the putative endoplasmic reticulum (ER) retention motif. Our finding will support diagnosis of male infertility. Northern blotting analysis of various human tissues showed that the transcript was 3 kb in length and was expressed exclusively in the testis. Using the fluorescence in situ hybridization (FISH) technique, human Calmegin gene was mapped to chromosome 4q28.3-q31.1.

Classification of 'activation' antibodies against integrin beta1 chain.

We compared the effects of two anti-beta1 integrin activating antibodies, TS2/16 and AG89, on K562 cell adhesion to fibronectin. Though both antibodies effectively induced cell adhesion, the EC50 for AG89 was more than 200-fold higher than that for TS2/16. Scatchard analysis of the data from [125I]Fab fragment binding to the cells revealed that the TS2/16 epitope is exposed constitutively on all the beta1 integrin molecules, while only 3% of the beta1 integrins on resting K562 cells bear the AG89 epitope. Calculation of the actual number of each antibody bound to the cell during the cell adhesion assay revealed that induction of cell adhesion can be accomplished by binding much fewer AG89 molecules compared to TS2/16. Thus, AG89 and TS2/16 represent distinct classes of anti-integrin activating antibodies that show completely different binding characteristics as well as different activation effects on the integrin molecule upon binding.

Effect of breeding bull on the fatty acid composition of the carcass lipid in steers of a beef breed cattle.

Samples of neutral lipid were taken from the carcasses of forty-eight Japanese Black steers, the progeny from three breeding bulls, on similar planes of nutrition and of the same age, and were analysed for fatty acid composition. The breeding bull seems to significantly affect the fatty acid composition of the lipid from the thoracic subcutaneous fatty tissue and the inter- and intra-muscular fatty tissues of the Longissimus dorsi muscle. No significant correlation between breeding bull and fatty acid composition of perinephric fatty tissue was found. There was an increase in total concentration of unsaturated fatty acids from internal to external sample locations.

Lead and aluminum bonding in Pb-AI metaphosphate glasses.

The bonding properties of cations in phosphate glasses determine many short- and medium-range structural features in the glass network, hence influencing bulk properties. In this work, Pb-Al-metaphosphate glasses (1 - x)Pb(PO(3))(2).xAI(PO(3))(3) with 0 < or = x < or = 1 were analyzed to determine the effect of the substitution of Pb by AI on the glass structure in the metaphosphate composition. The glass transition temperature and density were measured as a function of the Al concentration. The vibrational and structural properties were probed by Raman spectroscopy and nuclear magnetic resonance of (31)P, (27)AI, and (207)Pb. Aluminum incorporates homogeneously in the glass creating a stiffer and less packed network. The average coordination number for AI decreases from 5.9 to 5.0 as x increases from 0.1 to 1, indicating more covalent AI-O bonds. The coordination number of Pb in these glasses is greater than 8, showing an increasing ionic behavior for compositions richer in AI. A quantitative analysis of the phosphate speciation shows definite trends in the bonding of AIO(n) groups and phosphate tetrahedra. In glasses with x < 0.48, phosphate groups share preferentially only one nonbridging O corner with an AIO(n) coordination polyhedron. For x > 0.48 more than one nonbridging O can be linked to AIO(n) polyhedra. There is no corner sharing of O between AIO(n) and PbO(n) polyhedra nor between AIO(n) themselves throughout the compositional range. The PbO(n) coordination polyhedra show considerable nonbridging O sharing, with each O participating in the coordination sphere of at least two Pb. The bonding preferences determined for Al are consistent with the behavior observed in Na-AI and Ca-AI metaphosphates, indicating this may be a general behavior for ternary phosphate glasses.

On the effectiveness of whole spectral shape for vowel perception.

The formant hypothesis of vowel perception, where the lowest two or three formant frequencies are essential cues for vowel quality perception, is widely accepted. There has, however, been some controversy suggesting that formant frequencies are not sufficient and that the whole spectral shape is necessary for perception. Three psychophysical experiments were performed to study this question. In the first experiment, the first or second formant peak of stimuli was suppressed as much as possible while still maintaining the original spectral shape. The responses to these stimuli were not radically different from the ones for the unsuppressed control. In the second experiment, F2-suppressed stimuli, whose amplitude ratios of high- to low-frequency components were systemically changed, were used. The results indicate that the ratio changes can affect perceived vowel quality, especially its place of articulation. In the third experiment, the full-formant stimuli, whose amplitude ratios were changed from the original and whose F2's were kept constant, were used. The results suggest that the amplitude ratio is equal to or more effective than F2 as a cue for place of articulation. We conclude that formant frequencies are not exclusive cues and that the whole spectral shape can be crucial for vowel perception.

Characterization of development-specific, cell type-specific mouse testicular antigens using testis-specific polyclonal antibodies.

Antisera were raised by immunizing rabbits with mouse testicular germ cells, and absorbed in vivo by injection into castrated male whole mice to obtain a specific antiserum which reacted with mouse germ cells. The expression of mouse testis-specific antigenic macromolecules was then studied immunochemically with the antiserum. Approximately 20 antigenic macromolecules with molecular weights ranging from 26 to 110 kD were detected in the normal adult testis. At least 12 of these were differentiation-specific antigens appearing during development of germ cells, while others were expressed in testicular germ and/or somatic cells or detected only in mature spermatozoa. This technique for raising testis-specific antisera could be useful for isolation of cDNA clones encoding their antigens, as well as for investigation of the physiological roles of these molecules in germ cell differentiation at the molecular level.

Identification of differentiation antigens in mouse testicular germ cells recognized by monoclonal antibody TRA 55.

We have isolated a monoclonal antibody (mAb) TRA 55, which recognizes mouse testicular germ cells from mid-pachytene spermatocytes to the early stages of haploid spermatids during differentiation. Immunohistochemical analysis produced strong positive staining of the nuclei and faint staining in the cytoplasm of germ cells. At meiotic division, when the nuclear membrane disappeared, a specific positive signal could be observed on metaphase chromosomes. When germ cells produced haploid spermatids, antigenicity became suddenly weak and soon disappeared. TRA 55 did not react with testicular somatic cells, such as Sertoli cells or Leydig cells. Western blot analysis of the whole testis showed four positive bands with molecular weights of 43, 46, 49 and 55 kDa. Three bands of 43, 49 and 55 kDa, and a single band of 46 kDa were recovered in cytoplasmic and nuclear fractions of testicular germ cells, respectively. Chronological changes in the Western blot pattern indicated that these antigens became detectable in the testis at the age of 10 days. Furthermore, all antigens were resistant to periodate treatment, suggesting that the epitope was in an amino acid rather than a sugar moiety. These antigen molecules may play important roles in the differentiation of germ cells at the later stages of meiotic prophase and meiotic division in the mouse testis.

Isolation of a novel cDNA that encodes a protein localized to the pre-acrosome region of spermatids.

We have identified a novel cDNA clone, named AZ1, obtained from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5-azacytidine, a potent demethylating reagent. The amount of transcript increased with 5-azacytidine treatment of C3H10T1/2 cells and the transcript was highly expressed in mouse testis. As the mutant mouse jsd/jsd, which has a defect in germ cell maturation, barely expressed the transcript, the message was expected to be expressed specifically in spermatocytes. The mRNA was detected at significant levels in the testes from mice aged 16 days after birth, suggesting that its expression started at the pachytene spermatocyte stage. The elucidated nucleotide sequence contained a 2841-nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 107,254 Da. Specific antibodies raised against the fusion protein including glutathione S-transferase revealed an approximately 130-kDa band of a translation product in testis and in cultured cells transfected with AZ1 cDNA in the expression vector on Western-blot analysis. The protein was localized to the pre-acrosome region of round and elongated spermatids. However, it was not detected at a more advanced stage of spermatids, i.e. just before their release from Sertoli cells. This protein may play an important role in spermatogenesis.

The 'ligand-induced conformational change' of alpha 5 beta 1 integrin. Relocation of alpha 5 subunit to uncover the beta 1 stalk region.

Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-beta1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of beta1 subunits. The binding of a 125I-labeled AG89 Fab fragment to alpha5 beta1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated alpha5 beta1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only beta1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with non-functional beta1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant beta1 that cannot complex with alpha5. These data suggest that the AG89 epitope is masked by the alpha5 subunit. Ligand binding and integrin activation may uncover the beta1 stalk region by triggering a conformational shift of alpha5 relative to beta1.

Molecular cloning and characterization of meichroacidin (male meiotic metaphase chromosome-associated acidic protein).

We have isolated a cDNA clone encoding a germ cell specific protein from an expression cDNA library prepared from the mouse testis, using testis-specific polyclonal antibodies. Sequence analysis of the cDNA revealed that the deduced amino acid sequence consisted of 284 residues, including a nominal repeat structure in the N-terminal region. Northern blot analysis revealed the presence of a transcript of 1.3 kb exclusively expressed in the testis and ovary, but at relatively low levels in the ovary. In contrast, no other tissues and organs expressed significant levels of the transcript. Expression of the mRNA in the testis was first detected on day 14 in postnatal development. Western blot analysis showed the presence of the protein with a molecular weight of approximately 40 kDa and an isoelectric point of 4.9. The protein was exclusively found in the testis and ovary, but in a far lesser amount in the ovary as was the case with the transcript. Immunohistochemical examination revealed that the protein was predominantly present in the cytoplasm in pachytene spermatocytes through to round spermatids. However, during the disappearance of the nuclear envelope at both the first and second meiotic divisions, the protein was localized around the metaphase chromosomes and spindles. Because of this, the name meichroacidin which stands for male meiotic metaphase chromosome-associated acidic protein is proposed for this antigen. The highly regulated stage-specific expression of meichroacidin and its specific association with the metaphase chromosomes and spindles suggest that the protein plays important roles in male meiosis.

Behavioral compensations in a positional learning and memory task by aged monkeys.

The present experiment assessed learning and memory of a positional task by evaluating behavioral strategies as well as accuracy of a task in four young and four aged monkeys. They were tested in a delayed response (DR) task that has been widely used to study animal models of aging. The task consisted of two phases; an acquisition of the task and a positional memory test with five delay times (1-30 s). There was no clear difference between age groups in the number of trials needed for acquisition of the task. However, an analysis of behavior revealed differences in behavioral characteristics displayed during testing. The young monkeys showed various irrelevant behaviors during the execution of the task. In contrast, the aged monkeys consistently concentrated on the task exhibiting no behaviors irrelevant to the task. These results showed than the aged monkeys' performance was supported by a different behavioral strategy from the young monkeys. The results of the memory test were similar to those of the acquisition on the accuracy and the behavior. The aged monkeys depended on behavioral cues to preserve their positional memory, especially during the task. The present study suggests that cognitive impairments in aged monkeys can be compensated for by employing behavioral strategies.

A germ cell-specific nuclear antigen recognized by a monoclonal antibody raised against mouse testicular germ cells.

A monoclonal antibody (mAb TRA 104) raised against mouse testicular germ cells was able to recognize the nuclei of testicular germ cells at all the stages of differentiation from embryonic gonocytes to spermatids and did not react with any somatic cells. The antigen recognized by mAb TRA 104 was exclusively present in testicular extracts. The molecular weights and isoelectric point (pI) of the antigens determined by Western blotting analysis were 60-110 kDa and 7.2, respectively. This antigen(s) is referred to as a germ cell-specific nuclear antigen(s) (GENA) since GENA was first detected specifically in the genital ridge at around 12 days of gestation by Western blotting analysis. In the testis, the expression increased gradually until adulthood whereas it was lost in the ovary by postpartum day 5. Thus, GENA is a molecule(s) exclusively present in the nuclei of germ cells and may be a useful marker with which to study the mechanism of germ cell development and differentiation at the molecular level.

Cloning and characterization of a complementary deoxyribonucleic acid encoding haploid-specific alanine-rich acidic protein located on chromosome-X.

We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation.

Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells.

We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I-III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (for haploid germ cell-specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of Mr 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells.

Expression of selenoprotein-P messenger ribonucleic acid in the rat testis.

It has been suggested that a plasma protein, selenoprotein P, functions as an antioxidant and that its mRNA is expressed ubiquitously, including in the testis. To determine its physiological function, we have investigated the expression of selenoprotein-P mRNA in the rat testis. Northern blot analysis showed that selenoprotein P was exclusively expressed in the Leydig cell fraction. In situ hybridization experiments further supported this observation. Testes of rats administered ethylene dimethane sulfonate (EDS) were also examined by Northern blot analysis. Selective degeneration of Leydig cells by EDS treatment resulted in disappearance of selenoprotein-P mRNA from the testis. Furthermore, upon recovery, in association with regenerative differentiation of Leydig cells, reappearance of the selenoprotein-P mRNA was observed. These results indicated that selenoprotein-P mRNA was predominantly expressed in the interstitial Leydig cells.

Isolation and characterization of a haploid germ cell-specific novel complementary deoxyribonucleic acid; testis-specific homologue of succinyl CoA:3-Oxo acid CoA transferase.

We have isolated a cDNA clone encoding a mouse haploid germ cell-specific protein from a subtracted cDNA library. Sequence analysis of the cDNA revealed high homology with pig and human heart succinyl CoA:3-oxo acid CoA transferase (EC 2.8.3.5), which is a key enzyme for energy metabolism of ketone bodies. The deduced protein consists of 520 amino acid residues, including glutamate 344, known to be the catalytic residue in the active site of pig heart CoA transferase and the expected mitochondrial targeting sequence enriched with Arg, Leu, and Ser in the N-terminal region. Thus, we termed this gene scot-t (testis-specific succinyl CoA:3-oxo acid CoA transferase). Northern blot analysis, in situ hybridization, and Western blot analysis demonstrated a unique expression pattern of the mRNA with rapid translation exclusively in late spermatids. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals and gradually accumulated during spermiogenesis. It was also detected in the midpiece of spermatozoa by immunohistochemistry. The results suggest that the scot-t protein plays important roles in the energy metabolism of spermatozoa.

Molecular cloning and characterization of phosphatidylcholine transfer protein-like protein gene expressed in murine haploid germ cells.

We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.

Mapping of eight testis-specific genes to mouse chromosomes.

We previously identified eight testis-specific genes using antibodies raised against testicular germ cells. They are expressed during spermatogenesis and are presumed to be involved in testicular germ cell differentiation and sperm formation. We have mapped the genomic loci for these testis-specific genes using restriction fragment length variants in interspecific backcross mice. The calmegin gene (Clgn) was mapped to Chr 8. The synaptonemal complex protein gene 1 (Sycp1) probe hybridized with two sequences on different chromosomes; Sycp1-rs2 was mapped to Chr 3, whereas Sycp1-rs3 was mapped to Chr 7. The relaxin-like factor gene (Rlnl) was mapped to Chr 8, and collapsin response mediator protein 1 (Crmp1) was mapped to Chr 5. Three novel genes encoding testis-specific proteins A2 (Tsga2), A8 (Tsga8), and A12 (Tsga12) were mapped to chromosomes 3, X, and 10, respectively.