Coumarin glucosides from Cruciata taurica

Two new coumarin glycosides (1 and 2) along with two known coumarin glucosides, daphnin (3) and daphnetin glucoside (4) were isolated from the aerial parts of Cruciata taurica. The structures of the new compounds were elucidated by spectral methods and chemical means as 7-O-(6' -acetoxy-beta-D-glucopyranosyl)-8-hydroxycoumarin (1) and 7-O-[6 '-O-(3',4'-dihydroxycinnamoyl)-beta-D-glucopyranosyl]-8-hydroxycoumarin (2). The phylogenetic significance of coumarins in C. taurica was discussed.     

Possible cytoskeletal structures of rainbow trout sperm revealed by electron microscopic observation after detergent extraction

Despite the considerable research interest in fish sperm ultrastructure, little is known about the functions of different sperm cell components. Our electron microscopic study was aimed at identifying possible tissue-specific cytoskeletal structures in spermatozoa of rainbow trout Oncorhynchus mykiss (Teleostei, Salmoniformes, Salmonidae; formerly Salmo gairdneri). Based on the known resistance of the cytoskeleton to nonionic detergents, we compared the ultrastructure of unextracted and Triton-extracted sperm cells. Besides the nucleus, the centrioles and the axoneme, there were also other structures preserved in Triton-treated spermatozoa: the lateral extensions (sidefins) and a thin layer corresponding in position to the membrane-like structure underlying the midpiece plasma membrane in intact cells. Because of their stability, it could be hypothesized that these cytoplasmic components are likely to have cytoskeletal nature. They are possibly analogous to the well known tissue-specific cytoskeletal components of mammalian spermatozoa with periaxonemal and submitochondrial localization.     

Efficient gene delivery into murine ovarian cells by intraovarian injection of plasmid DNA and subsequent in vivo electroporation

We describe the use of direct injection of circular plasmid DNA and subsequent in vivo electroporation (EP) for efficient gene delivery to the ovarian cells, including follicular cells and oocytes of mice. When Trypan blue (TB) was injected into the central portion of an ovary by a glass micropipette, rapid dispersion of TB to each preantral and antral follicle was observed. Injections of lacZ-expressing plasmid DNA and subsequent in vivo EP resulted in transfection of follicles with efficiencies ranging from 8-60%, together with cells in the thecal portion of the ovary. Of the lacZ-positive follicles, some oocytes were also positive for lacZ activity. These findings suggest that a solution introduced inside the ovary is rapidly dispersed to each follicle. With this technique, we expect great progress in genetic engineering in murine ovary.     

Inhibition of Papaya latex papain by photosensitive inhibitors. 1-(4,5-dimethoxy-2-nitrophenyl)-2-nitroethene and 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene

1-(4,5-Dimethoxy-2-nitrophenyl)-2-nitroethene (1) was shown to be an irreversible inhibitor of papain (EC 3.4.22.2), causing a complete inhibition (120 min preincubation, pH 8.0), assuming that it attached to Cys-25 at the active site of the enzyme (while a short preincubation time caused activation). Only partial inhibition of papain was achieved, however, with 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (2), a compound synthesized in this work, which is also an irreversible inhibitor of papain. Since both compounds 1 and 2, and in each case of the inhibited enzyme, were 2-nitrobenzyl derivatives, they and the modified enzyme were expected to be photosensitive. Indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in a full recovery of the enzyme activity following inactivation with compound 1 (similar to our previous finding with -galactosidase) and up to 67% recovery following inhibition with compound 2.     

Norepinephrine transporter expression and function in noradrenergic cell differentiations

The classical view of norepinephrine transporter (NET) function is the re-uptake of released norepinephrine (NE) by mature sympathetic neurons and noradrenergic neurons of the locus ceruleus (LC; [1-3]). In this report we review previous data and present new results that show that NET is expressed in the young embryo in a wide range of neuronal and non-neuronal tissues and that NET has additional functions during embryonic development. Sympathetic neurons are derived from neural crest stem cells. Fibroblast growth factor-2 (FGF-2), neurotrophin-3 (NT-3) and transforming growth factor-1 (TGF-1) regulate NET expression in cultured quail neural crest cells by causing an increase in NET mRNA levels. They also promote NET function in both neural crest cells and presumptive noradrenergic cells of the LC. The growth factors are synthesized by the neural crest cells and therefore are likely to have autocrine function. In a subsequent stage of development, NE transport regulates differentiation of noradrenergic neurons in the peripheral nervous system and the LC by promoting expression of tyrosine hydroxylase (TH) and dopamine--hydroxylase (DBH). Conversely, uptake inhibitors, such as the tricyclic antidepressant, desipramine, and the drug of abuse, cocaine, inhibit noradrenergic differentiation in both tissues. Taken together, our data indicate that NET is expressed early in embryonic development, NE transport is involved in regulating expression of the noradrenergic phenotype in the peripheral and central nervous systems, and norepinephrine uptake inhibitors can disturb noradrenergic cell differentiation in the sympathetic ganglion (SG) and LC.     

Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules

Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Drugs that stabilize polysomes (emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (puromycin) promote the assembly of SGs. Moreover, emetine dissolves preformed SGs as it promotes the assembly of polysomes, suggesting that these mRNP species (i.e., SGs and polysomes) exist in equilibrium. We used green flourescent protein-tagged SG-associated RNA-binding proteins (specifically, TIA-1 and poly[A] binding protein [PABP-I]) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching shows that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. This unexpected result leads us to propose that mammalian SGs are sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes. A truncation mutant of TIA-1 (TIA-1DeltaRRM), which acts as a transdominant inhibitor of SG assembly, promotes the expression of cotransfected reporter genes in COS transfectants, suggesting that this process of mRNA triage might, directly or indirectly, influence protein expression.     

Spermatogenesis in the golden hamster during the first spermatogenic wave: a flow cytometric analysis

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36-38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88-90%) in the hamster occurred in the testis and only 10-12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205-211, 2000.     

SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation.     

Using HIV Surveillance Registry Data to Re-Link Persons to Care: The RSVP Project in San Francisco

Background

Persons with unsuppressed HIV viral load (VL) who disengage from care may experience poor clinical outcomes and potentially transmit HIV. We assessed the feasibility and yield of using the San Francisco Department of Public Health (SFDPH) enhanced HIV surveillance system (eHARS) to identify and re-engage such persons in care.

Methods

Using SFDPH eHARS data as of 4/20/2012 (index date), we selected HIV-infected adults who were alive, had no reported VL or CD4 cell count results in the past nine months (proxy for “out-of-care”) and a VL >200 copies/mL drawn nine to 15 months earlier. We prioritized cases residing locally for investigation, and used information from eHARS and medical and public health databases to contact them for interview and referral to the SFDPH linkage services (LINCS). Twelve months later, we matched-back to eHARS data to assess how HIV laboratory reporting delays affected original eligibility, and if persons had any HIV laboratory results performed and reported within 12 months after index date (‘new labs’).

Results

Among 434 eligible persons, 282 were prioritized for investigation, of whom 75 (27%) were interviewed, 79 (28%) could not be located, and 48 (17%) were located out of the area. Among the interviewed, 54 (72%) persons accepted referral to LINCS. Upon match-back to eHARS data, 324 (75%) in total were confirmed as eligible, including 221 (78%) of the investigated; most had new labs.

Conclusions

Among the investigated persons presumed out-of-care, we interviewed and offered LINCS referral to about one-quarter, demonstrating the feasibility but limited yield of our project. Matching to updated surveillance data revealed that a substantial minority did not disengage from care and that most re-engaged in HIV care. Verifying persons’ HIV care status with medical providers and improving timeliness of transfer and cross-jurisdictional sharing of HIV laboratory data may aid future efforts.     

P2Y2R Deficiency Attenuates Experimental Autoimmune Uveitis Development

We aimed to study the role of the nucleotide receptor P2Y₂R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y₂^+/+ and P2Y₂^-/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y₂^+/+ and P2Y₂^-/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4⁺ T lymphocytes from P2Y₂^+/+ and P2Y₂^-/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y₂^-/- mice as in P2Y₂^-/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y₂^-/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y₂^-/- and P2Y₂^+/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y₂^-/- T cells proliferate less and secrete less cytokines than the P2Y₂^+/+ one. We further found that antigen-presenting cells of P2Y₂^-/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y₂^-/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides.