Pancreastatin distribution and plasma levels in the pig

Pancreastatin is a peptide isolated from porcine pancreas which has insulin-suppressive actions in vitro and sequence homology with chromogranin A. Using radioimmunoassay and immunocytochemistry we investigated whether pancreastatin has a more widespread distribution and a possible endocrine role in the pig. Pancreastatin immunoreactivity was found in plasma, adrenal gland, pancreas, anterior pituitary and throughout the gastrointestinal tract. The immunoreactivity was colocalized with chromogranin immunoreactivity in endocrine cells and ultrastructurally (in the pancreas) to storage granules. Characterization of pancreastatin-like immunoreactivity, using gel permeation and high performance liquid chromatography, separated 3 different pancreastatin-like immunoreactive forms: one molecular form, indistinguishable from synthetic pancreastatin 1-49, was predominant in pancreas and thyroid and released into the circulation postprandially. However, a high dose (greater than 1 nmol/l) infusion of pancreastatin 33-49 (the biologically active moiety in vitro) into conscious pigs had no effect on either basal or glucose-stimulated insulin secretion.     

Hepatocytes sensitized to tumor necrosis factor-alpha cytotoxicity undergo apoptosis through caspase-dependent and caspase-independent pathways

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-alpha toxicity by repression of NF-kappaB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-alpha cytotoxicity by similar mechanisms, TNF-alpha-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IkappaB, that blocks NF-kappaB activation or following cotreatment with actinomycin D (ActD). TNF-alpha treatment of Ad5IkappaB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-alpha induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IkappaB/TNF-alpha-induced cell death but did not inhibit ActD/TNF-alpha-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IkappaB/TNF-alpha- and ActD/TNF-alpha-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IkappaB/TNF-alpha but not ActD/TNF-alpha treatment caused mitochondrial cytochrome c release. These results suggest that NF-kappaB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-alpha toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-alpha cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis.     

Respiratory function in cybrid cell lines carrying European mtDNA haplogroups: implications for Leber's hereditary optic neuropathy

The possibility that some combinations of mtDNA polymorphisms, previously associated with Leber's hereditary optic neuropathy (LHON), may affect mitochondrial respiratory function was tested in osteosarcoma-derived transmitochondrial cytoplasmic hybrids (cybrids). In this cellular system, in the presence of the same nuclear background, different exogenous mtDNAs are used to repopulate a parental cell line previously devoid of its original mtDNA. No detectable differences in multiple parameters exploring respiratory function were observed when mtDNAs belonging to European haplogroups X, H, T and J were used. Different possible explanations for the previously established association between haplogroup J and LHON 11778/ND4 and 14484/ND6 pathogenic mutations are discussed, including the unconventional proposal that mtDNA haplogroup J may exert a protective rather than detrimental effect.     

Prevention of nonspecific lysis in liposomal and erythrocyte immunoassay systems by small lipid vesicles and erythrocyte ghosts

Large unilamellar liposomes prepared by the reverse-phase evaporation method (REVs) were made immunoreactive by incorporating dinitrophenylaminocaproyl-phosphatidylethanolamine (DNP-Cap-PE) or 8-(3-carboxypropyl)-theophylline-dipalmitoylphosphatidylethanolamine (Th-DPPE) into the phospholipid bilayer. Specific lysis in the presence of anti-DNP-BSA and goat anti-theophylline serum respectively, was induced by adding guinea pig serum as source for complement to these liposomes. However, specific lysis was found to be compromised by high levels of nonspecific lysis as monitored by the release of the fluorescent aqueous-space marker 6-carboxyfluorescein. Nonspecific lysis could be prevented without affecting specific lysis by pretreatment of complement or incubation of the reaction mixture with small unilamellar liposomes (SUVs). SUVs of various lipid compositions produced the desired effect; however, when the fraction of negative charge in the SUVs was increased to 30 mol%, specific lysis was inhibited as well. In a similar assay system consisting of hemolysin-sensitized sheep red blood cells it was also found that nonspecific lysis could be inhibited by addition of erythrocyte ghosts to the incubation medium, although specific lysis was somewhat depressed. However, SUVs or REVs of a composition similar to sheep erythrocytes were ineffective indicating a more selective nature of complement-mediated immunoreaction with erythrocyte membranes than with synthetic bilayer membranes.     

Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number,oxidative phosphorylation and ROS detoxification pathways

Leber''s hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that modulate LHON penetrance must take into account also the exposure to environmental triggers such as tobacco smoke.Leber''s hereditary optic neuropathy (LHON) is among the most frequent mitochondrial diseases, affecting about 1 in 35 000–60 000 in Europe.^{1, 2} LHON is associated in over 90% of cases with one of three common mitochondrial DNA (mtDNA) point mutations affecting the Complex I subunit genes ND4 (m.11778G>A), ND1 (m.3460G>A) and ND6 (m.14484 T>C), usually occurring in homoplasmic fashion^{3, 4} (100% of mtDNA is mutant). This maternally inherited blinding disorder is caused by selective degeneration of retinal ganglion cells, particularly those originating the small axons composing the papillomacular bundle, which leads to optic atrophy.^{5, 6, 7} Clinically, a subacute loss of central vision develops in weeks/months, mostly affecting young adult men, with a peculiar pattern of fiber depletion⁸ and a relatively predictable natural history of visual function decline.⁹ Exceptions apply to age of onset, with childhood or late cases,^{10, 11} to propensity in recovering vision, more frequent with the m.14484 T>C mutation,¹² and to clinical expression that in a subset of patients may be more widespread.⁴The mtDNA mutations are necessary but not sufficient to cause LHON,¹³ with penetrance being on average about 50% for males and 10% for females. The association of specific mtDNA haplotypes of haplogroup J with the m.14484 T>C and m.11778G>A mutations has been consistently documented in patients of European descent, indicating that mtDNA background modulates to a certain extent disease penetrance.^{14, 15} However, in a prototypical LHON maternal lineage, despite all the individuals carry the homoplasmic mtDNA mutation regardless the haplogroup, only some develop the disease, pointing to further factors that may be genetic and environmental.¹⁶ Thus, male prevalence and incomplete penetrance remain the two investigated and problematic issues in LHON. Both issues have been recently mechanistically related to the efficiency of compensatory mitochondrial biogenesis.^{17, 18} Estrogens ameliorate mitochondrial dysfunction by activating mitochondrial biogenesis, suggesting that females are naturally protected during their fertile period.^{17, 19} Furthermore, by studying different experimental systems (blood cells, skeletal muscle, skin-derived fibroblasts and ocular tissue) we found that the unaffected mutation carriers had a significantly higher mtDNA copy number and mitochondrial mass compared with their affected relatives,¹⁸ indicating that efficiently increasing mitochondrial biogenesis may overcome the pathogenic effect of the mtDNA mutation. Recently, others obtained similar results in different LHON cohorts.²⁰ Notwithstanding, nuclear modifiers remain elusive. In particular, association of LHON with genetic variants was not consistent across different studies.^{18, 21} Similarly inconsistent was the association with chromosome X-linked loci, hypothesized to explain male prevalence.^{22, 23, 24}Several other factors have been implicated in LHON, including exposure to cigarette smoke, alcohol and chemical toxins, head trauma, acute physical illness, psychological stress, antiretroviral and antituberculosis drugs.^{4, 25} These and other environmental factors can have a triggering role in LHON pathogenesis. For example, in vitro exposure to 2,5 exanedione had a toxic effect on LHON cybrid cells, with an increased sensitivity if they harbored a haplogroup J background.²⁶ A major environmental trigger of LHON is cigarette smoke; Sadun et al.²⁷ and Kirkman et al.²⁵ showed that LHON penetrance is significantly increased in smokers, independently of gender and alcohol intake.In the current study, we explored further the effect of cigarette smoking in LHON, showing in white blood cells from patients of large LHON cohorts, and in skin-derived fibroblasts, that cigarette derivatives exert their toxicity by depressing mtDNA copy number and oxidative phosphorylation (OXPHOS). However, unaffected mutation carriers displayed the most efficient capacity for reactive oxygen species (ROS) detoxification, which was not hampered by exposure to cigarette derivatives.     

Lesion Load May Predict Long-Term Cognitive Dysfunction in Multiple Sclerosis Patients

Background

Magnetic Resonance Imaging (MRI) techniques provided evidences into the understanding of cognitive impairment (CIm) in Multiple Sclerosis (MS).

Objectives

To investigate the role of white matter (WM) and gray matter (GM) in predicting long-term CIm in a cohort of MS patients.

Methods

303 out of 597 patients participating in a previous multicenter clinical-MRI study were enrolled (49.4% were lost at follow-up). The following MRI parameters, expressed as fraction (f) of intracranial volume, were evaluated: cerebrospinal fluid (CSF-f), WM-f, GM-f and abnormal WM (AWM-f), a measure of lesion load. Nine years later, cognitive status was assessed in 241 patients using the Symbol Digit Modalities Test (SDMT), the Semantically Related Word List Test (SRWL), the Modified Card Sorting Test (MCST), and the Paced Auditory Serial Addition Test (PASAT). In particular, being SRWL a memory test, both immediate recall and delayed recall were evaluated. MCST scoring was calculated based on the number of categories, number of perseverative and non-perseverative errors.

Results

AWM-f was predictive of an impaired performance 9 years ahead in SDMT (OR 1.49, CI 1.12–1.97 p = 0.006), PASAT (OR 1.43, CI 1.14–1.80 p = 0.002), SRWL-immediate recall (OR 1.72 CI 1.35–2.20 p<0.001), SRWL-delayed recall (OR 1.61 CI 1.28–2.03 p<0.001), MCST-category (OR 1.52, CI 1.2–1.9 p<0.001), MCST-perseverative error(OR 1.51 CI 1.2–1.9 p = 0.001), MCST-non perseverative error (OR 1.26 CI 1.02–1.55 p = 0.032).

Conclusion

In our large MS cohort, focal WM damage appeared to be the most relevant predictor of the long-term cognitive outcome.     

The BDNF Val66Met Polymorphism Has Opposite Effects on Memory Circuits of Multiple Sclerosis Patients and Controls

Episodic memory deficits are frequent symptoms in Multiple Sclerosis and have been associated with dysfunctions of the hippocampus, a key region for learning. However, it is unclear whether genetic factors that influence neural plasticity modulate episodic memory in MS. We thus studied how the Brain Derived Neurotrophic Factor Val⁶⁶Met genotype, a common polymorphism influencing the hippocampal function in healthy controls, impacted on brain networks underlying episodic memory in patients with Multiple Sclerosis. Functional magnetic resonance imaging was used to assess how the Brain Derived Neurotrophic Factor Val⁶⁶Met polymorphism modulated brain regional activity and functional connectivity in 26 cognitively unimpaired Multiple Sclerosis patients and 25 age- and education-matched healthy controls while performing an episodic memory task that included encoding and retrieving visual scenes. We found a highly significant group by genotype interaction in the left posterior hippocampus, bilateral parahippocampus, and left posterior cingulate cortex. In particular, Multiple Sclerosis patients homozygous for the Val⁶⁶ allele, relative to Met⁶⁶ carriers, showed greater brain responses during both encoding and retrieval while the opposite was true for healthy controls. Furthermore, a robust group by genotype by task interaction was detected for the functional connectivity between the left posterior hippocampus and the ipsilateral posterior cingulate cortex. Here, greater hippocampus-posterior cingulate cortex connectivity was observed in Multiple Sclerosis Met⁶⁶ carriers relative to Val⁶⁶ homozygous during retrieval (but not encoding) while, again, the reverse was true for healthy controls. The Val⁶⁶Met polymorphism has opposite effects on hippocampal circuitry underlying episodic memory in Multiple Sclerosis patients and healthy controls. Enhancing the knowledge of how genetic factors influence cognitive functions may improve the clinical management of memory deficits in patients with Multiple Sclerosis.