A high field NMR study of the products ensuing from konjak glucomannan C(6)-oxidation followed by enzymatic C(5)-epimerization

Konjak glucomannan (KGM) is a water-soluble linear copolymer of (1-->4) linked beta-D-mannopyranosyl and beta-D-glucopyranosyl units. It has been selectively C6-oxidized by a 2,2,6,6-tetramethylpiperidin-1-oxy mediated reaction to obtain the corresponding uronan. Oxidized KGM has been treated with three different C-5 epimerases, AlgE4, AlgE6, and AlgE1, to obtain uronans with a various content of alpha-L-gulopyranuronate residues, namely, KGME4, KGME6, and KGME1. By use of 1D selective and 2D NMR techniques, a full assignment of the high field (600 MHz) NMR spectra of the purified native KGM and of the oxidized and epimerized derivatives has been obtained. Since in the anomeric region of the (1)H NMR spectrum of native KGM, diads sensitivity is present, the glucose-glucose, glucose-mannose, mannose-mannose, and mannose-glucose distribution has been obtained. In the (13)C spectrum of oxidized KGM, due to the presence of triad sensitivity on the C-4 resonance of glucuronic and mannuronic units, a better sequential investigation has been possible. As a result the average length of mannuronic blocks, N(M) is obtained. When AlgE4, AlgE6, and AlgE1 enzymes are used for the epimerization of oxidized KGM, the reaction products differ significantly both in the proportion and in the distribution of the mannuronic and guluronic residues. In epimerized KGM derivatives, a careful deconvolution of (1)H spectra allows the measurement of the degree of epimerization. In the case of KGME1 and KGME6, the average blocks length, N(G), of the guluronic blocks introduced in the polysaccharidic chain with the epimerization has also been calculated. Due to the shortness of mannuronic blocks in the oxidized KGM before the epimerization, N(G) in the epimerized compounds is also very low.     

Abnormal protein patterns in blood serum and cerebrospinal fluid detected by capillary electrophoresis

Capillary zone electrophoresis and high-resolution agarose gel electrophoresis were compared to detect protein components in serum and cerebrospinal fluid of patients. Both electrophoretic methods proved to be useful for detection of protein abnormalities (e.g., mono- and oligoclonal bands) in biological fluids, but capillary electrophoresis offered several important advantages, such as sample application without preliminary concentration, lack of staining procedures, and on-line evaluation of patterns. Furthermore, capillary electrophoresis exhibits shorter analysis time and high resolution with low baseline noise. The results were proven to be powerful in diagnosis and monitoring of dyscrasias in routine laboratory practice.     

Urocortin-deficient mice display normal stress-induced anxiety behavior and autonomic control but an impaired acoustic startle response

Corticotropin-releasing hormone (Crh) plays an important role in modulating physiological and behavioral responses to stress. Its actions are mediated through two receptors, Crhr1 and Crhr2. Urocortin (Ucn), a Crh-related neuropeptide and the postulated endogenous ligand for Crhr2, is a potential mediator of stress responses. We generated Ucn-deficient mice using embryonic stem cell technology to determine its role in stress-induced behavioral and autonomic responses. Unlike Crhr1- or Crhr2-deficient mice, Ucn-deficient mice exhibit normal anxiety-like behavior as well as autonomic regulation in response to stress. However, the mutant mice display an impaired acoustic startle response that is not due to an obvious hearing defect. Thus, our results suggest that Ucn does not play an essential role in stress-induced behavioral and autonomic responses. Ucn may modulate the acoustic startle response through the Ucn-expressing neuron projections from the region of the Edinger-Westphal nucleus.     

Involvement of the Rab27 binding protein Slac2c/MyRIP in insulin exocytosis

Rab27a is a GTPase associated with insulin-containing secretory granules of pancreatic beta-cells. Selective reduction of Rab27a expression by RNA interference did not alter granule distribution and basal secretion but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors of the GTPase revealed that the Rab27a-binding protein Slac2c/MyRIP is associated with secretory granules of beta-cells. Attenuation of Slac2c/MyRIP expression by RNA interference did not modify basal secretion but severely impaired hormone release in response to secretagogues. Although beta-cells express Myosin-Va, a potential partner of Slac2c/MyRIP, no functional link between the two proteins could be demonstrated. In fact, overexpression of the Myosin-Va binding domain of Slac2c/MyRIP did not affect granule localization and hormone exocytosis. In contrast, overexpression of the actin-binding domain of Slac2c/MyRIP led to a potent inhibition of exocytosis without detectable alteration in granule distribution. This effect was prevented by point mutations that abolish actin binding. Taken together our data suggest that Rab27a and Slac2c/MyRIP are part of a complex mediating the interaction of secretory granules with cortical actin cytoskeleton and participate to the regulation of the final steps of insulin exocytosis.     

Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C

Reactive oxygen species (ROS) formation plays a major role in diabetes-induced endothelial dysfunction, though the molecular mechanism(s) involved and the contribution of nitric oxide (NO) are still unclear. This study using bovine retinal endothelial cells was aimed at assessing (i) the role of oxygen-dependent vs. NO-dependent oxidative stress in the endothelial cell permeability alterations induced by the diabetic milieu and (ii) whether protein kinase C (PKC) activation ultimately mediates these changes. Superoxide, lipid peroxide, and PKC activity were higher under high glucose (HG) vs. normal glucose throughout the 30 d period. Nitrite/nitrate and endothelial NO synthase levels increased at 1 d and decreased thereafter. Changes in monolayer permeability to 125I-BSA induced by 1 or 30 d incubation in HG or exposure to advanced glycosylation endproduct were reduced by treatment with antioxidants or PKC inhibitors, whereas NO blockade prevented only the effect of 1 d HG. HG-induced changes were mimicked by a PKC activator, a superoxide generating system, an NO and superoxide donor, or peroxynitrite (attenuated by PKC inhibition), but not a NO donor. The short-term effect of HG depends on a combined oxidative and nitrosative stress with peroxynitrite formation, whereas the long-term effect is related to ROS generation; in both cases, PKC ultimately mediates permeability changes.     

VEGF expression is developmentally regulated during human brain angiogenesis

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor working as an endothelial cell-specific mitogen and exerting a trophic effect on neurons and glial cells, both these activities being essential during central nervous system vascularisation, development and repair. The vascularisation of human telencephalon takes place by means of an angiogenic mechanism, which starts at the beginning of corticogenesis and actively proceeds up to the last neuronal migration, when the basic scheme of the vascular network has been drawn. Our study focused on VEGF during this critical developmental period with the aim of identifying the cells that express VEGF and of correlating the events of angiogenesis with the main events of cerebral cortex formation. The results show that in fetal human brain VEGF protein is located on multiple cell types, cells proper to the nervous tissue, neuroepithelial cells, neuroblasts and radial glia cells, and non-neuronal cells, endothelial and periendothelial cells. In these cells VEGF expression appears developmentally regulated and is correlated with angiogenesis, which in turn responds to the high metabolic demands of the differentiating neocortex.     

Hospital Admission of Cancer Patients: Avoidable Practice or Necessary Care?

Background

Cancer patients are frequently admitted to hospital due to acute conditions or refractory symptoms. This occurs through the emergency departments and requires medical oncologists to take an active role. The use of acute-care hospital increases in the last months of life.

Patients and methods

We aimed to describe the admissions to a medical oncology inpatient service within a 16-month period with respect to patients and tumor characteristics, and the outcome of the hospital stay.

Results

672 admissions of 454 patients were analysed. The majority of admissions were urgent (74.1%), and were due to uncontrolled symptoms (79.6%). Among the chief complaints, dyspnoea occurred in 15.7%, pain in 15.2%, and neurological symptoms in 14.5%. The majority of the hospitalizations resulted in discharge to home (60.6%); in 26.5% the patient died and in 11.0% was transferred to a hospice. Admissions due to symptoms correlated with a longer hospital stay and a higher incidence of in-hospital death.

Conclusion

We suggest that hospital use is not necessarily a sign of inappropriately aggressive care: inpatient care is probably an unavoidable step in the cancer trajectory. Optimization of inpatient supportive procedures should be a specific task of modern medical oncology.     

An integrated assessment of histopathological changes of the enteric neuromuscular compartment in experimental colitis

Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4‐dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c‐Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS‐treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA‐positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c‐Kit‐ and TMEM16A/ANO1‐positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS‐induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.