Successful artificial insemination (AI) in the koala using neat and extended semen collected by electroejaculation (EE)

Presently AI in the koala has been based on the insemination of fresh undiluted semen collected with an artificial vagina (1). While this approach has been extremely successful, further refinement and implementation of AI for use with cryopreserved semen will require protocols that incorporate diluted semen collected by EE. Recent studies have shown that koala semen is likely to have an "ovulation factor" such that over-dilution may result in ovulation failure (2). The current study determined whether AI of EEed neat and/or diluted semen was capable of inducing a luteal phase and/or resulted in the production of pouch young. All koalas were inseminated in the breeding season between day 2 and 5 of oestrus and subsequently monitored for evidence of parturition (day 35) and return of oestrus. Successful induction of a luteal phase was based on evidence of an elevated progesterone concentration 28 days after insemination (2). All semen samples were collected by EE and seminal characteristics recorded (3). The diluent used for semen extension was Tris-citrate glucose (TCG) which contained antibiotics but no egg yolk (4). AI was conducted on conscious koalas using a "Cook koala insemination catheter" and a glass rod used to mimic penile thrusting (1). Three insemination treatments were used; (A) 1mL of undiluted semen (n = 9); (B) 2mL of 1:1 diluted semen (n = 9); and (C) 1 mL of 1:1 diluted semen (n = 9). The results of the AI trial are shown in Table 1. This study has shown that it is possible to use both neat and diluted semen (1:1; 1 or 2 mL) to successfully produce koala offspring at conception rates similar to those achieved following natural mating. Interestingly, dilution of semen had no apparent detrimental effect on induction of a luteal phase following AI.

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMR solution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide A beta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native A beta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length A beta peptides A beta(1-40) and A beta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which A beta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of A beta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation. (C) 2000 Academic Press.

Comparative evaluation of juvenoids for control of cat fleas (Siphonaptera : Pulicidae) in topsoil

The efficacy of a photostable formulation of methoprene and two photostable juvenoids, fenoxycarb and pyriproxyfen, and their residual activity in inhibiting the emergence of adult cat fleas, Ctenocephalides felis (Bouche), was studied in topsoil. Nursery pots composed of clay, peat, and plastic, and wooden flats were used to hold soil samples. Treated soil samples were exposed to sunlight during the 63-d study period. Methoprene was as effective as fenoxycarb and pyriproxyfen against cat fleas for up to 42 d in clay, peat, and plastic pots at a concentration of 64.56 mg (AI) /m(2) (6 mg [AI] /ft(2)), but its activity declined significantly thereafter. In contrast, fenoxycarb and pyriproxyfen showed strong residual activity for the entire 63 d. The activity of methoprene declined even more rapidly over time in wooden flats, while at the same concentrations the other two juvenoids showed significant residual activity for 63 d. Clay, peat, and plastic pots were therefore considered to be equally effective for evaluating the outdoor efficacy of juvenoids in comparison to the wooden flats. However, results obtained with wooden flats may be more realistic when testing residual activity of volatile chemicals such as methoprene. Fenoxycarb and pyriproxyfen showed strong efficacy and residual activity at concentrations of 8.07, 16.14, and 32.28 mg (AI) /m(2), whereas methoprene did not cause a significant reduction of adult emergence at levels below 64.56 mg (AI) /m(2). LC50 values for methoprene, fenoxycarb, and pyriproxyfen needed for preventing flea emergence when applied to topsoil were estimated to be 0.643, 0.031, and 0.028 ppm, respectively.

A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: Total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH2S)(28-29,56-57,76-77)]

The 101 residue protein early pregnancy factor (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH2CH2SH thiolate with XCH2CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH2CH2SH thiolate with a BrCH2CO-peptide than with a CICH2CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N-alpha-chloroacetyl, C-terminal thiolated peptide with a second N-alpha-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N-alpha-chloroacetyl peptides could be activated by halide exchange using saturated KI solutions to yield the highly reactive No-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or cassettes comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH2CH2SH] 13, ClAc-[EPF 30-55]-[NHCH2CH2SH] 14, and Ac-[EPF 1-27]-[NHCH2CH2SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH2S)(28-29,56-57,76-77)) 19 in 19% overall yield.