Role of endogenous brain-derived neurotrophic factor and sortilin in B cell survival

Brain-derived neurotrophic factor (BDNF), a major neuronal growth factor, is also known to exert an antiapoptotic effect in myeloma cells. Whereas BDNF secretion was described in B lymphocytes, the ability of B cells to produce sortilin, its transport protein, was not previously reported. We studied BDNF production and the expression of its receptors, tyrosine protein kinase receptor B and p75 neurotrophin receptor in the human pre-B, mature, and plasmacytic malignant B cell lines under normal and stress culture conditions (serum deprivation, Fas activation, or their combination). BDNF secretion was enhanced by serum deprivation and exerted an antiapoptotic effect, as demonstrated by neutralization experiments with antagonistic Ab. The precursor form, pro-BDNF, also secreted by B cells, decreases under stress conditions in contrast to BDNF production. Stress conditions induced the membranous expression of p75 neurotrophin receptor and tyrosine protein kinase receptor B, maximal in mature B cells, contrasting with the sequestration of both receptors in normal culture. By blocking Ab and small interfering RNA, we evidenced that BDNF production and its survival function are depending on sortilin, a protein regulating neurotrophin transport in neurons, which was not previously described in B cells. Therefore, in mature B cell lines, an autocrine BDNF production is up-regulated by stress culture conditions and exerts a modulation of apoptosis through the sortilin pathway. This could be of importance to elucidate certain drug resistances of malignant B cells. In addition, primary B lymphocytes contained sortilin and produced BDNF after mitogenic activation, which suggests that sortilin and BDNF might be implicated in the survival and activation of normal B cells also.     

Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of cAMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response.     

Catalytic activation of human glucokinase by substrate binding: residue contacts involved in the binding of D-glucose to the super-open form and conformational transitions

alpha-D-Glucose activates glucokinase (EC 2.7.1.1) on its binding to the active site by inducing a global hysteretic conformational change. Using intrinsic tryptophan fluorescence as a probe on the alpha-D-glucose induced conformational changes in the pancreatic isoform 1 of human glucokinase, key residues involved in the process were identified by site-directed mutagenesis. Single-site W-->F mutations enabled the assignment of the fluorescence enhancement (DeltaF/F(0)) mainly to W99 and W167 in flexible loop structures, but the biphasic time course of DeltaF/F(0) is variably influenced by all tryptophan residues. The human glucokinase-alpha-D-glucose association (K(d) = 4.8 +/- 0.1 mm at 25 degrees C) is driven by a favourable entropy change (DeltaS = 150 +/- 10 J.mol(-1).K(-1)). Although X-ray crystallographic studies have revealed the alpha-d-glucose binding residues in the closed state, the contact residues that make essential contributions to its binding to the super-open conformation remain unidentified. In the present study, we combined functional mutagenesis with structural dynamic analyses to identify residue contacts involved in the initial binding of alpha-d-glucose and conformational transitions. The mutations N204A, D205A or E256A/K in the L-domain resulted in enzyme forms that did not bind alpha-D-glucose at 200 mm and were essentially catalytically inactive. Our data support a molecular dynamic model in which a concerted binding of alpha-D-glucose to N204, N231 and E256 in the super-open conformation induces local torsional stresses at N204/D205 propagating towards a closed conformation, involving structural changes in the highly flexible interdomain connecting region II (R192-N204), helix 5 (V181-R191), helix 6 (D205-Y215) and the C-terminal helix 17 (R447-K460).     

Forty years trends in timing of pubertal growth spurt in 157,000 Danish school children

Background

Entering puberty is an important milestone in reproductive life and secular changes in the timing of puberty may be an important indicator of the general reproductive health in a population. Too early puberty is associated with several psychosocial and health problems. The aim of our study was to determine if the age at onset of pubertal growth spurt (OGS) and at peak height velocity (PHV) during puberty show secular trends during four decades in a large cohort of school children.

Methods and Findings

Annual measurements of height were available in all children born from 1930 to 1969 who attended primary school in the Copenhagen Municipality. 135,223 girls and 21,612 boys fulfilled the criteria for determining age at OGS and age at PHV. These physiological events were used as markers of pubertal development in our computerized method in order to evaluate any secular trends in pubertal maturation during the study period (year of birth 1930 to 1969). In this period, age at OGS declined statistically significantly by 0.2 and 0.4 years in girls and boys, respectively, whereas age at PHV declined statistically significantly by 0.5 and 0.3 years in girls and boys, respectively. The decline was non-linear with a levelling off in the children born between 1940 and 1955. The duration of puberty, as defined by the difference between age at OGS and age at PHV, increased slightly in boys, whereas it decreased in girls.

Conclusion

Our finding of declining age at OGS and at PHV indicates a secular trend towards earlier sexual maturation of Danish children born between 1930 and 1969. Only minor changes were observed in duration of puberty assessed by the difference in ages at OGS and PHV.     

Germline mutation in RNASEL predicts increased risk of head and neck, uterine cervix and breast cancer

THE BACKGROUND: Ribonuclease L (RNASEL), encoding the 2'-5'-oligoadenylate (2-5A)-dependent RNase L, is a key enzyme in the interferon induced antiviral and anti-proliferate pathway. Mutations in RNASEL segregate with the disease in prostate cancer families and specific genotypes are associated with an increased risk of prostate cancer. Infection by human papillomavirus (HPV) is the major risk factor for uterine cervix cancer and for a subset of head and neck squamous cell carcinomas (HNSCC). HPV, Epstein Barr virus (EBV) and sequences from mouse mammary tumor virus (MMTV) have been detected in breast tumors, and the presence of integrated SV40 T/t antigen in breast carcinomas correlates with an aggressive phenotype and poor prognosis. A genetic predisposition could explain why some viral infections persist and induce cancer, while others disappear spontaneously. This points at RNASEL as a strong susceptibility gene. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the implication of an abnormal activity of RNase L in the onset and development of viral induced cancers, the study was initiated by searching for germline mutations in patients diagnosed with uterine cervix cancer. The rationale behind is that close to 100% of the cervix cancer patients have a persistent HPV infection, and if a defective RNase L were responsible for the lack of ability to clear the HPV infection, we would expect to find a wide spectrum of mutations in these patients, leading to a decreased RNase L activity. The HPV genotype was established in tumor DNA from 42 patients diagnosed with carcinoma of the uterine cervix and somatic tissue from these patients was analyzed for mutations by direct sequencing of all coding and regulatory regions of RNASEL. Fifteen mutations, including still uncharacterized, were identified. The genotype frequencies of selected single nucleotide polymorphisms (SNPs) established in the cervix cancer patients were compared between 382 patients with head and neck squamous cell carcinomas (HNSCC), 199 patients with primary unilateral breast cancer and 502 healthy Danish control individuals. We found that the genotype frequencies of only one of the 15 mutations, the yet uncharacterized 5'UTR mutation rs3738579 differed significantly between cancer patients and control individuals (P-value: 4.43x10(-5)). CONCLUSION/SIGNIFICANCE: In conclusion, we have discovered an increased risk, a heterozygous advantage and thereby a protective effect linked to the RNASEL SNP rs3738579. This effect is found for patients diagnosed with carcinoma of the uterine cervix, HNSCC, and breast cancer thus pointing at RNASEL as a general marker for cancer risk and not restricted to familial prostate cancer.     

The good,the bad and the ugly: A neuropilin-2 story from normal to tumor-associated lymphangiogenesis

VEGF-C is a crucial player in lymphangiogenesis. Besides VEGFR-2 and VEGFR-3, it also binds NRP2. NRP2 enhances VEGF-C/VEGFR-3 effects in developmental lymphangiogenesis, but its role in adult and tumoral lymphangiogenesis is not known. In their study, Bagri and colleagues demonstrate that blocking NRP2 results in a decrease of metastasis formation, a phenomenon relying on tumoral lymphangiogenesis. Thus, they identified NRP2 as an attractive new target for modulating metastasis.Key words: VEGF-C, NRP2, VEGFR-3, tumoral lymphangiogenesis, metastasisBesides developmental and adult lymphangiogenesis, formation of new lymphatic vessels also occurs during tumor growth. This abnormal lymphangiogenesis enables tumor cells to escape from the solid tumor and to invade the body through the newly formed lymphatics. The resulting metastases are responsible for most cancer-associated deaths. Interestingly, inhibition of VEGF-C/VEGFR-3 axis has been shown to block metastasis formation in preclinical models.¹ However, treatment with VEGFR3_ECD may result in compromise of the normal lymphatic system leading to complications such as lymphedema. VEGF-C belongs to the VEGF family and is particularly involved in lymphangiogenesis. VEGF-C binds VEGFR-2 but mostly associates with high affinity with the third tyrosine kinase VEGF receptor, VEGFR-3.² Recently, it has been proved that VEGF-C also binds a receptor from the neuropilin family, NRP2. NRP2 associates with VEGFR-3, and binding of VEGF-C to NRP2 enhances VEGF-C/VEGFR-3-induced biological effects.³ NRP2 thus modulates developmental lymphangiogenesis, but its significance in adult or tumoral lymphangiogenesis remained unknown until a recent study by Bagri and colleagues who analyzed NRP2 function in tumor cell metastasis.⁴     

Modulation of Lck function through multisite docking to T cell-specific adapter protein

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr(280), Tyr(290), and Tyr(305) were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)(280) and Tyr(P)(290) phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)(505) phosphopeptide, whereas the TSAd Tyr(P)(305) peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20-30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is approximately 1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.     

Histological changes in rat duodenum mucosa after whole-body gamma irradiation.

In order to understand the mechanisms of intestinal injuries due to ionizing radiation, various groups of rats have been whole-body irradiated by gamma-rays at two dose rates (1 Gy/min and 1 Gy/hr), three doses (1, 2 and 4 Gy) and two post-irradiation times (24 and 48 hr). Duodenum samples of the animals were prepared for light microscopy, according to classical methods for histology and TUNEL reaction. A small number of morphological differences were observed within the mucosa between the two dose rates used. The extent and the number of lesions were more important at the slower dose rate (1 Gy/hr) and increased with the total dose. Clear cavities were seen inside the lamina propria which appeared like capillaries free of blood cells. The mitotic index calculated from crypt cells showed a regular decrease with the dose, which was exacerbated at 48 hr post-irradiation. On the other hand, the apoptotic index increased with the dose and the postirradiation time. Our results lead to hypothesize another mechanism of intestinal mucosa renewal allowing to explain mucosa denudations observed after radiotherapy. Thus we propose a new concept in which the duodenal mucosa renewal may occur by whole villi shedding into the duodenal lumen.