Noninvasive semen collection from an adult orangutan

Preservation of the genetic diversity of the captive orangutan, especially the wild-caught founders, is critical in maintaining a long-term population in zoological parks. One solution to the problem of maintaining maximum genetic diversity would be to initiate a program of artificial insemination for genetically underrepresented individuals through the banking and interinstitutional use of cryopreserved semen. However, little is known about basic orangutan semen characteristics, and current methodology is inadequate to support such a program. In this paper, we report the results of semen collection from an adult Sumatran orangutan (Pongo pygmaeus abelli), using an artificial vagina without anesthesia or electrical stimulation. A total of 27 ejaculates were evaluated during a 1-year period. The total and liquid volumes of the ejaculates at 1 h following collection were 6.1 ± 0.6 ml and 2.6 ± 0.4 ml, respectively (mean ± SEM). The liquid portion continued to exude semen for 2 h; however, 90% of the motile sperm was exuded within the first 30 min. The total number of sperm in the ejaculate was 164 ± 10⁶ ± 16.5, and the percentage of motile cells was 60 ± 2.7%. We conclude that the artificial vagina provides a promising technique for semen collection in the orangutan, and view these results as an initial step in developing methods for in vitro sperm capacitation, sperm cryopreservation, and artificial insemination. © 1993 Wiley-Liss, Inc.     

The modifying effect of gibberellic acid and kinetin on the growth abnormalities induced by 2,4-D in marrow seedlings and the possible involvement of endogenous ethylene

Swelling of the hypocotyl base induced by 2,4-D in seedlings of marrow was much reduced if GA₃ was also present in the incubation medium. At appropriate concentrations kinetin also counteracted this 2,4-D effect, although at higher concentrations appeared to be ineffective. GA₃ was also able to overcome the inhibitory effects of 2,4-D on extension growth in the hypocotyl but kinetin was much less effective in this case. None of the treatments employed was able to alleviate the inhibition of radicle extension induced by 2,4-D.Ethephon induced similar responses in the seedlings to those resulting from 2,4-D treatment, while treatment with a mixture of 2,4-D and CoCl₂ removed many of these growth abnormalities. These observations are interpreted as indicating that 2,4-D operates at least partly by stimulating the production of ethylene in the tissues. 2,4-D strongly inhibited the accumulation of endogenous gibberellin during the period of seedling development examined, but enhanced cytokinin levels during the later stages of the same period. The possibility of interactions at the biosynthetic level between gibberellins, cytokinins and ethylene and their involvement in the regulation of seedling development are discussed.     

The pharmacological actions of 5-hydroxytryptamine,FMRF-amide and substance P and their possible occurrence in the heart of the snail Helix aspersa L.

The actions of 5-hydroxytryptamine (5-HT), FMRF-amide and substance P were tested on the isolated heart of Helix aspersa. All three compounds were found to produce positive inotropic and chronotropic effects, the order of potency being 5-HT > substance P > FMRF-amide. However, whereas the responses to 5-HT and FMRF-amide were maximal within a few seconds, the response to substance P had a longer latency. Two other similar undecapeptides, eledoisin and physalaemin, were also tested and were found to have very similar actions to those of substance P. The effects of 5-HT and FMRF-amide could be separated using the 5-HT blockers methysergide and ketanserin, which had relatively little effect on the response to FMRF-amide.Immunohistochemical staining was carried out on sections through the Helix auricle and ventricle for 5-HT, FMRF-amide and substance P. Substantial 5-HT-like and substance P-like immunoreactivity was observed, apparently concentrated in nerve endings, but the level of FMRF-amide-like immunoreactivity was considerably lower. The fluorescence produced by all three compounds was evenly distributed throughout the heart tissue. HPLC analysis of heart tissue extract demonstrated a high level of 5-HT (about 8 μg/g wet weight) but a negligible catecholamine content.     

Evidence for conversion of N-Tyr-MIF-1 into MIF-1 by a specific brain aminopeptidase

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly·NH₂), an immunoreactive neuropeptide exhibiting saturable high affinity binding in rat brain was found to be converted into MIF-1 (Pro-Leu-Gly·NH₂) by a specific brain aminopeptidase present in rat brain homogenates or cytosol, but with low activity associated with synaptosomal plasma membranes and microsomes. Conversion occurred at a rate of 16 μmol per g w/wt per h and was unaffected by puromycin but inhibited by bestatin (I₅₀, 5 × 10^?5 M). Aminopeptidases purified from cytosolic fractions of rat brain (arylamidase), mouse brain (Mn²⁺-activated aminopeptidase) or porcine kidney (leucine aminopeptidase) were inactive towards N-Tyr-MIF-1 but degraded MIF-1 with release of Leu-Gly·NH₂ as detected by RP-HPLC procedures. Morphiceptin (Tyr-Pro-Phe-Pro·NH₂), a μ opioid agonist, also acted as a substrate for the N-Tyr-MIF-1 converting enzyme with cleavage of the Tyr-Pro bond. These tetrapeptides, but not MIF-1 or its N-blocked analogs, were degraded in vitro by a metalloendopeptidase purified from kidney membranes. Since dipeptide products were not detected for crude extracts, a significant role for brain metalloendopeptidase on turnover can be excluded. Thus the results point to the presence of a specific (X-Pro-degrading) aminopeptidase in brain cytosol as an enzyme responsible for converting N-Tyr-MIF-1 and inactivating morphiceptin.     

Phonotaxis in the painted reed frog (Hyperolius marmoratus)

Summary A detailed study was conducted of the three-dimensional accuracy of phonotaxis by femaleHyperolius marmoratus. This analysis involved videotape recordings of phonotactic approaches to an elevated loudspeaker through a three-dimensional grid. Females readily resolved the sound source elevation, but the jump error angles describing the precision of approach were considerably greater in this three-dimensional analysis than in the more conventional two-dimensional ground approach analysis. Extensive use was made of visual cues in elevated phonotactic approach and lateral head scanning prior to jumps, often accompanied by vertical changes in head orientation, was frequent. The ability of such small anurans to localize a sound source in both the horizontal and vertical plane is remarkable.On leave from the Section of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853, USA     

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. I. Synthesis of diphtheria toxin fragment A-S-S-human placental lactogen with methyl-5-bromovalerimidate.

In order to study the mechanism of entry of plant seed and bacterial toxins into mammalian cells, methods have been developed to synthesize artificial protein hybrid conjugates containing a moiety which binds to a cell membrane receptor and an active fragment of a toxin protein. Utilizing methyl-5-bromovalerimidate, a disulfide cross-linked conjugate of human placental lactogen (hPL) and diphtheria toxin fragment A (toxin A) was synthesized. The reagent was prepared from 5-bromovaleryl nitrile by Pinner synthesis and then used to amidinate hPL. The bromo group thus introduced was converted to S-sulfonate by nucleophilic displacement with 1 M aqueous sodium thiosulfate at room temperature overnight. The S-sulfonated hPL reacted readily with the-SH gorup of reduced toxin A to form a 1 mol/mol of disulfide conjugate in high yield. Thus when reduced toxin A was incubated with a 4-fold excess of the hPL S-sulfonate at 4 degrees and pH 6.5 for 120 h, a conjugate yield of 50% relative to the toxin A input was obtained. Homopolymer formation was negligible and the product was purified by gel filtration on Sephadex G-150. Purity of the conjugate estimated by quantitative analysis of sodium dodecyl sulfate gels was 90%. The toxin A-hPL conjugate retained the activities of both toxin A and hPL, as reported in the accompanying paper. This method of preparing protein hybrid conjugates appeared to have advantages over previous methods utilizing bifunctional reagents with respect to both yield and freedom from homopolymer formation.     

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen.

The biochemical and biologic properties of a purified disulfide conjugate of diphtheria toxin fragment A and human placental lactogen (toxin A-hPL) have been studied by (a) assaying the ADP-ribosyltransferase activity of the intact conjugate, (b) assaying the binding of the intact conjugate to mammary gland plasma membrane lactogenic receptors, and (c) assaying the effect of the conjugate on the rate of protein synthesis in rabbit mammary gland explants maintained in organ culture. The toxin A-hPL conjugate retains one-third of the NAD+:EF-2 ADP-ribosyltransferase activity of toxin A, and 26% of the hPL-binding activity to lactogenic receptors. Binding activity was demonstrated by radioreceptor assay and by assaying toxin A activity bound to membranes which was competitively displaced by excess hPL. Since the toxin A-hPL conjugate retained activities of its separate subunits, it could be regarded as a structural analogue of nicked diphtheria toxin with replacement of the original membrane-binding chain by another binding chain that is specific for lactogenic receptor. However, the conjugate failed to inhibit protein synthesis in organ-cultured mammary gland explants, although these were sensitive to native diphtheria toxin and could bind hPL. It is concluded from these results that the toxin A-hPL conjugate does not act as a functional analogue of diphtheria toxin with altered receptor specificity, and that the hPL receptor cannot mediate the entry of toxin A or toxin A-hPL from membrane-bound conjugate into the cytosol site of action of toxin A.     

Control of amino acid transport in the mammary gland of the pregnant mouse

The regulation of the uptake of the amino acid analog α-aminoisobutyric acid was studied in diced mammary glands from pregnant mice. Stimulation of uptake by insulin was not prevented by inhibitors of protein synthesis; protein synthesis inhibitors decreased uptake by 20%; this response occurred more promptly in insulintreated tissues. Elimination of extracellular amino acids led to a substantial increase in transport which was not abolished by inhibitors of protein synthesis. These results indicate that insulin does not increase amino acid transport in this system by altering synthesis and degradation of transport protein. They are consistent with a model in which the activity of the existing amino acid transport protein is subject to negative feedback regulation from the intracellular amino acid pool.     

Metabolic effects of salbutamol: comparison of aerosol and intravenous administration.

The effects of intravenous salbutamol (4 mug/kg) were compared with those of aerosol salbutamol (200 mug) in 10 asthmatic patients in a double-blind placebo-controlled study. Both methods of administration produced equal bronchodilatation. Intravenous salbutamol caused significant increases in plasma insulin and glucose levels and a fall in serum potassium concentration in addition to tachycardia and tremor, whereas aerosol salbutamol produced only a small transient increase in the plasma glucose level. The initially raised non-esterified fatty acid levels decreased significantly after aerosol and placebo but not after intravenous salbutamol.