Calcium mobilization by the plant estrogen ferutinin does not induce blood platelet aggregation

Platelet activation is closely associated with an increase in intracellular Ca(2+) concentration. Various compounds including Ca(2+) ionophores are able to trigger platelet aggregation by increasing intracellular Ca(2+) concentration in platelets. In the present study, we monitored the effect of the phytoestrogen ferutinin, which acts as a Ca(2+) ionophore in human blood platelets; its ionophore-like properties include upregulation of [Ca(2+)](in), activation of fibrinogen receptors and increased fibrinogen binding. Using spectrofluorometry and triple-color flow cytometry, we demonstrate that ferutinin increases [Ca(2+)](in) in both isolated platelets and platelets in whole blood from humans. This effect was almost completely blocked by the Ca(2+) chelator EGTA and was not sensitive to either Gd(3+) or econazole, which inhibit VOC and SOC channels, respectively. Nor was the effect sensitive to thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) ATPases. Ferutinin stimulated the expression of the active form of the GPIIb-IIIa complex and whole blood platelet aggregation only weakly and had no statistically significant effect on the binding of fibrinogen. These results demonstrate apparently inconsistent effects of ferutinin, which raises intraplatelet Ca(2+) concentration but fails to have an effect on spontaneous blood platelet aggregation. This pattern of responses may be caused by the combination of ferutinin's Ca(2+) ionophoric and estrogenic properties.     

Altered cellular distribution and subcellular sorting of gamma-tubulin in diffuse astrocytic gliomas and human glioblastoma cell lines

Centrosome amplification is a pivotal mechanism underlying tumorigenesis but its role in gliomas is underinvestigated. The present study specifically examines the expression and distribution of the centrosome-associated cytoskeletal protein gamma-tubulin in 56 primary diffuse astrocytic gliomas (grades II-IV) and in 4 human glioblastoma cell lines (U87MG, U118MG, U138MG, and T98G). Monoclonal anti-peptide antibodies recognizing epitopes in C-terminal or N-terminal domains of the gamma-tubulin molecule were used in immunohistochemical, immunofluorescence, and immunoblotting studies. In tumors in adults (n = 46), varying degrees of localization were detected in all tumor grades, but immunoreactivity was significantly increased in high-grade anaplastic astrocytomas and glioblastomas multiforme as compared to low-grade diffuse astrocytomas (p = 0.0001). A similar trend was noted in diffuse gliomas in children but the sample of cases was too small as to be statistically meaningful. Two overlapping patterns of ectopic cellular localization were identified in both primary tumors and glioblastoma cell lines: A punctate pattern, in which gamma-tubulin was partially co-distributed with pericentrin in the pericentriolar region, and a diffuse pattern, independent of pericentrin staining, denoting a soluble pool of gamma-tubulin. Cellular gamma-tubulin was detected in both soluble and insoluble (nocodazole-resistant) fractions of glioblastoma cells. Divergent localizations of gamma-tubulin and pericentrin suggest a differential distribution of these 2 centrosome-associated proteins in glioblastoma cell lines. Our results indicate that overexpression and ectopic cellular distribution of gamma-tubulin in astrocytic gliomas may be significant in the context of centrosome protein amplification and may be linked to tumor progression and anaplastic potential.     

Depletion of the Ras association domain family 1, isoform A-associated novel microtubule-associated protein, C19ORF5/MAP1S, causes mitotic abnormalities

Ras association domain family 1, isoform A (RASSF1A) is a novel tumor suppressor gene that is found to be inactivated in more than 40 types of sporadic cancers. In addition, mouse Rassf1a knockout models have an increased frequency of spontaneous and induced tumors. The mechanisms by which RASSF1A exerts its tumor suppression activities or the pathways it can regulate are not yet fully understood. Using yeast two-hybrid system, we have previously identified C19ORF5/MAP1S as the major RASSF1A-interacting protein. C19ORF5 has two conserved microtubule-associated regions and may function to anchor RASSF1A to the centrosomes. In this study, we have analyzed the cellular functions of C19ORF5. By using small interfering RNA-mediated depletion and time-lapse video microscopy, we show that C19ORF5 knockdown causes mitotic abnormalities that consist of failure to form a stable metaphase plate, premature sister chromatid separation, lagging chromosomes, and multipolar spindles. We also show that a fraction of C19ORF5 localizes to the spindle microtubules. Additionally, we show here that C19ORF5 localizes to the microtubule-organizing centers during microtubule regrowth after nocodazole washout. Knockdown of C19ORF5 disrupts the microtubule-organizing center and results in microtubule nucleation from several sites. Whereas the localization of pericentrin is not affected, alpha- and gamma-tubulin localization and sites of nucleation are greatly altered by C19ORF5 depletion. This may indicate that C19ORF5 plays a role in anchoring the microtubule-organizing center to the centrosomes. In addition, we show that the NH2 terminus of C19ORF5 is essential for this process. This novel role for C19ORF5 could explain the resulting mitotic abnormalities that occur on its depletion and can potentially provide an underlying mechanism for the frequent centrosome and microtubule abnormalities detected in several cancers.     

Cleavage of Protein Kinase D After Acute Hypoinsulinemia Prevents Excessive Lipoprotein Lipase�CMediated Cardiac Triglyceride Accumulation

OBJECTIVE

During hypoinsulinemia, when cardiac glucose utilization is impaired, the heart rapidly adapts to using more fatty acids. One means by which this is achieved is through lipoprotein lipase (LPL). We determined the mechanisms by which the heart regulates LPL after acute hypoinsulinemia.

RESEARCH DESIGN AND METHODS

We used two different doses of streptozocin (55 [d-55] and 100 [d-100] mg/kg) to induce moderate and severe hypoinsulinemia, respectively, in rats. Isolated cardiomyocytes were also used for transfection or silencing of protein kinase D (PKD) and caspase-3.

RESULTS

There was substantial increase in LPL in d-55 hearts, an effect that was absent in severely hypoinsulinemic d-100 animals. Measurement of PKD, a key element involved in increasing LPL, revealed that only d-100 hearts showed an increase in proteolysis of PKD, an effect that required activation of caspase-3 together with loss of 14-3-3ζ, a binding protein that protects enzymes against degradation. In vitro, phosphomimetic PKD colocalized with LPL in the trans-golgi. PKD, when mutated to prevent its cleavage by caspase-3 and silencing of caspase-3, was able to increase LPL activity. Using a caspase inhibitor (Z-DEVD) in d-100 animals, we effectively lowered caspase-3 activity, prevented PKD cleavage, and increased LPL vesicle formation and translocation to the vascular lumen. This increase in cardiac luminal LPL was associated with a striking accumulation of cardiac triglyceride in Z-DEVD–treated d-100 rats.

CONCLUSIONS

After severe hypoinsulinemia, activation of caspase-3 can restrict LPL translocation to the vascular lumen. When caspase-3 is inhibited, this compensatory response is lost, leading to lipid accumulation in the heart.Cardiac muscle has a high demand for energy and can use multiple substrates (1). Among these, glucose (∼30%) and fatty acid (∼70%) are the major sources from which the heart derives most of its energy (2). Fatty acid delivery and utilization by the heart involves 1) release from adipose tissue and transport to the heart after complexing with albumin (3), 2) provision through the breakdown of endogenous cardiac triglyceride (4), 3) internalization of whole lipoproteins (5), and 4) hydrolysis of circulating triglyceride-rich lipoproteins to fatty acids by lipoprotein lipase (LPL) positioned at the endothelial surface of the coronary lumen (6). The molar concentration of fatty acids bound to albumin is ∼10-fold less than that of fatty acids in lipoprotein triglycerides, (7) and LPL-mediated hydrolysis of triglyceride-rich lipoproteins is suggested to be the principal source of fatty acids for cardiac utilization (8). Coronary endothelial cells do not synthesize LPL (9). In the heart, this enzyme is produced in cardiomyocytes and subsequently secreted onto heparan sulfate proteoglycan (HSPG) binding sites on the myocyte cell surface (10). From here, LPL is transported onto comparable binding sites on the luminal surface of endothelial cells (11). At the lumen, LPL actively metabolizes the triglyceride core of lipoproteins; the released fatty acids are then transported into the heart.The earliest change that occurs in the type 1 diabetic heart is altered energy metabolism where in the presence of lower glucose utilization, the heart switches to using more fatty acids for energy supply (12). One means by which this is possible is through an increase in LPL at the coronary lumen. Using retrograde perfusion of the heart with heparin to displace vascular LPL, we found elevated LPL following diabetes (13–15). We determined that the increased enzyme is 1) not the result of increased gene expression (13), 2) unrelated to an increase in the number of endothelial HSPG binding sites (13), 3) associated with an acute reduction in insulin (within 60 min) (16), and 4) functionally relevant and capable of hydrolyzing lipoprotein triglycerides (17). More recently, we examined the contributions of the cardiomyocyte and endothelial cell in enabling this increased enzyme at the vascular lumen. Within the myocyte, LPL vesicle fission was regulated by protein kinase D (PKD) (18), whereas recruitment of LPL to the cardiomyocyte surface was controlled by stress kinases like AMP-activated protein kinase (AMPK) (19) and p38 mitogen-activated protein kinase (MAPK) that allowed for provision of an actin network that facilitated LPL movement (20). Translocation of LPL from the cardiomyocyte surface to the apical side of endothelial cells is then dependent on the ability of the endothelium to release heparanase (21,22), which enables myocyte HSPG cleavage and transfer of LPL toward the coronary lumen.Selective β-cell death and an ensuing diabetic state can be produced after a single intravenous dose of streptozotocin (STZ) (23). In Wistar rats, a dose-dependent increase in severity of diabetes is produced by 25–100 mg/kg STZ (24). After injection of 55 mg/kg (d-55), stable hyperglycemia develops within 24–48 h in concert with a ∼50% reduction in plasma insulin (16,24). Although these animals were insulin deficient, they did not require insulin supplementation for survival and did not develop ketoacidosis. In the absence of any changes in plasma fatty acids and triglycerides, these animals demonstrated an increase in coronary vascular LPL (13–15). Rats administered 100 mg/kg STZ (d-100) demonstrated intense β-cell necrosis, loss of 98% pancreatic insulin stores, and severely reduced plasma insulin (16). Compared to d-55 diabetic rats, these d-100 animals show a remarkable elevation of plasma fatty acids and triglycerides. Importantly, LPL activity decreases (14,25) in d-100 hearts, suggesting a potential mechanism to restrain LPL-derived fatty acids when the supply of this substrate from other reservoirs is in surplus. The objective of the present study was to determine the mechanisms by which the hypoinsulinemic heart limits its LPL-derived fatty acids under conditions of hyperlipidemia. Our data demonstrate that caspase-3 activation, by cleaving PKD, attempts to restrict the hydrolysis of circulating triglyceride by LPL to limit fatty acid provision and cardiac triglyceride overload. Thus, although caspase-3 inhibition could be protective in reducing cell death, its augmentation of LPL may induce profound cardiac triglyceride accumulation.     

Manometric tests of anorectal function in 90 healthy children: a clinical study from Kuwait

Background/Purpose

Anorectal manometry is a noninvasive test used to evaluate conditions like slow-transit constipation, anorectal outlet obstruction, and Hirschsprung disease and to assess postoperative results after Hirschsprung and anorectal malformations. This cross section study was designed to have normal manometric values of anorectal function in healthy children of different ages in Kuwait so that control values are available for comparisons with various pathological states.

Method

Anorectal manometry was conducted in 90 children aged 3 days to 12 years without any symptoms related to lower gastrointestinal tract. They were divided in 3 age groups (group 1—neonates up to 1 month, group 2—infants from 1 month to 1 year, and group 3—children more than 1 year). Water perfused system with anorectal catheter with 4 side holes was used to record length of anal canal or high-pressure zone, resting pressure of anal canal, and rectoanal inhibitory reflex (RAIR).

Result

Anorectal manometry was successfully done in all 90 children of different age groups without any complications. High-pressure zone or anal canal length was 1.67 ± 0.34 cm in neonates, 1.86 ± 0.6 cm in infants, and 3.03 ± 0.52 cm in children. Mean resting pressure of anal canal was 31.07 ± 10.9 mm Hg in neonates, 42.43 ± 8.9 mm Hg in infants, and 43.43 ± 8.79 mm Hg in children. Rectoanal inhibitory reflex was present in all of them. Mean RAIR threshold volumes of 9.67 ± 3.6, 14.0 ± 9.5, and 25.0 ± 11.6 mL was required for noenates, infants, and children, respectively.

Conclusion

Resting pressure of the anal canal, manometic anal canal length, and RAIR volume varies with the age. Normal values anorectal manometry at different age groups should be obtained to compare with pathological states of anorectum.     

Partial Splenic Embolization Versus Splenectomy for the Management of Hypersplenism in Cirrhotic Patients

Background  Hypersplenism occurs in patients with chronic liver disease, and splenectomy is the definitive treatment. However, the operation may be hazardous in patients with poor liver function. In recent years, partial splenic embolization (PSE) has been widely used in patients with hypersplenism and cirrhosis. This study was conducted to assess the safety and efficacy of PSE compared to splenectomy in the management of hypersplenism in cirrhotic patients. Methods  This study comprised 40 patients with hypersplenism secondary to cirrhosis. They were divided into two groups, each including 20 patients. The first group of patients were treated by PSE using polyvinyl alcohol particles to achieve embolization of at least 50% of the distal branches of the splenic artery. Postembolization arteriography and computed tomography were performed to document the extent of devascularization. Patients in the second group were treated by splenectomy with or without devascularization and left gastric ligation according to the presence or absence of esophageal varices. Results  There was marked improvement in platelet and leukocytic counts in both groups, and the counts remained at appropriate levels during the follow-up period. All patients in the first group had problems related to postembolization syndrome that abated by the first week. One patient in the first group died from myocardial infarction. No deaths occurred in the second group. Asymptomatic portal vein thrombosis developed in one patient in the first group that was treated with anticoagulation, and another patient developed splenic abscess treated by splenectomy with a good outcome. In the second group, three patients developed portal vein thrombosis, one of them being readmitted 4 months postoperatively with mesenteric vascular occlusion; that patient underwent a resection anastomosis with good outcome. Conclusions  Partial splenic embolization is an effective therapeutic modality for the treatment of hypersplenism secondary to chronic liver disease. It is a simple, rapid procedure that is easily performed under local anesthesia; and it allows preservation of adequate splenic tissue to safeguard against overwhelming infection.