Interim Report of the Committee on Histologic Mounting Media: Resinous Media

The Fisher “Permount” naphthalene polymer, the Hartman Leddon “H.S.R.” terpene polymer resin, a Monsanto polystyrene P-1, the Will Corporation “Diaphane” and “Green Diaphane”, and the du Pont “Lucite” methyl methacrylate polymer were examined, and the possibility of use of some other plastics was also explored. The first 5 mentioned were tested for color preservation of a variety of stains in comparison with Canada balsam and Clarite X. From this point of view polystyrene, the Hartman Leddon “H.S.R.” and the Fisher “Permount” resins were the most satisfactory, then the “Diaphanes”. Both “Permount” and “H.S.R.” show some yellowing. The H.S.R. with a melting point of 115°C, the Permount with 150°C. melting point, and the Polystyrene with a thermal denaturation point above 220°C. all excell Canada balsam in heat resistance. Trimethylbenzene, cymene and monoamylbenzene appear to be the best solvents for polystyrene. Mounts made in a solution of 20 g. polystyrene in 100 ml. trimethylbenzene can be packed flat slide to slide in 24 hours after mounting without sticking together.

This report is not intended to deprecate the use of other resinous mounting media which have not as yet been tested or compared with those mentioned herein.     

Degradation of Dynorphin-Related Peptides by the Puromycin-Sensitive Aminopeptidase and Aminopeptidase M

Abstract: The degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M was examined using these peptides as alternate substrate inhibitors. K _i determinations showed that both aminopeptidases exhibit a higher affinity for longer dynorphin-related peptides, i.e., K _i for dynorphin A-17 = 23–30 n M with the K _i increasing to 25–50 µ M for the enkephalin pentapeptides. Binding appears dependent not only on peptide length, but also on its sequence. With aminopeptidase M, as the peptide size increases from five to 10 amino acids, k _cat remains relatively constant; however, as the peptide size increases beyond a decapeptide, k _cat decreases significantly. With the puromycin-sensitive aminopeptidase, similar results were obtained except that k _cat was greatest for the pentapeptide. Thus, if one considers k _cat/ K _m as the relevant kinetic constant for estimating in vivo peptide hydrolysis, these results are consistent with the involvement of aminopeptidase M and the puromycin-sensitive aminopeptidase in the degradation of extended dynorphin-related peptides.     

Cellular Localization of Latent Murine Cytomegalovirus

Herpesviruses typically establish latent infection in their hosts. The cell(s) responsible for harboring latent virus, in most cases, is not known. Using immunofluorescence and PCR-in situ hybridization (PISH), a technique which combines the sensitivity of PCR with the localization and specificity of in situ hybridization, we provide the first direct evidence that endothelial cells are a major site of murine cytomegalovirus (MCMV) DNA in latently infected animals. These findings are consistent with existing knowledge of the biological behavior of CMV, in particular the transmission of latent CMV by solid organ and bone marrow transplantation, in both human and animal models. In addition, we have localized MCMV DNA in the lung alveolar macrophage and in bone marrow cells. Our findings confirm that bone marrow-derived hematopoietic cells are a site of CMV latency and further suggest that bone marrow may be a reservoir of infected progeny capable of migrating into the circulation and establishing latency in various tissues. These findings provide clearly needed insight into the site of latent infection which is central to an understanding of the mechanisms of reactivation.     

Preparation of Copolymers of Isobutyl Methacrylate and Styrene for Mounting Media

Details are given for the preparation of low molecular weight copolymers of isobutyl methacrylate and styrene for use in mounting microscopical sections between the slide and cover glass. Any refractive index in the range from 1.477 to 1.590 is available, depending upon the proportions of monomers used. A refractive index of 1.550 is recommended for general purposes, though other indices might be desirable in special cases including phase microscopy. The resin gives solutions in toluene of suitable fluidity at usable concentrations, adheres satisfactorily to glass, and has excellent light-transmission. The features which make it especially valuable are its optimum refractive index and its permanent water-whiteness.

Copolymers of isobutyl methacrylate and styrene may also be used to prepare plastic mounts of thick sections or gross specimens, which are optically cleared by the plastic mountant.     

Neuronal Localization of Pyroglutamate Aminopeptidase II in Primary Cultures of Fetal Mouse Brain

Pyroglutamate aminopeptidase II is a highly specific membrane-bound ectopeptidase proposed to inactivate thyrotropin releasing hormone (TRH) in brain extracellular space. Its activity was measured in primary cell cultures of fetal brain in an attempt to define its cellular localization. Enzyme activity was detected in hypothalamic or cortical cell membrane fractions from 4- to 12-day-old cultures. When proliferation of nonneuronal cells was abolished by cytosine arabinoside treatment, pyroglutamate aminopeptidase II specific activity was increased as compared to untreated cultures, the opposite was observed for pyroglutamate amino-peptidase I activity. Treatment of cortical cells with the neurotoxic agent glutamate reduced simultaneously pyroglutamate aminopeptidase II and glutamate decarboxylase activities. Glial cell cultures expressed pyroglutamate aminopeptidase I or glutamate synthase activities but not pyroglutamate aminopeptidase II. The data suggest that pyroglutamate aminopeptidase II is predominantly localized in neuronal cells. This is consistent with a role for pyroglutamate aminopeptidase II in TRH-ergic synaptic transmission.     

基于元胞自动机的生物性病原物类疾病传播模型

生物性病原物类疾病是单一植物群落中常见的一种传染病,它主要依靠风、雨和昆虫等外力传播病菌,传播过程中具有一定的潜伏性,元胞自动机是一种在时间和空间上离散,以每个个体状态的同步更新为前提来讨论系统整体性质的模型,本文利用其特点,将整个森林中的树木分为健康、潜伏期、发病期三类,以树木的营养状况和被感染时间长度为辅助变量,在考虑到树木得病后被治愈的情况后,建立了基于元胞自动机的树林生物性病原物类疾病传播模型．模拟结果表明,在不同的初始条件下,得病树所占的比例由疾病传播和被治愈的相对强度确定．疾病每次传播时,从爆发到被控制的过程具有相似的特点：随着恢复强度从小到大,初始阶段,被感染的树木迅速增加,接着进入传播速度相对缓慢的稳定过程,最终如果恢复机制足够强,则树林中的不健康树木会全部消失．     

Cellular Localization of Acetyl-Coenzyme A Synthetase in Yeast

In cells of Saccharomyces cerevisiae grown with glucose in standing cultures, the microsomal fraction had the highest specific activity for acetyl-coenzyme A synthetase and contained the greatest fraction of the total activity regardless of when the cells were harvested during growth. The addition of acetate did not affect the distribution of the enzyme, nor did subsequent aeration of such cells in phosphate buffer even in the presence of glucose, acetate, or succinate. In cells grown aerobically, however, the microsomal fraction had the highest specific activity and the greatest fraction of the total activity only until the cells reached the stationary phase. After this time, most of the activity was associated with the mitochondrial fraction. Finally, 3 or 4 days after inoculation, this fraction appeared to lose most of the enzyme to the microsomal and soluble fractions. Chloramphenicol, at concentrations that interfered with respiration but not with fermentation, prevented the association of acetyl-coenzyme A synthetase with the mitochondrial fraction in aerated cells, but it did not appreciably affect the large increases in enzyme activity observed during aerobic incubation. Cells grown with glucose under strict anaerobic conditions contained barely detectable amounts of acetyl-coenzyme A synthetase.     

Release of Aspartate and Glutamate Caused by Chloride Reduction in Synaptosomal Incubation Media

Release of the amino acid neurotransmitter candidates aspartate and glutamate from synaptosomes can be stimulated by reduction of [Cl-] in the incubation medium by replacement with the impermeant anions propionate and isethionate. Replacement by the permeant anion Br- is without effect on amino acid release. This release offers a biochemical method of studying the Cl-ion channel in CNS membrane.     

A Cell-Intrinsic Interferon-like Response Links Replication Stress to Cellular Aging Caused by Progerin

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Influence of Mounting Media on the Fading of Basic Aniline Dyes in Epoxy Embedded Tissues

A simple cytophotometric technique is used to quantitate stain fading of basic aniline dye-stained epoxy-embedded tissues mounted in six different commonly used mountants. Significant fading was detected with all six mountants, although rates varied. the lowest rate of fading was observed with immersion oil and the highest rate of fading with Canada balsam. No significant differences in fading rates of four synthetic mounting preparations were observed.     

Production of Aminopeptidase and Carboxypeptidase by Streptomyces peptidofaciens

Streptomyces peptidofaciens KY 2389, a new species isolated from a soil sample, exhibited the highest potencies in production of both aminopeptidase and carboxypeptidase among about 1,300 strains tested.

Optimum pH values of both aminopeptidase and carboxypeptidase for Leu-β-naphthyl-amide and Cbz-Gly-Leu were 8.0. Aminopeptidase was thermostable and the activity was not lost by treatment at 70°C for 1 hr, in the presence of Ca²⁺. Carboxypeptidase was heat-labile and over 50% of the activity was lost by treatment at 60°C for 1 hr. Of the synthetic peptides tested, Leu-Gly-Gly and Cbz-Gly-Leu were the most suitable substrates for amino-peptidase and carboxypeptidase, respectively.     

Effects of Mounting Media on Fading of Toluidine Blue and Pyronin G Staining in Epoxy Sections

Available mounting media cause fading of histological preparations over time. A study was designed to find the most suitable medium for durable mounting of Araldite embedded semithin sections of rabbit cerebral cortex stained with toluidine blue and pyronin G. Among four synthetic mounting media tested, only DePeX prevented fading of the sections during the first month. All mounting media tested helped preserve staining intensity after one month, since the fading rate after one year is only about half that in sections prepared without mounting medium. The average optical density of sections after one year was higher in preparations mounted with DePeX than in sections treated with the other mounting techniques tested in this study. After one year, the average optical density of sections mounted with DePeX had decreased approximately 20%.     

Inhibition of a Membrane-Bound Enkephalin-Degrading Aminopeptidase by Bestatin Analogs

Abstract: A variety of bestatin analogs were examined as potent inhibitors of a membrane-bound enkephalin-degrading aminopeptidase that was purified from monkey brain. Bestatinyl amino acid derivatives showed strong inhibition of this enzyme. The most effective was bestatin- l -Arg AcOH, with a K _i value of 0.21 × 10⁻⁸ M with Leu-enkephalin as substrate. It exhibited competitive kinetics and was about 100-fold more potent than bestatin. This compound seems to be useful for pharmacological and other studies.     

The N-Terminal Region of IFITM3 Modulates Its Antiviral Activity by Regulating IFITM3 Cellular Localization

Interferon-inducible transmembrane (IFITM) protein family members IFITM1, -2, and -3 restrict the infection of multiple enveloped viruses. Significant enrichment of a minor IFITM3 allele was recently reported for patients who were hospitalized for seasonal and 2009 H1N1 pandemic flu. This IFITM3 allele lacks the region corresponding to the first amino-terminal 21 amino acids and is unable to inhibit influenza A virus. In this study, we found that deleting this 21-amino-acid region relocates IFITM3 from the endosomal compartments to the cell periphery. This finding likely underlies the lost inhibition of influenza A virus that completes its entry exclusively within endosomes at low pH. Yet, wild-type IFITM3 and the mutant with the 21-amino-acid deletion inhibit HIV-1 replication equally well. Given the pH-independent nature of HIV-1 entry, our results suggest that IFITM3 can inhibit viruses that enter cells via different routes and that its N-terminal region is specifically required for controlling pH-dependent viruses.