The function of microglia associated with beta-amyloid deposits still remains a controversial issue. On the basis of recent ultrastructural data, microglia were postulated to be cells that form amyloid fibrils, not phagocytes that remove amyloid deposits. In this electron microscopic study, we examined the ability of microglia to ingest and digest exogenous amyloid fibrils in vitro. We demonstrate that amyloid fibrils are ingested by cultured microglial cells and collected and stored in phagosomes. The ingested, nondegraded amyloid remains within phagosomes for up to 20 days, suggesting a very limited effectiveness of microglia in degrading beta-amyloid fibrils. On the other hand, we showed that in microglial cells of classical plaques in brain cortex of patients with Alzheimer's disease, amyloid fibrils appear first in altered endoplasmic reticulum and deep infoldings of cell membranes. These differences in intracellular distribution of amyloid fibrils in microglial cells support our observations that microglial cells associated with amyloid plaques are engaged in production of amyloid, but not in phagocytosis.

Human brain short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) was found to catalyze the oxidation of 17␤-estradiol and dihydroandrosterone as well as alcohols. Mitochondria have been demonstrated to be the proper location of this NAD ؉ -dependent dehydrogenase in cells, although its primary structure is identical to an amyloid ␤-peptide binding protein reportedly associated with the endoplasmic reticulum (ERAB). This fatty acid ␤-oxidation enzyme was identified as a novel 17␤-hydroxysteroid dehydrogenase responsible for the inactivation of sex steroid hormones. The catalytic rate constant of the purified enzyme was estimated to be 0.66 min ؊1 with apparent K m values of 43 and 50 M for 17␤-estradiol and NAD ؉ , respectively. The catalytic efficiency of this enzyme for the oxidation of 17␤-estradiol was comparable with that of peroxisomal 17␤-hydroxysteroid dehydrogenase type 4. As a result, the human SCHAD gene product, a single-domain multifunctional enzyme, appears to function in two different pathways of lipid metabolism. Because the catalytic functions of human brain short chain L-3-hydroxyacyl-CoA dehydrogenase could weaken the protective effects of estrogen and generate aldehydes in neurons, it is proposed that a high concentration of this enzyme in brain is a potential risk factor for Alzheimer's disease.

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The tissue distribution, subcellular localization, and metabolic functions of human 17b-hydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase have been investigated. Human liver and gonads are abundant in this enzyme, but it is present in only negligible amounts in skeletal muscle. Its N-terminal sequence is a mitochondrial targeting sequence, but is not required for directing this protein to mitochondria. Immunocytochemical studies demonstrate that this protein, which has been referred to as ER-associated amyloid b-binding protein (ERAB), is not detectable in the ER of normal tissues. We have established that protocols employed to investigate the subcellular distribution of ERAB yield ER fractions rich in mitochondria. Mitochondria-associated membrane fractions believed to be ER fractions were employed in ERAB/Abbinding alcohol dehydrogenase studies. The present studies establish that in normal tissues this protein is located in mitochondria. This feature distinguishes it from all known 17b-hydroxysteroid dehydrogenases, and endows mitochondria with the capability of modulating intracellular levels of the active forms of sex steroids.

PrP 27-30, a unique protease-resistant protein associated with scrapie infectivity, derives from the proteolytic cleavage of a larger precursor encoded by a host gene. To identify sites of PrP biosynthesis, in situ hybridization was done using cloned PrP cDNA as a probe. In rodent brain, PrP mRNA was expressed in neurons, ependymal cells, choroid plexus epithelium, astrocytes, pericytes, endothelial cells and meninges of both scrapie-infected and uninfected animals. PrP mRNA was also detected in vitro in isolated brain microglia cells. Pulmonary cells and heart muscle cells contained high levels of this mRNA. Hybridization was not detected in spleen, confirming earlier RNA blot experiments indicating extremely low levels of PrP mRNA in this tissue. Results indicate that PrP mRNA is a normal component in a variety of non-neuronal tissues and may explain the origin of the amyloid plaques present in the subependymal region of scrapie-infected brain.

Topical microbicides are potentially an alternative method to vaccines for reducing the spread of herpes simplex virus (HSV). We have previously shown (S. Liu et al., Biochim. Biophys. Acta 1723:270-281, 2005) that the catechin (؊)-epigallocatechin gallate (EGCG) inactivates HSV at neutral pH; however, to function in the female genital tract EGCG must also be effective at acidic pH. EGCG inactivated HSV-1 and HSV-2 at pH 8.0 by 3 log 10 to 4 log 10 but was ineffective at pH 5.7. The EGCG digallate dimers theasinensin A, P2, and theaflavin-3,3-digallate (TF-3) inactivated both viruses by 3 log 10 to 4 log 10 at pH 5.7 and as much as 5 log 10 at pH 8.0. TF-3 inactivated HSV-1 and HSV-2 by 4 to 5 log 10 in the pH range of 4.0 to 5.7. Dimers with one gallate moiety had antiviral activity intermediate between the activities of EGCG and digallate dimers. Confocal and electron microscopy showed that theasinensin A did not damage Vero cells. All EGCG dimers inactivated enveloped viruses with class I, class II, and class III (HSV-1, HSV-2) fusion proteins more effectively than did monomeric EGCG. EGCG had no activity against the nonenveloped viruses tested, but TF-3 reduced the titer of 4 of 5 nonenveloped viruses by Х2 to 3.5 log 10 . Results also showed that HSV-1 glycoprotein B (gB) was aggregated more rapidly by theasinensin A than EGCG, which, when taken together with the nonenveloped virus data, suggests that dimers may inhibit the function of viral proteins required for infectivity. Digallate dimers of EGCG appear to have excellent potential as microbicidal agents against HSV at acidic and neutral pHs.Herpes simplex virus (HSV) is the leading cause of genital ulcers in the developed world (16). In the United States alone 40 to 60 million people are infected with HSV-2 (23). In some cities in North America and Europe, HSV-1, which is usually found orally, causes more than half of the new cases of genital herpes, particularly in women (16).In the absence of a fully effective HSV vaccine (1), topical microbicides represent an important potential strategy for preventing the sexual and mother-to-child spread of HSV. It has been shown for both HSV (33) and human immunodeficiency virus (HIV) (20) that the viral load in genital secretions is the chief predictor of viral transmission, suggesting that if a microbicide reduces the concentration of excreted HSV, the incidence of viral transmission will be reduced.(Ϫ)-Epigallocatechin gallate (EGCG), the primary catechin in green tea (5), has been previously shown in our laboratory to inactivate multiple clinical isolates of HSV-1 and HSV-2 (12). These studies were carried out at neutral pH. While efficacy at neutral pH would be fine for an anti-HSV compound utilized in the eye (14), a topical microbicide utilized vaginally (27) or on the skin (30) to reduce HSV transmission must function across a pH range that is more acidic than neutral. Vaginal pH will vary over a range of 3.9 to 5.7 (21) and then become more neutral in the presence of semen, rising to pH 6 to 7 (27). The p...

The fraction of cells capable of division was determined for 1) population of the human diploid cell strains, W138 after different numbers of subcultivations in uitro and 2 ) a single population of W138 cells at intervals throughout its entire in vitro lifespan. In both cases the percentage of cells capable of division decreased with increasing age in tissue culture. The rate and the magnitude of the decrease is sufficient to account for the limited in uitro lifespan reported by other investigators.Furthermore, the decrease in the fraction of cells capable of division in similar in some respects of senescence among human populations.After a period of active growth lasting several months, human diploid fibroblasts require a n increasingly longer time in which to become confluent monolayers. Ultimately, the cultures can no longer be maintained in tissue culture and are lost. This progressive decline has been interpreted as an expression of senescene at the cellular level (Hayflick, '65). In the studies reported here, we found that the fraction of nondividing cells in WI-38 culture increased throughout the entire lifespan of the culture in vitro, eventually reaching a level high enough to account for the loss of the culture. MATERIALS AND METHODSMonolayer cultures of WI-38 cells were prepared as previously described (Merz and Ross, '69).For subcultivation, cells were released from confluent monolayer cultures with 0.25% trypsin in Eagle's basal medium and aspirated to disperse clumps. The cell yield was divided into four parts, each of which was used to inoculate a new culture. The cultures were then incubated at 37°C in a n atmosphere of 5% CO, and 95% air until they reached confluency six to seven days later.The fraction of nondividing cells was determined, using suspensions of cells pre- equilibrated with a n atmosphere of 5% COz and 95% air. The dishes were incubated several hours to allow the cells to attach and flatten. The location of each single cell was then recorded, using a grid of 2-mm squares printed on the bottom of the dish as a reference. At 24-hour intervals thereafter, the number of cells at each location was recorded. RESULTSThe number of mapped cells that had not yet divided was recorded daily for seven days. Figure 1 shows the results obtained with three separate cultures of WI-38 cells at the fifteenth, thirty-second and forty-second passages, respectively. After a lag of approximately half a day, the number of single cells remaining undivided decreased for four to five days. Thereafter, there was no further division among the remaining single cells. It is these cells that constitute the nondividing fraction of the population. The percentage of nondividing cells increased with the passage level of the culture. The absence of division among these cells was not a reflection of a general decrease in the growth rate for all cells, since the remainder of the population continued to increase exponentially, even after division among the single cells had ceased (Merz and Ross, '69).

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