Effect of pentagastrin and gastrin-17 on the secretory function of the gastric glands

The regulation of the digestive glands of the stomach and pancreas in the body of animals and humans is provided by peptides, most of which are in various molecular forms. 10 molecular forms of peptides of the gastrin group and 5 peptides of the cholecystokinin (CCK) group have been identified, containing in their structure from 4 to 56 amino acids, the physiological role of which has been little studied. It has been proven that the liver removes up to 85% of short-chain peptides of the gastrin (pentagastrin) and cholecystokinin (CCK-8) groups.

Transmembrane 29 (Tmem29), a Newly Identified Molecule Showed Downregulation in Hypoxic-Ischemic Brain Damage

Transmembrane 29 (Tmem29) gene with unknown function is a gene located on the X chromosome of the mouse genome. The gene showed differential expression in the Vannucci neonatal hypoxic-ischemic mouse brain model. We found the gene expresses with different molecular forms, including a group of long non-coding RNA forming a family of transcripts. It was predominantly expressed in the testes, brain, and kidney of mouse. In vitro identification and functional characterization were carried out in Neuro2a cells. Using fluorescence microscopy, Tmem29 protein was found to be constitutively expressed in mouse cell lines of different origins. Oxygen glucose deprivation (OGD) induced apoptotic cell death in Neuro2a cells and was confirmed by activations of caspase 3. Tmem29 protein was found to be associated with cell death especially at the time points of caspase 3 activations. A similar response was obtained in glucose deprivation (GD) cultures suggesting Tmem29 response to a common mechanism induced by OGD and GD. Downregulation of Tmem29 was induced by OGD and GD, further validating its response to hypoxia-ischemia (HI) insults. Our findings contributed to further understanding of molecular events after hypoxic-ischemic insults and opens new avenues for developing protective and therapeutic strategies for hypoxic-ischemic encephalopathy or even pathological programmed cell death.

On the biological activity of cytokinin free bases and their ribosides

Main conclusion The free bases of cytokinins are the biologically active forms of the hormone while cytokinin ribosides become active only upon removal of the ribose residue. Abstract Cytokinins (CKs) belong to the classical plant hormones. They were discovered more than 65 years ago, but which molecular forms possess genuine CK activity is still matter of debate. Numerous studies support the view that only the free bases are the biologically active molecules. This standpoint has been challenged in a recent review (Nguyen et al. in Planta 254: 45, 2021) proposing that also CK ribosides may have genuine own CK activity. Here we critically discuss the pros and cons of this viewpoint considering the results of biological assays, CK binding studies, 3D structural data of CK-receptor interaction and mutant analyses. It is concluded that all types of study provide clear and convincing evidence only for biological activity of free bases and not ribosides; the latter are rather a transport form of the hormone without their own biological activity.

The molecular forms of BMP15 in a range of mammalian species

<p>BMP15 is an oocyte-secreted growth factor that is critical for ovarian follicular development and fertility in mammals. To improve our understanding of the species-specific effects of BMP15 on fertility, the aim of this study was to elucidate the molecular forms of BMP15 in mammals with high (rat and pigs) and low (sheep and deer) litter sizes. Western blotting experiments were undertaken using a monoclonal antibody directed against a semi-conserved area on the mature region of E.coli-expressed ovine BMP15. Within the oocyte lysate of all species tested, the predominant form was promature BMP15, although mature BMP15 was also present. Within oocyte-conditioned media of sheep, pigs and deer, the promature:mature BMP15 ratio dropped significantly from what was seen inside the oocyte, with promature BMP15 only slightly predominant in deer, and relatively equal amounts of each protein were present in pigs and sheep. No BMP15 was detected in the oocyte-conditioned media of rats. Cross-linking studies did not provide any evidence of BMP15/BMP15 homodimers or BMP15/GDF9 heterodimers in any species tested. Intra-oocyte levels of both promature and mature BMP15, from highest to lowest, were detected in deer, followed by sheep and pigs (similar), and then rats (significantly lower). In the oocyte-conditioned media, with the exception of the rat where BMP15 was absent, there was a similar pattern of promature BMP15 levels observed between species, whereas mature BMP15 levels showed no inter-species variation. In summary, the molecular forms and relative amounts of BMP15 protein differ across species. Generally, high BMP15 levels were associated with low litter size; however the pig is an exception. Furthermore, the molecular forms observed in this study differed from those reported in studies using recombinant BMP15 suggesting that recombinantly-expressed protein may not provide a fair representation of native BMP15.</p>

The Molecular Forms of GDF9 In A Range of Mammalian Species

<p>Growth Differentiation Factor (GDF) 9 is a member of the transforming growth factor β (TGFβ) superfamily that is exclusively expressed within and secreted from, the oocyte. This protein has generated much interest as it has been found to play a major role in follicular growth and maturation in mammals, and may be involved in determining litter size. Like most TGFβ family members, it is synthesised as a pre-pro-mature protein and is cleaved at various stages to allow the biologically active mature form to bind its Type II receptor. The aim of this study was to improve our understanding of the different molecular forms of GDF9 that are present within ovarian follicles of a range of mammalian species that differ in litter size. To achieve this aim, Western blotting experiments were performed to illustrate the molecular forms that were present within, and secreted from, the oocytes of rats, pigs, sheep and red deer. The detection of bands that represented the different molecular forms of GDF9 was undertaken using a monoclonal antibody that targeted a conserved region in the mature form of ovine GDF9. The predominant forms of GDF9 found within and secreted from the oocyte across the species were the promature and cleaved mature forms of GDF9. Densitometry analysis of the Western blots revealed that pig, sheep, and red deer had significantly more of the promature, than the mature, form within the oocyte. Conversely, there were no significant differences between the levels of promature and mature forms of GDF9 in the secreted media. Moreover, the levels of the specific molecular forms of GDF9 were not different between pigs, sheep and red deer. The levels of GDF9 in rat samples were low which may be due to a lower affinity of the monoclonal GDF9 antibody due to sequence differences between rat and ovine GDF9. Interestingly, applying a crosslinking reagent to the oocyte lysate and conditioned media samples revealed the appearance of a high molecular size band. The appearance of this band, which was more prominent in the rat and pig, was concomitant with the disappearance of the mature GDF9 band. The differential levels of these presumptive GDF9 multimers in these two species that have large litters may suggest that rat and pig mature GDF9 binds other oocyte secreted factors more readily than GDF9 from mono-ovulatory species. Importantly, no homo- or hetero- mature dimers of GDF9 were detected in any of the species studied. In summary, GDF9 was predominantly present as promature and cleaved mature forms both within the oocyte and in the secretions from the oocyte. Overall, these results indicated that the promature form was present in higher levels than the cleaved mature form. With the exception of the rat, there were no detectable species differences in the levels of the GDF9 forms within or secreted from the oocyte using Western blotting methodologies.</p>

The Molecular Forms of GDF9 In A Range of Mammalian Species

<p>Growth Differentiation Factor (GDF) 9 is a member of the transforming growth factor β (TGFβ) superfamily that is exclusively expressed within and secreted from, the oocyte. This protein has generated much interest as it has been found to play a major role in follicular growth and maturation in mammals, and may be involved in determining litter size. Like most TGFβ family members, it is synthesised as a pre-pro-mature protein and is cleaved at various stages to allow the biologically active mature form to bind its Type II receptor. The aim of this study was to improve our understanding of the different molecular forms of GDF9 that are present within ovarian follicles of a range of mammalian species that differ in litter size. To achieve this aim, Western blotting experiments were performed to illustrate the molecular forms that were present within, and secreted from, the oocytes of rats, pigs, sheep and red deer. The detection of bands that represented the different molecular forms of GDF9 was undertaken using a monoclonal antibody that targeted a conserved region in the mature form of ovine GDF9. The predominant forms of GDF9 found within and secreted from the oocyte across the species were the promature and cleaved mature forms of GDF9. Densitometry analysis of the Western blots revealed that pig, sheep, and red deer had significantly more of the promature, than the mature, form within the oocyte. Conversely, there were no significant differences between the levels of promature and mature forms of GDF9 in the secreted media. Moreover, the levels of the specific molecular forms of GDF9 were not different between pigs, sheep and red deer. The levels of GDF9 in rat samples were low which may be due to a lower affinity of the monoclonal GDF9 antibody due to sequence differences between rat and ovine GDF9. Interestingly, applying a crosslinking reagent to the oocyte lysate and conditioned media samples revealed the appearance of a high molecular size band. The appearance of this band, which was more prominent in the rat and pig, was concomitant with the disappearance of the mature GDF9 band. The differential levels of these presumptive GDF9 multimers in these two species that have large litters may suggest that rat and pig mature GDF9 binds other oocyte secreted factors more readily than GDF9 from mono-ovulatory species. Importantly, no homo- or hetero- mature dimers of GDF9 were detected in any of the species studied. In summary, GDF9 was predominantly present as promature and cleaved mature forms both within the oocyte and in the secretions from the oocyte. Overall, these results indicated that the promature form was present in higher levels than the cleaved mature form. With the exception of the rat, there were no detectable species differences in the levels of the GDF9 forms within or secreted from the oocyte using Western blotting methodologies.</p>

Oocyte-derived forms of ruminant BMP15 and GDF9 and a theoretical model to explain their synergistic response

<p>Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-secreted factors with well documented effects on ovarian follicular development and ovulation-rate. The aims of these studies were to: (i) identify the molecular forms of BMP15 and GDF9 that are produced and secreted by both the ovine and bovine oocyte using highly specific monclonal antibodies; (ii) assess the biological activity of some recombinant molecular forms of BMP15 and GDF9; (iii) visualise the various molecular forms using protein modelling techniques and; (iv) provide a hypothetical model of how oocyte-secreted form(s) of BMP15, GDF9 and their cell surface receptors may interact. Using genetic modifications and transformations of HEK293 cells, recombinant forms of ovine (o) BMP15, including a BMP15 (S356C) mutant capable of forming covalent dimers, and oGDF9 were produced. The bioactivity of these proteins was established using a rat granulosa cell proliferation bioassay. The specificity of the monoclonal antibodies MN2-61A (anti-BMP15) and 37A (anti-GDF9) used in these studies, and determination of the forms they recognise, was examined by Western blotting. The recombinant forms of oBMP15 were further interrogated by purification using both immobilised metal affinity chromatography (IMAC) and reverse phase HPLC. The BMP15 and GDF9 proteins produced and/or secreted by ovine and bovine oocytes, before and after in vitro incubation, were identified and compared with the molecular forms(s) of recombinant oBMP15 or oGDF9 using Western blotting under non-reducing, reducing and cross-linking conditions. The molecular forms of recombinant oBMP15 and oGDF9 comprise mainly mature monomers with a lesser amount of the uncleaved pro-mature form. Mature domains, in the dimeric mature form, were detected for oGDF9 and oBMP15 (S356C), but not oBMP15. These mature domains were almost entirely located within high molecular weight multimeric complexes, which likely also contain the pro-region. In contrast, BMP15 and GDF9 secreted from ruminant oocytes under in vitro conditions were found mainly in an unprocessed promature form, along with some fully processed mature domains that did not interact to form detectable mature homodimers or heterodimers. Throughout ovarian follicular development, BMP15 and GDF9 are co-expressed and it has been established that these two factors have synergistic effects on granulosa cell proliferation both in vitro and in vivo and also on follicular maturation and ovulation-rate in vivo. Moreover, the recombinant proteins oBMP15 and oGDF9 generated for this study, when added together, also demonstrated a synergistic effect in the granulosa cell proliferation assay but this was not observed for oBMP15 (S356C) and oGDF9. Currently, no adequate model has been proposed to explain how interactions between the cell membrane and forms of oocyte-derived BMP15 and GDF9 achieve their synergistic effects. To investigate this, two homology models of the promature BMP15 and GDF9 proteins were generated using promature porcine TGFB1 and human BMP9 as templates. These models, together with the previously determined forms of GDF9 and BMP15 produced by the ruminant oocyte, were used to visualise their potential interactions, both with each other and with their receptors. This report describes a model showing the possible interactions involved in a synergistic response. In this model, the mature domain is presented to the type II receptor by the proregion and heterodimers form at the level of the receptor. Differences, following heterodimerisation in the conformation and orientation between GDF9 and its type I receptor, as well as between type I and type II receptors, relative to that in homodimers, could explain how heterodimerisation leads to increased Smad3 phosphorylation and subsequent down-stream somatic cell responses.</p>

The molecular forms of BMP15 in a range of mammalian species

<p>BMP15 is an oocyte-secreted growth factor that is critical for ovarian follicular development and fertility in mammals. To improve our understanding of the species-specific effects of BMP15 on fertility, the aim of this study was to elucidate the molecular forms of BMP15 in mammals with high (rat and pigs) and low (sheep and deer) litter sizes. Western blotting experiments were undertaken using a monoclonal antibody directed against a semi-conserved area on the mature region of E.coli-expressed ovine BMP15. Within the oocyte lysate of all species tested, the predominant form was promature BMP15, although mature BMP15 was also present. Within oocyte-conditioned media of sheep, pigs and deer, the promature:mature BMP15 ratio dropped significantly from what was seen inside the oocyte, with promature BMP15 only slightly predominant in deer, and relatively equal amounts of each protein were present in pigs and sheep. No BMP15 was detected in the oocyte-conditioned media of rats. Cross-linking studies did not provide any evidence of BMP15/BMP15 homodimers or BMP15/GDF9 heterodimers in any species tested. Intra-oocyte levels of both promature and mature BMP15, from highest to lowest, were detected in deer, followed by sheep and pigs (similar), and then rats (significantly lower). In the oocyte-conditioned media, with the exception of the rat where BMP15 was absent, there was a similar pattern of promature BMP15 levels observed between species, whereas mature BMP15 levels showed no inter-species variation. In summary, the molecular forms and relative amounts of BMP15 protein differ across species. Generally, high BMP15 levels were associated with low litter size; however the pig is an exception. Furthermore, the molecular forms observed in this study differed from those reported in studies using recombinant BMP15 suggesting that recombinantly-expressed protein may not provide a fair representation of native BMP15.</p>

Oocyte-derived forms of ruminant BMP15 and GDF9 and a theoretical model to explain their synergistic response

<p>Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-secreted factors with well documented effects on ovarian follicular development and ovulation-rate. The aims of these studies were to: (i) identify the molecular forms of BMP15 and GDF9 that are produced and secreted by both the ovine and bovine oocyte using highly specific monclonal antibodies; (ii) assess the biological activity of some recombinant molecular forms of BMP15 and GDF9; (iii) visualise the various molecular forms using protein modelling techniques and; (iv) provide a hypothetical model of how oocyte-secreted form(s) of BMP15, GDF9 and their cell surface receptors may interact. Using genetic modifications and transformations of HEK293 cells, recombinant forms of ovine (o) BMP15, including a BMP15 (S356C) mutant capable of forming covalent dimers, and oGDF9 were produced. The bioactivity of these proteins was established using a rat granulosa cell proliferation bioassay. The specificity of the monoclonal antibodies MN2-61A (anti-BMP15) and 37A (anti-GDF9) used in these studies, and determination of the forms they recognise, was examined by Western blotting. The recombinant forms of oBMP15 were further interrogated by purification using both immobilised metal affinity chromatography (IMAC) and reverse phase HPLC. The BMP15 and GDF9 proteins produced and/or secreted by ovine and bovine oocytes, before and after in vitro incubation, were identified and compared with the molecular forms(s) of recombinant oBMP15 or oGDF9 using Western blotting under non-reducing, reducing and cross-linking conditions. The molecular forms of recombinant oBMP15 and oGDF9 comprise mainly mature monomers with a lesser amount of the uncleaved pro-mature form. Mature domains, in the dimeric mature form, were detected for oGDF9 and oBMP15 (S356C), but not oBMP15. These mature domains were almost entirely located within high molecular weight multimeric complexes, which likely also contain the pro-region. In contrast, BMP15 and GDF9 secreted from ruminant oocytes under in vitro conditions were found mainly in an unprocessed promature form, along with some fully processed mature domains that did not interact to form detectable mature homodimers or heterodimers. Throughout ovarian follicular development, BMP15 and GDF9 are co-expressed and it has been established that these two factors have synergistic effects on granulosa cell proliferation both in vitro and in vivo and also on follicular maturation and ovulation-rate in vivo. Moreover, the recombinant proteins oBMP15 and oGDF9 generated for this study, when added together, also demonstrated a synergistic effect in the granulosa cell proliferation assay but this was not observed for oBMP15 (S356C) and oGDF9. Currently, no adequate model has been proposed to explain how interactions between the cell membrane and forms of oocyte-derived BMP15 and GDF9 achieve their synergistic effects. To investigate this, two homology models of the promature BMP15 and GDF9 proteins were generated using promature porcine TGFB1 and human BMP9 as templates. These models, together with the previously determined forms of GDF9 and BMP15 produced by the ruminant oocyte, were used to visualise their potential interactions, both with each other and with their receptors. This report describes a model showing the possible interactions involved in a synergistic response. In this model, the mature domain is presented to the type II receptor by the proregion and heterodimers form at the level of the receptor. Differences, following heterodimerisation in the conformation and orientation between GDF9 and its type I receptor, as well as between type I and type II receptors, relative to that in homodimers, could explain how heterodimerisation leads to increased Smad3 phosphorylation and subsequent down-stream somatic cell responses.</p>

Utility of Plasma NGAL for the Diagnosis of AKI Following Cardiac Surgery Requiring Cardiopulmonary Bypass: A Systematic Review and Meta-analysis

The objective of this study was to assess the diagnostic value of plasma neutrophil gelatinase‑associated lipocalin (pNGAL) for the early diagnosis of acute kidney injury (AKI) in adult patients following cardiac surgery requiring cardiopulmonary bypass (CPB). Electronic databases and other resources were systematically searched for relevant studies. Risk of bias was assessed using the Quality Assessment for Diagnostic Accuracy Studies 2 (QUADAS-2) tool. Studies were assigned to a sub-group based on the timing of the pNGAL sample in relation to the cessation of CPB. These were <4 h, 4-8 h, 12 h or 24 h post-cessation of CPB. Summary values for sensitivity and specificity were estimated using the hierarchical summary receiver operator characteristic (ROC) curve model. A random-effects meta-analysis of each pair of sensitivity and specificity estimates from each included study was performed. In total, 3131 patients from 16 studies were included. When taken at 4-8 hours following CPB, pNGAL had superior performance for the diagnosis of AKI in the defined population when compared to earlier and later time points. Prediction regions and confidence intervals, however, demonstrated significant variability in pooled estimates of sensitivity and specificity. This is likely due to population and study design heterogeneity, lack of standardisation of assays and thresholds, and inability to distinguish the different molecular forms of NGAL. In conclusion, the diagnostic utility of pNGAL in this clinical setting is inconclusive and large individual studies of representative populations of cardiac surgery patients using assays that specifically detect NGAL in its monomeric form are required.