Phase separation in critical and off-critical fluids: specific experimental behaviours at criticality

The phase separation in fluids close to a critical point can be observed in the form of either an interconnected pattern (critical case) or a disconnected pattern (off-critical case). These two regimes have been investigated in different ways. First, a sharp change in pattern is shown to occur very close to the critical point when the composition is varied. No crossover has been observed between the t1 behaviour (interconnected) and a t1/3 behaviour (disconnected), where t is time. This latter growth law, which occurs in the case of compact droplets, will be discussed. Second, it has been observed that a growing interconnected pattern leaves a signature in the form of small droplets. The origin of such a distribution will be discussed in terms of coalescence of domains. No distribution of this kind is observed in the off-critical case.

Testis-specific histone (H1t) is not phosphorylated and has a weak interaction with chromatin

Soluble chromatin was prepared from rat testes after a brief micrococcal nuclease digestion. After adsorption onto hydroxylapatite at low ionic strength, the histone Hl subtypes were eluted with a shallow salt gradient of 0.3 M NaCl to 0.7 M NaCl. Histone Hlt was eluted at 0.4 MNaCl, while histones H1a and Hlc were eluted at 0.43 M NaCl and 0.45 M respectively. The extreme divergence of the amino acid sequence of the C-terminal half of histone Hlt, the major DNA binding domain of histone Hl, from that of the somatic consensus sequence may contribute to the weaker interaction of histone Hlt with the rat testis chromatin. Further, histone Hlt was not phosphorylated in vivo in contrast to histone Hla and Hlc, as is evident from the observation that histone Hlt lacks the SPKK motif recognized by the CDC-2kinase or the RR/KXS motif recognized by protein kinase A.

Thermodynamics of lectin-sugar interaction: Binding of sugars to winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBAI)

Combining site of WBAI is extended and encompasses all the residues of blood group A-reactive trisaccharide [GalNAcalpha3Galbeta4Glc]. Though both of the fucose residues of A-pentasaccharide [GalNAcalpha(Fucalpha2)3Galbeta(Fucalpha3)4Glc] do not directly interact, with the combining site they thermodynamically favour the interaction of GalNAcalpha3Galbeta4Glc part of the molecule by imposing a sterically favourable orientation of the binding epitope viz. GalNAcalpha3Galbeta4Glc of the saccharide. Binding of sugars is driven by enthalpy and is devoid of heat capacity changes. This together with enthalpy-entropy compensation observed for these processes underscore the importance of water reorganization as being one of the principal determinant of protein-sugar interactions.

Analysis of a conformation-specific epitope of the alpha subunit of human chorionic gonadotropin: Study using monoclonal antibody probes

Monoclonal antibodies (MAbs) have been used extensively for identification of sequence-specific epitopes using either the ELISA or/and IRMA methods, However, attempts to use MAbs for identification of conformation-specific epitopes have been very few as they are considered very labile. We have investigated the stability of conformation-specific epitopes of human chorionic gonadotropin (hCG) using a quantitative solid-phase radioimmnunoassay (SPRIA) technique. Several epitopes are stable to mild modification (chemical and proteolytic) conditions, and epitopes show differential stability for these modifications. Based on these observations, a monoclonal antibody (MAb 16) for an a-subunit-specific epitope of hCG has been used to monitor changes at the epitopic site (identified as epitope 16) on modification of hCG, using SPRIA with immobilized MAb 16. Modifications of amino groups, hydroxyl group of tyrosine as well as carboxyl group of Asp/Glu all bring about sufficient changes in the epitope integrity. Peptide bond hydrolysis at lysine residues damages the epitope, but not at arginine residues, Hydrolysis at tyrosine does not affect the epitope, though modification of the side-chain of tyrosine inactivates the epitope. Destruction of the epitope occurs on reduction of the disulphide bonds. Partial retention of the epitope activity is seen on modification of carboxyl or the epsilon-amino groups of lysine. Based on these results four to six amino acids have been identified to be at the epitopic site, and the data suggest that two peptide segments are brought together by the disulphide bond Cys10-Cys60 to form the epitope.

Developmental analysis of a female-specific 16S rRNA gene from mycetome-associated endosymbionts of a mealybug, Planococcus lilacinus

A clone showing female-specific expression was identified from an embryonic cDNA library of a mealybug, Planococcus lilacinus, In Southern blots this clone (P7) showed hybridization to genomic DNA of females, but not to that of males, However, P7 showed no hybridization to nuclei of either sex, raising the possibility that it was extrachromosomal in origin, In sectioned adult females P7 hybridized to an abdominal organ called the mycetome. The mycetome is formed by mycetocytes, which are polyploid cells originating from the polar bodies and cleavage nuclei that harbour maternally transmitted, intracellular symbionts. Electron microscopy confirmed the presence of symbionts within the mycetocytes, Sequence analysis showed that P7 is a 16S rRNA gene, confirming its prokaryotic origin, P7 transcripts are localized to one pole in young embryos but are found in the pole as well as in the germ band during later stages of development, P7 expression is detectable in young embryos of both sexes but the absence of P7 in third instar and adult males suggests that this gene, and hence the endosymbionts, are subject to sex-specific elimination. Copyright (C) 1997 Elsevier Science Ltd.

Comparative Nucleotide and Amino Acid Sequence Analysis of the Sequence-Specific RNA-Binding Rotavirus Nonstructural Protein NSP3

NSP3, an acidic nonstructural protein, encoded by gene 7 has been implicated as the key player in the assembly of the 11 viral plus-strand RNAs into the early replication intermediates during rotavirus morphogenesis. To date, the sequence or NSP3 from only three animal rotaviruses (SA11, SA114F, and bovine UK) has been determined and that from a human strain has not been reported. To determine the genetic diversity among gene 7 alleles from group A rotaviruses, the nucleotide sequence of the NSP3 gene from 13 strains belonging to nine different G serotypes, from both humans and animals, has been determined. Based on the amino acid sequence identity as well as phylogenetic analysis, NSP3 from group A rotaviruses falls into three evolutionarily related groups, i.e., the SA11 group, the Wa group, and the S2 group. The SA 11/SA114F gene appears to have a distant ancestral origin from that of the others and codes for a polypeptide of 315 amino acids (aa) in length. NSP3 from all other group A rotaviruses is only 313 aa in length because of a 2-amino-acid deletion near the carboxy-terminus, While the SA114F gene has the longest 3' untranslated region (UTR) of 132 nucleotides, that from other strains suffered deletions of varying lengths at two positions downstream of the translational termination codon. In spite of the divergence of the nucleotide (nt) sequence in the protein coding region, a stretch of about 80 nt in the 3' UTR is highly conserved in the NSP3 gene from all the strains. This conserved sequence in the 3' UTR might play an important role in the regulation of expression of the NSP3 gene. (C) 1995 Academic Press, Inc.

Comparative studies on the effects of specific immunoneutralization of endogenous FSH or LH on testicular germ cell transformations in the adult bonnet monkey (Macaca radiata)

PROBLEM: It is yet to be determined clearly whether the two hormones FSH and T act synergistically in the same cell type-the Sertoli cells-to control overall spermatogenesis or influence independently the transformation of specific germ cell types during spermatogenesis in the adult mammal. METHOD: Adult male bonnet monkeys specifically deprived of either FSH or LH using immunoneutralization techniques were monitored for changes in testicular germ cell transformation by DNA flow cytometry. RESULTS: FSH deprivation caused a significant reduction (>40%; P < 0.05) in [H-3] thymidine incorporation into DNA of proliferating 2C (spermatogonial) cells, a marked inhibition (>50%) in the transformation of 2C to primary spermatocytes (4C) and a concomitant, belated reduction (50%) in the formation of round spermatids (1C). In contrast, specific LH/T deprivation led to an immediate arrest in the meiotic transformation of 4C to 1C/HC leading to an effective and significant block (<90%; P < 0.01) in sperm production. CONCLUSION: Thus, LH rather than FSH deprivation has a more pronounced and immediate effect as the former primarily blocks meiosis (4C --> 1C/HC) which controls production of spermatids. These data provide evidence for LH/T and FSH regulating spermatogenic process in the adult primate by primarily acting at specific germ cell transformation steps.

Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus-specific cytotoxic T lymphocytes: Requirement for L3T4(+) T cells

The protective ability of cytotoxic T cells (CTL) raised in vitro against Japanese encephalitis virus (JEV) was examined by adoptive transfer experiments. Adoptive transfer of anti-JEV effecters by intracerebral (i.c.) but not by intraperitoneal (i.p.) or intravenous (i.v.) routes protected adult BALB/c mice against lethal i.c. JEV challenge. In contrast to adult mice, adoptive transfer of anti-JEV effecters into newborn (4-day-old) and suckling (8-14-day-old) mice did not confer protection. However, virus-induced death was delayed in suckling mice compared to newborn mice upon adoptive transfer. The specific reasons for lack of protection in newborn mice are not clear but virus load was found to be higher in newborn mice brains compared to those of adults and virus clearance was observed only in adult mice brains but not in newborn mice brains upon adoptive transfer. Specific depletion of Lyt 2.2(+), L3T4(+) or Thy-1(+) T cell populations before adoptive transfer abrogated the protective ability of transferred effecters. However, when Lyt 2.2(+) cell-depleted and L3T4(+) cell-depleted effecters were mixed and transferred into adult mice the protective activity was retained, demonstrating that both Lyt 2.2(+) and L3T4(+) T cells are necessary to confer protection. Although the presence of L3T4(+) T cells in adoptively transferred effector populations enhanced virus-specific serum neutralizing antibodies, the presence of neutralizing antibodies alone without Lyt 2.2(+) cells was not sufficient to confer protection.

Development of specific DNA probes and their usage in the detection of Plasmodium vivax infection in blood

The application of nucleic acid probes, in the detection of pathogenic micro-organisms, has become an integral part of diagnostic technologies. In this study, Plasmodium vivax-specific DNA probes have been identified by carrying out genomic subtractive hybridization. In this approach, the recombinant clones from a P. vivax genomic library are screened with radiolabelled human and P. falciparum DNA. The colonies which react with labelled P. falciparum and human DNA are eliminated and those which do not produce any autoradiographic signal have been subjected to further screening procedures. Three Fl vivax specific DNA probes have been obtained by these repeated screenings. Further analyses indicate that these probes are specific and sensitive enough to detect P. vivax infection in clinical blood samples when used in a non-radioactive DNA hybridization assay. (C) 1995 Academic Press Limited

Use of a specific aromatase inhibitor for determining whether there is a role for oestrogen in follicle/oocyte maturation, ovulation and preimplantation embryo development

The specific role of oestrogen in follicular maturation, ovulation and early embryonic development was investigated using Fadrozole (CGS 16949A), a non-steroidal aromatase inhibitor, to block oestrogen synthesis specifically and effectively in experimental animals. Induced and normal cyclical follicular maturation as well as normal and hCG/LH-induced ovulation were relatively unaffected by significantly depleting oestrogen in all animals (hamsters, rabbits, monkeys) studied other than rats. Fadrozole treatment significantly reduced the number of healthy antral follicles produced and the ovulatory response to exogenous hCG of immature rats primed with pregnant mares' serum gonadotrophin. The effect was specific, in that exogenously administered oestrogen reversed the blockade. Depletion of oestrogen, starting early in pro-oestrus in hamsters, had no effect on ovulation, oocyte maturation and fertilization, as normal implantation sites were seen on day 6 after coitus. In rabbits, oestrogen depletion during the periovulatory phase affected oviductal morphology and function. Although fertilization was not impaired, early embryo development did not appear to be normal. In monkeys, oestrogen depletion during the follicular phase did not lead to a block of follicular maturation or ovulation but resulted in a significant reduction in secretion of cervical mucus. Administration of either Fadrozole or Tamoxifen during the early luteal phase in cyclic monkeys that were allowed to mate prevented implantation and this appears to be due to impaired fertilization or faulty embryo development. These results suggest that, although there is a clear requirement for oestrogen to support the reproductive cycle in the female, the need for oestrogen in regulating specific events is species dependent.

Cloning, expression, purification, crystallization and preliminary X-ray studies of the mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis

The mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis has been cloned, expressed, purified and crystallized and the crystals have been characterized using X-ray diffraction. The Matthews coefficient suggests the possibility of two lectin domains in the triclinic cell. The amino-acid sequence of the domain indicates structural similarity to well characterized beta-prism II fold lectins.

Specific heat measurements on Ge-Se-In glasses

Bulk glasses of Ge(20)Se(80-x)ln(x) (O less than or equal to x less than or equal to 18) have been used for measurements of heat capacity at constant pressure (C-p) using a differential scanning calorimeter. These measurements reveal the chemical threshold in these glasses as a function of composition. The results are discussed in the light of microscopic phase separation in these glasses.

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro La (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2(d)) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K(k)D(d)) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K(k)D(d)) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.

Low-temperature specific heat of antiferromagnetic EuNi5P3 and mixed-valent EuNi2P2 in magnetic fields to 7 T

he specific heats of EUNi(5)P(3), an antiferromagnet, and EuNi2P2, a mixed-valence compound, have been measured between 0.4 and 30 K in magnetic fields of, respectively, 0, 0.5, 1, 1.5, 2.5, 5, and 7 T, and 0 and 7 T. In zero field the specific heat of EuNi5P3 shows a h-like anomaly with a maximum at 8.3 K. With increasing field in the range 0-2.5 T, the maximum shifts to lower temperatures, as expected for an antiferromagnet. In higher fields the antiferromagnetic ordering is destroyed and the magnetic part of the specific heat approaches a Schottky anomaly that is consistent with expectations for the crystal-field/Zeeman levels. In low fields and for temperatures between 1.5 acid 5 K the magnetic contribution to the specific heat is proportional to the temperature, indicating a high density of excited states with an energy dependence that is very unusual for an antiferromagnet. The entropy associated with the magnetic ordering is similar to R In8, confirming that only the Eu2+-with J=7/2, S=7/2, L=0-orders below 30 R. In zero field approximately 20% of the entropy occurs above the Neel temperature, consistent. with the usual amount of short-range order observed in antiferromagnets. The hyperfine magnetic field at the Eu nuclei in EUNi(5)P(3) is 33.3 T, in good agreement with a value calculated from electron-nuclear double resonance measurements. For EuNi2P2 the specific heat is nearly field independent and shows no evidence of magnetic ordering or hyperfine fields. The coefficient of the electron contribution to the specific heat is similar to 100 mJ/mol K-2.