A synergistic effect of albumin and fibrinogen on immunoglobulin-induced red blood cell aggregation

Therapeutic administration of immunoglobulins (Ig) has the potential to precipitate thrombotic events. This phenomenon may be explained by red blood cell (RBC) aggregation, which can be potentiated by Ig. The contribution of plasma albumin and fibrinogen to Ig-induced RBC aggregation is unclear. We examined RBC aggregation in three settings: 1) patients receiving therapeutic infusions of Ig; 2) patients receiving plasma supplemented in vitro with Ig; and 3) patients receiving RBC suspensions in standard buffer with varying concentrations of albumin, Ig, and fibrinogen. Ig infusion augmented aggregation of RBCs from patients with normal or high plasma levels of albumin but decreased aggregation in those with lower plasma albumin concentrations. In vitro, RBC aggregation was significantly increased only when all three components, fibrinogen, albumin, and Ig, were present at or above normal concentrations in the suspension but was unaffected when any one of the components was absent from the suspension. Our results suggest a three-way interaction among fibrinogen, Ig, and albumin that synergistically induces RBC aggregation in plasma. Understanding these interactions may help predict clinically important phenomena related to RBC aggregation, such as thrombotic complications of Ig infusion.     

Monozoic cysts of Hepatozoon canis

Small monozoic cysts found in the spleen of dogs infected with Hepatozoon canis are described from naturally and experimentally infected dogs. These forms of H. canis resemble cysts formed by other Hepatozoon species that infect frogs, lizards, and grey squirrels as intermediate hosts. The H. canis cyst stage differs in size and morphology from the large cysts of H. americanum, the second Hepatozoon species known to infect dogs.     

Voltage-Dependent Anion Channel Proteins in Synaptosomes of the Torpedo Electric Organ: Immunolocalization, Purification, and Characterization

In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca²⁺ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate–cytochrome-c oxidoreductase and antimycin-insensitive NADH–cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [¹⁴C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30–35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDAC1 (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.     

Emergence of Distinct Genotypes of Cryptosporidium parvum in Structured Host Populations

Cryptosporidium parvum is an apicomplexan parasite that infects humans and ruminants. C. parvum isolated from cattle in northeastern Turkey and in Israel was genotyped using multiple polymorphic genetic markers, and the two populations were compared to assess the effect of cattle husbandry on the parasite's population structure. Dairy herds in Israel are permanently confined with essentially no opportunity for direct herd-to-herd transmission, whereas in Turkey there are more opportunities for transmission as animals range over wider areas and are frequently traded. A total of 76 C. parvum isolates from 16 locations in Israel and seven farms in the Kars region in northeastern Turkey were genotyped using 16 mini- and microsatellite markers. Significantly, in both countries distinct multilocus genotypes confined to individual farms were detected. The number of genotypes per farm was higher and mixed isolates were more frequent in Turkey than in Israel. As expected from the presence of distinct multilocus genotypes in individual herds, linkage disequilibrium among loci was detected in Israel. Together, these observations show that genetically distinct populations of C. parvum can emerge within a group of hosts in a relatively short time. This may explain the frequent detection of host-specific genotypes with unknown taxonomic status in surface water and the existence of geographically restricted C. hominis genotypes in humans.     

Neurochemical Evidence for Agmatine Modulation of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) Neurotoxicity

Agmatine treatment is known to exert neuroprotective effects in several models of neurotoxic and ischemic brain and spinal cord injuries. Here we sought to find out whether agmatine treatment would also prove to be neuroprotective in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease. Concomitant daily treatment (intraperitoneal injections) with agmatine (100 mg/kg for 5 days) and MPTP (40 mg/kg for 2 days) exacerbated MPTP-related toxicity as evidenced by a larger reduction in dopamine uptake into striatal synaptosomes (42.4% as compared to 58.3% of control, respectively). In contrast, agmatine treatment commencing after MPTP, produced partial protection (31%) against MPTP dopaminergic toxicity. The findings implicate agmatine in mechanisms regulating MPTP neurotoxicity, but underscore the characteristic neuroprotective efficacy of agmatine when applied after the insult.     

A New Fungal Diterpene Induces VDAC1-dependent Apoptosis in Bax/Bak-deficient Cells

The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release. Diphenylamine-2-carboxilic acid, an inhibitor of VDAC1 conductance and oligomerization, inhibited cyathin-R-induced VDAC1 oligomerization and apoptosis. Similarly, Bcl-2 overexpression conferred resistance to cyathin-R-induced apoptosis and VDAC1 oligomerization. Silencing of VDAC1 expression prevented cyathin-R-induced apoptosis. Finally, cyathin-R effectively attenuated tumor growth and induced apoptosis in Bax/Bak-deficient cells implanted into a xenograft mouse model. Hence, this study identified a new compound promoting VDAC1-dependent apoptosis as a potential therapeutic option for cancerous cells lacking or presenting inactivated Bax/Bak.     

Endothelial Matrix Assembly during Capillary Morphogenesis: Insights from Chimeric TagRFP-Fibronectin Matrix

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.