Limb development in chick embryos: cyclic AMP-dependent protein kinase activity, cyclic AMP, and prostaglandin concentrations during cytodifferentiation and morphogenesis

The effects of prostaglandin E2 (PGE2) on cyclic AMP (cAMP) concentrations of chick limb bud cells obtained from limbs at various stages of development were investigated. In addition, endogenous concentrations of PGE2 were examined in whole limbs from comparable stages. Prior to either chondrogenesis or myogenesis (stages 20-23), cells were more responsive to PGE2, in terms of cAMP levels, than those of differentiated phenotypes, obtained at stages 25-28. This greater responsiveness to PGE2 of undifferentiated cells was correlated with endogenous concentrations of PGE2 which were significantly higher in undifferentiated limbs than in limbs containing differentiated cartilage and muscle. Cyclic AMP-dependent protein kinase (PKA) activity was detectable in cell homogenates at each stage examined and did not appear to change in cAMP dependency at any stage. The majority (80-85%) of total enzyme activity was localized in soluble fractions of cell homogenates while the residual activity was localized to membrane-enriched, particulate fractions. The results demonstrate that both responsiveness of limb mesenchyme to PGE2 and endogenous concentrations of PGE2 are maximal prior to cytodifferentiation of limb tissues. The presence of cAMP-dependent protein kinase in these undifferentiated cells supports a regulatory role for both PGE2 and a cAMP-protein phosphorylation system in the differentiation of limb tissues.     

Protospirura kaindiensis n. sp. (Spirura: Spiruridae) and other helminths from Pseudohydromys (Muridae: Hydromyinae) from Papua New Guinea

One cestode and 3 species of nematodes are recorded from Pseudohydromys murinus and Pseudohydromys occidentalis (Muridae: Hydromyinae), from Papua New Guinea, for the first time. Heterakis fieldingi (Ascaridida: Heterakidea) has previously been known from Australia. Odilia sp. resembles Odilia praeputialis in the orientation of the synlophe and the number and size of ridges but differs in the length of spicule and lack of a praepuce. Protospirura kaindiensis n. sp. (Spirurida: Spiruridae) is readily distinguished from all other members of the genus by the number and arrangement of caudal papillae and the length of the spicules.     

Methionine sulfoximine supplementation enhances productivity in GS–CHOK1SV cell lines through glutathione biosynthesis

In Lonza Biologics' GS Gene Expression System?, recombinant protein‐producing GS–CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine‐free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER‐resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS–CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS–CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb‐producing GS–CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight‐fold and the concentration in harvest medium by two‐fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process‐driven method for increasing mAb productivity from GS–CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17–25, 2017     

The life history of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island wallaby (Macropus eugenii): development and hatching of the egg and the freeliving stages

Smales L. R. The life history of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island Wallaby (Macropus eugenii): development and hatching of the egg and the free living stages. International Journal for Parasitology7: 449–456. Labiostrongylus eugenii (Trichonematidae) occurs in the stomach of the Kangaroo Island Wallaby. Egg morphology is similar to that of other strongyloids. When incubated at 25°C embryogenesis is completed in about 30 h. An incomplete moult occurs within the egg, and larvae hatch at a sheathed second-stage $\text{43}\text{1}\text{2}\text{–83}\text{1}\text{2}$ h later. Development occurred at all temperatures between 2° and 37°C with an optimum about 25°C and an upper limit near 37°C. The hatching process is very rapid, taking about 2 min. It is signalled by increased larval activity followed by a change in shell permeability. The larva hatches at that pole of the shell which has become plastic.The sheathed second-stage larva measures 659.50 ± 22.54 μm by 27.98 ± 1.22 μm. Its internal structures are concealed by a mass of opaque granules which were demonstrated as neutral lipid by oil red O staining. A second incomplete moult at 3–4 days results in a doubly sheathed infective larva from which the lipid gradually disappears. The mouth never appears patent and the larvae neither feed nor grow but rather decrease in size with age. Optimal temperatures for larvae range between 15°–25°C with 37°C about the upper limit. The significance of this developmental pattern is discussed.     

Binding of cyclic AMP and cyclic GMP to renal cortical homogenates. Relationship with phosphorylation

This study examined the binding of both cyclic AMP and cyclic GMP to receptor proteins in particulate and soluble subfractions of renal cortical homogenates from the golden hamster. The binding of both nucleotides was compared to subsequent effects of both nucleotides on the phosphorylation of histone from identical fractions. Cyclic AMP binding and cyclic AMP-dependent protein kinase activity predominated in the cytosol, with some binding and enzyme activity also detected in particulate fractions. Cyclic GMP and cyclic GMP-dependent protein kinase activity could only be demonstrated in cytosolic fractions and represented only 20-30% of cyclic AMP-dependent activity in this fraction. Binding of both nucleotides was highly specific, however, cyclic AMP showed some interaction with cyclic GMP binding. Evidence suggesting that each nucleotide interacts with a specific protein kinase was as follows: both the binding activity of the cyclic nucleotides and their combined protein kinase activity show additivity; cyclic AMP and cyclic GMP binding activity could be separated on sucrose gradients; cyclic AMP and cyclic GMP protein kinase activity could be separated with Sephadex G-100 chromatography, after preincubation of homogenate supernatants with either cyclic AMP or cyclic GMP. The results demonstrate the presence of both cyclic AMP- and cyclic GMP-dependent protein kinase in renal cortex.     

Gorgorhynchoides pseudocarangis n. sp. (Acanthocephala: Isthmosacanthidae) from Pseudocaranx dentex (Carangidae) in southeast Queensland,Australia, with comments on the Isthmosacanthidae

Gorgorhynchoides pseudocarangis n. sp. (Isthmosacanthidae), is described from the intestine of the white trevally Pseudocaranx dentex (Bloch & Schneider) (Carangiformes: Carangidae) collected in Moreton Bay, Queensland, Australia. The new species has a proboscis armature of 27–28 rows of 16–17 hooks. It is most similar morphologically to Gorgorhynchoides bullocki Cable & Marafachisi, 1970 and Gorgorhynchoides gnathanodontos Smales, 2014 but differs from the former in having a longer proboscis with more rows of hooks, ventral hooks 6/7–12 with notched tips and trunk spines which do not extend onto the anterior bulbous swelling, and from the latter in having a longer proboscis, ventral hooks 6/7–12 with notched tips, more circles of trunk spines, larger eggs and a proboscis armature with all hooks lacking manubria. Previous molecular phylogenetic analyses have shown that the genus Serrasentis Van Cleave, 1923 is sister to Gorgorhynchoides Cable & Linderoth, 1963, although some have failed to resolve these two lineages in separate monophyletic clades. We performed novel single-gene and concatenated phylogenetic analyses using cox1 mtDNA, 18S and 28S rDNA gene-sequences, resolving Gorgorhynchoides and Serrasentis in monophyletic sister clades and demonstrating that Gorgorhynchoides pseudocarangis n. sp. is phylogenetically distinct from related species for which molecular sequence data are available. We view the previous amendment of the Isthmosacanthidae to include the genera Golvanorhynchus Noronha, Fabio & Pinto, 1987, Gorgorhynchoides, Isthmosacanthus Smales 2014 and Serrasentis, and the transfer of the family to the Polymorphida, as the most satisfactory classification at present, although additional molecular evidence would provide greater stability.

     

The effect of formulation variables on protein stability and integrity of a model IgG4 monoclonal antibody and translation to formulation of a model ScFv

Objectives

There are a number of blockbuster monoclonal antibodies on the market used for the treatment of a variety of diseases. Although the formulation of many antibodies is achieved in ‘platform’ formulations, some are so difficult to formulate that it can result in an inability to develop a finished drug product. Further, a large number of antibody-inspired or-based molecules are now being developed and assessed for biotherapeutic purposes and less is understood around the required active protein drug concentrations, excipients and additives required in final product formulations.

Results

We investigated the effect of formulation variables (pH, buffer composition, glycine and NaCl concentration, time and temperature of accelerated stability studies) on antibody solubility/aggregation and activity using a Plackett–Burman Experimental Design approach. We then used the findings from this study and applied these to the formulation of a single chain variable fragment (ScFv) molecule. Our data shows that prediction of ScFc stability from a model monoclonal antibody could be achieved although further formulation optimization was required. Mass spectrometry analysis confirmed changes to the mass and hence authenticity of both the model antibody and ScFv under formulation conditions that did not provide appropriate conditions for protection of the molecules.

Conclusions

The role of the different formulation conditions on maintaining protein integrity is described and using mass spectrometry shows that protein integrity is compromised under particular conditions. The implications for predicting successful formulations for protein molecules is discussed and how antibody formulations could be used to predict formulation components for novel antibody based molecules.

     

An electrophoretic and morphological analysis of Labiostrongylus (Labiosimplex) bancrofti (Johnston & Mawson, 1939) (Nematoda: Cloacinidae), from macropodid marsupials,with the description of two new species

Allozyme electrophoresis was used to compare specimens of Labiostrongylus (Labiosimplex) bancrofti from two species of Australian macropodids, Macropus dorsalis and M. parryi, with a related species, L. (Labiomultiplex) uncinatus which also infests M. dorsalis. Each nematode was characterised genetically at 18 enzyme loci. The level of fixed genetic differences detected between L. (Ls.) bancrofti from M. parryi and M. dorsalis (83%) is equivalent to that when each is compared to the morphologically distinct species L. (Lm.) uncinatus (89–94%), demonstrating unequivocally that the taxon L. (Ls.) bancrofti represents at least two species, one in M. parryi and one in M. dorsalis. In addition, morphological evidence from additional specimens collected from M. parryi suggested the existence of a third sibling species in this group. All three species differ in the shape of the spicule tips; L. (Ls.) bancrofti has longer spicules than either of the two new species. L. (Ls.) quasibancrofti n. sp. has smaller cephalic papillae, larger oesophago-intestinal diverticula, a larger genital cone and a longer female tail than L. (Ls.) turnbulli n. sp. The taxon L. (Ls.) bancrofti consists, therefore, of three species, L. (Ls.) turnbulli in M. parryi, L. (Ls.) quasibancrofti in M. dorsalis, and L. (Ls.) bancrofti found in both host species, as well as in four species of rock wallabies.