Inappropriate laboratory testing in internal medicine inpatients: Prevalence, causes and interventions.

BACKGROUND: To reduce overutilization of laboratory testing many interventions have been tried, but selecting the most effective intervention for a given setting is challenging. To be sustainable, interventions need to align with healthcare providers' needs and daily practices. This study aimed to assess the extent of overutilization and the perspectives of healthcare providers, which may be used to guide the choice of intervention. METHODS: The extent of inappropriate laboratory testing in internal medicine inpatients was evaluated using a database. Surveys and focus groups were used to investigate healthcare providers' perceptions on its causes and solutions. RESULTS: On average, patients had 5.7 laboratory orders done during the first week of admission, whereas guidelines advise performing laboratory testing no more than twice per week. Repeat testing of normal test results occurred in up to 85% of patients. The frequency of laboratory testing was underestimated by survey responders, even though the majority of responders (78%) thought that laboratory tests are ordered too frequently. Residents were considered to be most responsible for laboratory test ordering.The primary causes of overutilization discussed were personal factors, such as a lack of awareness and knowledge, as well as feelings of insecurity. Regarding possible solutions, residents generally recommended educational interventions, whereas specialists tended to favour technical solutions such as lockouts. CONCLUSION: Inappropriate laboratory testing is common in internal medicine. The most important causes are a lack of awareness and knowledge, especially in residents. The intervention most favoured by residents is education, suggesting educational interventions may be most applicable.

Breast conserving surgery for extensive DCIS using multiple radioactive seeds.

BACKGROUND AND OBJECTIVES: Breast conserving surgery (BCS) can be challenging for large regions of ductal carcinoma in situ (DCIS), resulting in high rates of positive resection margins. Radioactive seed localization (RSL) using multiple radioactive iodine (125I) seeds can be used to bracket extensive DCIS (eDCIS). The goal of this study was to retrospectively compare the use of a single or multiple 125I seeds in RSL to enable BCS in patients with eDCIS. METHODS: All patients with eDCIS (area of ≥3.0 cm) who underwent either single or multiple-seed RSL between January 2008 and December 2016 were included. Patient, tumor and surgery characteristics were compared between both groups. Primary outcome measures were positive resection margin and re-operation rates. RESULTS: Respectively 48 and 58 patients with eDCIS underwent single- and multiple-seed RSL and subsequent BCS. The rate of positive resection margin (focal and more than focal) with single-seed RSL was 47.9%, compared to 29.3% with multiple-seed RSL (p = 0.06). The re-operation rate was 39.6% with single-seed RSL and 20.7% in the multiple-seed RSL group (p = 0.05). CONCLUSION: Multiple-seed RSL enables bracketing of large areas of DCIS, with the potential to decrease the high rate of positive resection margins in this patient group.

Is an unfavourable cardiovascular risk profile a risk factor for vasomotor menopausal symptoms? Results of a population-based cohort study.

OBJECTIVE: Evidence suggests an association between vasomotor menopausal symptoms (VMSs), i.e. hot flushes and night sweats, and cardiovascular disease. However, the causal pathway is unclear. We investigated whether an unfavourable cardiovascular risk profile is a risk factor for VMS later in life. DESIGN: Retrospective cohort study. SETTING: Women aged 50-70 from the general population. POPULATION: The Prospect-European Prospective Investigation into Cancer and Nutrition (Prospect-EPIC) cohort is a population-based cohort of women who enrolled between 1993 and 1997. Follow-up questionnaires were sent at 5-year intervals for 15 years. Women who returned the third questionnaire, answered questions regarding lifetime VMS and did not report VMS prior to baseline were included in this study (n = 1295). METHODS: At baseline, the Framingham Risk Score (FRS) was determined. We used logistic regression analysis to calculate odds ratios (ORs) for the association between baseline FRS and incident VMS. MAIN OUTCOME MEASURE: Incident VMS. RESULTS: At baseline (mean age ± standard deviation, 52.2 ± 3.6 years), 21.2% had a FRS > 10%. During follow-up, 40.2% of women reported the onset of VMS. Adjusted for body mass index, physical activity, education and alcohol consumption, each point increase in FRS was associated with a decreased incidence of VMS [OR, 0.94 (95% CI, 0.91-0.97)]. Additional adjustment for menopausal status attenuated the OR to null [OR, 0.98 (95% CI, 0.95-1.01)]. None of the separate FRS variables were associated with VMS after adjustment for age. CONCLUSIONS: In our cohort, an unfavourable cardiovascular risk profile was not associated with VMS, and therefore we found no evidence for the involvement of a vascular mechanism in the etiology of VMS.

Vitamin D status is not associated with inflammatory cytokine levels during experimental human endotoxaemia.

Vitamin D has been shown to modulate innate immune responses in vitro and ex vivo; however, human in-vivo data are lacking. At high latitudes, seasonal vitamin D deficiency is common due to alternating ultraviolet (UV)-B radiation exposure. In the present study, we investigated whether levels of 25 hydroxyvitamin D(3) [25(OH)D(3) ] and its active metabolite 1,25 dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] are subject to seasonal variation and whether plasma levels of these vitamin D metabolites correlate with the in-vivo cytokine response during experimental human endotoxaemia [administration of lipopolysaccharide (LPS) in healthy volunteers]. Plasma levels of 25(OH)D(3) and 1,25(OH)(2) D(3) were determined in samples obtained just prior to administration of an intravenous bolus of 2 ng/kg LPS (derived from Escherichia coli O:113) in 112 healthy male volunteers. In the same subjects, plasma levels of the inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 were analysed serially after endotoxin administration. Plasma levels of 1,25(OH)(2) D(3) , but not 25(OH)D(3) , were subject to significant seasonal variation, with lower levels in autumn and winter. 25(OH)D(3) and 1,25(OH)(2) D(3) levels did not correlate with plasma cytokine responses. Furthermore, 25(OH)D(3) deficient subjects (< 50 nmol/l) displayed an identical cytokine response compared with sufficient subjects. In conclusion, plasma levels of vitamin D are not correlated with the LPS-induced TNF, IL-6 and IL-10 cytokine response in humans in vivo. These findings question the direct role of vitamin D in modulation of the innate immune response.

Transduction of E13 murine neural precursor cells by non-immunogenic recombinant adeno-associated viruses induces major changes in neuronal phenotype.

Neural precursor cells (NPCs) provide a cellular model to compare transduction efficiency and toxicity for a series of recombinant adeno-associated viruses (rAAVs). Results led to the choice of rAAV9 as a preferred candidate to transduce NPCs for in vivo transplantation. Importantly, transduction promoted a neuronal phenotype characterized by neurofilament M (NFM) with a concomitant decrease in the embryonic marker, nestin, without significant change in glial fibrillary acidic protein (GFAP). In marked contrast to recent studies for induced pluripotent stem cells (iPSCs), exposure to rAAVs is non-immunogenic and these do not result in genetic abnormalities, thus bolstering the earlier use of NPCs such as those isolated from E13 murine cells for clinical applications. Mechanisms of cellular interactions were explored by treatment with genistein, a pan-specific inhibitor of protein receptor tyrosine kinases (PRTKs) that blocked the transduction and differentiation, thus implying a central role for this pathway for inducing infectivity along with observed phenotypic changes and as a method for drug design. Implantation of transduced NPCs into adult mouse hippocampus survived up to 28 days producing a time line for targeting or migration to dentate gyrus and CA3-1 compatible with future clinical applications. Furthermore, a majority showed commitment to highly differentiated neuronal phenotypes. Lack of toxicity and immune response of rAAVs plus ability for expansion of NPCs in vitro auger well for their isolation and suggest potential therapeutic applications in repair or replacement of diseased neurons in neurodegeneration.

Recent advances on neuronal caspases in development and neurodegeneration.

In view of a large and growing literature, this overview emphasizes recent advances in neuronal caspases and their role in cell death. To provide historical perspective, morphology and methods are surveyed with emphasis on early studies on interleukin converting enzyme (ICE) as a prototype for identifying zymogen subunits. The unexpected homology of ICE (caspase-1) to Caenorhabditis elegans death gene CED-3 provided early clues linking caspases to programmed cell death, and led later to discovery of bcl-2 proteins (CED-9 homologs) and 'apoptosis associated factors' (Apafs). Availability of substrates, inhibitors, and cDNAs led to identification of up to 16 caspases as a new superfamily of unique cysteine proteinases targeting Asp groups. Those acting as putative death effectors dismantle neurons by catabolism of proteins essential for survival. Caspases degrade amyloid precursor protein (APP), presenilins (PS1, PS2), tau, and huntingtin, raising questions on their role in neurodegeneration. Brain contains 'inhibitors of apoptosis proteins' (IAPs) survivin and NAIP associated also with some neuronal disorders. Apoptotic stress in neurons initiates a chain of events leading to activation of distal caspases by pathways that remain to be fully mapped. Neuronal caspases play multiple roles for initiation and execution of cell death, for morphogenesis, and in non-mitotic neurons for homeostasis. Recent studies focus on cytochrome c as pivotal in mediating conversion of procaspase-9 as a major initiator for apoptosis. Identifying signaling pathways and related events paves the way to design useful therapeutic remedies to prevent neuronal loss in disease or aging.

The importance of folic acid.

Folic acid is necessary for cell development; for the metabolism of specific biochemical reactions in the body, such as the conversion of homocysteine to methionine; and for the metabolism of specific anticonvulsant drugs. Folic acid has an interrelationship with vitamin B12. A deficiency of folate increases the risk of NTDs, as well as contributing to hyperhomocystinemia, a condition associated with increased cardiovascular disease and NTDs. For the prevention of NTDs, it is recommended that a woman of childbearing age consume a daily folate intake of 400 micrograms; however, the average dietary folate intake is half that amount, and the FDA folate fortification of cereal grains adds only 100 micrograms daily. The woman in her childbearing years does not meet the recommendation with dietary and food fortification. Periconceptional folic acid supplementation is essential, because the neural tube closes 23 to 27 days after conception. Therefore, a multiple vitamin containing folic acid is the practical solution at present if the food fortification is not increased. The bioavailability of folate in the vitamin preparation is approximately double that of dietary folate. Most preparations contain 400 micrograms of folic acid, and if the woman took a multiple vitamin (400 micrograms of folate) in addition to her diet (230 micrograms of folate), she would not exceed 1000 micrograms (1 mg) daily, which is considered the upper limit of daily folate ingestion by dietary fortification and supplementation before the masking of vitamin B12 becomes a concern. However, in this group of patients, pernicious anemia is rare. Regarding cardiovascular disease in men and women, there are no long-term studies showing the benefit of folic acid in reducing the homocysteine level. At present, there are only estimations. However, they should not be ignored. Although it is not the current standard of practice, adding a multiple vitamin containing folic acid to the regimen of men and women starting anticonvulsant medication should be considered in order to prevent the folate lowering observed with such commonly used drugs as PHT and carbamazepine. Women in childbearing years should be on a folic acid supplement when taking an anticonvulsant drug. In general, it appears that all men and women would benefit from increased folate intake. This can be accomplished through vitamin supplementation when there is compliance. However, if the food fortification for folate is increased in the future, then the issue of vitamin supplementation will have to be readdressed.

Drugs and smoking.

Generally, one should keep in mind that if a smoker takes a drug that is metabolized primarily by the CYP 1A2 system, an increased dose may be required. The patient should be closely monitored if he or she is taking several medications that may inhibit or induce the CYP 1A2 isoenzyme. Hepatic enzyme metabolism does not completely explain the effects of smoking on drugs, however, because smokers may have different physiologic responses to pain and sedation that depend on changes in plasma protein binding and in the rate and efficacy of subcutaneous absorption. Most of the few sex/gender-analyzed pharmacokinetic studies in smokers occurred after 1995. It appears that the 1993 changes at the National Institutes of Health and the Food and Drug Administration regarding inclusion of women in clinical trials enabled these examinations to take place. Because smokers--who make up nearly a quarter of the U.S. adult population--tend to consume more medications than nonsmokers, this social habit should be considered one of the most important to impact on drug metabolism in both sexes.

Drug and environmental factors associated with adverse pregnancy outcomes. Part II: Improvement with folic acid.

OBJECTIVE: To provide a comprehensive review of periconceptional folic acid supplementation and factors affecting folate supplementation trials. DATA SOURCES: A MEDLINE search was conducted through December 1997. Additional sources were obtained from Current Contents and citations from the references obtained. Search terms included folate, folic acid, neural tube defect, spina bifida, and anencephaly. STUDY SELECTION: Relevant animal and human studies examining the effects of folate were reviewed. DATA EXTRACTION: Data collected included: type of study, folate dosing, dietary folate intake, serum and red blood cell folate concentrations, type of defect(s) studied, vitamin usage, parental risk factors, factors affecting trial results. DATA SYNTHESIS: Nine key factors have been identified that affect outcomes of folic acid supplementation trials. Daily doses of 0.8 mg decreased the occurrence and doses of 4 mg decreased the recurrence of neural tube defects in randomized clinical trials. Since lower folic acid doses were effective in nonrandomized trials, research is needed to determine the lowest effective dosage. Other benefits involving pregnancy outcome are suggested. CONCLUSIONS: Women of childbearing age should take a daily folic acid supplement to reduce the risk of pregnancies resulting in infants with a neural tube defect and other potential adverse pregnancy outcomes. Further health benefits from folic acid supplementation are reviewed in Part III of this series.

Drug and environmental factors associated with adverse pregnancy outcomes. Part III: Folic acid: pharmacology, therapeutic recommendations, and economics.

OBJECTIVE: To review folic acid's mechanism of action, adverse effects, therapeutic recommendations, compliance, and cost. DATA SOURCES: A MEDLINE search was conducted through December 1997. Additional sources were obtained from Current Contents and citations from the references obtained. Search terms included folate, folic acid, neural tube defect, homocysteine, and methylenetetrahydrofolate reductase. STUDY SELECTION: Animal and human studies examining the effects of folate were reviewed. DATA EXTRACTION: Data collected included mechanism of action, safety issues, dosing recommendations, compliance with recommendations, and economics. DATA SYNTHESIS: Folic acid decreases neural tube defect risk through an effect on methionine-homocysteine metabolism. In addition, increased folate intake may reduce cardiovascular morbidity and mortality. Since toxicity is minimal, everyone can potentially benefit from increased folate consumption. To help achieve this, the Food and Drug Administration has mandated that cereal grain be fortified with 140 micrograms of folic acid per 100 g of grain, which will add approximately 0.1 mg of folate to the average diet. Studies recommend supplementing with 0.2 mg to promote optimal homocysteine concentrations and for preventing neural tube defects. CONCLUSIONS: Despite fortification, most women will still receive less folate than the 0.4 mg/d recommended by the Public Health Service. All population groups would benefit from increased folate intake. Current studies indicate 200 micrograms/d may be the minimum effective amount of fortification needed for normalizing homocysteine concentrations and preventing a significant number of neural tube defects; thus, a higher level of food fortification may be warranted.

Drug and environmental factors associated with adverse pregnancy outcomes. Part I: Antiepileptic drugs, contraceptives, smoking, and folate.

OBJECTIVE: Part I of this review examines the relationship between antiepileptic drugs (AEDs) and pregnancy outcomes. Drug-induced folate deficiency and the role of AED metabolism are emphasized. Part II will discuss periconceptional folate supplementation for prevention of birth defects. Part III will discuss the mechanism of folate's protective effect, therapeutic recommendations, compliance, and cost. DATA SOURCES: A MEDLINE search was conducted for journal articles published through December 1997. Additional sources were obtained from Current Contents and citations from the references obtained. Search terms included phenytoin, carbamazepine, phenobarbital, primidone, valproic acid, oral contraceptives, clomiphene, drug-induced abnormalities, spina bifida, anencephaly, neural tube defect, folate, folic acid, and folic acid deficiency. STUDY SELECTION: Relevant animal and human studies examining the effects of AEDs, smoking, and oral contraceptives on folate status and pregnancy outcome are reviewed. DATA EXTRACTION: Studies and case reports were interpreted. Data extracted included dosing, serum and red blood cell folate concentrations, teratogenicity of anticonvulsant medications, metabolism of AEDs and folate, and genetic susceptibility to AED-induced teratogenicity. DATA SYNTHESIS: Low serum and red blood cell folate concentrations are associated with adverse pregnancy outcomes. Decreases in serum folate are seen with AEDs, oral contraceptives, and smoking. Since similar birth defects are observed with multiple AEDs, metabolism of aromatic AEDs to epoxide metabolites and genetic factors may play a role in teratogenesis. CONCLUSIONS: Adequate prepregnancy planning is essential for women who have epilepsy. Women receiving folate-lowering drugs may be at increased risk of adverse pregnancy outcomes. Therefore, epileptic women contemplating pregnancy should be treated with the minimum number of folate-lowering drugs possible and receive folic acid supplementation.

The semisynthetic glycosphingolipid LIGA20 potently protects neurons against apoptosis.

LIGA20 (a semisynthetic GM1 with n-dichloroacetyl sphingosine) having antinecrotic properties was evaluated as an antiapoptotic agent using cerebellar granule cells as a neuronal model. Neurons exposed to medium containing 5 mM K+ in absence of serum (apoptotic medium) for up to 22 h resulted in 65% cell death, accompanied by oligonucleosomal laddering, and a 15-fold-increase in caspase-3 activity. The relationships between times required for enzyme activation and cell death suggests a linkage between these two events. Pretreatment with 10 microM LIGA20 for 10 min significantly attenuated the aforementioned apoptotic changes. LIGA20 also attenuated apoptosis induced by Pb2+, ethanol, and C6-ceramide. Data show that LIGA20 is a versatile neuroprotective agent protecting cells from multiple pathways of cell death.

Activation of caspase-3 and apoptosis in cerebellar granule cells.

Caspase-3 activity increased dramatically in cytosolic extracts of rat cerebellar granule cells exposed to apoptotic conditions (basal medium Eagle (BME) containing 5 mM K+ without serum) when assayed with Ac-DEVD-amc, but not with Ac-YVAD-afc, a preferred substrate for caspase-1. This provided a basis to examine relationships between enzyme activity and cell viability for purposes of selecting an optimal time for comparing neuroprotective agents or strategies. Exposure of neurons to an apoptotic medium containing 5 mM K+ in absence of serum led to a rapid 5- to 10-fold increase in caspase-3 within 2-4 hr but without significant cell loss, or morphological alterations. Exposure to apoptotic medium followed by replacement with maintenance medium containing 25 mM K+ and serum led to a rapid fall in caspase-3 and prevention of cell death. This strategy was not effective after 13 hr exposure despite a large fall in enzyme activity. These temporal changes infer systems for rapid enzyme turnover and/or activation of cytoplasmic components linked to later DNA degradation. The effects of cycloheximide point to requirements for protein synthesis, and those of Glu exclude a caspase-3 dependent pathway for necrotic cell damage. Brief treatment with 10 microM LIGA20, an anti-necrotic agent, also attenuated cell loss and caspase-3 activity, indicating a broad spectrum of neuroprotection. Rapid and long-lasting effects, together with its biophysical properties, suggest that this semisynthetic ganglioside acted upstream at or near a membrane site. As such, gangliosides provide useful agents to further probe pathways relevant to neuronal death in culture.

X-linked female band heterotopia-male lissencephaly syndrome.

We report a family with band heterotopia in a mother and daughter and lissencephaly in a son (X-linked inheritance pattern). Postmortem examination of the boy revealed classical lissencephaly and, among other findings, simplified and discontinuous inferior olives without inferior olivary heterotopia. The absence of inferior olivary heterotopia may distinguish X-linked lissencephaly from other conditions with classic lissencephaly such as Miller-Dieker syndrome.

Failure of standard magnetic resonance imaging in patients with refractory temporal lobe epilepsy.

OBJECTIVE: To compare the sensitivity of standard magnetic resonance imaging (MRI) scans done outside an epilepsy center with that of special protocol MRI scans done at an epilepsy center in delineating relevant lesions of the temporal lobe. SUBJECTS: Eighty-four consecutive patients who had temporal lobe resections for refractory temporal lobe epilepsy between January 1, 1993, and February 1, 1996. DESIGN: The reports of findings on standard MRI scans done outside an epilepsy center were compared with the findings of special protocol MRI scans done with 1.5-mm T1-weighted coronal and 3-mm T2-weighted coronal images (no gaps) on a 1.5-T system. Both sets of MRI findings were compared with findings on histologic examination of the resected tissue. RESULTS: Of the 84 patients, 51 had standard MRI scans done outside an epilepsy center; of these, there were 34 patients with normal results, 10 with tumors, 2 with vascular malformations, 2 with hippocampal atrophy, 2 with unclassified abnormalities, and 1 with cortical malformation. In 32 of the 34 patients with normal results of an MRI scan done outside an epilepsy center, abnormalities were found on our special protocol MRI scans. These included hippocampal atrophy in 27 patients, tumors in 2, and cortical malformations in 1. Additionally, all 17 of the abnormalities detected on the standard MRI scans done outside the epilepsy center were identified on our special protocol MRI scans. Important pathologic abnormalities of the temporal lobe were identified in 16 (35%) of the 46 patients with standard MRI scans done outside an epilepsy center and in 44 (96%) with our special protocol MRI scans. In the 29 patients for whom adequate surgical specimens were available and results of standard MRI scans were normal, our special protocol MRI scans showed the abnormality in 27 (93%). CONCLUSIONS: Conventional neuroimaging studies are inadequate for diagnosing hippocampal sclerosis although they fairly readily detect low-grade tumors and vascular malformations. Magnetic resonance imaging scans for the evaluation of patients with refractory temporal lobe epilepsy should be done with a special temporal lobe protocol and read by physicians experienced with the findings in hippocampal sclerosis. Health care dollars are wasted on neuroimaging done for refractory temporal lobe epilepsy outside epilepsy centers.

Pseudoprogression of cerebral cavernous angiomas: the importance of proper magnetic resonance imaging technique.

Magnetic resonance imaging (MRI) is the method of choice to detect cerebral cavernous angiomas. Specifically tailored MRI studies are necessary to evaluate patients with cerebral cavernous angiomas. The patient described here, for whom serial MRIs performed on different scanners falsely suggested rapid disease progression, illustrates the point.

Brain cathepsin B but not metalloendopeptidases degrade rAPP751 with production of amyloidogenic fragments. Comparison with synthetic peptides emulating beta- and gamma-secretase sites.

Lysosomal cathepsin B but not L degraded rAPP751 to yield C-terminal 19-25 kDa fragments containing beta A4, reinforcing the view that acidic proteases participate in endosomal-lysosomal processing to yield amyloidogenic fragments in situ. This mechanism is consistent with fragmentation of endogenous APPs within clathrin-coated vesicles (CVs) by vesicular hydrolases, with the appearance of C-terminal amyloidogenic fragments following incubation at pH 6.5. A neutral endopeptidase resembling NEP 24.11 (PS-NEP) purified from detergent extracts of human brain degraded rAPP751; however, breakdown was not blocked robustly by metal chelators or phosphoramidon, suggesting the presence of an alternative processing enzyme. Effects of other inhibitors showed that breakdown was mediated by serine-protease-like component(s). A phosphoramidon-insensitive metalloendopeptidase (PI-NEP) partially purified from rat brain P2 using detergents, and resembling NEP 24.15, showed no activity towards rAPP751. Peptides containing putative beta- or gamma-secretase sites were synthesized for purposes of examining their metabolism by the brain enzymes. Those containing beta-secretase sites were hydrolysed at one or more sites by the four enzymes, but only PI- and PS-NEP acted at the Met-Asp site of Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg.NH2. In the case of substrates containing the gamma-site, these two categories of enzymes were the only ones degrading N-Ac-Ile-Ala.NH2. These data imply that the brain metalloendopeptidases, while inactive towards intact precursors, may be involved in turnover of intermediates containing beta- or gamma-sites.

Amyloid precursor protein is enriched in axolemma and periaxolemmal-myelin and associated clathrin-coated vesicles.

The amyloid precursor protein (APP) is widely distributed within the CNS, where it is expressed in both neurons and glia. We have isolated axolemma and periaxolemmal-myelin from rat brain and have determined by Western blot that APPs, Mr 100-110 kDa, are major constituents of these membrane. Isolation of axolemma, periaxolemmal-myelin, and compact myelin show that while APP represents 1 and 0.6% of the proteins of these respective membranes, it is absent from compact myelin. These results indicate that APP transported down the axon is deposited at sites in the axolemma as well as the synapse, and that within the myelin complex, APP is targeted to the periaxolemmal domain. Both axolemma and periaxolemmal-myelin contained a 10.5 kDa APP peptide which, based on reactivity with anti-C-terminal APP antibodies but not with anti-N-terminal antibody, appears to be a membrane-associated C-terminal fragment. Western blots with antibodies to Alzheimer precursor-like proteins (APLP) indicate that APP immune reactivity is not a result of cross reactivity with APLPs. Isolation of axolemma from human autopsy material showed nearly identical results with a clear enrichment, relative to homogenate, of APP Mr 100-110 and the 10.5 kDa C-terminal peptide. The demonstration of APP in axolemma and periaxolemmal-myelin was replicated in membrane isolated from bovine brain. Bovine studies were extended to analysis of white matter clathrin-coated vesicles; these data show that coated vesicles isolated from white matter, under conditions that previous studies indicate are largely endocytic vesicles, contain levels of APP comparable to that found in axolemma and periaxolemmal-myelin. In addition, these vesicles contain cysteinyl and aspartyl proteases. Incubation of axolemma with cathepsin B at pH 6.0 caused a rapid loss in the immune reactivity of APP Mr 100-110 and Mr 10.5 when analyzed with antibodies to APP672-695. This appears to be the result of hydrolysis within the epitope and not proteolysis of APP or the C-terminal peptide, since no loss of reactivity was observed when analyzed with antibodies to sites more distal to the C-terminus. Thus, cathepsin B hydrolyses membrane bound APP close to the C-terminus and may be a useful tool for altering C-terminal APP function.

Hydrolysis of amyloid precursor protein-derived peptides by cysteine proteinases and extracts of rat brain clathrin-coated vesicles.

Amyloid precursor proteins (APPs) and C-terminal fragments were colocalized with cysteine proteinase-like enzymes in purified rat brain clathrin-coated vesicles. Vesicular extracts degraded beta A4(12-28), yielding a product profile similar to that of purified rat brain cathepsin B. Cathepsin B degraded this peptide sequentially, with initial cleavage occurring at Val18-Phe19 and Phe19-Phe20 followed by release of dipeptides. Enzyme also hydrolyzed beta A4(1-40) at Phe19-Phe20 bond but at lower rates, likely due to aggregate formation. An octapeptide analogue of the domain adjacent to beta A4 (N-Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-NH2) was also hydrolyzed by brain cathepsins B and L, and metalloendopeptidase 24.11. Enzymes acted at multiple sites, but only 24.11 cleaved the Met-Asp bond, thus resembling a proposed beta-secretase. Data imply that clathrin-coated vesicles contain cysteine-like proteinases capable of initiating the processing of APP or its fragments.