Structure-specific amyloid precipitation in biofluids.

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. Here we report a method to capture amyloid-containing aggregates in human biofluids in an unbiased way, a process we name amyloid precipitation. We use a structure-specific chemical dimer, a Y-shaped, bio-inspired small molecule with two capture groups, for amyloid precipitation to increase affinity. Our capture molecule for amyloid precipitation (CAP-1) consists of a derivative of Pittsburgh Compound B (dimer) to target the cross ß-sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we demonstrate that we can target the amyloid structure of all protein aggregates present in human cerebrospinal fluid, isolate them for analysis and then characterize them using single-molecule fluorescence imaging and mass spectrometry. Amyloid precipitation enables unbiased determination of the molecular composition and structural features of the in vivo aggregates formed in neurodegenerative diseases.

Passive transfer of allergic encephalomyelitis in rats: a tool for drug mechanism studies and detecting late-acting immunosuppressants.

1. A strategy is described for evaluating drugs against different phases in the development of an auto allergic disease, experimental allergic encephalomyelitis. It is based on a cell transfer technique whereby the disease is passively transferred with lymphoid cells from actively immunized donor rats to normal syngeneic rats = passive recipients. Drugs may be applied in vivo to either the cell donors or the cell recipients or to cells in vitro whilst in transit; their efficiency being determined by the severity of the passive disease (weight loss, paralysis) in the recipients. 2. Examples are given illustrating the application of these techniques to: (a) evaluating the lymphocyte-deactivating activity of various nitrogen mustards in vitro; (b) recognizing drugs, e.g. gold derivatives, clofazimine, etc. that are not conventional immunosuppressant (or cytostatic) agents which, when given to the recipient animals, may prevent the expression of the adopted disease; (c) comparing some known immunosuppressants for potency, duration of action, etc.; (d) demonstrating the versatility of cycloleucine, ICI-47,776, etc. 3. Some merits of the strategy are discussed vis a vis using the local graft-versus-host reaction in rats to search for new drugs.

Cord blood immune biomarkers in small for gestational age births.

Fetal growth restriction is a risk factor for development of adulthood diseases, but the biological mechanism of this association remains unknown. Limited biomarkers have been studied in settings of preterm birth and maternal inflammation, but the relationship between a wide range of immune biomarkers and fetal growth has not been studied. The hypothesis of this study was that fetal growth restriction is associated with altered immune biomarker levels. We examined the relationship between small for gestational age (SGA) status and 27 umbilical cord blood immune biomarkers. This study was part of a large-scale cohort study of preterm birth and low birth weight conducted at Boston Medical Center, an inner city, predominantly minority patient population. Growth status was determined based on birth weight standardized to an internal reference. There were 74 SGA births and 319 appropriate for age (AGA) births with complete clinical and biomarker data. Adjusting for covariates and using AGA as reference, SGA births had lower levels of log IL-1ß (ng/l; ß -0.38, 95% CI -0.57, -0.19, P < 0.01), log BDNF (ß -0.29, 95% CI -0.55, -0.03, P < 0.05) and log NT-3 (ß -0.46, 95% CI -0.77, -0.15, P < 0.01). No associations were found between other biomarkers and SGA. In conclusion, three biomarkers were selectively associated with SGA status. Our results provide information that could be used to guide additional studied aimed at determining mechanisms that contribute to fetal growth.

Stroke Belt initiative: the Tennessee experience.

African Americans have a higher rate of stroke than other U.S. population groups. As part of the 11-state "Stroke Belt" region, Tennessee has the fifth highest death rate from stroke in the country. In 1993, a two-year community-based project was initiated to reduce the incidence of stroke and its associated risk factors among African Americans. Three counties, Shelby (Memphis), Davidson (Nashville), and Hamilton (Chattanooga), were selected as project sites because of their large African American populations. Specific objectives of the project were to promote risk factor awareness in African Americans, assist African American churches and community groups in developing and implementing intervention programs, and build the capacity for intervention programs within African American communities by collaborating with a variety of community organizations. This article describes the program's approach, which included using both adults and youth as mentors. In addition, it presents major accomplishments and lessons learned in project implementation.

Preferential looking in clinical practice: a year's experience.

Preferential Looking (PL) is now a well established laboratory method of measuring visual acuity in preverbal children. We have evaluated the feasibility of its routine use in clinical practice. We present our methods and results obtained in 80 normal children and 36 children with visual disorder and discuss the problems encountered in applying this test.

Analysis of transcripts homologous to acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase induced in rat liver by methylclofenapate.

Methylclofenapate is a potent peroxisome proliferating agent and liver carcinogen in rats. Animals exposed to daily oral doses (2.5 mg/kg body wt.) for a 21-day period were studied to determine the levels of mRNA homologous to peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme in total liver RNA. Northern blotting revealed transcripts of approximately 3.8 and 3.3 kilobases (kb), homologous to acyl-CoA oxidase and the bifunctional enzyme, respectively. Levels of these transcripts began to rise at approximately 4 h after the initial dose of the agent, and reached maximum induction (35- and 60-fold, respectively, in excess of control levels) at 2-8 days after the start of the study. The kinetics of induction for acyl-CoA oxidase mRNA resembled those of palmitoyl-CoA oxidase activity, and the induction of mRNA preceded the expression of enzyme activity, further supporting a transcriptional control model of induction of the peroxisomal enzymes. The levels of mRNA induction for the peroxisomal enzymes were higher in the present study than those reported elsewhere for single doses of peroxisome proliferating agents and probably reflect the increased tissue levels achievable in long term carcinogenesis studies.

Regulation of cell shape in the Cloudman melanoma cell line.

We show that Cloudman melanoma cells undergo rapid arborization in response to [Nle4,D-Phe7]alpha-melanocyte-stimulating hormone, a potent analogue of alpha-melanocyte stimulating hormone (alpha-MSH). The arbors were established by extension of processes and resembled dendrites. We used this system to study the regulation of cell shape. alpha-MSH is known to induce increases in cAMP levels, and agents such as forskolin and isobutylmethylxanthine that led to increased cAMP also caused arborization. However, equally dramatic arbors were formed after incubation with the protein kinase C inhibitor H-7 [1-(5-isoquinolinesulfonyl)-alpha-methyl-piperazine]. Phorbol diesters that activate protein kinase C led to cell rounding and antagonized alpha-MSH. The actions of protein kinase C cannot be rationalized in terms of indirect effects on cAMP: neither H-7 nor phorbol diesters alone altered cAMP levels, nor did they affect the increase in cAMP induced by MSH. We show also that MSH produced longer-term effects that cannot be mimicked by cAMP. Specifically, even in the continued presence of alpha-MSH, arborization was followed by morphological reversal to the unstimulated flattened configuration within 2 hr. (This did not occur with other agents that increase cAMP or with H-7.) Most importantly, whereas MSH-induced arborization occurred in the presence of cycloheximide, actinomycin D, or in enucleated cells, the reversal of arborization did not. Thus, MSH induced a program of rapid shape change that was dependent on new protein synthesis and gene transcription.

Resistance of mouse lymphoma L5178YAII cells to alkylation with methylmethane sulphonate resides in a late step of excision repair.

The mutant mouse lymphoma cell line (L5178YAII), resistant to X-rays, ultraviolet light and alkylating agents, was reinvestigated in an attempt to establish the nature of the mutation. These cells were compared with P388 mouse lymphoma cells, which exhibit normal sensitivity to these mutagens. A series of studies was conducted to compare DNA alkylation and strand breakage with cell survival after exposure of the two cell lines to methylmethane sulphonate. It was found that neither the degree of alkylation nor the removal of the common alkylation products was correlated with the different sensitivities observed in these cell lines. A correlation was established between cell killing and the production of long-lived strand breaks. P388 cells were found to accumulate twice as many long-lived strand breaks compared to L5178YAII cells, at equal levels of alkylation. This suggested that long-lived strand breaks were the major toxic lesions. Further experiments indicated that these long-lived strand breaks were produced by a process consistent with excision repair. Evidence is also presented that indicates that the mutation in L5178YAII cells that is responsible for their resistance may occur in ligase activity or its associated ADP-ribosyl transferase system.

Effects of hydroxyurea consistent with the inhibition of repair synthesis in ultraviolet irradiated HeLa cells.

Many studies on DNA repair using established in vitro cell cultures employ conditions of low serum, nutrient deprivation, and blockade with hydroxyurea (HU) to reduce the background levels of DNA replication. There are some reports in the literature which indicate that HU inhibits DNA repair. In the present study the effects of HU on strand breaks and repair synthesis in UV irradiated Hela cells were investigated using a combined strand break and UDS assay. In conditions of low serum and arginine deprivation, HU produced effects consistent with the inhibition of the repair synthesis step in excision repair. Although the conditions used in this study are severe the results suggest that HU may have qualitatively similar effects in other studies which employ its use to detect repair synthesis.

An investigation of DNA-repair phenomena induced by 4-chloromethylbiphenyl (4CMB).

The ability of 4CMB to induce repair phenomena has been investigated in 3 types of assay. No significant unscheduled DNA synthesis or repair replication was found, although the compound gave a weakly positive dose-related response in strand-break assays, with 250 microgram/ml producing significant amounts of strand breaks. These data indicate that 4CMB is a weakly genotoxic agent.

Muscle adenylate deaminase deficiency. Report of six new cases.

We describe six adult patients (five men and one woman) out of 364 whose muscle biopsy specimens disclosed muscle adenylate deaminase deficiency. Two men had an associated dermatomyositis and another man had an associated progressive systemic sclerosis. Although the patients were different clinically, all complained of muscular weakness or poor exercise tolerance. The occurrence of muscle adenylate deaminase deficiency in both sexes suggests a possible autosomal mode of inheritance.

Adjuvant polyarthritis. V. Induction by N-acetylmuramyl-L-alanyl-D-isoglutamine, the smallest peptide subunit of bacterial peptidoglycan.

N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), an apparently nonimmunogenic bacterial peptidoglycan-derived small peptide, was found to induce a polyarthritis the rat similar to that induced by Freund's complete adjuvant when injected in the form of an oil emulsion. An oil emulsion of its isomer, N-acetylmuramyl-L-alanyl-L-isoglutamine, which unlike MDP has no immunostimulatory activity, failed to induce the disease.

Adenylosuccinase in human muscular dystrophy.

The level of adenylosuccinase was measured in muscle from patients with Duchenne and other major forms of progressive muscular dystrophies and certain related neuromuscular diseases. The activity was found to be unaltered in all diseases that were examined.

Role of thymus for N-acetyl muramyl-L-alanyl-D-isoglutamine-induced polyarthritis and granuloma formation in euthymic and athymic nude rats or in neonatally thymectomized rats.

A synthetic adjuvant, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), produced extremely severe polyarthritis with almost 100% incidence in Rowett euthymic rnu/+ rats, but the same dose of MDP (100 microgram) did not produce the disease in athymic rnu/rnu rats. Five hundred micrograms of MDP or 0.2 mg of heat-killed Mycobacterium bovis BCG, however, produced mild and transient polyarthritis in nude rats with very low incidence. We have not yet succeeded in reconstituting the disease susceptibility of nude rats by using thymus cells from normal rnu/+ rats. After intradermal inoculation of 100 microgram of MDP, nude rats developed small granulomas with a little necrosis and very few multinucleated giant cells only in the regional lymph nodes, whereas, in addition to the development of polyarthritis, euthymic rnu/+ rats developed typical granuloma with massive necrosis accompanied by numerous polymorphonuclear leukocytes and sparse multinucleated giant cells in the regional lymph nodes. Thymus cell-reconstituted rnu/rnu rats developed granuloma with sparse giant cells, relatively large areas of necrosis, and many polymorphonuclear leukocytes. Neonatal thymectomy may depress adjuvant-induced arthritis in the high-responder Lewis rats and enhance the disease development in the low-responder F344 rats. These findings suggested that (i) thymus plays an important role in promoting the development of MDP-induced arthritis; (ii) MDP-induced granuloma formation does not require thymus functions; (iii) the thymus functions may however be involved in the development of massive necrosis surrounded by considerable polymorphonuclear leukocyte infiltration, the mechanisms of which remain to be determined; and (iv) there is no direct correlation between granuloma formation and development of adjuvant arthritis.

Methylthioadenosine nucleosidase in normal and dystrophic human muscle.

Human skeletal muscle homogenate was found to contain a nucleosidase that catalyzes the hydrolysis of 5'-methylthioadenosine, a known inhibitor of many methyl transfer reactions. When the levels of methylthioadenosine nucleosidase in muscle of patients were compared with those of controls, no significant alterations in its activity were noted in patients with various forms of muscular dystrophies, polymyositis and certain denervating diseases.

Muscle fructose 1,6-diphosphatase deficiency associated with an atypical central core disease.

A 25-year-old woman with a non-familial congenital nonprogressive myopathy was found to have atypical core-like lesions in type 1 muscle fibers. Typical core lesions (approximately 13 micrometers in diameter) and smaller, PAS positive (4.1 micrometers in diameter) atypical core were associated with a predominant type 1 fibre myopathy. A specific deficiency of fructose 1, 6-diphosphatase was found with normal values for nine other muscle glycolytic and mitochondrial marker enzymes. The data provide evidence for a specific muscle enzyme deficiency in a patient with atypical central core disease.