A proposed neural pathway for vocalization in South African clawed frogs,Xenopus laevis

Vocalizations of South African clawed frogs are produced by contractions of laryngeal muscles innervated by motor neurons of the caudal medulla (within cranial nerve nucleus IX-X). We have traced afferents to laryngeal motor neurons in male and female frogs using retrograde axonal transport of horseradish peroxidase conjugated to wheat germ agglutinin (HRP-WGA). After iontophoretic injection of HRP-WGA into n. IX-X, retrogradely labelled neurons were seen in the contralateral n. IX-X, in rhombencephalic reticular nuclei, and in the pre-trigeminal nucleus of the dorsal tegmental area (DTAM) of both males and females.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Androgen-induced alterations in vocalizations of femaleXenopus laevis: modifiability and constraints

Summary We examined effects of exogenous androgen (testosterone and dihydrotestosterone) on vocalizations of ovariectomized, adult female South African clawed frogs,Xenopus laevis. When paired with sexually active males, all ovariectomized females exhibited ticking, the unreceptive or release call. Ticking consists of low amplitude, regularly spaced clicks with a mean interclick interval of 154 ms. When androgen-treated and paired with sexually active males, these ovariectomized females also exhibited an aberrant call (atypical ticking) in which click multiples replaced the single clicks of ticking. Mean ICI's for atypical ticking were 37 ms for click doublets and 22 ms for click quadruplets. Androgen treatment decreased the total time spent vocalizing (typical and atypical ticking) by ovariectomized females.All androgen-treated females were then tested repeatedly with sexually receptive females in an attempt to elicit the male-typical vocalization, mate calling. Six of 17 females did not vocalize at all, even when gonadotropin injected. Eight females gave rapid (mean ICI, 36 ms) trains of clicks in an irregular temporal pattern (tick-like calls). Three females gave brief trills with alternating fast and slow components. Comparison of mate calllike vocalizations of androgen-treated females to mate calling of males reveals that calls in females are considerably shorter in duration (female: 0.32 min versus male: 45 min) and slower in tempo (ICI's; fast trill, female: 21 ms, male: 14 ms; slow trill, female: 36 ms, male: 28 ms). Incomplete masculinization of the vocal pattern of females by androgen treatment in adulthood may be due to developmental constraints on the modifiability of the neurons and muscles responsible for calling.Abbreviations C cholesterol - DHT dihydrotestosterone - HCG human chorionic gonadotropin - IBI interburst interval - ICI interclick interval - ovx ovariectomized - T testosterone     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

An optimized live bacterial delivery vehicle safely and efficaciously delivers bacterially transcribed therapeutic nucleic acids

There is an unmet need for delivery platforms that realize the full potential of next-generation nucleic acid therapeutics. The in vivo usefulness of current delivery systems is limited by numerous weaknesses, including poor targeting specificity, inefficient access to target cell cytoplasm, immune activation, off-target effects, small therapeutic windows, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we characterize the safety and efficacy of a delivery platform comprising engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli SVC1) for intracellular cargo delivery. SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to allow escape of their cargo from the phagosome, and to have minimal immunogenicity. We describe SVC1's ability to deliver short hairpin RNA (shRNA), localized SVC1 administration to various tissues, and its minimal immunogenicity. To validate the therapeutic potential of SVC1, we used it to deliver influenza-targeting antiviral shRNAs to respiratory tissues in vivo. These data are the first to establish the safety and efficacy of this bacteria-based delivery platform for use in multiple tissue types and as an antiviral in the mammalian respiratory tract. We expect that this optimized delivery platform will enable a variety of advanced therapeutic approaches.     

Studies on the characteristics of alginate gels in relation to their use in separation and immobilized applications

Studies on diffusion of NAD and hemoglobin from calcium and barium gels are reported where alginate grade, concentration, and gel dimensions were varied. These show that NAD diffusion characteristics are unaffected by alginate and ion concentrations; however, hemoglobin diffusion is affected by alginate concentration. Both hemoglobin and NAD diffusion patterns were shown to be affected by alginate gel dimensions. Studies are reported that show that polymannuronic alginate gels posses good porosity characteristics while polyguluronic alginates from gels with lower porosity, specifically with respect to high-molecular-weight compounds. These findings are discussed with the view to the use of alginate gels for immobilization, solids separation, and diffusion chromatography techniques.     

Effect of rIFN-gamma and IL-2 treatments in mouse and nude rat infections with Toxoplasma gondii.

Mice and nude rats lethally infected with T. gondii and treated with recombinant rat interferon-gamma (rIFN-gamma) or recombinant human interleukin-2 (rIL-2) were protected against death, when compared with untreated infected controls. In mice rIFN-gamma and rIL-2 played an important role in "prophylactic treatment", but not in "curative therapy". The survival rate was 42% in mice treated with 3 doses of 20,000 U of rIFN-gamma at days -2, -1, 0 before challenge and up to 66% in mice treated with 3 doses of 10,000 U of rIFN-gamma at days -2, 0, +2 before and after infection. Whereas the survival rate was 33% in mice that received 3 doses of 500 U rIL-2 at days -2, -1, 0 before infection, or -2, 0, +2 before and after infection respectively, up to 50% of the mice treated with 3 doses of 1,000 U rIL-2 at days -2, -1, 0 survived. In nude rats rIFN-gamma had a slight effect in "prophylactic treatment", whereas rIL-2 was active only in "curative treatment". The survival rate was 25% both in nude rats treated with doses of 400,000 U of rIFN-gamma at days -3, 0 before challenge, or with doses of 5,000 U of rIL-2 at days +2, +6, +9 after infection. These results lead us to hypothesise that the mechanism by which the lymphokine treatment exerts a protective effect on Toxoplasma infected mice is different from that on nu/nu rats. We conclude that these cytokines may play a notable role in modulating the host's immune defence against T. gondii infection.     

Protection of mice and nude rats against toxoplasmosis by a multiple antigenic peptide construction derived from Toxoplasma gondii P30 antigen.

The first part of this work presents the sequence of the first 20 NH2 terminus residues obtained from P30, the major surface Ag of Toxoplasma gondii, purified by HPLC. A synthetic peptide (P30 48-67) has been prepared both in linear form and as a multiple antigenic peptide (MAP) construct. Immunization of mice and rats with the P30 48-67 MAP in the presence of IFA induces high levels of IgG antibodies that recognize both the linear peptide and the MAP construct in ELISA, and P30 in Western blots of NP-40-extracted tachyzoite Ag. Because these sera are negative in immunofluorescence assays with whole tachyzoites, it seems that IgG antibodies induced by P30 48-67 MAP, although recognizing the denatured structure, are unable to recognize the native protein. However, the protective effect of both constructs has then been studied in mice and nude rats. Whereas immunization of mice with the monomeric peptide does not confer any protection against oral infection with 1200 cysts of T. gondii 76K strain (mortality within 11 days), 40% of mice immunized with the MAP construct survived up to 75 days after infection. Nude rats were passively transferred with 5 x 10(4) T lymphocytes from P30 48-67 MAP-immunized Fischer rats before infection with 5 x 10(4) RH strain tachyzoites. They survived up to 40 days after infection and raised an intense IgG antibody response against P30, whereas nude rats transferred with control lymphocytes died within 21 days. This shows that immunization with P30 48-67 MAP also induces an efficient T cell immune response. The present work confirms the recently demonstrated role of P30 in protective immunity and shows the interest of peptide octameric constructions as inducers of partially protective immune responses in toxoplasmosis, as already demonstrated in schistosomiasis.     

Hormone effects on male sex behavior in adult South African clawed frogs, Xenopus laevis

Intact, adult male Xenopus laevis were injected with human chorionic gonadotropin (HCG) and tested with intact HCG-primed females. Under these conditions, males displayed high levels of sex behavior (clasping of females). By 2 weeks after castration, these males had ceased clasping. Testosterone and dihydrotestosterone reinstated clasping in male castrates. Following removal of testosterone or dihydrotestosterone pellets, castrated males ceased to clasp. No male was ever observed to clasp following estradiol implanted in pellets or in silastic capsules. In experiments on castrated, adult, female Xenopus laevis, both testosterone and testosterone propionate pellets reliably produced male sex behavior in the form of clasping. The clasping of testosterone-implanted female and male castrates was very similar in form and duration. The behavioral effectiveness of testosterone in both sexes and the ineffectiveness of estradiol in eliciting clasping is paralleled by autoradiographic localization of sex steroids in brain where the distribution of testosterone-concentrating, cells is the same for males and females, but different from the distribution of estradiol-concentrating cells.