Is Intracranial Atherosclerosis an Independent Risk Factor for Cerebral Atrophy? A Retrospective Evaluation

Background

Our purpose was to study the association between the intracranial atherosclerosis as measured by cavernous carotid artery calcification (ICAC) observed on head CT and atrophic changes of supra-tentorial brain demonstrated by MRI.

Methods

Institutional review board approval was obtained for this retrospective study incorporating 65 consecutive patients presenting acutely who had both head CT and MRI. Arterial calcifications of the intracranial cavernous carotids (ICAC) were assigned a number (1 to 4) in the bone window images from CT scans. These 4 groups were then combined into high (grades 3 and 4) and low calcium (grades 1 and 2) subgroups. Brain MRI was independently evaluated to identify cortical and central atrophy. Demographics and cardiovascular risk factors were evaluated in subjects with high and low ICAC. Relationship between CT demonstrated ICAC and brain atrophy patterns were evaluated both without and with adjustment for cerebral ischemic scores and cardiovascular risk factors.

Results

Forty-six of the 65 (71%) patients had high ICAC on head CT. Subjects with high ICAC were older, and had higher prevalence of hypertension, diabetes, coronary artery disease (CAD), atrial fibrillation and history of previous stroke (CVA) compared to those with low ICAC. Age demonstrated strong correlation with both supratentorial atrophy patterns. There was no correlation between ICAC and cortical atrophy. There was correlation however between central atrophy and ICAC. This persisted even after adjustment for age.

Conclusion

Age is the most important determinant of atrophic cerebral changes. However, high ICAC demonstrated age independent association with central atrophy.

     

Binding to CD20 by Anti-B1 Antibody or F(ab')2 is sufficient for induction of apoptosis in B-cell lines

CD20 is a B-cell-specific cell surface protein expressed on mature B lymphocytes and is a target for monoclonal antibody therapy for non-Hodgkin's lymphoma (NHL). Though clear clinical efficacy has been demonstrated with several anti-CD20 antibodies, the mechanisms by which the antibodies activate CD20 and kill cells remain unclear. Proposed mechanisms of action include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and induction of apoptosis. In this report we compared the activity of two anti-CD20 antibodies, Anti-B1 Antibody (tositumomab) and rituximab (C2B8), in a variety of cellular assays using a panel of B-cell lines. Anti-B1 Antibody showed a low level of activity in a CDC assay against complement-sensitive B-cell lines, Ramos and Daudi. We found that there is an inverse correlation between the expression of CD55 and CD59 and CDC mediated by either Anti-B1 Antibody or rituximab. Rituximab was more potent at inducing CDC when compared to Anti-B1 Antibody. Using Raji cells as target cells and human peripheral blood leukocytes as effector cells, Anti-B1 Antibody was a potent inducer of ADCC. The activities of Anti-B1 Antibody and rituximab were nearly identical in the ADCC assay. In addition, Anti-B1 Antibody showed direct induction of apoptosis in all B-cell lines tested. In general, crosslinking Anti-B1 Antibody with a goat anti-mouse Ig did not further enhance the percentage of cells undergoing apoptosis. Importantly, a F(ab')(2) fragment of Anti-B1 Antibody induced apoptosis, while the Fab fragment did not, indicating that the Fc region was not required and dimerization of CD20 may be sufficient for induction of apoptosis. In contrast, rituximab, which binds to an overlapping epitope on CD20 with a three-fold lower affinity than Anti-B1 Antibody, did not efficiently induce apoptosis in the cell lines tested in the absence of crosslinking. In conclusion, these two anti-CD20 antibodies have overlapping, but distinct mechanisms of action on B-cell lines.     

Specific localization of scallop gill epithelial calmodulin in cilia

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.     

Early events in the cellular formation of proparathyroid hormone

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.     

Photoinactivation of B-type monoamine oxidase by a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolite

The reaction of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) with monoamine oxidase from a variety of tissues including rat and monkey brain, bovine liver, and human placenta and platelets was found to yield, as a primary product, a reactive photosensitive substance with an absorbance maximum at 345 nm which is not the cation 1-methyl-4-phenylpyridinium ion previously reported as a monoamine oxidase-MPTP metabolite in vivo and in vitro. Our results suggest that the 1-methyl-4-phenyl-pyridinium ion is probably only generated in subsequent nonenzymatic transformations of this reactive monoamine oxidase metabolite. This substance was found to specifically inactivate the B-form of monoamine oxidase by a photo-induced mechanism and to react directly with NADPH and dopamine. Properties of the metabolite and potential significance of its reactions to MPTP neurotoxicity are discussed.     

Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B

This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Evidence for para Dechlorination of Polychlorobiphenyls by Methanogenic Bacteria

When microorganisms eluted from upper Hudson River sediment were cultured without any substrate except polychlorobiphenyl (PCB)-free Hudson River sediment, methane formation was the terminal step of the anaerobic food chain. In sediments containing Aroclor 1242, addition of eubacterium-inhibiting antibiotics, which should have directly inhibited fermentative bacteria and thereby should have indirectly inhibited methanogens, resulted in no dechlorination activity or methane production. However, when substrates for methanogenic bacteria were provided along with the antibiotics (to free the methanogens from dependence on eubacteria), concomitant methane production and dechlorination of PCBs were observed. The dechlorination of Aroclor 1242 was from the para positions, a pattern distinctly different from, and more limited than, the pattern observed with untreated or pasteurized inocula. Both methane production and dechlorination in cultures amended with antibiotics plus methanogenic substrates were inhibited by 2-bromoethanesulfonic acid. These results suggest that the methanogenic bacteria are among the physiological groups capable of anaerobic dechlorination of PCBs, but that the dechlorination observed with methanogenic bacteria is less extensive than the dechlorination observed with more complex anaerobic consortia.