Michael H. Herbstreith

Endogenous apolipoprotein E suppresses LPS-stimulated microglial nitric oxide production

THE human apolipoprotein (apo) E4 isoform is associated with an increased risk for Alzheimers disease (AD) and poor prognosis after acute CNS injury. Addition of human apoE inhibits murine microglial activation in culture, suggesting that microglia might be an important physiological target of apoE. In the present study, we examined the role of endogenous murine apoE in modulating microglial nitric oxide (NO) production following lipopolysaccharide (LPS) stimulation. Brain cultures from apoE-deficient mouse pups showed enhanced NO production relative to cultures from wild-type mice and from transgenic mice expressing the human apoE3 isoform, demonstrating that endogenous apoE produced by glial cultures is capable of inhibiting microglial function. ApoE produced within the brain may suppress microglial reactivity and thus alter the CNS response to acute and chronic injury.

Cystatin C Mutation in an Elderly Man With Sporadic Amyloid Angiopathy and Intracerebral Hemorrhage

BACKGROUND Cerebral amyloid angiopathy (CAA) with intracerebral hemorrhage (ICH) occurs both sporadically and as a result of mutations in either cystatin C or the amyloid precursor protein. ICH due to cystatin C mutations typically occurs in young people of Icelandic origin. CASE DESCRIPTION We report a case of sporadic CAA with ICH in an elderly Croatian man with a mutation in cystatin C identical to that found in Icelandic hereditary cerebral hemorrhage with amyloidosis. CONCLUSIONS This is the first case report of sporadic CAA associated with the same mutation causing hereditary cerebral hemorrhage with amyloidosis of the Icelandic type. Sporadic CAA may thus be associated with genetic mutations in some patients. The frequency of these mutations is yet to be determined.

Increased phosphorylated components of erythrocyte membrane spectrin band II with reference to Duchenne muscular dystrophy

Increased erythrocyte band II phosphorylation has been demonstrated in patients with Duchenne muscular dystrophy (DMD). Band II has been purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing of purified band II from which SDS has been removed reveals heterogeneity. Band II migrates as multiple bands on isoelectric focusing gels and as a single band on SDS-polyacrylamide gels. The increased phosphorylation in DMD does not occur uniformly in all components of band II. Highly phosphorylated components of band II are present in DMD in which only minimal phosphorylation occurs in controls. These results suggest the presence of an abnormal band II substrate in erythrocyte membranes from DMD patients.

Vesicles with Transport Capability Isolated from Cultured Fibroblasts

Plasmalemmas from cultured human skin fibroblasts, isolated by a simple and reproducible method, can be converted to vesicles which are capable of active transport of aminoacid when glutathione is included within the vesicles. In the isolation, the plasmalemmas are stabilized with a Ricinus lectin, with preservation of the classic plasmalemma enzymes. The procedure has been applied successfully to a number of normal and abnormal human skin fibroblasts including those of myotonia dystrophica and progeria victims, and to the lung fibroblast WI-38. As part of a study of the characteristics of transport enzymes related to aging and to the muscular dystrophies in cultured fibroblasts, it was desirable to simplify the system by the use of vesicles prepared from the fibroblast plasmalemmas. The procedure described below is similar in some respects to that applied to another membranous system in the use of a lectin to stabilize the plasmalemma structure. The use of other stabilizing agents such as heavy metals and surfactant polymers which react with the membranes, but could compromise the reliability of the enzyme assays, was avoided. Since the focus of this study was on the enzymic systems of transport, the examination of facilitated diffusion or exchange was excluded. The well defined glutathione-dependent mechanism of aminoacid transport was examined to verify the competence of the vesicles for active transport and to confirm their sidedness. Other enzymes of transport, the ATPases, and membrane marker enzymes were also determined.

Genetic linkage studies of chromosome 17 RFLPs in von Recklinghausen neurofibromatosis (NF1)

Recent localization of the gene for von Recklinghausen neurofibromatosis (NF1) to chromosome 17 has led to studies to identify additional tightly linked probes that can be used in defining the primary genetic defect in NF1. We have examined and obtained blood for DNA linkage studies on over 250 individuals from 10 multigeneration neurofibromatosis families. We have analyzed 130 members in 7 families with the available chromosome 17 NF1 linked probes, pE51, D17S71, and D17Z1, as well as two probes generated from our own chromosome 17/19 enriched library (LDR92, LDR152A). Tight linkage was found between NF1 and the centromeric probe D17Z1 (theta = 0.04) and between NF1 and D17S71 (theta = 0.08). A definite recombinant was seen for the D17Z1 marker, which previously had not exhibited crossingover. Chromosome 17 DNA markers pE51, LDR92, and LDR152A gave slightly positive scores, which were not statistically significant.

Localization of Cloned Unique DNA to Three Different Regions of Chromosome 19: Screen for Linkage Probes for Myotonic Dystrophy

Screening polymorphic DNA probes for linkage to myotonic dystrophy (DM) and to other reported chromosome 19 (CH19) genes will develop a linkage map for human CH19. We report here the assignment of 3 cloned unique DNA sequences to 3 distinct regions of CH19. The novel use of 35S-labeled probes facilitated the rapid localization of the gene for the third complement factor (C3) to 19p13.2 by in situ hybridization. Metaphase chromosomes were from normal peripheral lymphocytes as well as from a fibroblast line containing a 15;19 translocation which permitted clear identification of CH19 regions of localization. Two random clones isolated from a plasmid library of human F-group enriched chromosomal DNA (D19S5 and D19S6) were in like manner assigned to 19p1.2 and 19q13.2 to 19qter, respectively.

Identification of abnormally [32P]‐phosphorylated cyanogen bromide cleavage product of erythrocyte membrane spectrin in Duchenne muscular dystrophy

We measured [32P]-phosphorylation of erythrocyte membrane spectrin band 2 peptides from patients with Duchenne muscular dystrophy. Erythrocyte ghosts were prepared and subjected to [32P]-phosphorylation by endogenous protein kinase incubations. Purified spectrin was then cleaved by cyanogen bromide, and the resulting peptides were analyzed by electrophoresis on 5%/15% SDS polyacrylamide stacking slab and tube gel systems. More than 50% of the incorporated [32P] was associated with a single band, CN-A, with an apparent molecular weight of 23 kilodaltons. The Coomassie blue-stained peptides were identical in patients and controls. Band CN-A represented approximately 2% of the total peptide protein but was more [32P]-phosphorylated in patients than in controls.

Dystrophin mRNA Processing in the Canine Homologue of Duchenne Muscular Dystrophy: An Authentic Model for Gene Therapy

Duchenne muscular dystrophy (DMD) is a fatal, X-linked, recessive disease of humans that afflicts 1 in 3500 live-born males (Koenig et al. 1988). Approximately two thirds of DMD patients carry detectable deletions in the gene encoding dystrophin (Walker et al. 1989; Koenig et al. 1987), which is a muscle cytoskeletal protein (Koenig et al. 1988). Dystrophin protein (Hoffmann et al. 1988) and transcript (Oronzi Scott et al. 1988) are either absent or severely deficient in DMD patients (Koenig et al. 1988).