Mesotocin and vasotocin in the brain of the lizard Gekko gecko

Summary The distribution of mesotocin and vasotocin was studied in the brain of the lizard Gekko gecko with antisera specific for either peptide. Both mesotocinergic and vasotocinergic perikarya are found in the paraventricular and supraoptic nuclei of the hypothalamus, whereas vasotocinergic neurons are exclusively present in the bed nucleus of the stria terminalis and in a cell group of the rhombencephalon. The distributional pattern of the mesotocinergic fibers corresponds closely to that of the vasotocinergic fibers. However, throughout the entire brain the mesotocinergic innervation is less dense than the vasotocinergic innervation. No sex differences are present in the mesotocinergic fiber system.Abbreviations acc nucleus accumbens - bst bed nucleus of the stria terminalis - bv blood vessel - dB diagonal band of Broca - dc dorsal cortex - dth dorsolateral thalamic nucleus - lc lateral cortex - me median eminence - oc optic chiasma - ot optic tract - pag periaqueductal grey - pvn paraventricular nucleus - rc rhombencephalic cell group - sep septum - son supraoptic nucleus - tect mesencephalic tectum - vth ventrolateral thalamus     

Dimerization of papain induced by mercuric chloride and a bifunctional organic mercurial.

The bifunctional mercurial meso-1,4-bis(acetatomercuri)-2,3-diethoxybutane and mercuric chloride are capable of dimerizing papain, by the attachment of the thiol group of two molecules of papain to each molecule of reagent. This is evident from the titration data, gel filtration and sedimentation equilibrium. The conformational change of papain necessary for this reaction is discussed.     

Is HPA axis reactivity in childhood gender-specific? A systematic review

Background

In adults, hypothalamus–pituitary–adrenal (HPA) axis activity shows sexual dimorphism, and this is thought to be a mechanism underlying sex-specific disease incidence. Evidence is scarce on whether these sex differences are also present in childhood. In a meta-analysis, we recently found that basal (non-stimulated) cortisol in saliva and free cortisol in 24-h urine follow sex-specific patterns. We explored whether these findings could be extended with sex differences in HPA axis reactivity.

Methods

From inception to January 2016, PubMed and EMBASE.com were searched for studies that assessed HPA axis reactivity in healthy girls and boys aged ≤18 years. Articles were systematically assessed and reported in the categories: (1) diurnal rhythm, (2) cortisol awakening response (CAR), (3) protocolled social stress tests similar or equal to the Trier Social Stress Test for children (TSST-C), (4) pharmacological (ACTH and CRH) stress tests, and (5) miscellaneous stress tests.

Results

Two independent assessors selected 109 out of 6158 records for full-text screening, of which 81 studies (with a total of 14,591 subjects) were included. Studies showed that girls had a tendency towards a more variable diurnal rhythm (12 out of 29 studies), a higher CAR (8 out of 18 studies), and a stronger cortisol response to social stress tests (9 out of 21 studies). We found no evidence for sex differences in cortisol response after a pharmacological challenge or to miscellaneous stress tests.

Discussion

Sex differences in HPA axis reactivity appear to be present in childhood, although evidence is not unequivocal. For a better evaluation of sex differences in HPA axis reactivity, standardization of protocols and reports of stress tests is warranted.

     

Isolation and properties of particles containing the reactioncenter complex of Photosystem II from spinach chloroplasts

By density gradient centrifugation of the 80000 × g supernatant of digitonintreated spinach chloroplasts two main green bands and one minor green band were obtained. The purification and properties of the particles present in the main bands, which were shown to be derived from Photosystem I and Photosystem II, have been described previously; those of the particles in the minor fraction will be described in the present paper.

After purification, these particles show Photosystem II activity but are devoid of Photosystem I activity. They have a high chlorophyll a/chlorophyll b ratio and are enriched in β-carotene and cytochrome b₅₅₉. At liquid nitrogen temperature, photoreduction of C550 and photooxidation of cytochrome-b₅₅₉ can be observed. At room temperature, cytochrome b₅₅₉ undergoes slight photooxidation.

These properties indicate that this particle may be the reaction-center complex of Photosystem II. It is suggested that, in vivo, the Photosystem II unit is made up of a reaction-center complex and an accessory complex, the latter being found in one of the main green bands of the density gradient.     

Determination of Changes in the Phosphorylation State of the Neuron-Specific Protein Kinase C Substrate B-50 (GAP43) by Quantitative Immunoprecipitation

To determine changes in the degree of phosphorylation of the protein kinase C substrate B-50 in vivo, a quantitative immunoprecipitation assay for B-50 (GAP43, F1, pp46) was developed. B-50 was phosphorylated in intact hippocampal slices with 32Pi or in synaptosomal plasma membranes with [gamma-32P]ATP. Phosphorylated B-50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti-B-50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the incorporation of 32P into B-50 was quantified by densitometric scanning of the autoradiogram. Only a single 48-kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B-50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified 32P-labelled B-50 added to slice homogenates or synaptosomal plasma membranes was greater than 95%; and (2) modulation of B-50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50 values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4 beta-phorbol 12,13-dibutyrate stimulated B-50 phosphorylation, whereas 4 alpha-phorbol 12,13-didecanoate was inactive. Thus, we conclude that the B-50 immunoprecipitation assay is suitable to monitor changes in B-50 phosphorylation in intact neuronal tissue.     

Longterm quiescent cells in the aged human subventricular neurogenic system specifically express GFAP‐δ

A main neurogenic niche in the adult human brain is the subventricular zone (SVZ). Recent data suggest that the progenitors that are born in the human SVZ migrate via the rostral migratory stream (RMS) towards the olfactory bulb (OB), similar to what has been observed in other mammals. A subpopulation of astrocytes in the SVZ specifically expresses an assembly‐compromised isoform of the intermediate filament protein glial fibrillary acidic protein (GFAP‐δ). To further define the phenotype of these GFAP‐δ expressing cells and to determine whether these cells are present throughout the human subventricular neurogenic system, we analysed SVZ, RMS and OB sections of 14 aged brain donors (ages 74‐93). GFAP‐δ was expressed in the SVZ along the ventricle, in the RMS and in the OB. The GFAP‐δ cells in the SVZ co‐expressed the neural stem cell (NSC) marker nestin and the cell proliferation markers proliferating cell nuclear antigen (PCNA) and Mcm2. Furthermore, BrdU retention was found in GFAP‐δ positive cells in the SVZ. In the RMS, GFAP‐δ was expressed in the glial net surrounding the neuroblasts. In the OB, GFAP‐δ positive cells co‐expressed PCNA. We also showed that GFAP‐δ cells are present in neurosphere cultures that were derived from SVZ precursors, isolated postmortem from four brain donors (ages 63‐91). Taken together, our findings show that GFAP‐δ is expressed in an astrocytic subpopulation in the SVZ, the RMS and the OB. Importantly, we provide the first evidence that GFAP‐δ is specifically expressed in longterm quiescent cells in the human SVZ, which are reminiscent of NSCs.     

Differential effects of gemcitabine on ribonucleotide pools of twenty-one solid tumour and leukaemia cell lines

To gain a more detailed insight into the metabolism of 2', 2'-difluoro-2'-deoxycytidine (dFdC, gemcitabine, Gemzar) and its effect on normal ribonucleotide (NTP) metabolism in relation to sensitivity, we studied the accumulation of dFdCTP and the changes in NTP pools after dFdC exposure in a panel of 21 solid tumour and leukaemia cell lines. Both sensitivity to dFdC and accumulation of dFdCTP were clearly cell line-dependent: in this panel of cell lines, the head and neck cancer (HNSCC) cell line 22B appeared to be the most sensitive, whereas the small cell lung cancer (SCLC) cell lines were the least sensitive to dFdC. The human leukaemia cell line CCRF-CEM accumulated the highest concentration of dFdCTP, whereas the non-SCLC cell lines accumulated the least. Not only the amount of dFdCTP accumulation was clearly related to the sensitivity for dFdC (R=-0.61), but also the intrinsic CTP/UTP ratio (R=0.97). NTP pools were affected considerably by dFdC treatment: in seven cell lines dFdC resulted in a 1.7-fold depletion of CTP pools, in two cell lines CTP pools were unaffected, but in 12 cell lines CTP pools increased about 2-fold. Furthermore, a 1.6-1.9-fold rise in ATP, UTP and GTP pools was shown in 20, 19 and 20 out of 21 cell lines, respectively. Only the UTP levels after treatment with dFdC were clearly related to the amount of dFdCTP accumulating in the cell (R=0.64 (P<0.01)), but not to the sensitivity to dFdC treatment. In conclusion, we demonstrate that besides the accumulation of dFdCTP, the CTP/UTP ratio was clearly related to the sensitivity to dFdC. Furthermore, the UTP levels and the CTP/UTP ratio after treatment were related to dFdCTP accumulation. Therefore, both the CTP and UTP pools appear to play an important role in the sensitivity to dFdC.     

Cell Specific Cytotoxicity and Structure-Activity Relationship of Lipophilic 1-B-D-Arabinofuranosylcytosine (Ara-C) Derivatives

Abstract

Lipophilic derivatives of ara-C were developed with the aim to improve drug penetration and retention in solid tumors. Ara-C was esterified at the 5′-position with fatty acids (16–22 C-atoms, 0–3 double bonds). The derivatives were inactive in cell lines with various forms of ara-C and 2′,2′-difluorodeoxycytidine (dFdC, gemcitabine) resistance, including deoxycytidine kinase (dCK) deficiency. The activity in the parent cell lines correlated negatively with chain length and positively with double bonds.