Sulpholipid metabolism in developing chicken retina.

Glycolipid analysis of chicken retina and brain indicated the presence of cerebroside, cerebroside 3-sulphate and sulphogalactosylglycerolipid In retina, the ratio of cerebroside to cerebroside 3-sulphate was approximately half compared to brain. During chicken retina ontogenesis the ratio of cerebroside 3-sulphate to sulphogalactosylglycerolipid increased rapidly and in the adult animal, the amount of cerebroside 3-sulphate was 14 times higher than that of sulphogalactosylglycerolipid. The activity of PAPS: cerebroside sulphotransferase and arylsulphatase A in developing chicken retina indicated that the general ontogenic profiles of retinal PAPS: cerebroside sulphotransferase and arylsulphatase A were similar to those obtained for the brain. Both the enzymes showed the highest activity just before hatching. The significance of occurrence of sulpholipids in retina is discussed.     

Gold nanoparticles–conjugated quercetin induces apoptosis via inhibition of EGFR/PI3K/Akt–mediated pathway in breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

Epidermal growth factor plays a major role in breast cancer cell proliferation, survival, and metastasis. Quercetin, a bioactive flavonoid, is shown to exhibit anticarcinogenic effects against various cancers including breast cancer. Hence, the present study was designed to evaluate the effects of gold nanoparticles–conjugated quercetin (AuNPs‐Qu‐5) in MCF‐7 and MDA‐MB‐231 breast cancer cell lines. Borohydride reduced AuNPs were synthesized and conjugated with quercetin to yield AuNPs‐Qu‐5. Both were thoroughly characterized by several physicochemical techniques, and their cytotoxic effects were assessed by MTT assay. Apoptotic studies such as DAPI, AO/EtBr dual staining, and annexin V‐FITC staining were performed. AuNPs and AuNPs‐Qu‐5 were spherical with crystalline nature, and the size of particles range from 3.0 to 4.5 nm. AuNPs‐Qu‐5 exhibited lower IC₅₀ value compared to free Qu. There was a considerable increase in apoptotic population with increased nuclear condensation seen upon treatment with AuNPs‐Qu‐5. To delineate the molecular mechanism behind its apoptotic role, we analysed the proteins involved in apoptosis and epidermal growth factor receptor (EGFR)–mediated PI3K/Akt/GSK‐3β signalling by immunoblotting and immunocytochemistry. The pro‐apoptotic proteins (Bax, Caspase‐3) were found to be up regulated and anti‐apoptotic protein (Bcl‐2) was down regulated on treatment with AuNPs‐Qu‐5. Additionally, AuNPs‐Qu‐5 treatment inhibited the EGFR and its downstream signalling molecules PI3K/Akt/mTOR/GSK‐3β. In conclusion, administration of AuNPs‐Qu‐5 in breast cancer cell lines curtails cell proliferation through induction of apoptosis and also suppresses EGFR signalling. AuNPs‐Qu‐5 is more potent than free quercetin in causing cancer cell death, and hence, this could be a potential drug delivery system in breast cancer therapy.     

Purification and properties of human placenta arylsulphatase A.

1. Arylsulphatase A (arylsulphate sulphohydrolase E.N. 3.1.6.1) has been purified 7200-fold from human placenta using concanavalin A Sepharose chromatography. 2. Ultracentrifugation studies indicated that the purified enzyme was homogenous with respect to sedimentation coefficient and molecular weight and has a molecular weight of 102000. 3. The purified enzyme could hydrolyze cerebroside 3-sulphate, seminolipid and sulphogalactosylsphingosine under identical conditions. 4. The kinetic parameters for the hydrolysis of all sulphate esters used in the present study were the same. 5. Both seminolipid and sulphogalactosylsphingosine were competitive inhibitors for the hydrolysis of cerebroside-3-sulphate with an inhibition constant of 0.2 mM. 6. Kinetic parameters, metal ion effect and heat inactivation profile of enzyme suggest that the same active site of enzyme is responsible for the hydrolysis of cerebroside 3-sulphate, seminolipid and sulphogalactosylsphingosine.     

Surgically induced cryptorchidism-related degenerative changes in spermatogonia are associated with loss of cyclic adenosine monophosphate-dependent phosphodiesterases type 4 in abdominal testes of rats

The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.     

Brain phospholipases A2: a perspective on the history

The phospholipases A2 (PLA2) belong to a large family of enzymes involved in the generation of several second messengers that play an important role in signal transduction processes associated with normal brain function. The phospholipase A2 family includes secretory phospholipase A2, cytosolic phospholipase A2, calcium-independent phospholipase A2, plasmalogen-selective phospholipase A2 and many other enzymes with phospholipase A2 activity that have not been classified. Few attempts have been made purify and characterize the multiple forms of PLA2 and none have been fully characterized and cloned from brain tissue. A tight regulation of phospholipase A2 isozymes is necessary for maintaining physiological levels of free fatty acids including arachidonic acid and its metabolites in the various types of neural cells. Under normal conditions, phospholipase A2 isozymes may be involved in neurotransmitter release, long-term potentiation, growth and differentiation, and membrane repair. Under pathological conditions, high levels of lipid metabolites generated by phospholipase A2 are involved in neuroinflammation, oxidative stress, and neural cell injury.     

Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements

Hantaviruses infect human endothelial and immune cells, causing two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). We have identified key signaling elements termed immunoreceptor tyrosine-based activation motifs (ITAMs) within the G1 cytoplasmic tail of all HPS-causing hantaviruses. ITAMs direct receptor signaling within immune and endothelial cells and the presence of ITAMs in all HPS-causing hantaviruses provides a means for altering normal cellular responses which maintain vascular integrity. The NY-1 G1 ITAM was shown to coprecipitate a complex of phosphoproteins from cells, and the G1 ITAM is a substrate for the Src family kinase Fyn. The hantavirus ITAM coprecipitated Lyn, Syk, and ZAP-70 kinases from T or B cells, while mutagenesis of the ITAM abolished these interactions. In addition, G1 ITAM tyrosines directed intracellular interactions with Syk by mammalian two-hybrid analysis. These findings demonstrate that G1 ITAMs bind key cellular kinases that regulate immune and endothelial cell functions. There is currently no means for establishing the role of the G1 ITAM in hantavirus pathogenesis. However, the conservation of G1 ITAMs in all HPS-causing hantaviruses and the role of these signaling elements in immune and endothelial cells suggest that functional G1 ITAMs are likely to dysregulate normal immune and endothelial cell responses and contribute to hantavirus pathogenesis.     

Molecular interaction of acrylonitrile and potassium cyanide with rat blood

The interaction of acrylonitrile (VCN) with rat blood has been investigated at the molecular level in an attempt to understand the possible mechanism of its toxicity. The results obtained were compared to those with potassium cyanide (KCN), a compound known to liberate cyanide (CN^?) in biologic conditions. The radioactivity derived from K¹⁴CN was eliminated faster than that from [1-¹⁴C]VCN. Up to a maximum of 94% of ¹⁴C from VCN in erythrocytes was detected covalently bound to cytoplasmic and membrane proteins, whereas 90% of the radioactivity from KCN in erythrocytes was found in the heme fraction of hemoglobin. Determination of specific activity showed that binding occurred more in vivo than in vitro which indicated that the VCN molecule was bioactivated inside erythrocytes. These results indicate that KCN interacts mainly through CN^? liberation and binding to heme, whereas VCN, which binds to cytoplasmic and membrane proteins, may cause damage to red cells by mechanisms other than release of CN^?.     

Octopamine receptors in the honeybee (Apis mellifera) brain and their disruption by RNA-mediated interference

Octopamine plays important neuromodulatory roles in the honeybee brain. Accordingly, mRNA from a recently identified honeybee octopamine receptor (AmOA1) is distributed throughout the brain. We have evaluated the occurrence of AmOA1 in the antennal lobe (AL) as well as rest of the brain (RB) by western blotting using an antiserum raised against a peptide selected from AmOA1 sequence. In addition to an expected band (78 kDa in the AL), one additional band (72 kDa) was identified from the AL and four bands (48, 60, 72 and 78 kDa) were observed in the RB. These bands were also recognized with antiserum against a different peptide segment from an octopamine receptor ortholog from the fruitfly (OAMB). Significant sequence identity with the peptide segment used to generate the antiserum was only found with OAMB and its splice variants in fruitfly; it was less conserved in other biogenic amine receptors from honeybee and other insects. Furthermore, western blot analysis performed on brains with dsRNA-treated antennal lobes showed a decrease in the intensity of all four bands. This suggests that AmOA1 antiserum specifically recognizes one or more types of AmOA1 receptors in the honeybee brain. We extend our earlier study of RNAi to quantify the rate of spread of dsRNA from a localized injection to other neuropils.     

Glycogen synthase kinase-3 acts upstream of ADP-ribosylation factor 6 and Rac1 to regulate epithelial cell migration

Cell sheet movement during epithelial wound closure is a complex process involving collective cell migration. We have found that glycogen synthase kinase-3 (GSK-3) activity is required for membrane protrusion and crawling of cells at the wound edge and those behind it in wounded Madin-Darby canine kidney (MDCK) epithelial cell monolayers. RNA interference-based silencing of GSK-3alpha and GSK-3beta expression also results in slowed cell sheet migration, with the effect being more pronounced with knockdown of GSK-3beta. Both GSK-3alpha and GSK-3beta are in activated states during the most active phase of cell migration. In addition to having a positive control or permissive, rather than negative, function in MDCK cell migration, GSK-3 appears to act upstream of the small GTPases ADP-ribosylation factor 6 (ARF6) and Rac1. Expression of constitutively active ARF6 restores a protrusive, migratory phenotype in cells treated with GSK-3 inhibitors. It does not, however, restore to normal levels the directional polarization of cells behind the wound edge toward the wound area, implying the existence of a separate ARF6-independent branch of the GSK-3 pathway that regulates proper wound-directed polarization of these cells. Finally, inhibition of GSK-3 also strongly reduces activation of Rac1 and cell scatter in response to hepatocyte growth factor/scatter factor, which triggers dispersal and migration of cells in monolayer culture as fibroblast-like individual cells, a mode of epithelial cell motility distinct from the collective migration of wound closure.