Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Predator avoidance behaviour in the buffy-headed marmoset,Callithrix flaviceps

The predator avoidance behaviour of a free-ranging group of buffy-headed marmosets,Callithrix flaviceps, was recorded in detail during the course of a long-term study of behavioural ecology at the Fazenda Montes Claros, southeastern Brazil. Four distinct patterns of predator avoidance behaviour, each with specific vocalisations, were recognised and are described here. The selection and use of sleeping sites by the study group are also described. An analysis of the records indicates that these small monkeys are generally most vulnerable to predation by aerial raptors. Variations in the frequency of alarm calls also indicate that the marmosets tend to be more vigilant at higher levels in the forest and when the leaf cover is less extensive. The implications of group size and social structure for both the evolution and the efficacy of the anti-predator behaviour of marmosets are also discussed.     

Digestive enzymes in midgut cells,endo-and ectoperitrophic contents,and peritrophic membranes of Spodoptera frugiperda (lepidoptera) larvae

In the midgut of Spodoptera frugiperda larvae, subcellular fractionation data suggest that aminopeptidase and part of amylase, carboxypeptidase A, dipeptidase, and trypsin are bound to the microvillar membranes; that major amounts of soluble dipeptidase, cellobiase, and maltase are trapped in the cell glycocalyx; and finally that soluble carboxypeptidase, amylase, and trypsin occur in intracellular vesicles. Most luminal acetylglucosaminidase is soluble and restricted to the ectoperitrophic contents. Aminopeptidase occurs in minor amounts bound to membranes both in the ectoperitrophic contents and incorporated in the peritrophic membrane. Amylase, carboxypeptidase A, and trypsin are found in minor amounts in the ectoperitrophic contents (both soluble and membrane-bound) and in major amounts in the peritrophic membrane with contents. Part of the activities recovered in the last mentioned contents corresponds to enzyme molecules incorporated in the peritrophic membrane. The results suggest that initial digestion is carried out in major amounts by enzymes in the endoperitrophic space and, in minor amounts, by enzymes immobilized in the peritrophic membrane. Intermediate and final digestion occur at the ectoperitrophic space or at the surface of midgut cells. The results also lend support to the hypothesis that amylase and trypsin are derived from membrane-bound forms, are released in soluble form by a microapocrine mechanism, and are partly incorporated into the peritrophic membrane. © 1994 Wiley-Liss, Inc.     

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N -Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.     

The chimeric cytokine Hyper-IL-6 enhances the efficiency of lentiviral gene transfer in hepatocytes both in vitro and in vivo

Lentiviral vectors have been used for gene transfer into the liver but their ability to efficiently transduce quiescent hepatocytes remains controversial. Lentivirus-mediated gene transfer is more efficient in cycling cells. We determine the effect of H-IL6 in the lentiviral transduction. The lentiviral vector was used to transduce HepG2 cells and mice liver cells, previously treated with H-IL6. The highest transduction level was observed in HepG2 cells treated with 30 ng/mL H-IL6 and in the mice that received 4 μg H-IL6. Our results suggest that H-IL6 is an inducer of lentiviral gene transfer into the liver cells without any toxicity.     

High-resolution mapping reveals linkage between genes in common bean cultivar Ouro Negro conferring resistance to the rust,anthracnose, and angular leaf spot diseases

Key message

Co-segregation analysis and high-throughput genotyping using SNP, SSR, and KASP markers demonstrated genetic linkage between Ur-14 and Co-3 ⁴ /Phg-3 loci conferring resistance to the rust, anthracnose and angular leaf spot diseases of common bean.

Abstract

Rust, anthracnose, and angular leaf spot are major diseases of common bean in the Americas and Africa. The cultivar Ouro Negro has the Ur-14 gene that confers broad spectrum resistance to rust and the gene cluster Co-3 ⁴ /Phg-3 containing two tightly linked genes conferring resistance to anthracnose and angular leaf spot, respectively. We used co-segregation analysis and high-throughput genotyping of 179 F_2:3 families from the Rudá (susceptible) × Ouro Negro (resistant) cross-phenotyped separately with races of the rust and anthracnose pathogens. The results confirmed that Ur-14 and Co-3 ⁴ /Phg-3 cluster in Ouro Negro conferred resistance to rust and anthracnose, respectively, and that Ur-14 and the Co-3 ⁴ /Phg-3 cluster were closely linked. Genotyping the F_2:3 families, first with 5398 SNPs on the Illumina BeadChip BARCBEAN6K_3 and with 15 SSR, and eight KASP markers, specifically designed for the candidate region containing Ur-14 and Co-3 ⁴ /Phg-3, permitted the creation of a high-resolution genetic linkage map which revealed that Ur-14 was positioned at 2.2 cM from Co-3 ⁴ /Phg-3 on the short arm of chromosome Pv04 of the common bean genome. Five flanking SSR markers were tightly linked at 0.1 and 0.2 cM from Ur-14, and two flanking KASP markers were tightly linked at 0.1 and 0.3 cM from Co-3 ⁴ /Phg-3. Many other SSR, SNP, and KASP markers were also linked to these genes. These markers will be useful for the development of common bean cultivars combining the important Ur-14 and Co-3 ⁴ /Phg-3 genes conferring resistance to three of the most destructive diseases of common bean.

     

Cell recruitment and cytokines in skin mice sensitized with the vaccine adjuvants: saponin, incomplete Freund's adjuvant, and monophosphoryl lipid A

Vaccine adjuvants are substances associated with antigens that are fundamental to the formation of an intense, durable, and fast immune response. In this context, the use of vaccine adjuvants to generate an effective cellular immune response is crucial for the design and development of vaccines against visceral leishmaniasis. The objective of this study was to evaluate innate inflammatory response induced by the vaccine adjuvants saponin (SAP), incomplete Freund's adjuvant (IFA), and monophosphoryl lipid A (MPL). After a single dose of adjuvant was injected into the skin of mice, we analyzed inflammatory reaction, selective cell migration, and cytokine production at the injection site, and inflammatory cell influx in the peripheral blood. We found that all vaccine adjuvants were able to promote cell recruitment to the site without tissue damage. In addition, they induced selective migration of neutrophils, macrophages, and lymphocytes. The influx of neutrophils was notable at 12 h in all groups, but at other time points it was most evident after inoculation with SAP. With regard to cytokines, the SAP led to production of interleukin (IL)-2, IL-6, and IL-4. IFA promoted production of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, IL-17, IL-4, and IL-10. We also observed that MPL induced high production of IL-2, TNF-α, and IFN-γ, in addition to IL-6, IL-17, and IL-10. In peripheral blood, values of certain cell populations in the local response changed after stimulation. Our data demonstrate that the three vaccine adjuvants stimulate the early events of innate immune response at the injection site, suggesting their ability to increase the immunogenicity of co-administered antigens. Moreover, this work provides relevant information about elements of innate and acquired immune response induced by vaccine adjuvants administered alone.     

Further evidence for cortical control over replication of the macronucleus and basal bodies in Stentor

This paper presents further evidence that the cortex controls macronuclear replication and basal body production during the cell cycle of Stentor. At the onset of cell division, basal body production occurs on the ventral side of the cell to form an oral primordium; this structure develops slowly into the oral apparatus destined for the posterior daughter cell. Meanwhile, a series of morphological changes in the macronucleus (coalescence, elongation, nodulation) doubles the number of nodes in preparation for division. When a cell undergoing oral development is grafted to a morphostatic cell of equal size, oral development is usually induced in the morphostatic component and the two members of the graft complex eventually become synchronized with respect to macronuclear morphology. However, primordium induction and nuclear synchronization usually do not occur when the 2 members of the graft complex are separated by cortical discontinuities which heal gradually, even though the graft components demonstrably share a common endoplasm throughout the experiment. These results suggest that the cell surface may control the replication of organelles such as the macronucleus and basal bodies which are normally kept “in step” with the cell cycle in such a way that they are not lost or reproduced too frequently.     

Leprosy in a prison population: A new active search strategy and a prospective clinical analysis

BackgroundThis study evaluates an active search strategy for leprosy diagnosis based on responses to a Leprosy Suspicion Questionnaire (LSQ), and analyzing the clinical, immunoepidemiological and follow-up aspects for individuals living in a prison population.MethodsA cross-sectional study based on a questionnaire posing 14 questions about leprosy symptoms and signs that was distributed to 1,400 prisoners. This was followed by dermatoneurological examination, anti-PGL-I serology and RLEP-PCR. Those without leprosy were placed in the Non-leprosy Group (NLG, n = 1,216) and those diagnosed with clinical symptoms of leprosy were placed in the Leprosy Group (LG, n = 34).FindingsIn total, 896 LSQ were returned (64%), and 187 (20.9%) of the responses were deemed as positive for signs/symptoms, answering 2.7 questions on average. Clinically, 1,250 (89.3%) of the prisoners were evaluated resulting in the diagnosis of 34 new cases (LG), based on well-accepted clinical signs and symptoms, a new case detection rate of 2.7% within this population, while the NLG were comprised of 1,216 individuals. The confinement time medians were 39 months in the LG while it was 36 months in the NLG (p>0.05). The 31 leprosy cases who responded to the questionnaire (LSQ+) had an average of 1.5 responses. The symptoms “anesthetized skin area” and “pain in nerves” were most commonly mentioned in the LG while “tingling, numbness in the hands/feet”, “sensation of pricks and needles”, “pain in nerves” and “spots on the skin” responses were found in more than 30% of questionnaires in the NLG. Clinically, 88.2% had dysesthetic macular skin lesions and 97.1% presented some peripheral nerve impairment, 71.9% with some degree of disability. All cases were multibacillary, confirming a late diagnosis. Anti-PGL-I results in the LG were higher than in the NLG (p<0.0001), while the RLEP-PCR was positive in 11.8% of the patients.InterpretationOur findings within the penitentiary demonstrated a hidden prevalence of leprosy, although the individuals diagnosed were likely infected while living in their former communities and not as a result of exposure in the prison. The LSQ proved to be an important screening tool to help identify leprosy cases in prisons.     

Tropical Bacillus Strains Inoculation Enhances Maize Root Surface Area,Dry Weight,Nutrient Uptake and Grain Yield

Journal of Plant Growth Regulation - The rising demand for agricultural commodities in developing countries has put increasing pressure on land resources for higher yields, with associated growth...