INTRANUCLEAR AGGREGATES OF FERRITIN IN LIVER CELLS OF MICE TREATED WITH SACCHARATED IRON OXIDE : Their Possible Relation to Nuclear Protein Synthesis

Several months following parenteral injections of saccharated iron oxide into DBA/2J mice, granules rich in iron were found in nuclei of scattered parenchymal liver cells as well as in the cytoplasm. As seen in the light microscope, the intranuclear granules were brown; most of them measured between 0.5 µ and 1 µ in cross-section. They gave positive Prussian blue tests, and were not selectively stainable with pyronine. Electron micrographs of the granules showed closely packed aggregates of ferritin molecules, occasionally in paracrystalline order. The intranuclear collections were often surrounded by bands of material of moderate opacity. Scattered ferritin molecules and collections of such molecules were also present in the cytoplasm of many liver cells, but there seemed to be no quantitative relationship between intranuclear and cytoplasmic ferritin. Liver cells from untreated control mice failed to reveal intranuclear deposits of ferritin. Although the site of origin of the intranuclear aggregates of ferritin is unknown, the findings suggest the possibility that under suitable circumstances ferritin synthesis may take place within nuclei of liver cells—perhaps induced by the presence of colloidal iron.     

Mast cell restricted mouse and human tryptase·heparin complexes hinder thrombin-induced coagulation of plasma and the generation of fibrin by proteolytically destroying fibrinogen

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-β. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-β· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.     

Electron cytochemical analysis ofChlamydomonas polysaccharides

Conventionally prepared thin sections ofChlamydomonas reinhardtii grown photosynthetically or grown on acetate in the dark showed a pyrenoid surrounded by starch plates located centrally within the chloroplast. Light-grown cells contained few additional granules, whereas dark-grown cells had numerous large deposits. The PATAg technique of Thiéry was used to study the chemical nature of these deposits. Dense silver deposition was observed over the granules and plates in both types of cells; dark-grown cells had numerous, large areas of PATAg-reactive material. Other regions of the cells did not have reactive material. Enzymatic digestion with amylases showed that most, but not all, of the PATAg-reactive material was starch. Several small, granular areas remained in both light and dark-grown cells, even after extensive digestion. These results show that growth conditions affect patterns of starch storage and thatChlamydomonas has amylase-insensitive carbohydrate-containing material as well.     

New insights into early sequential PrPsc accumulation in scrapie infected mouse brain evidenced by the use of streptomycin sulfate

To investigate the amplifying potentialities of streptomycin sulfate in the immunohistochemical (IHC) detection of the abnormal prion protein (PrPsc), we used a sequential brain sampling from C506M3 scrapie strain inoculated C57Bl/6 mice. The weekly removed brains, from 7 to 63 days post intra-cranial inoculation were analysed using PrPsc IHC. The introduction of streptomycin sulfate, a technique developed for accurate cellular and regional mapping of PrPsc deposition in several animal TSEs, revealed a substantial amplifying effect and a clear specific PrPsc detection as early as 28 days post inoculation. The location of the first detected PrPsc deposits suggests a possible involvement of the cerebrospinal fluid in the early dissemination of the infectious agent. The meaning of these newly accessible PrPsc deposits is discussed in relation to a possible nascent form of PrPsc molecules detected in situ for the first time. Altogether, these findings argue that this method can be highly useful to study the early stages after infection with prion agents.     

The receptor for mouse hepatitis virus in the resistant mouse strain SJL is functional: implications for the requirement of a second factor for viral infection.

The SJL mouse strain is resistant to infection by some strains of the murine coronavirus mouse hepatitis virus (MHV), such as JHM and A59. The block to virus infection has been variously attributed to defects in virus receptors or virus spread. Since the cellular receptors for MHV, mmCGM1 and mmCGM2, have recently been identified as members of the carcinoembryonic antigen family, we reexamined the possible defectiveness of the MHV receptors in SJL mouse strain. Cloning and sequencing of the cDNAs of both mmCGMs RNAs from SJL mice revealed that they were identical in size to those of the susceptible C57BL/6 (B6) mouse. There was some sequence divergence in the N terminus of the mmCGM molecules between the two mouse strains, resulting in a different number of potential glycosylation sites. This was confirmed by in vitro translation of the mmCGM RNAs, which showed that the glycosylated mmCGM2 of SJL was smaller than that of B6 mice. However, transfection of either mmCGM1 or mmCGM2 from SJL mice into MHV-resistant Cos 7 cells rendered the cells susceptible to MHV infection. The ability of the SJL mmCGM molecules to serve as MHV receptors was comparable to that of those from B6. These molecules are expressed in SJL mouse brain and liver in a similar ratio and in amounts equivalent to those in the B6 mouse. Furthermore, we demonstrated that an SJL-derived cell line was susceptible to A59 but resistant to JHM infection. We concluded that the MHV receptor molecules in the SJL mouse are functional and that the resistance of SJL mice to infection by some MHV strains most likely results from some other factor(s) required for virus entry or some other step(s) in virus replication.     

Localization of selenium deposits in meristematic cells of Allium sativum L. roots treated with selenium salts

Ultrastructural analysis of garlic roots treated for 24 h with sodium selenate or sodium selenite at the concentrations 80, 160, 320 microM revealed the presence of selenium deposits in meristematic cells. They appeared as small and large granules or aggregates of electron-dense material. Many small granules were localised in plastids but some in mitochondria, endoplasmic reticulum as well as in Golgi apparatus, nucleus and cytoplasm. Sometimes the large granules were seen in cytoplasm but aggregates of electron-dense material only in vacuoles. It seems possible that these deposits represent a non-dissolved form of selenium, i.e. elemental selenium or its complexes with other ions.     

Mouse senile amyloidosis. ASSAM amyloidosis in mice presents universally as a systemic age-associated amyloidosis.

Amyloid deposition in 11 inbred strains of mice (A/J, SJL/J, DDD, C57BL/6J, B10.BR, C57BL/10, B10A/SgSn, C3H/HeMs, B10A(5R), DBA/2 and C57BL/6Cr5/c) was studied using the peroxidase antiperoxidase (PAP) method and antisera against ASSAM and murine protein AA. Among the 170 mice examined, in 77 (45.3%) from the nine strains other than C3H/HeMs and DBA/2, there was evidence of spontaneous amyloid deposits in routine histological sections. Immunohistochemical studies using 54 mice with amyloid deposition, demonstrated ASSAM deposition in 45 mice (83.3%) in all nine strains, although the incidence and intensity of the deposition differed somewhat between strains. SJL/J and A/J had ASSAM deposits from the age of 8 months and the incidence increased with advancing age. In the other seven strains, ASSAM was first deposited at an older age than in the SJL/J and A/J strains. In A J, C57BL/6J, C57BL/10, B10.BR, B10A(5R) and C57BL/6Cr5/c, protein AA often coexisted with ASSAM. The distribution pattern of the ASSAM deposits was similar to that observed among the SAM strains. Thus, ASSAM is an ubiquitously distributed senile amyloid protein in the mouse. Determination of the molecular type of apoA-II, a serum precursor of ASSAM, among all 11 strains using the polymerase chain reaction (PCR) revealed the SAM-P/1 type apoA-II variant in SJL/J and A/J strains with a high susceptibility to ASSAM deposition. We concluded from this study that amino acid substitution in precursor apoA-II may be responsible for the early onset and severe amyloid deposition in the mouse.     

Inclusion bodies in pinealocytes of the cotton rat (Sigmodon hispidus)

Pinealocytes of the cotton rat (Sigmodon hispidus) often contain large (2-6 micron diameter) intracytoplasmic inclusions, the function of which is not known. These inclusions may represent nucleolus-like bodies, mineral deposits, secretory products or viral inclusions. In this study these inclusions were classified as type A, B or C inclusions based on the amount of electron-dense material interspersed within the finely granular material comprising the bulk of these inclusions. Each type of inclusion was analyzed by X-ray microanalysis and enzymatic proteinaceous digestion. X-ray microanalysis of these inclusions differed both quantitatively and semiquantitatively from that of human or gerbil pineal concretions, the latter two of which are extracellular deposits. Pronase, a proteolytic enzyme, digested the electron-dense material only after longer times of tissue exposure to this enzyme in contrast to the easily digested, finely granular matrix-like material of these inclusions. Such intrapinealocytic inclusions have only been observed in the cotton rat. Their functional significance remains unknown.     

Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimer's disease

To better understand amyloid-beta (Abeta) metabolism in vivo, we assessed the concentration of Abeta in the CSF and plasma of APP(V717F) (PDAPP) transgenic mice, a model that develops age-dependent Alzheimer's disease (AD)-like pathology. In 3-month-old mice, prior to the development of Abeta deposition in the brain, there was a highly significant correlation between Abeta levels in CSF and plasma. In 9-month-old-mice, an age at which some but not all mice have developed Abeta deposition, there was also a significant correlation between CSF and plasma Abeta; however, the correlation was not as strong as that present in young mice. In further exploring CSF and plasma Abeta levels in 9-month-old mice, levels of CSF Abeta were found to correlate highly with Abeta burden. Analysis of the CSF: plasma Abeta ratio revealed a selective two-fold increase in plaque versus non-plaque bearing mice, strongly suggesting a plaque-mediated sequestration of soluble Abeta in brain. Interestingly, in 9-month-old mice, a significant correlation between CNS and plasma Abeta was limited to mice lacking Abeta deposition. These findings suggest that there is a dynamic equilibrium between CNS and plasma Abeta, and that plaques create a new equilibrium because soluble CNS Abeta not only enters the plasma but also deposits onto amyloid plaques in the CNS.     

beta-Amyloid peptide vaccination results in marked changes in serum and brain Abeta levels in APPswe/PS1DeltaE9 mice, as detected by SELDI-TOF-based ProteinChip technology.

Although the pathogenesis of Alzheimer's disease (AD) is not fully understood, growing evidence indicates that the deposition of beta-amyloid (Abeta) and the local reactions of various cell types to this protein play major roles in the development of the disease. Immunization with the Abeta 1-42 peptide has been reported to decrease Abeta deposits in the brains of mutant amyloid precursor protein (APP/V717F) transgenic (tg) mice (Schenk et al. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 1999;400:173-177). We have replicated this finding in APPswe/PS1DeltaE9 tg mice, which also develop Abeta deposits in the brain. The immunized animals developed high titers of antibodies against Abeta 1-42 in serum, and Abeta deposits in the brains were significantly reduced. Using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry and ProteinChip((R)) technology, we detected trends toward increased soluble Abeta peptide in the brain and a decrease in assayable Abeta peptide in the serum of immunized compared with control animals. This last finding raises the possibility that anti-Abeta antibodies in the periphery sequester Abeta peptides or target them for degradation and in this way contribute to the enhanced Abeta clearance from the brain in immunized animals.     

Seeded strain‐like transmission of β‐amyloid morphotypes in APP transgenic mice

The polymorphic β‐amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral β‐amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop β‐amyloid depositions that differ in morphology, binding of amyloid conformation‐sensitive dyes, and Aβ40/Aβ42 peptide ratio. To determine the nature of such β‐amyloid morphotypes, β‐amyloid‐containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced β‐amyloid deposition with the morphological, conformational, and Aβ40/Aβ42 ratio characteristics of β‐amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced β‐amyloid deposits with the characteristics of β‐amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced β‐amyloid deposits, although less prominent, and the induced deposits were similar to the β‐amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated Aβ in APP transgenic mice can be maintained by seeded conversion.     

The role of platelet factor 4 in local and remote tissue damage in a mouse model of mesenteric ischemia/reperfusion injury

The robust inflammatory response that occurs during ischemia reperfusion (IR) injury recruits factors from both the innate and adaptive immune systems. However the contribution of platelets and their products such as Platelet Factor 4 (PF4; CXCL4), during the pathogenesis of IR injury has not been thoroughly investigated. We show that a deficiency in PF4 protects mice from local and remote tissue damage after 30 minutes of mesenteric ischemia and 3 hours of reperfusion in PF4-/- mice compared to control B6 mice. This protection was independent from Ig or complement deposition in the tissues. However, neutrophil and monocyte infiltration were decreased in the lungs of PF4-/- mice compared with B6 control mice. Platelet-depleted B6 mice transfused with platelets from PF4-/- mice displayed reduced tissue damage compared with controls. In contrast, transfusion of B6 platelets into platelet depleted PF4-/- mice reconstituted damage in both intestine and lung tissues. We also show that PF4 may modulate the release of IgA. Interestingly, we show that PF4 expression on intestinal epithelial cells is increased after IR at both the mRNA and protein levels. In conclusion, these findings demonstrate that may PF4 represent an important mediator of local and remote tissue damage.     

Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA₂β), which is encoded by the PLA2G6 gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA₂β-knockout (KO) mice as well as in PLA2G6-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA₂β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA₂β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA₂β-KO mice. DMT1 and IRPs were significantly upregulated in PLA2G6-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in PLA2G6-KD cells. These results suggest that the genetic ablation of iPLA₂β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction.     

Aberrant immune responses in a mouse with behavioral disorders

BTBR T+tf/J (BTBR) mice have recently been reported to have behaviors that resemble those of autistic individuals, in that this strain has impairments in social interactions and a restricted repetitive and stereotyped pattern of behaviors. Since immune responses, including autoimmune responses, are known to affect behavior, and individuals with autism have aberrant immune activities, we evaluated the immune system of BTBR mice, and compared their immunity and degree of neuroinflammation with that of C57BL/6 (B6) mice, a highly social control strain, and with F1 offspring. Mice were assessed at postnatal day (pnd) 21 and after behavioral analysis at pnd70. BTBR mice had significantly higher amounts of serum IgG and IgE, of IgG anti-brain antibodies (Abs), and of IgG and IgE deposited in the brain, elevated expression of cytokines, especially IL-33 IL-18, and IL-1β in the brain, and an increased proportion of MHC class II-expressing microglia compared to B6 mice. The F1 mice had intermediate levels of Abs and cytokines as well as social activity. The high Ab levels of BTBR mice are in agreement with their increased numbers of CD40(hi)/I-A(hi) B cells and IgG-secreting B cells. Upon immunization with KLH, the BTBR mice produced 2-3 times more anti-KLH Abs than B6 mice. In contrast to humoral immunity, BTBR mice are significantly more susceptible to listeriosis than B6 or BALB/c mice. The Th2-like immune profile of the BTBR mice and their constitutive neuroinflammation suggests that an autoimmune profile is implicated in their aberrant behaviors, as has been suggested for some humans with autism.     

Association among Amyloid Plaque,Lipid, and Creatine in Hippocampus of TgCRND8 Mouse Model for Alzheimer Disease

Amyloid peptide (Aβ) aggregation in the brain is a characteristic feature of Alzheimer disease (AD). Previously, we reported the discovery of focally elevated creatine deposits in brain tissue from TgCRND8 mice, which express double mutant (K670N/M671L and V717F) amyloid protein precursor. In this study, frozen hippocampal tissue sections from 5-, 8-, 11-, 14-, and 17-month old TgCRND8 and littermate control mice were examined with Fourier transform infrared microspectroscopy to explore the distribution of lipid, creatine, and dense core plaque deposits. Lipid distribution throughout the hippocampus was similar in transgenic (Tg) and non-Tg littermates at all ages. Dense core plaques were always found to lie within a thin (30–50 μm) lipid envelope, confirmed by imaging through serial sections. Creatine deposits were found in all TgCRND8 mice; the extent of deposition increased with age. Minor creatine deposits appeared in the oldest littermate controls. Distribution in the serial sections showed moderate correlation between layers, slightly disturbed by the freeze/thaw process. Creatine deposits in Tg mice were not specifically co-localized with plaques or lipid halos. The dimension of the lipid envelope is comparable with that of the diffuse halo of nonaggregated amyloid, implying a dynamic association in vivo, postulated to have a significant role in the evolving neurotoxicity.     

Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease

Alzheimer's disease (AD) may result from the accumulation of amyloid-beta (Abeta) peptides in the brain. The cysteine protease cathepsin B (CatB) is associated with amyloid plaques in AD brains and has been suspected to increase Abeta production. Here, we demonstrate that CatB actually reduces levels of Abeta peptides, especially the aggregation-prone species Abeta1-42, through proteolytic cleavage. Genetic inactivation of CatB in mice with neuronal expression of familial AD-mutant human amyloid precursor protein (hAPP) increased the relative abundance of Abeta1-42, worsening plaque deposition and other AD-related pathologies. Lentivirus-mediated expression of CatB in aged hAPP mice reduced preexisting amyloid deposits, even thioflavin S-positive plaques. Under cell-free conditions, CatB effectively cleaved Abeta1-42, generating C-terminally truncated Abeta peptides that are less amyloidogenic. Thus, CatB likely fulfills antiamyloidogenic and neuroprotective functions. Insufficient CatB activity might promote AD; increasing CatB activity could counteract the neuropathology of this disease.     

Proenkephalin: A general pathway for enkephalin biosynthesis in animal tissues

Guinea pig adrenal, brain, and myenteric plexus have been shown to contain many polypeptides that yield free enkephalins on digestion with trypsin and carboxypeptidase B. The enkephalin-containing polypeptides (ECPs) range from 500 to >20,000 daltons and show similarities in their chromatographic behavior to the ECPs present in the chromaffin granules of the bovine adrenal medulla. Furthermore, the heptapeptide [Met]enkephalin-Arg⁶-Phe⁷, that is now known to represent the carboxyl terminal sequence of the proenkephalin found in bovine adrenal medulla (Gübler et al. (1982) Nature (London), in press), was identified in all three guinea pig tissues. It appears that processing of a proenkephalin similar to the one in adrenal medulla represents a general pathway for enkephalin biosynthesis in animal tissues.     

Interaction of collagen with hydrophobic protein granules in the egg capsule of the dogfish scyliorhinus canicula

The egg capsule of the dogfish is a composite material containing collagenous fibrils and 2 mum spherical hydrophobic protein granules. The latter appear to owe much of their hydrophobicity to an exceptionally high tyrosine content (approximately 20% of total amino acid residues). The hydrophobic component appears to form as an emulsion in the secretory granules of the D and E zone gland cells of the nidamental gland. Droplets of the hydrophobic material appear to become coated with remarkably regular layers of radially-arranged collagen molecules which form a series of concentric, evenly spaced layers around each hydrophobic granule. Numerous disclinations were seen where the layers around adjacent granules interfered with one another. The layers are thought to represent a lamellar liquid crystalline phase previously described for this collagen (Knight et al., 1993). The fine structural appearance of the concentric layers and evidence for radial arrangement of collagen molecules within them is compatible with the suggestion that the layers are built from a dumbbell-shaped unit approximately 35 nm long with hydrophobic groups concentrated at the ends. This unit may represent a dumbbell-shaped molecule or an oligomer of two or more molecules lying parallel with one another in a head-to-tail arrangement. Such a unit can be readily incorporated into models for the micellar, hexagonal columnar and final fibrillar phases previously described for this collagen (Knight et al., 1993). Evidence from the TEM study of stretched egg capsule wall suggests that there is a mechanical interaction between the hydrophobic granules and the collagen fibrils in the fully formed material. We suggest that the radial, concentric layered arrangement of collagen molecules is established by hydrophobic interactions within the liquid crystalline material and locked into place by oxidative covalent cross-linking to give a 3-dimensional cross-linked meshwork of collagen fibrils and hydrophobic granules. The latter arrangement helps to account for the high tensilestrength and toughness of this material.     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.