Transient appearance of Strongylocentrotus purpuratus Otx in micromere nuclei: Cytoplasmic retention of SpOtx possibly mediated through an α-actinin interaction

At the 16-cell stage, the sea urchin embryo is partitioned along the animal-vegetal axis into eight mesomeres, four macromeres, and four micromeres. The micromeres, unlike the other blastomeres, are autonomously specified to produce skeletogenic mesenchymal cells and are also required to induce the vegetal-plate territory. A long-held belief is that micromeres inherit localized maternal determinants that endow them with their cell autonomous behavior and inducing capabilities. Here, we present evidence that an orthodenticle-related protein, SpOtx appears transiently in nuclei of micromeres but not in nuclei of mesomeres and macromeres. At later stages of development, SpOtx was translocated into nuclei of all cells. To address the possibility that SpOtx was retained In the cytoplasm at early developmental stages we searched for cytoplasmic proteins that interact with SpOtx. A proline-rich region of SpOtx resembling an SH3-binding domain was used to screen an embryo cDNA expression library, and a cDNA clone was isolated and shown to be α-actinin. A yeast two-hybrid analysis showed a specific interaction between the proline-rich region of SpOtx and a putative SH3 domain of the sea urchin α-actinin. Because micromeres lack an actin-based cytoskeleton, the results suggested that, at the vegetal pole of the 16-cell stage embryo, SpOtx was translocated into micromere nuclei, whereas in other blastomeres SpOtx was actively retained in the cytoplasm by binding to α-actinin. The transient appearance of SpOtx in micromere nuclei may be associated with the specification of micromere cell fate. © 1996 Wiley-Liss, Inc.     

Novel hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling pathway in endothelial cells

Background  

The growth of new blood vessels in adult life requires the initiation of endothelial cell migration and proliferation from pre-existing vessels in addition to the recruitment and differentiation of circulating endothelial progenitor cells. Signals emanating from growth factors and the extracellular matrix are important in regulating these processes.     

Involvement of Manduca sexta peptidoglycan recognition protein-1 in the recognition of bacteria and activation of prophenoloxidase system

Although the importance of peptidoglycan recognition proteins (PGRPs) in detecting bacteria and promoting immunity is well recognized in Drosophila melanogaster and other insect species, such a role has not yet been experimentally established for PGRPs in the tobacco hornworm, Manduca sexta. In this study, we purified M. sexta PGRP1 from the baculovirus-insect cell expression system, tested its association with peptidoglycans and intact bacteria, and explored its possible link with the prophenoloxidase activation system in larval hemolymph. Sequence comparison suggested that PGRP1 is not an amidase and lacks residues for interacting with the carboxyl group of meso-diaminopimelic acid-peptidoglycans (DAP-PGs). M. sexta PGRP1 gene was constitutively expressed at a low level in fat body, and the mRNA concentration became much higher after an injection of Escherichia coli. Consistently, the protein concentration in larval plasma increased in a time-dependent manner after the immune challenge. Purified recombinant PGRP1 specifically bound to soluble DAP-PG of E. coli but not to soluble Lys-type PG of Staphylococcus aureus. In addition, this recognition protein completely bound to insoluble PGs from Micrococcus luteus, Bacillus megaterium and Bacillus subtilis, whereas its association with the bacterial cells was low even though their peptidoglycans are exposed on the cell surface. After PGRP1 had been added to plasma of naïve larvae in the absence of microbial elicitor, there was a concentration-dependent increase in prophenoloxidase activation. Phenoloxidase activity, as usual, increased after the plasma was incubated with peptidoglyans or bacterial cells. These increases became more prominent when insoluble M. luteus or B. megaterium PG or soluble E. coli PG and PGRP1 were both present. Statistic analysis suggested a synergistic effect caused by interaction between PGRP1 and these PGs. Taken together, these results indicated that PGRP1 is a member of the M. sexta prophenoloxidase activation system, which recognizes peptidoglycans from certain bacteria and initiates the host defense response. The unexplained difference between the purified PGs and intact bacteria clearly reflects our general lack of understanding of PGRP1-mediated recognition and how it leads to proPO activation.     

Feasibility of using different approaches for recruiting younger twins to establish a population based twin register in Sri Lanka.

Identifying twins for a population-based register can be achieved through birth records or community surveys. We studied the feasibility and effectiveness of different methods of identifying and recruiting twins to establish a population based register. To trace twins a population survey was carried out using an interviewer administered questionnaire. We also inspected the birth registration certificates at a divisional secretariat reported from a specified hospital between the years of 1985-1997 and compared it to the birth register of this same hospital. To recruit twins a random sample of 75 twin pairs (150 twins) identified at the Divisional Secretariat were contacted through the post and 25 twin pairs (50 twins) were personally visited. The prevalence of twins was 6.5 twins per 1000 people in the area surveyed. The twinning rate at the hospital was 18.92 twins per 1000 births. A discrepancy of 38 multiples births between the hospital labour room records and those registered at the DS was noted. The response from the postal invitation for recruitment was 59% and the response from the personal invitation was 68%. (Difference 9.4% 95% CI; 7.06-11.73). Community survey and systematic inspection of birth records either at the hospital or the birth registration office was an effective method to trace twins. Once traced, personal contact was more effective than the postal invitation for recruitment of younger twins. A cost-effective approach would be to use a postal coverage followed by personal contact for non-responders. The alternative method, community coverage, would have financial implications.     

A simple filtration device for separating nonadherent microorganisms from mammalian cells in in vitro studies on microbial adherence

In adherence studies, the removal of nonadherent microorganisms is essential for the valid enumeration of microorganisms that adhere to host cells. Although filtration devices are available commercially for the removal of nonadherent microorganisms, these are expensive and not reusable. In this article, we describe a simple, inexpensive, and reusable filtration device composed of two chambers of nylon, a nylon membrane of desired pore size, a rubber washer, and supporting stainless steel mesh. The device was effective in in vitro adherence assays for removing nonadherent endospores of Rhinosporidium seeberi from human buccal epithelial cells, providing valid counts of adherent microorganisms.     

Challenges in recruiting older twins for the Sri Lankan twin registry.

The National Twin Registry of Sri Lanka was established in 1997 as a volunteer register. To extend it to a population-based register, we examined the effectiveness of tracing older twins by inspecting birth records and recruiting them by postal invitation and in-person contact. Birth records at a divisional secretariat reported from 2 maternity hospitals between the years of 1954-1970 were scrutinised to identify a random sample of twins. These hospitals had the highest twin delivery rates for the whole country. We identified 620 twins and a questionnaire was mailed to them. Research assistants visited a cohort of non-respondents (71) in the postal survey. These 620 twins were identified after perusing 20700 birth records. The twinning rate was estimated at 29.95 ([620/20700] x 1000) twins per 1000 registered births (CI 27.63-32.27). In the postal survey, 37 (12%) responded and 62 letters were returned (20%). Both twins were still alive in 20 pairs, one was still alive in 15 pairs, and both twins were dead in 2 pairs. During field visits, 42 (59.2%) addresses were located. Information was available on 16 twin pairs. Both twins were alive in 8 pairs, one each in 4 pairs, and both were dead in 4 pairs and at least one twin was traced in 10 pairs (14%). Both the postal and the field survey gave a low yield. This finding is different from tracing younger twins born between 1985-1997 by using the same methods. Migration, urbanization and development in the country might have affected tracing older twins from the birth record addresses, which were decades old.     

Adsorption Mechanism of Cr(VI) onto Coir Pith

Reductive adsorption of Cr(VI) on coir pith (hereafter CP) was examined as a function of pH, ionic strength, and temperature. The CP contains 1.33 meq g^{? 1} phenolic, 0.43 meq g^{? 1} of lactonic, and 0.35 meq g^{? 1} carboxylic sites. Thus the CP surface is enriched with electron-donating oxygen functionalities. As evidenced by infrared (IR) spectroscopy, the Cr(VI) → Cr(III) conversion is facilitated by CP sites that are enriched with O─ O functional groups. The adsorption of reduced Cr(VI) was found to occur via C─ O─ functional groups first forming innersphere complexes with the CP surface, yielding keto (> C═ O) groups on the CP surface. The reductive adsorption of Cr(VI) was almost completed within 3 to 4 h, and it was dependent on pH and background ionic strength, yielding the highest monolayer coverage (9.56E-7 mol m^{? 2}) at pH 3.7 in 0.1 M NaNO₃. The Γ_Cr(III) followed the order with respect to the ionic strength: Γ_{0.1 M} > Γ_{0.01 M} > Γ_{0.001 M}. The initial rate constant, k _i , increased with temperature as k _i ^{313 K} > k _i ^{303 K} > k _i ^{293 K} > k _i ^{283 K}.     

Establishing a twin register in Sri Lanka.

Nearly all twin registers are based in developed countries and there is no twin register in the developing world. Our objectives were to initiate the process of establishing a nationwide twin register in Sri Lanka by starting a volunteer register first and working towards a population-based register. Regular newspaper advertisements, feature articles, radio talks, and television programmes were used to publicise a competition for twins, their parents/relatives and friends requesting them to participate by sending in details of twins. The competition ran from 28 March 1997 for a period of 3 months. It offered prizes for three winners selected by drawing lots. Advertisements highlighted the objective of the competition as establishing a twin register for future research and emphasised that informed consent would be obtained for individual research projects. Those who registered comprise 4602 twin pairs (same sex: male--1564, female--1885; different sex--1153), 80 sets of triplets (same sex: male--17, female--31; different sex--42) and two sets of quadruplets (different sex). The oldest twins, triplets, quadruplets are 85, 46, and 5 years old, respectively; 88.0% of twins are less than 30 years old. Although others have previously used media publicity to enrol twins in twin registers, we believe this to be the first time that twins have been enrolled through competition. We have more young twins, and our gender and zygosity proportions after applying Weinburg's rule do not match the proportions expected from a volunteer twin sample. Establishing a twin register for research purposes has proved possible in a developing country.     

Two Extracellular Matrices From Oocytes of the Marine Shrimp Sicyonia ingentis that Independently Mediate Only Primary or Secondary Sperm Binding

During spawning, female Sicyonia ingentis simultaneously release ova and stored nonmotile sperm and mix them externally to initiate gamete interaction. Sperm bind to a thin vitelline envelope (VE) via their anterior appendage and within seconds are induced to undergo acrosomal exocytosis. The sperm penetrate the VE and become secondarily bound to the surface coat (SC), a glycocalyx on the oocyte surface. In this study, both extracellular matrices were isolated from S. ingentis oocytes. Isolated VEs mediated only primary sperm binding (i.e., before the acrosome reaction), while the isolated SCs mediated only secondary sperm binding (i.e., after acrosomal exocytosis). Isolated S. ingentis VEs were used to characterize primary sperm binding activity. The two extracellular matrices differ morphologically and possess different polypeptide profiles. Soluble fractions of isolated VEs inhibited primary sperm binding in a concentration dependent manner, and immunolocalization of VE components demonstrated highly localized VE binding sites at the tip of the sperm anterior appendage by which sperm bind eggs. Extensive Pronase digestion of VE components did not affect sperm binding activity of solubilized VE components, while complete deglycosylation with trifluoromethanesulfonic acid destroyed sperm binding activity. However, neither alkaline treatment nor enzyme digestion using glycosidases specific for asparagine and serine/ threonine linked oligosaccharides affected sperm binding activity.     

Comparative genomic analysis of the <Emphasis Type="Italic">Tribolium</Emphasis> immune system

Background  

Tribolium castaneum is a species of Coleoptera, the largest and most diverse order of all eukaryotes. Components of the innate immune system are hardly known in this insect, which is in a key phylogenetic position to inform us about genetic innovations accompanying the evolution of holometabolous insects. We have annotated immunity-related genes and compared them with homologous molecules from other species.