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Biomedical subjects

G Shaw

Publications and source records attributed to G Shaw.

At least 109 records · Page 6Linked to original sources
PubMed1997

Cytokinin-derived cyclin-dependent kinase inhibitors: synthesis and cdc2 inhibitory activity of olomoucine and related compounds.

Cyclin-dependent kinases (cdk) have recently raised considerable interest in view of their essential role in the regulation of the cell division cycle. The structure-activity relationships of cdk inhibition showed that the 1, 3; and 7 positions of the purine ring must remain free, probably for a direct interaction, in which it behaves as a hydrogen bond acceptor. Olomoucine (6-(benzylamino)-2-[(2-hydroxyethyl)amino]-9-methylpurine, OC), roscovitine (6-(benzylamino)-2(R)-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpur ine), and other N6,2,9-trisubstituted adenines were found to exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. Removal or change of the side chain at position 2 or the hydrophobic group at position 9 dramatically decreased the inhibitory activity of olomoucine or roscovitine. Inhibition of cdk with OC and related compounds clearly arrests cell proliferation of many tumor cell lines at G1/S and G2/M transitions and also triggers apoptosis in the target tumor cells in vitro and in vivo. Thus, from a pharmacological point of view, OC may represent a model compound for a new class of antimitotic and antitumor drugs.

Animals↗
PubMed1997

Müllerian duct regression in a marsupial, the tammar wallaby.

Müllerian duct regression is first apparent in male pouch young of the tammar wallaby (Macropus eugenii) 6-7 days after birth and, as in eutherian mammals, is characterised by a condensation of the periductal mesenchyme into a whorl around the ductal epithelial cells. A decrease in the density of the extracellular matrix was observed in the region of the whorl. In contrast to eutherian mammals no changes were observed in the mean outer diameter of the Müllerian duct during the early stages of regression. The time at which these mesenchymal changes occur corresponds to the period of Müllerian inhibiting substance secretion in the postnatal tammar testis.

Animals↗
PubMed1997

Characterization of additional casein kinase I sites in the C-terminal "tail" region of chicken and rat neurofilament-M.

In previous studies we have identified Ser502, Ser528, and Ser534 as target sites in chicken neurofilament middle molecular mass protein (NF-M) for casein kinase I (CKI) in vitro and have shown that these sites are also phosphorylated in vivo. We now make use of a combination of molecular biological and protein chemical techniques to show that two additional in vivo phosphorylation sites in chicken NF-M, Ser464 and Ser471, can also be phosphorylated by CKI in vitro. These two sites are conserved in higher vertebrate NF-M molecules, and recombinant protein constructs containing the homologous rat NF-M peptides can be phosphorylated by CKI in vitro, suggesting that phosphorylation of these sites is conserved at least in higher vertebrates. The two new sites are adjacent to a conserved peptide sequence (VEEIIEET-V) found once in higher vertebrate NF-M molecules and twice in lamprey NF-180. Variants of this sequence are also found in neurofilament low and high molecular mass proteins (NF-L and NF-H) and alpha-internexin, and in mammalian NF-L are known to be associated with in vivo phosphorylation sites. We speculate that CKI phosphorylation in general, and these sites in particular, may be important in neurofilament function.

Amino Acid Sequence↗
PubMed1997

Evidence for a local fetal influence on myometrial oxytocin receptors during pregnancy in the tammar wallaby (Macropus eugenii).

Mesotocin (MT), the oxytocin-like peptide of the tammar wallaby (Macropus eugenii) is important for delivery of live young. The tammar mesotocin receptor (MTR) was first characterized using the iodinated oxytocin receptor antagonist [125I]d(CH2)5 [Tyr(Me)2, Tyr4, Orn8, Tyr-NH(2)9]-vasotocin. MTR concentrations were then measured in matched samples of gravid and nongravid myometrium and median vagina at different stages of the 26-day pregnancy. MTR concentrations in both the gravid and nongravid myometrium changed significantly (ANOVA, p < 0.01) during pregnancy. There was no difference in MTR concentrations between uteri on Days 8-22. From Day 23 of pregnancy, MTR concentrations in the gravid myometrium increased (615.8 +/- 144.0 fmol/mg protein), whereas in the nongravid myometrium, they remained unchanged (248.6 +/- 65.5 fmol/mg protein). Receptor concentrations were high in the gravid myometrium during the last 3 days of pregnancy but decreased significantly in the nongravid myometrium. In the median vagina, MTR concentrations were low compared with myometrial tissues and did not increase at term. Changes in MTR concentrations paralleled changes in uterine responsiveness to exogenous MT in vitro. Our data show that MTR concentrations and the responsiveness to MT differ between the gravid and nongravid myometrium during pregnancy. The increase in MTRs in the gravid myometrium and the decrease in the nongravid suggest that different factors influence these receptors in the separate uteri, independent of systemic influence.

Animals↗
PubMed1997

The influence of the anti-androgen flutamide on early sexual differentiation of the marsupial male.

The development of the prostate and the normal descent of the testes in the tammar wallaby (Macropus eugenii) were influenced by treatment with the non-steroidal anti-androgen flutamide. Male pouch young were treated daily from day 9 to day 45, or from day 20 to day 45, of pouch life. Prostate development was inhibited in both treatment groups to a similar extent. Since prostatic buds do not form until day 25 of pouch life, these results suggest that there is a window of androgen sensitivity operating between day 20 and day 25 of pouch life. The number of prostatic buds was significantly lower, but despite the duration of treatment there was never complete abolition of prostate development. Although testes had descended to the same position in treated and control pouch young, inguinal hernias developed in three of four animals treated with flutamide from day 9. These data demonstrate that virilization of the male reproductive tract in this marsupial is dependent on a relatively brief exposure to androgens. Blocking androgen receptor action interferes with normal development of the inguinal canal, which suggests that it is this aspect of inguinoscrotal testicular descent that is androgen dependent.

Androgen Antagonists↗
PubMed1997

Migration of primordial germ cells to the developing gonadal ridges in the tammar wallaby Macropus eugenii.

Primordial germ cells (PGCs) of the tammar wallaby Macropus eugenii have a distinctive morphology and stain positively for alkaline phosphatase. PGCs are identifiable in embryos with 12 somites, on about day 17 of the 26.5 day gestation period, when they are located in all three germ layers of the developing embryo and in the endoderm of the bilaminar and vascular (trilaminar) yolk sac membranes. PGCs are positive for alkaline phosphatase (ALP) at least between days 17 and 22 of pregnancy. In whole mounts on day 17, three groups of cells positive for ALP occur: about 40 just caudal to the neural tube, and about 20 distributed on either side of the last three somites. By day 21, there are about 150 PGCs in the newly formed gonadal ridges and 275 in the mesenteries. On days 21-22, there are PGCs in the umbilical mesoderm, the dorsal mesentery and the coelomic angles between the dorsal mesentery and the mesonephroi. On day 22, most ALP-positive PGCs are located in the dorsal mesentery, where they occur in groups. They apparently do not migrate through the hindgut endoderm, but occasional PGCs are seen in sites such as the mesonephros, the adrenals, the blood vessels of the yolk sac and in the vicinity of the dorsal aorta and dorsal nerve cord. Between day 23 and day 25, 1 day before birth, most of the 3200-4000 PGCs complete their migration to the gonadal ridges. Although there are marked differences between embryogenesis of tammars and mice, development and the pattern of migration of PGCs in this marsupial mammal are similar to that of eutherian mammals.

Animals↗
PubMed1996

The pleckstrin homology domain of human beta I sigma II spectrin is targeted to the plasma membrane in vivo.

We have examined the in vivo targeting potential of the Pleckstrin Homology (PH) domain from human beta I sigma II spectrin using a novel Aequoria victoria green fluorescent protein (GFP) fusion vector constructed from a human codon optimized cDNA. This vector efficiently expresses both GFP and the GFP spectrin fusion protein in COS7 and other cell lines. GFP expressed alone shows only diffuse cytoplasmic staining which is not associated with the plasma membrane. In contrast the GFP-beta I sigma II spectrin PH domain fusion protein localizes under the plasma membrane of transfected COS7 cells in vivo. Fixation of cells transfected with GFP alone in -20 degrees C methanol results in the removal of all specific fluorescence. In contrast cells transfected with the GFP-beta I sigma II spectrin construct and fixed -20 degrees C methanol continue to show strong membrane fluorescence, consistent with a role for the spectrin PH domain in membrane localization in vivo.

Amino Acid Sequence↗
PubMed1996

Mechanism of bracken fern carcinogenesis: evidence for H-ras activation via initial adenine alkylation by ptaquiloside.

Bracken fern (Pteridium spp.) causes cancer of the oesophagus and the urinary bladder in cattle and sheep. Ptaquiloside (PT) is believed to be the carcinogenic principle which alkylates DNA when activated to its unstable dienone form (APT) under alkaline conditions. In this report we present evidence for the presence of PT-DNA adducts in the ileum of bracken fem-fed calves using the 32P-postlabelling assay. H-ras mutations were also observed in the ileum using single strand conformation polymorphism (SSCP) technique. Mutations corresponding to adenine to pyrimidine transversions in the codon 61 of H-ras were identified by the cycle sequencing method. In vitro DNA alkylation studies showed that APT alkylated H-ras primarily at the adenines. In addition, the rate of depurination of alkylated adenine was sequence dependent. Investigation of DNA template activity using a plasmid DNA showed that DNA synthesis by T7 DNA polymerase was terminated by the presence of all alkylated bases but certain apurinic sites allowed the DNA synthesis to continue. These results suggest that initial alkylation of adenine by PT in codon 61 followed by depurination and error in DNA synthesis lead to activation of H-ras proto-oncogene.

Adenine↗
PubMed1996

The pleckstrin homology domain: an intriguing multifunctional protein module.

Pleckstrin homology (PH) domains are a family of compact protein modules defined by sequences of roughly 100 amino acids. These domains are common in vertebrate, Drosophila, C. elegans and yeast proteins, suggesting an early origin and fundamental importance to eukaryotic biology. Many enzymes which have important regulatory functions contain PH domains, and mutant forms of several such proteins are implicated in oncogenesis and developmental disorders. Numerous recent studies show that PH domains bind various proteins and inositolphosphates. Here I discuss PH domains in detail and conclude that they form a versatile family of membrane binding and protein localization modules.

Agammaglobulinemia↗
PubMed1996

Testosterone control of male-type sexual behavior in the tammar wallaby (Macropus eugenii).

In the tammar wallaby, Macropus eugenii, the expression of male-type sexual behavior is apparently determined by the activating effects of testicular hormones in adulthood. The incidence of male-type copulatory behavior and sexual checking behavior was compared in intact (control) males, control females, testosterone-treated females, and three groups of males castrated either postnatally (24-26 days of age), prepubertally (14.5 months of age), or in adulthood. All three groups of castrated male wallabies showed a very low incidence of male sexual behavior in adult life, comparable to that shown by the untreated females. Adult female wallabies with 100-mg testosterone implants showed a high incidence of male sexual behavior which was indistinguishable from that shown by intact males. The results suggest that sex differences in male-type behavior in the tammar wallaby are due to short-term inductive effects of testosterone acting on a sexually indifferent brain. There is no evidence of any long-term organizational effects of testosterone acting in fetal or neonatal life on the neural pathways controlling male-type sex behavior in this marsupial mammal.

Animals↗
PubMed1996

Sexual differentiation of the urogenital system of the fetal and neonatal tammar wallaby, Macropus eugenii.

In male tammar wallabies, the scrotum is the first organ to become sexually differentiated, 4-5 days before birth (day 22 of gestation). This is followed by enlargement of the gubernaculum and processus vaginalis one day before birth. However the indifferent gonad does not show any signs of testicular cord formation or androgen production until later, at around the time of birth; this is more pronounced at 2 days post-partum (p.p.), when the testis takes on a characteristic rounded appearance. Primordial germ cells proliferate throughout the testis at this time, although the testis does not become significantly heavier than the ovary until around 80 days p.p.. In females, the appearance of the mammary glands is the first sign of sexual differentiation 4-5 days before birth. The indifferent gonad first shows signs of developing an ovarian cortex and medulla 7 days after birth. The migrating germ cells are confined to the cortex, and first start to enter meiosis about 25 days after birth. The Wolffian (mesonephric) ducts are patent to the urogenital sinus in fetuses at day 21 of gestation. In the female they have started to regress by 10 days p.p. and only rudiments remain by day 25 p.p.. The Müllerian (paramesonephric) ducts develop adjacent to the cranial pole of the mesonephros at about day 25 of gestation and grow caudally to meet the urogenital sinus between days 2 and 7 p.p.. The Müllerian duct of the female develops a prominent ostium abdominale by day 9 p.p., but this structure has completely regressed in males by day 13 p.p.. The testis and ovary both migrate caudally, together with the adjacent mesonephros, at about day 10 p.p.. The ovaries remain around the level of lumbar vertebra 4 after about day 7 p.p., while the testes continue to descend. The testes enter the internal inguinal ring at about day 25 p.p., about the time that prostatic buds first appear in the urogenital sinus, and are in the inguinal canal from days 25 to 36 p.p.. They enter the scrotum at around day 36 p.p., and testicular descent is complete by days 65-72 p.p.. Melanin develops in the tunica vaginalis 72 days after birth. The overall development of the urogenital system in this marsupial is similar to that of eutherians but the sequence of events differs, with some aspects of genital differentiation preceding gonadal differentiation, apparently because they are directly controlled by X-linked genes, rather than indirectly controlled by gonadal steroids.

Animals↗
PubMed1996

Compartmentation of alpha-internexin and neurofilament triplet proteins in cultured hippocampal neurons.

Intermediate filaments comprise an integral part of the neuronal cytoskeleton. However, little is known about their function, and there remains some uncertainty about their precise subcellular localization. We examined the timing of expression and distribution of alpha-internexin, neurofilament triplet proteins and peripherin using immunocytochemistry in cultured hippocampal neurons. alpha-Internexin immunostaining was present in all neurons at all developmental stages. Immunostaining appeared as long filaments in axons and short fragments in dendrites which extended into dendritic spines. The presence of alpha-internexin in dendritic spines was confirmed in situ by electron microscopy of rat hippocampal tissue sections and suggests that this intermediate filament may serve as a link between cytoskeletal elements in dendritic shafts and spines. In culture, immunostaining using antibodies against individual triplet protein subunits indicated that light (NF-L) and middle (NF-M) subunits were first expressed in cells shortly after the initiation of axonal outgrowth. Expression of the heavy (NF-H) subunit occurred a few days later. Although timing and localization of expression did not correlate with the initiation of axonal or dendritic processes, it was coincident with periods of rapid outgrowth. Triplet proteins were more abundant in axons and appeared to be incorporated into lengthier filaments than in dendrites. Highly phosphorylated NFH/M immunoreactivity was polarized to axons after 6 days in culture. The distribution of one NF-H epitope was restricted to GABAergic neurons in mature cultures, suggesting a cell-type specific modification. Peripherin was not detectable at any time in hippocampal cultures. Our results show that intermediate filaments are integral components of the neuronal cytoskeleton of cultured hippocampal neurons throughout development. Furthermore, the localization of alpha-internexin suggests that it may be involved in the formation or maintenance of dendritic spines.

Animals↗
PubMed1996

Loss of particulate contaminants from plant canopies under wet and dry conditions.

There is a requirement for data describing the loss with time of particulate contamination from plant canopies. Measurements were made of the loss rates of monodispersed silica spheres (three sizes, with Mass Median Aerodynamic Diameters (MMADs) 1.9, 5.3 and 8.4 microm) from wheat (Triticum aestivum) and broad bean (Vicia faba) canopies. The spheres were labelled with tracers detectable by Instrumental Neutron Activation Analysis (INAA). Canopies were contaminated under realistic turbulence conditions in a wind tunnel, then removed to sheltered and exposed field sites or to a glasshouse containing a rain simulator. Samples were taken periodically, and the level of contamination per plant determined by INAA. Statistical analysis of the resulting data suggested an offset exponential loss model, with a residue of deposit that is not lost over time. Loss half-lives in the order of 1-2 days were obtained for an exposed wheat crop and 3-4 days for a partially sheltered wheat crop, with permanent residues of initial deposit for the exposed crop of 4-8%, and for the partially sheltered crop of 22-52%. A broad bean crop under glasshouse conditions showed loss half-lives of 0.5-1.5 days with residues of 22-26% initial contamination. A double exponential loss model also fitted the data well in some cases, and it is possible that a slow loss of the residual deposit occurs, being masked by noise in the current data set.

Journal Article↗
PubMed1996

Localization of sites in the tail domain of the middle molecular mass neurofilament subunit phosphorylated by a neurofilament-associated kinase and by casein kinase I.

We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKl) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKl inhibitor CKl-7, (2) the similarity in substrate specificity of NFAK and authentic CKl, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKl under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKl. We have also identified Ser502, SER528, and Ser536 as phosphorylation sites by NFAK/CKl in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKl phosphorylation consensus sequences, and Ser528 and Ser536 and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser502 nor Ser536 has been identified previously as NF-M phosphorylation sites.

Amino Acid Sequence↗
PubMed1996

The uterine environment in early pregnancy in the tammar wallaby.

In tammar wallabies, Macropus eugenii, the uterine environment plays a key role in regulating development, because during the first two-thirds of gestation an acellular mucoid coat and shell prevent direct cell-cell contact between the endometrium and embryonic cells. This control is seen very clearly in the facultative lactational diapause of tammars. Removal of the suckled pouch young during the breeding season terminates diapause, leading to a distinct increase in metabolic activity of the embryo. By Day 4, oxidative metabolism of glucose has substantially increased, providing a four-fold increase in ATP production. By Day 5, RNA synthesis has increased. These changes are dependent on progesterone-induced changes in uterine secretions. By Day 3, there is greater progesterone secretion by the corpus luteum and, by Day 4, uterine protein synthesis has increased. The nature of the uterine regulatory factor is still not known. There are changes in some uterine proteins, but no detectable change in ionic components of the uterine fluid. Only one defined potential regulator, platelet-activating factor, has been identified, the concentration of which increased during reactivation. The influence of the steroid hormones progesterone and oestradiol on the uterus and diapausing embryo, and other changes that occur later in development, are also discussed in the present review.

Animals↗
PubMed1996

A role for glucocorticoids in parturition in a marsupial, Macropus eugenii.

Dexamethasone treatment induces premature birth in tammar wallabies. Treatment was administered at one of three times between 1200 h on Day 24 and 0930 on Day 25, and birth occurred 22.8 +/- 0.5 h later, significantly earlier than the time of birth for controls, which was 47.7 +/- 2.3 h after treatment. The neonates from treated females were significantly lighter than control neonates (360 +/- 9 vs. 413 +/- 5 mg), and 60% of these died within 12 h of birth, suggesting that premature birth can lead to neonatal mortality. None of the control neonates died. The patterns of secretion of prolactin, prostaglandin F2 alpha-metabolite (PGFM), and progesterone of control and treated animals around the time of birth were similar. A transient pulse of PGFM was coincident with birth while prolactin levels in plasma increased before, and progesterone concentrations fell steeply immediately after, parturition in both groups of animals. The only difference between control and treated animals was in the timing of the hormonal events, which, along with birth, was significantly advanced by the treatment. We conclude that cortisol may play a role in triggering parturition in this marsupial species.

Animals↗