The muscle protein dysferlin accumulates in the Alzheimer brain

Dysferlin is a transmembrane protein that is highly expressed in muscle. Dysferlin mutations cause limb-girdle dystrophy type 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin has also been described in neural tissue. We studied dysferlin distribution in the brains of patients with Alzheimer disease (AD) and controls. Twelve brains, staged using the Clinical Dementia Rating were examined: 9 AD cases (mean age: 85.9 years and mean disease duration: 8.9 years), and 3 age-matched controls (mean age: 87.5 years). Dysferlin is a cytoplasmic protein in the pyramidal neurons of normal and AD brains. In addition, there were dysferlin-positive dystrophic neurites within Aβ plaques in the AD brain, distinct from tau-positive neurites. Western blots of total brain protein (RIPA) and sequential extraction buffers (high salt, high salt/Triton X-100, SDS and formic acid) of increasing protein extraction strength were performed to examine solubility state. In RIPA fractions, dysferlin was seen as 230–272 kDa bands in normal and AD brains. In serial extractions, there was a shift of dysferlin from soluble phase in high salt/Triton X-100 to the more insoluble SDS fraction in AD. Dysferlin is a new protein described in the AD brain that accumulates in association with neuritic plaques. In muscle, dysferlin plays a role in the repair of muscle membrane damage. The accumulation of dysferlin in the AD brain may be related to the inability of neurons to repair damage due to Aβ deposits accumulating in the AD brain.
Introduction
Aggregation of neuronal proteins is a common mechanistic theme in neurodegenerative diseases [13]. Whether due to point mutations or post-translational modification, normally soluble proteins are altered and form insoluble fibrillar aggregates [18]. Alzheimer’s disease (AD), the most common form of dementia, is characterized pathologically by abundant diffuse and neuritic plaques throughout most cortical regions. Amyloid β-protein (Aβ) is a 39–43 amino acid peptide cleaved from a larger precursor protein (amyloid precursor protein or APP) that deposits extracellularly as senile plaques either as loose, nonfibrillar diffuse plaques, or as a more compacted, fibrillar form, with dystrophic neurites coursing through the plaque [25]. Other aggregated proteins found in the AD brain include the microtubule-associated protein tau [11] as neurofibrillary tangles and the presynaptic protein α-synuclein [13] in the form of Lewy bodies.
The alteration, deposition and aggregation of amyloid proteins may be an ill-fated response to the disease process or themselves lead to a cascade of cellular responses ultimately resulting in neuronal dysfunction and death.
Nonbrain protein aggregates have been described in muscle disease. For example, amyloid-like deposits have been described in inclusion body myositis [2]. Other protein aggregates found in muscle disease include desmin-related myopathies, actinopathies and myosinopathies [30]. Aggregates containing these proteins have not been described in brain disease. Another muscle protein associated with myopathy is dysferlin. Dysferlin is a plasma membrane and cytoplasmic vesicle-associated protein implicated in three adult-onset muscle diseases (limb-girdle dystrophy, type 2B, Miyoshi myopathy and distal anterior compartment myopathy)[4, 9, 22, 24]. Although described in peripheral nerve [30] and demonstrated by Western blot in human and mouse brain [21], there is little known about the function of dysferlin in the brain or its role in neurodegenerative disease. We describe the pattern of dysferlin expression in the normal and AD brain and examine dysferlin accumulation in association with amyloid deposition in the brains of AD patients.
Materials and methods
Case materials
Case materials were derived from research participants in a longitudinal study of healthy aging and dementia who were studied postmortem. The diagnostic criteria are consistent with probable AD reported by the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer’s Disease and Related Disorders Association [26] and confirmed by autopsy to meet high probability of AD according to National Institute of Aging-Reagan criteria [28]. The clinical dementia rating (CDR) was used to determine the presence or absence of dementia and stage its severity [27]. The global CDR is derived by a synthesis of individual ratings in six cognitive and functional categories where CDR = 0 indicates no dementia and CDR = 0.5, 1, 2, or 3 corresponds to very mild, mild, moderate, or severe dementia [27]. The CDR is closely correlated with the presence of dementia pathology at autopsy [14]. The Washington University Human Studies Committee approved all procedures.
Twelve brains were examined. Three cases were rated as nondemented (CDR 0) with a mean age of 87.5 years. Nine AD brains were examined; three each of mild (CDR 1), moderate (CDR 2) and severe (CDR 3). The mean age of the demented cases was 85.9 years with mean disease duration of 8.9 years (Table 1). Control cases were all Braak stage I or II [6]. Sampling during postmortem exam was done from 29 different cortical, subcortical and brainstem regions. Paraffin-embedded sample for this study were taken from midfrontal, cingulate and entorhinal regions; three sections were stained for each antibody with appropriate controls [15]. Biochemistry was performed on midfrontal gyrus adjacent to the section taken for immunohistochemistry for each case.
Table 1Sample characteristics and findings at postmortem examinationCaseAge (years)SexCDRDisease duration (years)Brain wt (g) Braak scoresCERAD criteriaNIA–Reagan criteriaNeurofibrillaryAmyloid192F0–1,120IIA––285F0–1,100IA––392F0–1,210IA––485F181,200IVCDefiniteHigh599M121,230VBDefiniteHigh695M1141,360VCDefiniteHigh781M216790VICDefiniteHigh891M2111,580VBDefiniteHigh990M271,170IVBDefiniteHigh1086M3101,310VICDefiniteHigh1179F38770VICDefiniteHigh1288M3111,130VICDefiniteHigh
Immunohistochemistry
Immunohistochemistry was done as previously described [14, 15]. Briefly, serial 6 μm sections of paraformaldehyde-fixed, paraffin-embedded tissue were prepared and incubated overnight at 4°C with antibodies against dysferlin (Novacastra Laboratories), tau (17026, gift from J. Trojanowski), and amyloid β-protein (6E10, Signet). Sections were developed by avidin–biotin complex method (Vector Laboratories) for immunohistochemistry with DAB (brown), Vector Red (pink) and Vector SG (blue) as chromogens. Alexa-Red and Green (Jackson Laboratories) antibodies were used for immunofluorescence. Sections were examined with a Nikon E800 Research microscope, and images were digitized and viewed with AnalySIS analytic software (Soft Imaging Systems, GmbH).
Western blots
Western blotting and biochemical fractionation were performed as described previously [16]. Briefly, two sections of gray matter (0.3 g) from the midfrontal cortex of AD and normal age-matched control brains were dissected from underlying white matter. After dissection, gray matter was divided into two samples (Fig. 1). One sample was homogenized in RIPA (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 50 mM Tris, pH 8, 20 mM, NaF, 2 mM EGTA, 0.5% levamisole, 1 mM NaVO4) to estimate total dysferlin. The other sample was homogenized and subjected to sequential extraction buffers of increasing protein extraction strength. For each buffer, pellets were washed twice and supernatants pooled. Samples were first homogenized in 2 ml/gram of tissue of high-salt (HS) buffer (50 mmol/l Tris, pH 7.4, 750 mmol/l NaCl, 10 mmol/l NaF, 5 mmol/l ethylenediaminetetraacetic acid with protease inhibitors) and centrifuged at 100,000g for 30 min. The pellets were re-extracted and the supernatants were pooled. The pellets were sequentially extracted twice with 2 ml/gram of HS buffer/1% Triton X-100 (HS/T) and once with 1 ml/gram sodium dodecyl sulfate (SDS) sample buffer (1% SDS, 10% sucrose, 10 mmol/l Tris, pH 6.8, 1 mmol/l ethylenediaminetetraacetic acid, 40 mmol/l dithiothreitol). The pellets were extracted with 0.67 ml/g 70% formic acid (FA) and disrupted with two sequential 2-s sonication bursts. FA was evaporated in a vacuum centrifuge (Eppendorf). SDS sample buffer (0.67 ml/g) was added to the dried pellets, followed by vigorous vortex, and the pH was adjusted with NaOH. SDS sample buffer was added to the HS and HS/Triton fraction and all of the samples were boiled for 5 min. The FA samples were centrifuged at 13,000g for 5 min to remove insoluble debris. Five microliters of each fraction was loaded in separate lanes for SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with antibodies again dysferlin. The immune complexes were visualized with the use of the ECL Plus kit (Amersham) according to the manufacturer’s protocol. The autoradiographs obtained were scanned and the band intensity quantified utilizing TotalLab software (Nonlinear Dynamics, Newcastle on Tyne, UK). Statistical analyses were performed by analysis of variance using SPSS statistical software.
Fig. 1Schematic representation of sequential protein extraction buffers. Samples are divided into equal portions. The first sample is homogenized in RIPA buffer and blotted to estimate total dysferlin levels. The other sample is homogenized in sequential extraction buffers of increasing protein extraction strength. The first extraction is in high-salt (HS) buffer. The supernatants are saved and the pellet is extracted with HS buffer/1% Triton X-100 (HS/T) buffer. After saving the supernatants, the resultant pellet is homogenized in sodium dodecyl sulfate (SDS) sample buffer. Resulting pellets are finally extracted with 70% formic acid (FA). Supernatants are blotted to estimate differing levels of dysferlin solubility with the HS fraction representing the most soluble phase and the FA fraction representing the most insoluble phase
Results
Localization of dysferlin in normal (CDR 0) brain
Microscopic evaluation of the dysferlin staining in the normal (CDR 0) brain localized expression to the cytoplasm of large pyramidal neurons of layers III, V and VI in the neocortex (Fig. 2a), layer II neurons of the entorhinal cortex, CA 1–3 neurons in the hippocampus and large projection neurons in the amygdala (not shown). Dysferlin was not localized to astrocytes, endothelial cells or microglia (arrowheads in Fig. 2b). Little to no dysferlin neuritic staining was seen in control cases (Braak stage I or II).
Fig. 2Localization of dysferlin in brains of nondemented subjects. a, b Demonstrates the localization of dysferlin immunoreactivity in the brain. There is a neuronal expression of dysferlin localized in cytoplasm of large pyramidal (layers III, V, VI) neurons in the frontal cortex at low power (a). Little to no neuritic staining was detected. Dysferlin expression is confined to neurons (arrow, DAB brown in b) and is not seen in glial cells stained with GFAP (small arrowheads, Vector Red pink in b). Magnification of (a) is 100× and the scale bar in (a) = 50 μm. Magnification of (b) is 400× and the scale bar in (b) = 20 μm
Dysferlin in the AD brain
Dysferlin neuritic accumulation is found throughout the AD brain and in double-label studies it is localized in Aβ neuritic plaques in the neocortex (Fig. 3a). Double-labeled studies demonstrate co-localization of dysferlin (brown in Fig. 3b) and Aβ (pink in Fig. 3b) in the hippocampus.
Fig. 3Localization of dysferlin with amyloid pathology in the AD brain. a, b Demonstrates the location of the dysferlin accumulation in the AD brain with double-labeled studies. a Diffuse dysferlin accumulation (DAB, brown) and Aβ (DAB-nickel, black) in the frontal cortex. Using VectorRed (pink, Vector Laboratories) as the chromogen for Aβ and DAB (brown, Vector Laboratories) as the chromogen for dysferlin, the pathologic deposition is localized as neuritic pathology in Aβ neuritic plaques in the neocortex hippocampus (b). Magnification of (a) is 100× and the scale bar in (a) = 20 μm. Magnification of (b) is 200× and the scale bar in (b) = 20 μm
Dysferlin neuritic pathology occurs in conjunction with tau neuritic pathology
In Fig. 4, we demonstrate that neurites in plaques may comprise tau, dysferlin or both. Triple-labeled studies of midfrontal (Fig. 4a), cingulate (Fig. 4b) and entorhinal (Fig. 4c) cortex illustrate the relationship of tau (pink, arrowheads) and dysferlin (brown, arrows) within an Aβ plaque (blue). Double-labeled immunofluorescent studies (Fig. 4d–f) demonstrate that tau (green in 4d) and dysferlin (red in Fig. 4e) have both distinct and overlapping (yellow in Fig. 4f) epitopes in neuritic plaques. Dysferlin does not appear to co-localize with the more abundant tau dystrophic neurites.
Fig. 4Localization of dysferlin with tau pathology in the AD brain. a–f Demonstrates the location of the dysferlin and tau accumulation in the AD brain with triple-labeled immunohistochemistry studies (a–c) and double-labeled immunofluorescence studies (d–f). a Midfrontal, b cingulate and c entorhinal demonstrate tau (Vector Red, pink, arrowheads), dysferlin (DAB, brown, arrows) and Aβ (Vector SG, blue). Note the distinction between tau-positive and dysferlin-positive structures. Double-labeled immunofluorescence studies demonstrated neurites within amyloid plaques. Neurites may comprise tau (green, d), dysferlin (red, e) or overlapping epitopes (yellow in merged image of f). Magnification of (a–f) is 600× and the scale bar in (d) = 20 μm
Biochemistry
Western blot analysis following RIPA extraction demonstrated the presence of dysferlin as 230–272 kDa bands in all brains examined (Fig. 5a); however, there was a decrease in the total dysferlin content in CDR 3 (severe AD) brains compared to CDR 0 (nondemented). There is a progressive decrease in total dysferlin with progressive disease (r = 0.998, P = 0.001). Western blot analysis following serial extraction of proteins demonstrated a change in the solubility state of dysferlin across the spectrum of dementia (Fig. 5b). No dysferlin could be demonstrated in the HS fraction. In contrast to the control CDR 0 brains, however, there was a significant reduction of dysferlin in the HS/T fraction in the demented cases (CDR 1 and 2) and was undetectable at the CDR 3 level of severity (F = 2,991, P < 0.001). In the SDS-soluble fraction from the demented cases, there was an accumulation of 230–272 kDa bands with dementia progression, that is a more insoluble form of dysferlin was found in severe CDR 3 brains compared to milder stages of dementia (CDR 1) or in nondemented individuals (F = 3,183, P < 0.001). Dysferlin did not aggregate or accumulate in the FA-soluble fractions of the control or AD brains.
Fig. 5Western Blot analysis of dysferlin in nondemented and AD brains. a Nondemented (CDR 0) and AD brains (mild = CDR 1, moderate = CDR 2, and severe = CDR 3) were extracted in RIPA as described in the Materials and methods. Five microliters of each extraction were loaded into separate lanes of a 12% SDS-polyacrylamide gel electrophoresis and after transfer to nitrocellulose membranes were probed with antibodies against dysferlin. The blot demonstrates the presence of dysferlin in both normal and AD brains with declining levels of protein across disease progression. Band intensities were quantified and compared in a bar graph. There is a linear decrease in total dysferlin associated with disease progression (r = 0.998, P = 0.001). b The same brains were sequentially extracted with HS, HS/T, SDS and FA as described in the Materials and methods section as loaded onto 12% SDS-polyacrylamide gel electrophoresis as described earlier. Dysferlin was not seen in either the HS or FA fractions. Dysferlin was demonstrated prominently in HS/T fraction in the CDR 0 brains, with a significant reduction (F = 2,991, P < 0.001) of dysferlin in the HS/T fraction in the demented cases (CDR1 and 2) and was nearly undetectable in severe AD (CDR 3) In contrast, the SDS fraction representing insoluble phases of dysferlin demonstrated significant increases (F = 3138, P < 0.001) in the 230–272 kDa bands as dementia progressed from mild (CDR 1) to severe (CDR 3)
Discussion
Dysferlin is a new protein described in the AD brain associated with neuritic plaques. Dysferlin is widely expressed in the brain and is found in intracytoplasmic compartments in pyramidal neurons in normal brains. Dysferlin begins to accumulate in dystrophic neurites in the AD brain at mild stages of dementia (CDR 1). Dysferlin is found at all stages of disease with greater accumulation in more advanced disease (CDR 3).
Significantly, our Western blot analysis demonstrated dysferlin in RIPA-extracted fraction in all cases; however, in the serial extracted fractions an alteration in the solubility state of dysferlin was found in demented cases (CDR > 1) compared with nondemented cases. This coincides with the finding of neuritic pathology by immunohistochemistry. The HS/T fraction represents the transmembrane cytoskeletal fraction of normal dysferlin and decreases with advanced stages of dementia. This may represent the loss of membrane integrity and alteration in cytoskeleton structure. In contrast, the SDS-soluble fraction represents more insoluble forms of the protein suggesting aggregation of dysferlin may be related to disease progression. This alteration of solubility and accumulation is seen in other neurodegenerative diseases (e.g. Parkinson’s disease, multiple system atrophy, dementia with Lewy bodies) [15, 31].
Dysferlin is a member of ferlin family, homologous to fer1 protein in C. elegans [5]. It is highly expressed in muscle but little is known about the role of the protein in the brain. Several members of the ferlin family have been mapped. Dysferlin is found on chromosome 2p13 [1] and deficiency is associated with three clinical phenotypes: Limb-Girdle muscular dystrophy, type 2B, Miyoshi myopathy and distal anterior compartment myopathy [20, 22, 24]. Another member of the ferlin family, Otoferlin is mapped to chromosome 2p23 and is associated with autosomal recessive nonsyndromic deafness [31]. Two other members of the ferlin family have been described: Myoferlin (10q24) [7] and FerlL4 (20q11) [12]. Myoferlin is a type II transmembrane protein upregulated in Duchenne muscular dystrophy and may have a role in muscle regeneration and repair [10]. The function of Fer1L4 is unknown.
Dysferlin is thought to play a role in muscle membrane maintenance and repair [3, 4, 9, 12, 19]. In C. elegans, the Fer1 protein plays a role in vesicle trafficking and membrane fusion and can bind to phospholipids in a calcium-dependent manner [22]. In dysferlin null-mice, muscle cells are defective in repairing membrane disruptions leading to degeneration [4, 21, 23]. If dysferlin has a similar membrane repair function in neurons, then it is possible that the deposition of dysferlin in AD may be related to the inability of neurons to repair damage due to accumulating Aβ pathology throughout the progression of AD. The findings reported suggest that there may be common mechanisms of membrane repair in degenerative diseases of muscle (e.g. dystrophinopathies and sarcoglycanopathies) and brain (e.g. AD).
The aggregation of normally soluble proteins in fibrillar lesions is the neuropathologic hallmark of many neurodegenerative diseases. Whether the aggregates or their precursors are the actual toxic species is still under debate [8]; however, it is likely that the conversion of proteins from a soluble to insoluble state impairs the long-term viability of neurons. In addition, because dysferlin appears to accumulate in conjunction with Aβ deposition and disease progression it may be able to serve as a marker of disease progression. Continued efforts aimed at understanding abnormalities in the misfolding of protein leading to neurodegenerative disease such as Aβ and tau, or proteins associated with aggregation such as dysferlin will provide insights into disease mechanisms underlying neurological disorders characterized by abundant filamentous lesions.

dysferlin
alzheimer’s disease
amyloid β-protein
protein aggregation
tau protein
neurodegeneration


Concordance between school outcomes and developmental follow-up results of very preterm and/or low birth weight children at the age of 5 years

Introduction Long-term follow-up studies have revealed a high frequency of developmental disturbances in preterm survivors of neonatal intensive care who were formerly considered to be non-disabled. These developmental disturbances interfere with the acquisition of everyday skills and, in particular, with normal school functioning.
Introduction
The survival rate of very preterm (VPT < 32 completed weeks of gestation) and very low birth weight (VLBW <1500 g) children has steadily improved during the last decades, but the prevalence of major disabilities or handicaps in these children has remained stable. Follow-up studies have shown that VPT and VLBW infants are at risk with regard to neurological dysfunctioning such as cerebral palsy and mental retardation [6, 17, 31, 33]. Even more children of these groups experience difficulties in intellectual, speech and language skills as well as clumsiness and attention and behaviour problems, all of which can affect school functioning [4, 12, 13, 16, 19, 24, 26].
Significant/severe neurosensory dysfunctioning is usually detected early, with the result that children with major handicaps often attend a school for special education from school entry. Minor developmental impairments and disabilities, however, frequently go undetected until school age (4 years in The Netherlands). These children start their school career in mainstream education. Once at school, however, certain learning problems may become apparent during the first 2 years (the kindergarten years), but more often these appear only when the children start formal academic training [15, 22, 34]. Several reasons may explain this phenomenon of 'growing into educational deficits' [2, 9, 18, 20, 27].
Mild developmental disturbances usually pose no problem for the child until greater demands have to be met at school or when more detailed evaluations are made. In addition, the cumulative effect of the failure to acquire basic skills and the resulting declining motivation may play an important role. In The Netherlands, schools are focused strongly on the early detection of children who need special assistance. After 1 year of education, at the age of 5, teachers usually have a good impression of both the cognitive and language development of their children and of their motor skills and behaviour. Standardized tests are used to assess children who are not doing well at school, and the data from these tests are used to underpin the need for special help for these children. Additionally, many neonatal intensive care units (NICUs) have follow-up programmes for premature children in which standardized assessments of different domains enable an overall evaluation of the child's development.
Within the framework of follow-up research we assessed a group of VPT and VLBW children at the age of 5 years in different developmental domains. Perinatal, social and economic data as well as information on school functioning were available. This provided the opportunity to compare school outcomes and developmental follow-up outcomes, assuming that developmental skills mediate the effects of biological and social risk factors on school performance [35]. We studied the degree of agreement between follow-up assessments and school outcomes as well as the most important characteristics of the children (perinatal, developmental and socio-economic) in this context. Our aim was to obtain a understanding of the (potential) developmental disturbances in order to facilitate an adequate and timely signalling of children who need special help to meet their developmental capacities.
Methods
Study population
The study population consisted of 768 infants of less than 32 weeks of gestation and/or weighing less than 1500 g who were born between October 1992 and December 1994 and treated in three Dutch neonatal intensive care units: the Radboud University Nijmegen Medical Centre, the Academic Medical Centre, Amsterdam, and the Máxima Medical Centre, Veldhoven. Mortality before the age of 5 years was 131 (17%). Forty-six children (6%) were excluded because they had participated in another study. Thirty-three children (4%) with severe or moderate cerebral palsy, blindness, mental retardation, chromosomal abnormalities, inborn errors of metabolism, personality developmental disorders and/or attendance at special schools or institutions were excluded. Sixty children (8%) of non-Dutch parents were excluded from the analyses since school problems could be caused by language and cultural problems. As a result, 498 children (65%) qualified for the study.
School performance and socio-economic situation
A questionnaire was mailed to the parents of the participating children 1 month before follow-up assessments were to be carried out; these were returned when the family attended the outpatient clinic for the assessment. The paediatrician initiated the follow-up assessment by checking the questionnaire and exploring the answers with the family in more depth. Within the framework of the present study we analysed questions addressing school performance and educational level of the parents (low, middle, high). School outcome was defined in two categories: (1) normal (mainstream education without problems, which means no remedial teaching or other forms of extra help) or (2) problematic (mainstream education with remedial teaching or special mainstream education; i.e. schools for children with mild learning, behavioural or cognitive problems). The parents were asked to fill in the school items together with the schoolteacher. The educational level of the parents was used in this study as a measure of the social and economic situation (SES) because it is the best indicator in predicting school outcomes in The Netherlands [5].
Perinatal data
Perinatal data were prospectively collected during admission in the NICU, stored in the NICU databases and retrieved for data analysis. The following individual characteristics and perinatal variables were considered: gender, multiple birth, gestational age, birth weight, small for gestational age (SGA: birth weight below the 10th centile), low 5-min Apgar score (<7), intracranial haemorrhage grade 3 and 4 (ICH) [32], periventricular leucomalacia (PVL), bronchopulmonary dysplasia (BPD; oxygen dependency at 36 weeks postconceptional age or at 28 days of life), ante- or postnatal use of steroids and length of stay in NICU.
Assessment at the age of 5 years
Clinical assessments were carried out on the health and neurological functioning of the children (not reported in this article) and four developmental domains: cognition, language, motor functioning and behaviour. A paediatrician, a child psychologist and a paediatric physical therapist assessed the children. Appointments were scheduled at random.
Cognitive development was assessed with the Revised Amsterdam Children’s Intelligence Test (RAKIT) for children aged 4–11 years (short version). This version has a correlation of 0.93 with the full-scale test. The concurrent validity with the Wechsler Intelligence Scale for Children-Revised (WISC-R) is 0.86 for total IQ (Intelligent Quotient) [3].
This test included logical reasoning, word knowledge, visual-motor integration and word fluency. It also included visual synthesis for children younger than 5.2 years and visual analysis and memory for children aged 5.2 years or older. The norm score (IQ score) of the test is 100 with a standard deviation (SD) of 15. All scores higher or equal to 85 are classified as normal, while scores below 1 or 2 SD’s are classified as mild or severe cognitive problems, respectively.
The Dutch Language Screening Test assessed language and speech. This test consists of 39 items covering the use of vocabulary, comprehension, memory and production of language, use of plurals, prepositions and pronunciations. The total score varies from 0 to 52, with a score ≤18 considered to be normal, that of 19–25 considered to indicate mild problems and that of >25 to indicate severe problems [7]. The Movement Assessment Battery for Children (Movement ABC) was used to assess motor skills. A total motor impairment score (range: 0–40) was computed by summing the scores on all motor tasks. A total score ≤10.5 (15th centile) was considered to be normal, from 11.0 through to 17.0 (5th centile) considered to indicate children at risk and >17.0 to indicate abnormal motor development [11, 23].
To assess behavioural outcomes, we used the Child Behaviour Checklist for children aged 4–18 years (CBCL). This is a standardized parental questionnaire used to describe the skills and behavioural problems of children. It comprises 113 descriptions of behavioural problems, with each description scored as: 0 (not true), 1 (somewhat or sometimes true) or 2 (very true or often true). A total problem score is obtained by summing all items. The severity of the behaviour problems can be the borderline and clinical cut-off point, corresponding with standardized norm scores of 60 and 63, respectively [1, 30]. A normal score is <60, a mild problematic score is ≥60 and ≤63, and a severe problem score is >63.
Statistical analysis
In order to compare the results of the follow-up assessments and school outcomes, we cross-tabulated categorical data originating from the individual developmental test results of the children with school outcomes (normal or problematic). Normal follow-up outcomes were defined as four normal developmental scores or only one mild developmental problem score. Problematic follow-up outcomes were defined as two mild developmental problem scores, one severe developmental problem score or two or more severe developmental problem scores.
Cross-tabulation was also used to describe the relation between the follow-up assessment outcomes and the school outcomes and the nominal variables (chi-square for testing). A one-way analysis of variance was used to assess within each of the follow-up outcomes the differences between the two types of school outcomes on the interval variables (F test for testing). The general linear model (GLM) was used for multivariate testing of the differences between the two school outcome groups within each of the two follow-up outcome groups.
Results
Of the 498 children included in this follow-up study, 143 (29%) were not assessed for various reasons (Table 1). Consequently, all outcome data, including developmental data, were collected on 355 children at the age of 5 years. No significant differences were found in perinatal data between assessed, partly assessed and unassessed children, with the exception of multiple births in that they were overrepresented in the assessed group [14].
Table 1Eligible and assessed children nPercentagenPercentageCohort 1992–1994768100  Died13117  Excluded because of participation in an other study466  Excluded because of severe handicap and/or in Special Schools334  Children of non-Dutch parents608Eligible49865498100  Address unknown255  Moved outside the country51  Treated in another hospital61  Impossibility to make a convenient appointment296  Refusal by the parents388  Assessment not fully performed408Assessed35571
Mutual correlations between child characteristics and perinatal variables were present but rather low. The percentage of children with all follow-up assessment results being normal or with only one mild developmental problem score was 64% (n=228); 36% (n=127) of the children had at least two mild problems or one or more severe problems according to the four developmental tests. No school problems were reported for 222 children (63%), while 133 children (37%) were reported to school problems as reflected in the need for special help (remedial teaching or other forms of extra help or specialized mainstream education). One hundred and seventy-five children (49% of the study group) had normal school outcomes and normal follow-up outcomes and 80 children (23% of the study group) had problematic follow-up outcomes and problematic school outcomes. These results show that for 72% (n=255) of the children there was a positive relation between school outcomes and the results of the follow-up assessments. Fifty-three children (15% of the study group) had school problems, while their developmental test scores were normal, and 47 children (13% of the study group) did not receive any extra help at school, although they had test scores that fell in the problematic range. However, for 100 children (28%) there was no concordance between the school outcomes and follow-up assessment results (Table 2). A significant difference (χ2=66.44, p=0.00) was found between the two school outcome groups, with more developmental problems in the school problem group. Children who received special help at school although they had obtained normal follow-up assessment results, differed significantly from the children with normal follow-up results without special assistance. This subgroup comprised more boys and SGA children who had lower Movement ABC scores (motor skills) and, in particular, lower language screening test scores.
Table 2Follow-up outcomes versus school outcomesSchool outcomes:Normal: n=222 (100%)Problematic: n=133 (100%)Total: n=355 (100%)Follow-up outcomes:  Normal: n=228 (64%)   Four normal developmental scores124 (56%)26 (20%)   One mild developmental problem score51 (23%)27 (20%)  Subtotal175 →49%53 →15%  Problematic: n=127 (36%)   Two mild developmental problem scores8 (4%)10 (7%)   One severe developmental problem score36 (16%)54 (41%)   Two or more severe developmental problem scores3 (1%)16 (12%)  Subtotal47 →13%80 →23%
For children who did not receive any extra help at school, even though problematic follow-up assessments were found, the results differed significantly from those who did receive extra help. This subgroup was less preterm, had higher Apgar scores, had had BPD less often and was treated less frequently with steroids during the neonatal period. Their scores on the Movement ABC, the language screening test and, in particular on the RAKIT (intelligence test) were better (Tables 3 and 4). Within each of the follow-up assessment groups (normal or problematic assessment results) the MANOVA showed significant differences between the two school outcome groups (F=3.47, p<0.01, df=6.22 and F=3.29, p<0.01, df=6.12, respectively).
Table 3Means, standard deviations (SD) and one-way analyses of variance for normal and problematic school outcomes Follow-up outcomesSchool outcomesNormalProblematicFMeanSDMeanSDGestational ageNormal30.081.8530.501.542.33Problematic30.692.0129.902.303.82*Birth weightNormal1286.24319.201314.28307.09.32Problematic1225.09306.431241.22364.36.06NICU stayNormal27.2022.3325.0716.53.42Problematic26.9120.8236.9437.172.89RAKIT IQNormal104.8211.77102.3910.641.83Problematic95.1712.1986.7412.9213.19***Movement ABCNormal6.114.317.814.236.44*Problematic15.307.7118.389.103.80*Language scoreNormal8.294.6810.785.7410.48***Problematic11.557.8615.488.486.73*CBCL Total problem scoreNormal47.228.8348.068.25.38Problematic55.3210.7055.0512.06.02*p<0.05, ***p<0.001Table 4Comparison of child's characteristics, perinatal data and parents’ education with normal and problematic school outcomes Follow-up outcomesSchool outcomesχ2NormalProblematicPercentagePercentageMaleNormal42574.10*Problematic6463.01Multiple birthNormal35441.43Problematic3236.20SGANormal23374.29*Problematic4253.25Apgar scoreNormal1572.27Problematic47318.73*ICHNormal502.55Problematic46.21PVLNormal22.04Problematic261.09BPDNormal46.24Problematic0168.40**Neonatal steroidsNormal10.31Problematic0115.62*Parents education-highNormal4236.73Problematic39262.15Parents education-middleNormal4751.11Problematic3848.90Parents education-lowNormal1113.18Problematic2326.10*p<0.05, **p<0.01
Discussion
The developmental skills of children are largely expressed in school functioning [25]. Therefore, it would be expected that there is a great concordance between the assessments of the NICU follow-up teams and school teams with respect to the identification of children who need special assistance at school. The results of this study support this expectation with respect to children with good developmental outcome assessments and those with obvious problematic developmental outcomes. While the latter group mainly consisted of children with very low gestational ages and/or serious perinatal problems, some were being faced with the direct adverse consequences of the use of neonatal steroids [36]. It is likely that the problems of these children and the genuine concern of parents and teachers has offered them the extra attention and assistance they required.
Children with relatively normal developmental outcomes who, notwithstanding this, received extra help at their schools, still had language and/or motor problems that apparently interfered with school functioning, thereby causing schoolteachers to provide them with extra attention and help. The overrepresentation of boys in this group, as in both groups with problematic developmental outcomes, is a frequent finding [8] and because their behavior is often more demonstrative there is a stronger need to deal with. More children who were SGA were also found in this group. A number of these were still small at the age of 5 years, which may have led to overprotection or the conviction that the child needs more time or help to be ready for reading, writing or arithmetic [10, 21]. Further research should reveal if the problems of this latter group of children are attributable to not being ready for learning (pre) academic skills or to still being too playful, or whether it concerns serious learning and developmental problems.
The limited number of children with developmental problems who were not signalled by the school did not have special characteristics with the exception of having slightly better developmental outcomes and/or having not been born extremely premature, having not been subjected to neonatal steroids and/or having had a somewhat less complicated neonatal period. It is possible that their developmental problems did not interfere with school functioning or they were able to compensate for them, but it is also possible that their parents and/or teachers did not signal their problems adequately. Parents are sometimes very relieved that the first difficult and worrisome years are over and unconsciously cut themselves off from new problems or both parents and teachers lower their expectations and demands because of overprotection. We did not find that less well-educated parents had more difficulties in mobilizing extra help for their children than relatively well-educated parents. Children of the former displayed developmental problems more often, but they received extra help and assistance in the same ratio as children of relatively more highly educated parents. Based on this line of reasoning a possible overrepresentation of children of less well-educated parents in the unassessed group would not change the degree of concordance between the follow-up results and school outcomes.
One may question whether our developmental tests were sensitive enough to register minor neurological signs. In the overall study the paediatrician did a neurological examination (the modified Touwen examination) [28]. This examination classified 120 (34%) of the children participating in this study with neurological problems (disabling cerebral palsy, non-disabling cerebral palsy, minimal neurological dysfunction and gross motor retardation). Ninety-five (93%) of these children were also classified as problematic by one or more of the other instruments. While these neurological data provide a more complete picture, they do not improve the degree of concordance between school outcome and follow-up outcome (70%).
Of the preterm children tested at age 5 years, one-half were found to have problems in the developmental assessments or in school functioning. This outcome confirms the findings of others that follow-up research in more than one developmental domain in combination with school outcomes shows a more problematic outcome picture [29].
Schools are doing quite well in identifying children with and without developmental problems, but the need for longitudinal multidisciplinary follow-up programmes in different developmental domains remains. Information that promotes the understanding of the development of these children during their school career provides schools and parents with more tools for early detection and will facilitate the design and evaluation of intervention programmes.

school outcome
low birth weight children
premature children
school performance
developmental outcome
very preterm children


The association of heart rate recovery immediately after exercise with coronary artery calcium: the coronary artery risk development in young adults study

We tested whether slower heart rate recovery (HRR) following graded exercise treadmill testing (GXT) was associated with the presence of coronary artery calcium (CAC). Participants (n = 2,648) ages 18–30 years at baseline examination underwent GXT, followed by CAC screening 15 years later. Slow HRR was not associated with higher odds of testing positive (yes/no) for CAC at year 15 (OR = 0.99, p = 0.91 per standard deviation change in HRR). Slow HRR in young adulthood is not associated with the presence of CAC at middle age.
Introduction
The prognostic value of slow heart rate recovery (HRR) after exercise in predicting cardiovascular disease (CVD) events has been established [5, 11, 12, 13, 15]. Initial increases in heart rate with exercise are due to parasympathetic withdrawal while sympathetic activation is responsible for heart rates greater than a 100 beats/minute. In the first two minutes following cessation of exercise, the rapid decrease in heart rate is principally determined by parasympathetic reactivation [1, 8]. Although slow HRR is associated with less autonomic nervous system responsiveness, the underlying mechanisms linking slow HRR to increased cardiovascular morbidity are not well understood. It is possible that slow HRR is associated with a higher susceptibility for atherosclerosis. Prior studies of patients referred for cardiac angiography for suspected ischemic heart disease (IHD) suggest an association between slow HRR and higher atherosclerotic burden [15]. Further, slow HRR has been observed to be associated with several risk factors for atherosclerosis [2, 3, 10]. However, the relationship between HRR and atherosclerosis in the general population has not been studied using a prospective study design. In a cohort of healthy young adults, we investigated whether slow HRR following a graded exercise treadmill test (GXT) was associated with the presence of coronary artery calcium (CAC), a marker of subclinical atherosclerosis, when assessed 15 years later.
Methods and statistical analysis
The Coronary Artery Risk Development in Young Adults study (CARDIA) is a longitudinal study designed to investigate the origins of cardiovascular disease in young adulthood [6]. Beginning in 1985, 5,115 African-American and Caucasian individuals [(African-American (52%) and women (54%)] ages 18–30 were recruited at sites in Birmingham, Alabama; Chicago, Illinois; Minneapolis, Minnesota; and Oakland, California. All participants gave informed consents prior to enrollment.
At baseline, a symptom-limited maximal GXT was administered using the Balke protocol [14]. The test included nine 2-minute stages of increasing difficulty with participants encouraged to exercise to exhaustion, followed by a recovery period at a speed of 3.2 km/hour at 0% grade. HRR was defined as the difference between the maximum HR and HR at 2-minutes into recovery [10]. Participants were ineligible for exercise testing if they were on cardioactive medications, had a resting systolic or diastolic pressure >160 or >100 mmHg, or were febrile at time of examination. The rate of energy expenditure for the completion GXT was estimated and reported in metabolic equivalents (METs), as previously described [14]. Information on physical activity was collected by interview using a standardized questionnaire.
At the year 15 follow-up examination, returning participants (n = 3,043) underwent coronary artery computed tomography (CT) scanning for the measurement of CAC. Mean HRR did not differ between those who did and did not return to the year 15 examination (42.8 versus 42.5 bpm, respectively; p = 0.55). Details of the scanning procedures have been described elsewhere [4]. Briefly, using standardized protocols, two scans were obtained for each participant (1–2 minutes apart) using electron beam CT scanners at the Chicago and Oakland sites and multidetector-row CT scanners at the Birmingham and Minneapolis sites. Calcium scores were calculated across each coronary artery and then summed across all the arteries. The final CAC score of positive scans was calculated as the mean of the two CAC scores obtained from each of the scans.
Participants were sequentially excluded from this analysis for the following reasons: use of medications that affect heart rate (HR) (n = 27), unavailable GXT or CAC data (n = 661), missing data on blood pressure, lipids, glucose, or smoking (n = 353), pregnancy (n = 27) or those absent at the year 15 exam (n = 1,399). Following exclusions, 2,648 participants remained.
Baseline characteristics were compared across sex-specific HRR tertiles. For continuous variables, test for linear trend was performed with HRR as a continuous variable using linear regression models. The Cochran-Armitage test was used to check for linear trend in binomial proportions across the HRR categories. Next, logistic regression was used to estimate the odds of the presence of CAC (defined as a CAC score >0) in relation to year 0 HRR (independent variable). HRR was modeled both as a continuous variable and in tertiles (fastest HRR tertile as the reference). Statistical significance was determined at P < 0.05. All analyses were conducted using SAS version 9.1 (SAS Institute Inc, Cary, NC).
Results
Demographic characteristics of the study sample, by 2-minute HRR tertiles, are presented in Table 1. Mean 2-minute HRR (standard deviation) for men and women were 44.3 (11.4) and 41.7 (11.5) bpm, respectively. Participants with slower HRR had less favorable GXT performance characteristics, a higher resting heart rate (both at year 0 and year 15) and reported less physically activity (both at year 0 and year 15).
Table 1Baseline characteristics according to tertiles of 2-minute heart rate recovery (N = 2,648)aBaselineBaseline sex-specific HRR tertiles1 (slow)23 (fast)P trendNumber of women479463521NANumber of men392391402NAHRR (median [min, max])Women31.0 (1.0, 36.0)41.0 (37.0, 45.0)51.0 (46.0, 116.0)NAMen34.0 (−6.0, 39.0)44.0 (40.0, 48.0)54.0 (49.0, 97.0)NAAge (years)25.4 (3.5)25.1 (3.6)25.0 (3.6)0.003African-American, N (%)360 (41.3)383 (44.9)435 (47.1)0.014Body Mass Index kg/m224.9 (5.2)24.1 (4.2)23.8 (4.0)<0.001Resting heart rate (bpm) at year 071.5 (11.4)69.3 (10.2)66.1 (9.7)<0.001Resting heart rate (bpm) at year 1568.3 (10.8)68.2 (12.1)66.5 (11.3)<0.001Maximum heart rate (bpm)180.1 (15.8)180.0 (14.6)181.0 (13.9)0.001Estimated METs at peak exercise11.8 (3.0)12.1 (2.7)12.5 (2.7)<0.001Total physical activity score (exercise units) at year 0375.8 (273.0)428.6 (317.5)459.7 (307.3)<0.001Total physical activity score (exercise units) at year 15325.4 (278.1)346.9 (288.0)382.4 (296.6)<0.001aValues are expressed as mean (standard deviation) unless otherwise indicated. HRR = heart rate recovery. NA = not applicable. METs = metabolic equivalents
The prevalence of CAC in the study sample was 9.0% (n = 239). Mean HRR at year 0 did not differ between those who had positive CAC scores versus those who had a CAC score of 0 at year 15 (42.8 bpm, for both). The unadjusted odds ratio (OR) for having a CAC score >0 for those in the slowest HRR tertile compared to those in the fastest HRR tertile was not significantly greater than 1.00 (Table 2). Similar findings were observed when HRR was studied as a continuous variable.
Table 2Unadjusted odds ratio (95% confidence interval) for the presence of coronary artery calcium (score >0) at year 15 by baseline 2-minute heart rate recoveryTotal sampleCaucasian menCaucasian womenAfrican-American menAfrican-American womenOdds Ratio (95% CI) for threshold models based on HRR tertilesTertile 1 (slow)1.13 (0.81–1.57)0.90 (0.54–1.50)1.12 (0.53–2.33)1.11 (0.52–2.36)1.44 (0.60–3.49)Tertile 21.20 (0.87–1.66)0.90 (0.54–1.50)0.90 (0.41–2.01)1.55 (0.78–3.09)1.37 (0.57–3.32)Tertile 3 (fast)1.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)Odds Ratio (95% CI) for HRR as continuous variablePer SD change in HRRa0.99 (0.87–1.13)1.04 (0.84–1.28)0.86 (0.65–1.15)0.95 (0.70–1.30)0.88 (0.60–1.27)HRR = heart rate recovery. CAC = coronary artery calcium. CI = confidence interval. SD = standard deviation aSD of HRR was 11.5 bpm
In secondary analysis, similar findings were observed when the presence of higher CAC burden (defined as a score >100 [n = 34]) was studied, as well as when quartiles or quintiles of 2-minute HRR were analyzed. Lastly defining HRR at 1-minute into recovery also did not result in any association between HRR and CAC.
Conclusion
Slower HRR in young adulthood is not associated with the presence of CAC when assessed 15 years later in middle age (average age 40 years). Moreover, mean HRR at baseline did not differ between those with and without measurable CAC at year 15. While previous studies have examined the relationship of HRR with CAD events this study is first to investigate the relationship of HRR with a measure of subclinical atherosclerosis.
Slow HRR has been associated with higher incident all-cause mortality, sudden cardiac death (SCD), and CVD events; however, the underlying mechanisms that link these relationships together are not known [5]. Morshedi-Meibodi et al. [12] using data from the Framingham Heart Study (FHS) observed slow HRR to be associated with coronary heart disease events (defined as acute coronary syndromes or SCD), suggesting a possible relationship between slow HRR and ischemic processes. Similarly, slow HRR has been observed to be associated with several risk factors for atherosclerosis [2, 3, 10]. In contrast to findings from the FHS, Jouven et al. [9] observed slow HRR to be related only to SCD and not death from myocardial infarction. Due to over lapping and discrepancy in clinical end points used in prior HRR studies, it cannot be determined whether slow HRR is associated with atherosclerosis or an increased susceptibility to lethal cardiac arrhythmias.
The ability of coronary CT to assess the global burden of atherosclerosis in young adults is uncertain, and CAC is only a subset of atherosclerosis, representing calcified plaques which tend to be more stable than their lipid-rich counterparts [7]. Despite these limitations, our study suggests that slow HRR in young adulthood is not related to subclinical atherosclerosis at middle age, which supports the hypothesis that slow HRR is associated with mechanisms related to cardiac arrhythmia rather than atherosclerosis.

heart rate
heart rate recovery
coronary artery calcium
autonomic nervous system
atherosclerosis
sudden cardiac death


Substrate specificity of a long-chain alkylamine-degrading Pseudomonas sp isolated from activated sludge

A bacterium strain BERT, which utilizes primary long-chain alkylamines as nitrogen, carbon and energy source, was isolated from activated sludge. This rod-shaped motile, Gram-negative strain was identified as a Pseudomonas sp. The substrate spectrum of this Pseudomonas strain BERT includes primary alkylamines with alkyl chains ranging from C3 to C18, and dodecyl-1,3-diaminopropane. Amines with alkyl chains ranging from 8 to 14 carbons were the preferred substrates. Growth on dodecanal, dodecanoic acid and acetic acid and simultaneous adaptation studies indicated that this bacterium initiates degradation through a Calkyl–N cleavage. The cleavage of alkylamines to the respective alkanals in Pseudomonas strain BERT is mediated by a PMS-dependent alkylamine dehydrogenase. This alkylamine dehydrogenase produces stoichiometric amounts of ammonium from octylamine. The PMS-dependent alkylamine was found to oxidize a broad range of long-chain alkylamines. PMS-dependent long-chain aldehyde dehydrogenase activity was also detected in cell-free extract of Pseudomonas strain BERT grown on octylamine. The proposed pathway for the oxidation of alkylamine in strain BERT proceeds from alkylamine to alkanal, and then to the fatty acid.
Introduction
Primary fatty amines contain a nitrogen atom attached to one long alkyl chain. Commercial primary alkylamines are usually mixtures of homologs because the sources of the hydrophobic groups are fatty acids derived from palm oil, coconut oil or tallow. The alkyl chains therefore vary in both chain length and degree of unsaturation. Alkylamines are primarily introduced into the environment through emissions during industrial production. The alkylamines produced in high volumes are toxic to aquatic organisms (Newsome et al. 1991; Schultz et al. 1991; Finlay and Callow 1997). Microbial degradation of primary fatty amines is therefore important for removal in biological wastewater treatment systems and to maintain low environmental concentrations.
Yoshimura et al. (1980) studied the biodegradability of various fatty amines in the MITI test. The MITI test assesses the ready biodegradability by determining the biological oxygen demand (OECD 1992). Ratios of the biological oxygen demand and theoretical oxygen demand >0.6 were achieved in MITI tests, demonstrating the susceptibility of alkylamines (C8 to C18) to biodegradation (Yoshimura et al. 1980). Dodecylamine was also demonstrated to be biodegradable in another ready biodegradability test, i.e. Closed Bottle test (OECD 1992). The biodegradation of dodecylamine started immediately and reached ∼80% within a week (van Ginkel et al. 1995). The results obtained in ready biodegradability tests are far more satisfactory when supported by pure culture studies. Until now two pure cultures of bacteria have been shown to degrade primary fatty amines (Yoshimura et al. 1980; Selig et al. 1999). Possible intermediates of alkylamine degradation were not identified in these studies. Therefore, much remains to be learned about the substrate specificity of long-chain alkylamine degrading micro-organisms and the biodegradation pathway.
The aim of this study was to investigate how alkylamines are degraded by micro-organisms using a pure culture capable of utilizing alkylamines as sole carbon and energy source. Substrate specificities of the pure culture and cell-free extracts catalysing the initial degradation steps are reported.
Materials and methods
Chemicals
Samples of octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, octadecylamine cocoamine and tallowamine (Armeen®) were provided by Akzo Nobel Surfactants, Stenungsund, Sweden. All other chemicals were of reagent-grade quality and obtained from Sigma Aldrich, Zwijndrecht, The Netherlands or Akcross Organics, Geel, Belgium. The biochemicals were purchased from Boehringer, Mannheim, Germany.
Activated sludge
Activated sludge used as inoculum was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This activated sludge plant treats predominantly domestic wastewater.
Media
The mineral salts medium used for isolation and growth experiments contained the following in 1 l of deionized water; 1.55 g K2HPO4, 0.85 g NaH2PO4, 0.5 g NH4Cl, 0.1 g MgSO4 · 7H2O and 0.1 ml trace solution described by Vishniac and Santer (1957). The medium was sterilized by autoclaving for 20 min, together with any added growth substrate (1 g l−1). The volatile primary amines, i.e. octylamine and decylamine were added after autoclaving the mineral salt medium. When these volatile primary amines were used as growth substrate, silica gel was added to give a final concentration of 32 g l−1. Silica gel was added to reduce the concentration of alkylamines in the water phase.
Enrichment, isolation and growth
Aerobic long-chain alkylamine degrading micro-organisms were isolated from an enrichment culture developed from activated sludge. The enrichment culture was obtained in batch culture flasks containing 200 ml mineral salts medium and 1.0 g l−1 dodecylamine inoculated with 5 ml of activated sludge. After five subcultures a bacterium in this culture was streaked to purity on agar plates containing mineral salts medium, 1.0 g l−1 dodecylamine and 15 g l−1 agar. The cultures and agar plates were incubated at 30°C. The growth rate of strain BERT was recorded by measuring the increase in turbidity in culture flasks with a Hach Ratio XR turbidimeter (Hach, Loveland, CO, USA). The ability of the strain to grow on other substrates was tested using similar media in which dodecylamine was substituted with possible intermediates or other fatty amine derivatives.
Culture conditions and preparation of washed cell suspensions and cell-free extracts
Cells were grown on octylamine as sole nitrogen, carbon and energy source in a continuous culture. This continuous culture was run at 30°C in a 2-l fermentor (Applikon, Schiedam, The Netherlands) with a working volume of 1.0 l. The impeller speed was 500 rpm. The mineral salt medium was pumped continuously into the fermentor at a rate of 1.0 l day−1 by means of a peristaltic pump. Octylamine was introduced separately to the fermentor with a syringe pump and teflon tube giving a final concentration of 1.0 g l−1. This set-up was required because of transport of the alkylamine through silicone tubes. Cells were also cultivated in the fermentor with acetate, octanal and octanoate under the same conditions but with a mineral salts medium containing the respective organic compounds. The pH of the reactor was maintained at 7.
Experiments on the nitrogen balance were also conducted in the fermentor fed with a mineral salts medium without ammonium chloride. The nitrogen recovery was estimated from the amount of nitrogen leaving the fermentor as biomass, ammonium and total nitrogen. The total nitrogen represents the residual substrate and/or water-soluble nitrogen-containing compound formed during the biodegradation process.
Cells were harvested from the continuous cultures by centrifugation at 25,000g for 10 min and washed three times with 15 mM phosphate buffer, pH 7.0. Washed cells (10 ml) were disrupted by a French pressure cell at 35,000 psi (Thermo IEC, Needham Heights, MA, USA) After one pass the insoluble matter was precipitated at 50,000g for 20 min.
Oxygen consumption
The endogenous and substrate-dependent oxygen uptake rates by washed cell suspensions were determined in a polarographic oxygen monitor (Yellow Springs Instruments, Yellow Springs, OH, USA). The polarographic oxygen monitor consisted of a thermostated vessel with a magnetic stirrer, and an oxygen electrode to measure oxygen depletion. Cells were resuspended in phosphate buffer at 30°C in a final volume of 5 ml. After 5 min necessary to determine the endogenous respiration, the substrate-dependent respiration was measured by injection of 0.1 ml of a 1.0 g l−1 substrate solution into the vessel. Suspensions of alkylamines with more than ten carbons and octanal were heated prior to injection.
Enzyme assays
All enzyme assays were performed at 30°C. Spectrophotometric enzyme assays were carried out in a Shimadzu UV 160A spectrophotometer (Shimadzu, Kyoto, Japan) in 1 cm light-path cuvettes. The oxygen uptake was measured with a polarographic oxygen monitor (Yellow Springs Instruments).
Alkylamine dehydrogenase (phenazinium methyl sulfate (PMS)-dependent) was assayed by recording the oxygen uptake in a polarographic oxygen monitor. The reaction mixture (volume 5 ml) contained 0.8 mM PMS and 0.7 g l−1 protein in a phosphate buffer (15 mM; pH 7). The reaction was started by injecting alkylamine in the reaction vessel at a concentration of 4.0 mM. Alkylamine dehydrogenase (PMS-dependent) activity was also determined by colorimetric detection of ammonium in a reaction vessel containing 8.0 mM octylamine, 0.8 mM PMS and 0.5 g l−1 protein in a phosphate buffer (15 mM; pH 7). The enzyme assays were carried out at least twice and the difference in specific activity was less than 20%.
Alkylamine dehydrogenase (NAD(P)-dependent) activity was spectrophotometrically assayed by measuring the increase of NAD(P)H at 340 nm. The reaction mixture consisted of 1.3 mM NAD(P), a phosphate buffer (15 mM; pH 7) and cell-free extract (1.4 g l−1 protein) in a total volume of 3 ml. The alkylamine dehydrogenase (NAD(P)-dependent) activity was measured under anaerobic conditions to minimize NADH oxidase activity. For that purpose the contents of the cuvettes were stoppered with Suba Seal® septa (Sigma Aldrich, Zwijndrecht, The Netherlands) and flushed with nitrogen gas.
Aldehyde dehydrogenase activity was also measured by spectrophotometric and a respirometric assays. Aldehyde dehydrogenase (PMS-dependent) activity was assayed respirometrically. The reaction mixture (volume 5.0 ml) contained a phosphate buffer (15 mM; pH 7), 0.8 mM PMS, cell-free extract (0.7 g l−1 protein). The reaction was started by injecting octanal in the reaction vessel giving 4.0 mM.
Aldehyde dehydrogenase (NAD(P)-dependent) activity was determined by measuring the increase in absorbance at 340 nm. Reaction mixtures contained phosphate buffer (15 mM; pH 7), 0.15 μM aldehyde, 1.0 mM NAD and 0.1 mM KCN and enzyme solution in a volume of 3 ml.
Analyses
Ammonium was determined colorimetrically by forming indophenol blue with hypochlorite and salicylate in the presence of sodium nitroferricyanide as catalyst (Verdouw et al. 1978).
Protein was quantified by bicinchoninic acid method. Cells were first lysed by incubating at 95°C with 1.0 M NaOH. The protein concentration was estimated by using the Bio-Rad Protein assay kit with bovine serum albumin as standard protein. Dissolved organic carbon and total nitrogen were quantified with a Shimadzu TOC apparatus (Shimadzu).
Octylamine was analysed by ion chromatography with a Dionex DX-120 (Dionex, Sunnyvale, CA, USA). The Dionex DX-102 was equipped with an IonPac CS14 (4 mm) analytical column, a 25 μl sample loop, a CSRS-I in the external water mode and a CDM-3 flow-through conductivity cell with a DS4 detection stabilizer. The DX-120 was operated at a column temperature of 20°C and a detector temperature of 35°C. The eluent was deionized water with 5.0 mM methylsulphonic acid and 2.5% acetonitrile. The eluent flow rate was 1.0 ml min−1.
The dry weight of the micro-organisms was determined by washing and concentrating a known volume from the continuous culture through centrifugation (25,000 g for 30 min). The concentrated biomass on a preweighed watch glass was dried for 1.5 h at 104°C.
Results
Isolation and characterization
A culture capable of growth on dodecylamine was readily enriched from activated sludge through repeated transfers of cells to fresh mineral medium containing dodecylamine. Dodecylamine plates streaked with dilutions of this enrichment culture enabled the isolation of bacteria. The predominant isolate BERT was a Gram-negative, motile, non-spore-forming rod, 2.0–3.5 μm long and 0.6–0.8 μm wide. Oxidase, catalase, urease and alcohol dehydrogenase were present. The strain was able to utilize the following substrates: glucose, phenylacetate, citrate, malate, mannose mannitole and gluconate. It did not grow on maltose, trehalose, m-inositol, citraconate, erytritol, sorbitol, d-xylose, d-tartrate and l-arabinose. The strain was not capable of reducing nitrate. The identity of the primary alkylamine degrading bacterium was determined by the profile of cellular fatty acids and partial 16S rRNA gene sequencing. The profile of the cellular fatty acids is typical for the RNA group I of the genus Pseudomonas. The partial sequences have shown a similarity of 99.3% to Pseudomonas putida, P. plecoglosscida and P. alcaligenes. These characteristics only allow to place strain BERT within the RNA group I of the genus Pseudomonas.
Growth
A number of fatty amine derivatives were tested for their ability to support growth of Pseudomonas strain BERT. The strain was found to be able to grow on a wide range of long-chain primary amines, i.e. octylamine (in the presence of silica gel), decylamine (in the presence of silica gel), dodecylamine, tetradecylamine, octadecylamine, cocoamine oleylamine and tallowamine as sole source of carbon. Coco-1,3-diaminopropane and dodecyl-1,3-diaminopropane—both in the presence of silica gel—were also used as carbon and energy source by Pseudomonas strain BERT whereas dodecyldimethylamine, didodecylamine, didodecymethylamine, dodecyltrimethylammonium chloride did not support growth. Mineral salts media amended with possible intermediates of dodecylamine degradation such as dodecanal, dodecanoic acid and acetate supported growth of Pseudomonas strain BERT.
Depletion of dodecylamine to non-detectable concentrations from the nitrogen-free mineral salts medium in batch cultures and the concurrent growth of the isolate clearly demonstrate that this isolate also used alkylamines as its nitrogen source (data not shown).
Octylamine was used as growth substrate in a number of experiments because this amine is soluble in water at a concentration of 1.0 g l−1. Use of octylamine allowed, for instance, accurate measurement of the increase in turbidity due to growth. From the growth curve, a doubling time of 4 h was estimated (data not shown). Growth yield obtained with Pseudomonas strain BERT was 0.4 g dry weight of cells per g of octylamine utilized. The fate of the nitrogen of octylamine in terms of ammonium, biomass-nitrogen, nitrogen-containing soluble microbial products and residual octylamine was assessed in a continuous culture. Total nitrogen, ammonium and biomass-nitrogen were measured in triplicate. Approximately 25 ± 3% of the nitrogen was recovered as biomass-nitrogen. Octylamine-nitrogen was converted for 60 ± 4% into ammonium. Less than 1% of the nitrogen in the effluent was present as octylamine. Approximately 10 ± 2% of the nitrogen was recovered as water-soluble nitrogen-containing products.
Respiration experiments
The alkylamine-degrading isolate was grown on a number of different substrates as sole source of carbon after which respiration rates of washed cell suspensions were examined with a variety of substrates in order to identify possible intermediates of the degradation pathway (Table 1). Washed cell suspensions of Pseudomonas strain BERT grown with octylamine were capable of oxidizing octylamine, octanoate octanal and acetate. Whole cells of Pseudomonas strain BERT also showed octylamine-dependent oxygen uptake when grown with octanal, and octanoate. However, strain BERT grown with acetate did not respire octylamine. Finally, Pseudomonas strain BERT grown on octanal, octanoate and actate displayed significant activities with the alkanal and alkanoate tested (Table 1).
Table 1Oxidation of various potential intermediates of alkylamine degradation by washed cell suspensions of strain BERT grown on octylamine, octanal, octanoate and acetateSubstrateGrowth substrateOctylamineOctanalOctanoateAcetatenmol min−1 mg−1 proteinOctylamine8525440Octanal6243660Octanoate84539041Acetate39354150Rates of oxygen uptake are expressed as nmoles O2 min−1 mg−1 protein after correction for endogenous respiration. The endogenous respiration of the strain grown on octylamine, octanal, octanoate and acetate were 8, 4, 8 and 6 nmol O2 min−1 mg protein−1, respectively
The ability of octylamine-grown strain BERT to oxidize alkylamines was also evaluated (Table 2). Octylamine-grown cells were capable of oxidizing all alkylamines with alkyl chains ranging from 3 to 18 carbon atoms. The highest activity was observed when hexylamine, octylamine and decylamine were added to the reaction vessel. The oxidation rate of nonylamine was comparable to the activity found with octylamine. Propylamine was respired at a low rate. Low activities were also detected with alkylamines with more than 14 carbon atoms. All other fatty amine derivatives except for dodecyl-1,3-diaminopropane were not oxidized by strain BERT grown on octylamine (Table 2).
Table 2Oxidation of various alkylamines by washed cell suspensions and alkylamine dehydrogenase (PMS-dependent)SubstrateWashed cell suspensionCell-free extractnmol min−1 mg−1 proteinButylamine145Hexylamine8830Octylamine8533Decylamine7634Dodecylamine6731Tetradecylamine5735Hexadecylamine3118Octadecylamine125Nonylamine8535Propylamine73Methylamine00Didecylamine00Decyldimethyamine00Decyltrimethylammonium 00Dodecyl-1,3-diaminopropane165Rates of oxygen uptake are expressed as nmoles O2 min−1 mg−1 protein. The rates for the washed cell suspensions are corrected for the endogenous respiration of octylamine-grown cells, i.e. 8 nmol O2 min−1 mg protein−1
Enzymatic activities
Activities of enzymes that may be involved in alkylamine metabolism were tested in cell-free extracts prepared from cells grown on octylamine. Extracts of octylamine–grown cells released ammonium in stoichiometric amounts from octylamine in the presence of PMS (Fig. 1). In another experiment the presence of PMS induced the consumption of 0.08 mM oxygen upon the addition of 0.17 mM octylamine. The activity of the PMS-dependent alkylamine dehydrogenase with dodecylamine was 31 nmol min−1 mg−1 protein. NAD and NADP could not replace the artificial electron acceptor. The substrate specificity of cell-free extracts catalysing the conversion of alkylamines was studied by measuring the oxygen consumption in the presence of PMS. The enzyme was active towards alkylamines with alkyl chains with 8–18 carbons. Propylamine, nonylamine and dodecyl-1,3-diaminopropane also acted as substrate for the enzyme. Among the primary alkylamines examined, the most preferred were alkylamines with alkyl chains length ranging from 8 to 12 carbons. Methylamine secondary, tertiary and quaternary fatty amine derivatives were no adequate substrates (Table 2). Demonstration of alkanal dehydrogenase activity in cell extracts was achieved by incubating cell-free extracts with octanal and PMS and measuring the oxygen consumption. An activity of 19 nmol O2 min−1 mg−1 protein was measured. Alkanal dehydrogenase activity was not detected with other electron acceptors, i.e. NAD and NADP, despite repeated attempts.
Fig. 1Formation of ammonium from 0.78 mM octylamine by cell-free extract of octylamine-grown cells in the presence (open square) and absence (filled square) of PMS. The protein concentration in the assay was 0.5 g l−1
Discussion
The fate of long-chain alkylamines in the environment is largely dependent on the ability of micro-organisms to metabolize these compounds. Although degradation of alkylamines in OECD ready biodegradability tests has been observed (Yoshimura et al. 1980; OECD 1992; van Ginkel et al. 1995), little is known about the intermediates in the biodegradation process and the enzymes involved in the biodegradation. In this paper, we describe the properties of the newly isolated Pseudomonas strain BERT, which is capable of degrading long-chain alkylamines. Yoshimura et al. (1980) also isolated a Pseudomonas strain. Both Pseudomonas strains degraded primary alkylamines with varying alkyl chain lengths. Pseudomonas species are noted for their metabolic diversity and are often isolated from enrichments designed to identify bacteria that degrade anthropogenic organic compounds. Another species known to utilize a long-chain alkylamine as sole source of carbon and energy is from the genus Rhodococcus isolated from top soil (Selig et al. 1999).
A nitrogen mass balance obtained in a continuous culture already, strongly indicated that octylamine a water soluble long-chain alkylamine is completely (ultimately) biodegradable because almost all of the octylamine-nitrogen was converted into biomass-nitrogen and ammonium. Other evidence of complete degradation of alkylamines has been obtained through the proposed biodegradation pathway. The biodegradation of alkylamines was assumed to proceed either via a Calkyl–N cleavage or oxidation at the far-end of the alkyl chain. Based on the induction pattern, and PMS-dependent dehydrogenase activity with alkylamines, the biodegradation of alkylamines is suspected to involve a cleavage of the Calkyl–N bond, which yield the respective alkanals and ammonium. The alkanals are oxidized by a PMS-dependent alkanal dehydrogenase to the respective fatty acids. The induction of long-chain alkanal dehydrogenase activity by growth of the bacterium on octylamine demonstrates that alkanals are significant intermediates. A PMS-dependent long-chain alkanal dehydrogenase was also detected in Acinetobacter calcoaceticus HO1-N grown on hexadecane and hexadecanol (Fox et al. 1992). At present it is unknown if both the alkanal and the alkylamine are dehydrogenated by the same enzyme in Pseudomonas strain BERT. Fatty acids produced by the alkanal dehydrogenase are channelled into the β-oxidation cycle (Ratledge 1994). The degradative pathway shown in Fig. 2 is inducible because octylamine is not oxidized by acetate-grown cells (Table 1). The degradation pathway is quite similar to those described for alkyltrimethylammonium salts, alkyldimethylamines and alkylbis(2-hydroxyethyl)amines (van Ginkel 1996, 2003). This biodegradation pathway provides evidence of total mineralization of alkylamines as indicated by the nitrogen mass balance and the ready biodegradability test results (Yoshimura et al. 1980; van Ginkel et al. 1995).
Fig. 2Proposed pathway for the degradation of alkylamines in Pseudomonas strain BERT; (1) alkylamine dehydrogenase, (2) aldehyde dehydrogenase. The fatty acids formed are channelled into the β-oxidation cycle
PMS, an artificial electron acceptor is generally used to assay enzymes known as quinoprotein enzymes. Physiological electron acceptors are cytochrome c, protein-bound haem and ubiquinone. Methylamine dehydrogenases (de Beer et al. 1980; Duine et al. 1990), short-chain alkylamine dehydrogenases (Shinagawa et al. 1988) and a long-chain alkanal dehydrogenase (Fox et al. 1992) have been identified as quinoproteins. Methylamine dehydrogenases catalyse the oxidative deamination of methylamine to methanal and ammonium (McIntire et al. 1990). The PMS dependent removal of ammonium from alkylamines in strain BERT may be catalysed by an enzyme similar to methylamine dehydrogenases although no activity towards methylamine was detected (Table 2). Zhu et al. (2000) demonstrated that the conversion of one amino acid of methylamine dehydrogenase altered the substrate preference of methylamine dehydrogenase to a large extent. The preferred substrates of the mutated enzyme are alkylamines with at least seven carbons. Long-chain alkylamines are probably also oxidized by an alkylamine dehydrogenase isolated from a butylamine-grown Pseudomonas sp. although the highest activity was found with butylamine (Shinagawa et al. 1988). A quinoprotein was also found in a P. putida catalysing the deamination of propylamine and butylamine both naturally occurring alkylamines (Adachi et al. 1998). Only a long-chain alkylamine dehydrogenase is required to convert anthropogenic alkylamines into compounds, which can enter an existing pathway. This enzyme may have evolved from methylamine dehydrogenases or short-chain alkylamine dehydrogenases involved in the degradation of naturally occurring amines.
A major feature of Pseudomonas strain BERT is its broad substrate specificity with respect to primary alkylamines. Activities of washed cell suspensions and alkylamine dehydrogenase activities in cell-free extracts demonstrate that alkylamines with alkyl chain lengths ranging from 6 to 10 carbons are preferred. Odd alkyl chain amines were also respired by Pseudomonas strain BERT. Water-insoluble long-chain alkylamines are oxidized at a lower rate by washed cell suspensions and alkylamine dehydrogenase of strain BERT. This probably reflects the low bioavailability of the long-chain alkylamines. Limited bioavailability of long-chain alkylamines (C12 to C18) can also be concluded from the non-toxicity of the alkylamines to strain BERT at a concentration of 1.0 g l−1. In batch cultures growth of Pseudomonas strain BERT on octylamine and decylamine was only possible in the presence of silica gel. Silica gel reduces the toxicity of fatty amine derivatives through adsorption onto the silica gel (van Ginkel et al. 1992; Selig et al. 1999).
The ease with which Pseudomonas strain BERT was isolated and the high growth rate of strain BERT are consistent with studies showing ready biodegradability of many primary amines (Yoshimura et al. 1980; van Ginkel et al. 1995). Based on the biodegradation pathway and the broad substrate specificity, significant differences in the biodegradability of alkylamines with varying chain lengths are not expected. However, degradation of primary alkylamines does differ in ready biodegradability tests (Yoshimura et al. 1980). This can be explained by biocidal effects and limited bioavailability. Reading across of the ready biodegradability test results enabled by this study reveals that all long-chain alkylamines—both saturated and unsaturated—are readily biodegradable. Future research should focus on identifying the intermediates and purifying the enzymes involved in the degradation of alkylamines.

substrate specificity
long-chain alkylamines
pseudomonas sp
biodegradation pathway