Differentiation of the melanotrophic cells of rat pituitary primordium in organotypic culture in defined medium

Summary Organotypic cultures, in defined medium, of pituitary primordia obtained from 15-day-old rat fetuses were performed in order to study the in vitro differentiation of melanotrophic cells. The morphological and ultrastructural features of the transplants resembled those of the gland developing in vivo. In situ hybridization on semi-thin sections, using a ³⁵S-labelled oligonucleotide probe, revealed pro-opiomelanocortin-mRNA-containing cells on the first day of culture in the anterior lobe and after 2–3 days in the intermediate lobe. Immunoperoxidase labelling of adjacent sections showed that the same cells reacted with antibodies against -melanocyte-stimulating hormone (MSH), ₃ and adrenocorticotropic hormone in both lobes. The pro-opiomelanocortin-mRNA-containing cells formed progressively conspicuous areas in the intermediate lobe, which was almost uniformly labelled after 6 days. In the anterior lobe, these cells remained scattered in small cell groups, and colloidal gold immunolabelling showed the progressive disappearance of MSH labelling from the secretory vesicles in cells exhibiting morphological features of adult corticotrophic cells. Both the MSH content of the explants and MSH release into the culture medium increased with time. Treatment with the dopamine agonist bromocriptine induced a strong dose-dependent decrease in MSH secretion, which was significant after 3 days in culture, indicating that dopamine D₂ receptors are able to regulate hormonal release of melanotrophic cells at early stages. This system constitutes a suitable model for further studies of factors controlling cell differentiation and cellular interactions involved in histogenesis.     

Habitat and population size of the coelacanth Latimeria chalumnae at Grand Comoro

Synopsis In 1987 and 1989 coelacanths were observed for the first time in their natural habitat with the help of submersibles. Coelacanths were found between 150–253 m depth, their preferential depth seems to be around 200 m; the water temperature ranged between 16.5–22.8° C. During the day coelacanths aggregate in small non-aggressive groups in sheltered lava-caves. Caves might be a limiting factor for distribution. At night they leave the caves for hunting by drifting singly along the steep lava slopes. They migrate between different caves located within a large home range covering more than 8 km coastline. Coelacanths are site-attached, some for a period of at least 2 years. Our own observations and earlier catch records show that only the west coast of Grand Comoro is a suitable coelacanth habitat with more structural complexity and prey fish abundance than other coastlines of the island. From our survey we estimated a total coelacanth population off Grand Comoro to be 150–210 individuals; a saturated population would be 370–510 individuals. This small relict population seems to be stable. International protection of coelacanths against commercial interests is needed     

Interaction between prostaglandin E2 and leukotriene D4 on the excretion of electrolytes by the dog kidney in vivo

Recent experiments indicate that prostaglandin E₂ potentiates the vasodilatory properties of leukotrienes in the skin microcirculation. The present experiments were undertaken to study the effect of leukotriene D₄ and prostaglandin E₂ on renal hemodynamics and urinary electrolytes in the dog. Experiments were performed in three groups of anesthetized Mongrel dogs: the first group was studied under hydropenia, whereas the two remaining groups were studied during water diuresis with (Group 3) or without indomethacin (Group 2). LTD₄ (100ng/min) and PGE₂ (3ug/min) were infused in the left renal artery to minimize systemic effects of these compounds. LTD₄ alone failed to influence urinary sodium excretion in all 3 groups. In Group 1, urinary sodium increased from 77 ± 6 to 393 ± 74uEq/min during PGE₂, and further increased to 511 ± 52uEq/min during LTD₄ + PGE₂. No change occured in the contralateral right kidney. In this group, glomerular filtration as well as renal plasma flow were not statistically influenced. In Group 2, the same phenomenon was observed for urinary sodium. The combined infusion of LTD₄ + PGE₂ increased urinary sodium without significant changes in glomerular filtration and renal plasma flow. Finally, in Group 3, indomethacin was shown to reduce the natriuretic effects of LTD₄ and PGE₂: during PGE₂ alone, urinary sodium increased from 90 ± 14 to 260 ± 66uEq/min, and only rose from 80 ± 10 to 175 ± 19uEq/min during the combined infusion of LTD₄ and PGE₂. In groups 2 and 3, free water clearance was utilized as an index of sodium chloride reabsorption in the thick ascending limb: this parameter increased from 2.35 ± 0.25 to 4.70 ± 0.30ml/min, while urinary volume was increasing from 3.55 ± 0.25 to 10.05 ± 0.65ml/min, during LTD₄ + PGE₂. Indomethacin, administered in Group 3, (3mg/kg/hr) again abolished the effect of combined PGE₂ + LTD₄. These results indicate a potentiating effect of leukotriene D₄ on the PGE₂-induced natriuresis in the anesthetized dog. These phenomena occured in the absence of significant changes in renal hemodynamics, therefore suggesting a direct tubular effect of these arachidonic acid metabolites. Finally, the water diuresis experiments suggest a proximal site of action of PGE₂ and LTD₄.     

Use of DNA fingerprinting to determine parentage in muskrats (Ondatra zibethicus).

The detection of high levels of genetic variability by DNA fingerprinting probes has allowed researchers to accurately assess relatedness. Multiple-mating strategies are characteristic of the mating systems of small mammals. As such, techniques that provide an accurate indication of how individuals are related genetically is of great importance to assess the mating system of a species. In this study, we applied the DNA fingerprinting technique to captive and wild muskrats (Ondatra zibethicus) to determine its usefulness for parentage analysis in wild populations. We found that DNA digested with the restriction enzyme Hae III and probed with Jeffrey's minisatellite 33. 15 identified a large amount of polymorphism in both groups of muskrats. The DNA fingerprinting technique correctly assessed parentage within the captive group. In the wild population, paternity was assigned between two adult males based on diagnostic fragments and similarity of banding patterns. The likelihood that paternity could be misassigned to a full sibling was high in this free-ranging population. However, because natal dispersal in muskrats is male biased, it is unlikely that two brothers would associate with the same female.     

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca²⁺ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca²⁺ binding to the SARS-CoV-2-FP, the role of Ca²⁺ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca²⁺-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca²⁺-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca²⁺ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca²⁺ ions. These findings provide novel mechanistic insights regarding the role of Ca²⁺ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.     

Soil carbon saturation: concept,evidence and evaluation

Current estimates of soil C storage potential are based on models or factors that assume linearity between C input levels and C stocks at steady-state, implying that SOC stocks could increase without limit as C input levels increase. However, some soils show little or no increase in steady-state SOC stock with increasing C input levels suggesting that SOC can become saturated with respect to C input. We used long-term field experiment data to assess alternative hypotheses of soil carbon storage by three simple models: a linear model (no saturation), a one-pool whole-soil C saturation model, and a two-pool mixed model with C saturation of a single C pool, but not the whole soil. The one-pool C saturation model best fit the combined data from 14 sites, four individual sites were best-fit with the linear model, and no sites were best fit by the mixed model. These results indicate that existing agricultural field experiments generally have too small a range in C input levels to show saturation behavior, and verify the accepted linear relationship between soil C and C input used to model SOM dynamics. However, all sites combined and the site with the widest range in C input levels were best fit with the C-saturation model. Nevertheless, the same site produced distinct effective stabilization capacity curves rather than an absolute C saturation level. We conclude that the saturation of soil C does occur and therefore the greatest efficiency in soil C sequestration will be in soils further from C saturation.

Microarray-based oncogenic pathway profiling in advanced serous papillary ovarian carcinoma

Introduction

The identification of specific targets for treatment of ovarian cancer patients remains a challenge. The objective of this study is the analysis of oncogenic pathways in ovarian cancer and their relation with clinical outcome.

Methodology

A meta-analysis of 6 gene expression datasets was done for oncogenic pathway activation scores: AKT, β-Catenin, BRCA, E2F1, EGFR, ER, HER2, INFα, INFγ, MYC, p53, p63, PI3K, PR, RAS, SRC, STAT3, TNFα, and TGFβ and VEGF-A. Advanced serous papillary tumours from uniformly treated patients were selected (N = 464) to find differences independent from stage-, histology- and treatment biases. Survival and correlations with documented prognostic signatures (wound healing response signature WHR/genomic grade index GGI/invasiveness gene signature IGS) were analysed.

Results

The GGI, WHR, IGS score were unexpectedly increased in chemosensitive versus chemoresistant patients. PR and RAS activation score were associated with survival outcome (p = 0.002;p = 0.004). Increased activations of β-Catenin (p = 0.0009), E2F1 (p = 0.005), PI3K (p = 0.003) and p63 (p = 0.05) were associated with more favourable clinical outcome and were consistently correlated with three prognostic gene signatures.

Conclusions

Oncogenic pathway profiling of advanced serous ovarian tumours revealed that increased β-Catenin, E2F1, p63, PI3K, PR and RAS –pathway activation scores were significantly associated with favourable clinical outcome. WHR, GGI and IGS scores were unexpectedly increased in chemosensitive tumours. Earlier studies have shown that WHR, GGI and IGS are strongly associated with proliferation and that high-proliferative ovarian tumours are more chemosensitive. These findings may indicate opposite confounding of prognostic versus predictive factors when studying biomarkers in epithelial ovarian cancer.