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Journal ArticleDOI

Developmental changes and polymorphism in human alcohol dehydrogenase

01 Feb 1971-Annals of Human Genetics (Blackwell Publishing Ltd)-Vol. 34, Iss: 3, pp 251-271
TL;DR: Human alcohol dehydrogenase has been investigated by spectrophotometry assay and by starch‐gel electrophoresis and it is shown that the former is superior to the latter in terms of specificity and purity.
Abstract: Ann. H u m . Genet., Lond. (1971), 34, 251 Printed in Great Britain Developmental changes and polymorphism in human alcohol dehydrogenase BY MOYRA SMITH, D. A. HOPKINSON AND HARRY HARRIS M.R.C. Human Biochemical Genetics Unit, Galton Laboratory, University College London I n man, alcohol dehydrogenase (alcohol: NAD oxidoreductase E.C. 1 . 1 . 1 . 1 ) occurs princi- pally in liver, though low levels of activity have aIso been found in lung, kidney and the gastro- intestinal tract (Moser, Papenberg & von Wartburg, 1968). Evidence for at least three distinct isozymes has been obtained by chromatography of liver extracts on CM cellulose (Blair & Vallee, 1966) and also by electrophoresis (Moser et al. 1968; Pikkarainen & Raiha, 1969; Murray & Motulsky, 1970). Von Wartburg, Papenberg & Aebi (1965) reported that certain individuals have an atypical form of alcohol dehydrogenase associated with an increased level of activity. The usual and atypical forms of the enzyme were shown to differ markedly in pH activity curves with ethanol as substrate. The pH optimum for the usual form was found to be pH 10.8 and for the atypical form pH 8.5. The enzymes also differed in the relative rates at which they oxidized various other alcohols, and in the degree of inhibition produced by various metal binding agents. On the other hand no significant differences were observed in Michaelis constants for the substrates ethanol or acetaldehyde or for the corresponding coenzymes NAD or NADH. Also the pH activity curve with acetaldehyde as substrate was essentially the same for both enzymes, having an optimum at pH 6.0-6.5. A simple screening test to distinguish the usual from the atypical enzyme in crude liver homo- genates was designed (von Wartburg et al. 1965). This involves determining the ratio of the activity at pH 11.0 to that at pH 8-8 with ethanol as substrate under standard conditions. The usual enzyme gives a value for this ratio greater than 1.0, and the atypical enzyme less than 1.0. In a survey of 59 liver samples from different individuals in Switzerland, 12 were found to have the atypical alcohol dehydrogenase, and in another series of 50 individuals from London, 2 were found to be atypical (von Wartburg & Schiirch, 1968). The atypical enzyme occurred in indi- viduals varying from 16 to 82 years of age. Pikkarainen & Raiha (1967) reported that alcohol dehydrogenase activity in liver is low during foetal life and reaches adult levels about 5 years after birth. Changes in electrophoretic pattern have also been noted during development (Pikkarainen & Raiha, 1969; Murray & Motulsky, 1970). In the earliest stages only a single isozyme is observed but later further iso- zymes appear. I n adult liver individual variations in the relative contribution of the different isozymes to the total activity have been noted (von Wartburg & Schiirch, 1968), but no clear electrophoretic differences between the usual and atypical alcohol dehydrogenases as determined by the ratio of activity at pH 11.0 and pH 8.8 were detected. The present paper is concerned with a study of human alcohol dehydrogenase in which liver, lung, kidney and intestinal material from foetuses, infants and adults has been examined. The en- zyme has been investigated both by spectrophotometric assay at different pH’s and by starch-gel

Summary (1 min read)

Cilt.2 No.1 2014

  • Bu gelişmeler sonucunda laik modern devlet Avrupa'da 16-17.
  • Görüldüğü üzere batı dünyasında laiklik; kilise ile dünyevi-siyasi otoriteler arasındaki mücadelenin, bir başka deyişle Hıristiyan dini geleneğinin tarihsel pratiğinin sonucudur.
  • Bu nedenle laiklik bağlamında Hıristiyanlık ve İslamiyet arasındaki farklılık temelinden hareketle yapılan yorumlara da eleştirel yaklaşmak gerekir.
  • Ancak bu yorum İncil'deki "Hiç kimse iki efendiye kulluk edemez" vb. gibi tam ters yöndeki birçok hükmün ihlal edilmesine dayalı eksik bir yorumdur.
  • Dolayısıyla bu geleneğin ortaya çıkardığı din-devlet ilişkisi modeli hiçbir zaman "din devleti" modeli değildir; "devlet dini" modelidir.

2. OSMANLI İMPARATORLUĞU DÖNEMİNDE LAİKLİK

  • Boyunca, siyasi alanın dışında laikliğin yerleşebileceği felsefi-kültürel temeller, hemen hemen hiç yoktur.
  • Ancak bu dönemdeki siyasi laiklik; devleti, kağıt üzerinde laikleştirmeye başlamıştır.
  • Siyasal alanda yapılan bu düzenlemeler, Cumhuriyet'e kadar devam eden ikiliklerin bir cephesini oluşturur.
  • Ancak, bu sınırlılıklara rağmen Cumhuriyet aydınları laiklik prensibini devletin anayasal temeline yerleştirebilmişlerdir.
  • Türk İslami'nin esnek İslami yorumlar üretme geleneği, ulemanın asırlar boyu devletin bir uzvu ve tamamlayıcısı olması, Sünni İslam'ın aktivist bir muhalefet Cilt.2 No.1 2014 geleneğinin bulunmaması ve Osmanlı döneminde yaşanan siyasi laiklik bunu mümkün kılan unsurlar arasında zikredilebilir.

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Title
Developmental changes and polymorphism in human alcohol dehydrogenase.
Permalink
https://escholarship.org/uc/item/5p67k47p
Journal
Annals of human genetics, 34(3)
ISSN
0003-4800
Authors
Smith, M
Hopkinson, DA
Harris, H
Publication Date
1971-02-01
DOI
10.1111/j.1469-1809.1971.tb00238.x
Copyright Information
This work is made available under the terms of a Creative Commons Attribution
License, availalbe at https://creativecommons.org/licenses/by/4.0/
Peer reviewed
eScholarship.org Powered by the California Digital Library
University of California

Ann.
Hum.
Genet.,
Lond.
(1971),
34,
251
Printed
in
Great Britain
251
Developmental changes and polymorphism in
human alcohol dehydrogenase
BY
MOYRA SMITH, D.
A.
HOPKINSON
AND
HARRY HARRIS
M.R.C.
Human Biochemical Genetics Unit, Galton Laboratory,
University College London
In man, alcohol dehydrogenase (alcohol: NAD oxidoreductase E.C.
1.1.1.1)
occurs princi-
pally in liver, though low levels
of
activity have aIso been found in lung, kidney and the gastro-
intestinal tract (Moser, Papenberg
&
von Wartburg,
1968).
Evidence for
at
least three distinct
isozymes has been obtained by chromatography
of
liver extracts on CM cellulose (Blair
&
Vallee,
1966)
and also by electrophoresis (Moser
et
al.
1968;
Pikkarainen
&
Raiha,
1969;
Murray
&
Motulsky,
1970).
Von Wartburg, Papenberg
&
Aebi
(1965)
reported that certain individuals have an atypical
form of alcohol dehydrogenase associated with an increased level of activity. The usual and
atypical forms of the enzyme were shown to differ markedly in pH activity curves with ethanol
as substrate. The pH optimum for the usual form was found to be pH
10.8
and for the atypical
form pH
8.5.
The enzymes also differed in the relative rates at which they oxidized various other
alcohols, and in the degree of inhibition produced by various metal binding agents. On the
other hand no significant differences were observed in Michaelis constants for the substrates
ethanol or acetaldehyde
or
for the corresponding coenzymes NAD or NADH. Also the pH
activity curve with acetaldehyde as substrate
was
essentially the same for both enzymes,
having an optimum at pH
6.0-6.5.
A
simple screening test to distinguish the usual from the atypical enzyme in crude liver homo-
genates was designed (von Wartburg
et al.
1965).
This involves determining the ratio of the
activity at pH
11.0
to that
at
pH
8-8
with ethanol as substrate under standard conditions. The
usual enzyme gives
a
value
for
this ratio greater than
1.0,
and the atypical enzyme less than
1.0.
In
a
survey of
59
liver samples from different individuals in Switzerland,
12
were found to have
the atypical alcohol dehydrogenase, and in another series of
50
individuals from London,
2
were
found to be atypical (von Wartburg
&
Schiirch,
1968).
The atypical enzyme occurred in indi-
viduals varying from
16
to
82
years of age.
Pikkarainen
&
Raiha
(1967)
reported that alcohol dehydrogenase activity in liver is low
during foetal life and reaches adult levels about
5
years after birth. Changes in electrophoretic
pattern have also been noted during development (Pikkarainen
&
Raiha,
1969;
Murray
&
Motulsky,
1970).
In the earliest stages only
a
single isozyme is observed but later further iso-
zymes appear. In adult liver individual variations in the relative contribution of the different
isozymes to the total activity have been noted (von Wartburg
&
Schiirch,
1968),
but no clear
electrophoretic differences between the usual and atypical alcohol dehydrogenases as determined
by the ratio of activity at pH
11.0
and pH
8.8
were detected.
The present paper is concerned with
a
study of human alcohol dehydrogenase in which liver,
lung, kidney and intestinal material from foetuses, infants and adults has been examined. The en-
zyme has been investigated both by spectrophotometric assay at different
pH’s
and by starch-gel

252
M.
SMITH,
D.
A.
HOPKINSON
AND
H.
HARRIS
electrophoresis. The findings suggest that
at
least three distinct gene loci may be concerned in
determining the structure
of
the various alcohol dehydrogenase isozymes in man, and that they
are active to markedly different degrees in different stages of development in different tissues.
MATERIALS
AND METHODS
Foetal samples
(9-22
weeks gestation) were obtained from therapeutic abortions. Other samples
were obtained from autopsies, and were mainly from adults but some came from premature
infants and
a
few from young children. The autopsy samples were generally taken'between
12
and
48
hr. after death, the body having been refrigerated in the meanwhile. The samples if
not examined immediately were stored at
-20"
C. Care was taken to avoid repeated freezing and
thawing
of
samples since this was found to lead to a rapid decay of alcohol deliydrogenase activity.
The tissues examined in detail were liver, lung, kidney and intestine. Although traces of alcohol
dehydrogenase activity probably occur in certain other tissues, the activity was found to be much
too low to be investigated by the techniques used here.
Assay of alcohol dehydrogenase was carried out by the method described by von Wartburg
et
al.
(1965)
using ethanol as substrate. In each case an assay was carried out at pH
8.8
and also
at
pH
11.0.
For
assays of the enzyme
in
liver, the tissue was homogenized in
9
volumes of 0.1~
phosphate buffer
pH
7.0
and the debris removed by centrifugation at
3000
rev./min. for
15
min.
;
0.2
ml. of the supernatant was added to
2.8
ml.
of
the reaction mixture, giving
a
final volume of
3
ml. which contained
1.6
x
10-3~ NAD and
1.6
x
1OW2~
ethanol in
3.3
x
M
sodiumpyrophos-
phate buffer
pH
8.8
or
3.3
x
lop2
M
glycine/NaOH buffer pH
11-0.
The reaction was followed in a
Gilford spectrophotometer at
340
mp and at
25"
C.
A
blank reaction without ethanol was also
run. The enzyme activity was expressed as the change in optical density per minute per gram
of tissue. The assays on lung tissue required more concentrated extracts,
so
here the tissue was
homogenized in
2
vol. of phosphate buffer and after centrifugation
0-2
ml. of the supernatant
was used in the reaction mixture.
For electrophoresis the tissue was homogenized with an equal volume of water in the case of
liver.
For
lung, intestine and kidney the samples were generally homogenized directly without
any addition of water. After centrifugation the supernatant was subjected to horizontal starch-
gel electrophoresis the inserts being made with filter paper (Whatman no.
17).
The gel buffer
was
0.025
M
Tris/HCl
at
pH
8.6
and contained
4
x
M
NAD. The bridge buffer was
0.3
M
Tris/HCl
pH
8-6.
Suitably insulated metal cooling plates through which chilled water
(5-10"
C.)
circulated were used to control the gel temperature during electrophoresis. Electrophoresis was
either carried out for
4-5
hr.
at
12
V./cm.
or
for
16
hr.
at
5
V./cm.
Following electrophoresis the gels were sliced and stained using an agar overlay. Alcohol
dehydrogenase was detected with
a
reaction mixture containing
20
mg.
NAD,
10
mg. tetra-
zolium salt MTT,
2
mg. phenazine methosulphate and
0.1
ml. ethanol in
25
ml.
0.05
M
Tris/HCl
buffer at pH
8-6
mixed with
25
ml.
2
yo
aqueous agar
at
55'
C.
The gels were incubated at
37"
C.
for
1-2
hr.
RESULTS
(a) Activity determinations Liver
Alcohol dehydrogenase was assayed at both pH
8.8
and pH
11.0
in
222
different liver samples
using ethanol as substrate. The samples came from individuals of both sexes and covered a

Changes
in
human alcohol dehydrogenase
253
wide range of ages:
56
were from early foetuses
(9-22
weeks gestation),
37
from premature
infants and infants less than
1
year old, and the remainder were adults over
20
years of age.
Fig.
1
shows a plot of activity at pH
11.0
against activity at pH
8-8,
each point representing a
separate individual. Because of the very wide range of activities observed at both pH's
a
log. scale has been used. The points appear to be distributed into two distinct groups. In the
larger group the activity at pH 11.0 is greater than
at
pH
8-8.
In
the smaller group the opposite
is the case. Within each group there is
a
good correlation between activity
at
pH
8.8
and activity
at pH 11.
15
10
8
6
5
4
3
0
-2
v-
I,
CI
x
>
LI
.-
.-
r
:I
I
2
0.8
0.6
0.5
0.4
0.3
0.2
0.1
0
0
-*
. .
00
.
.
.*
..
..
'0
.
v
0'
0.
@o
P
:
.
.O*
.
0' 0
A
A
A
A
A
O.
tl
O
'0
0
OeO
00
0
0
.
A
A
A
A
A
AA
Adults and infants:
pH
ratio >1.0
A
Adults and infants:
pH
ratio
<1.0
0
Foetuses
.
A
I
A
I
I1111
I
I
I
I
IIIII
I I
I
0.2
0.3
0.4
0.50.6
0.8
1
2
3
456
810
20 30
40
ADH
activity at
pH
8.8
Fig.
1.
Plot
on
log.
scale
of
alcohol dehydrogenase activity (0.D.3ao/min./G tissue)
at
pH
11
against
activity
at
pH
8.8
in liver samples from
129
adults,
37
premature infmts and infants between term
and ono year and
56
foetuses. Each point represents
a
separate individual.
The samples with greater activity at pH
8.8
presumably represent those which have the
'atypical' alcohol dehydrogenase described by von Wartburg
et
al.
(1965). This interpretation
has been confirmed in several cases by determining the full pH activity curves. 'Atypical'
samples with higher activities at pH
8.8
were found to have pH optima close to this value whereas
other samples were found to have pH optima close to pH
11.0.

254
M.
SMITH,
D.
A.
HOPKINSON
AND
H. HARRIS
The
pH
activity ratio (activity at pH ll.O/activity
at
pH
8.8)
which was suggested by von
Wartburg
as
a simple discriminant between the two types of enzyme has been calculated for
each sample. The distribution
of
values of this ratio in the series of samples
is
shown in Fig.
2.
Pig.
2
(a)
shows the distribution
of
the values
of
this ratio in
166
samples obtained from adults
24
22
20
18
16
14
12
10
8-
6-
4-
=
2-
'p
-0-
c:
Y)
-
d
.-
-
-
-
-
-
-
-
-
la1
Adults
._
%
0.2 0.6
1.0
1.4
1.8
2.2 2-6 3.0 3.4
3.8
4.2
Foetuses
10
8
6
4
2
0.2 0.6
1.0
1.4
1.8
2-2 2.6 3.0
3.4
3.8 4.2
pH
activity ratio
Fig.
2.
Distribution
of
alcohol dehydrogenase
pH
activity ratios (activity at pH Il/activity at
pH
8.8)
in liver samples from
(a)
129
adults and
37
premature infants and infants between term
arid
one
yoar
of
age,
(b)
56
foetuses. Each square represents one individual.
and infants.
It
is clearly bimodal. Sixteen samples can be seen to have the atypical pH ratio
phenotype, and
150
to have the usual pH ratio phenotype. The atypical phenotype was found
in both
sexes
and over a wide
range
of ages. The youngest was in
a
premature infant of about
28
weeks gestation, and the oldest in an individual
of
90
years.
Fig.
2
(b)
shows the distribution
of
pH ratios in fifty-six samples from young foetuses
(9-22
weeks gestation). Here all the values fall into the range
of
the usual pH ratio phenotype. The

Citations
More filters
Book ChapterDOI
TL;DR: This chapter describes the advances with an emphasis on the structures of the alcohol dehydrogenases and the relationship between structure and function, and establishes that mammalian alcohol dehydrogensases have a distant evolutionary link to both the yeast and bacterial enzymes.
Abstract: Publisher Summary This chapter describes the advances with an emphasis on the structures of the alcohol dehydrogenases and the relationship between structure and function Yeast and mammalian alcohol dehydrogenase differ in substrate specificity and rate of catalytic activity The classic yeast enzyme is more specific for acetaldehyde and ethanol, which is consistent with its recognized physiological Significance to participate in alcohol fermentation at the end of the glycolytic pathway Enzyme forms with other functions and properties also occur in yeast The mammalian enzymes have broad substrate specificity and, even with primary alcohols, the maximum activity is not observed with ethanol Alcohols including ethanol, produced in the intestinal tracts mainly by bacterial actions, are found in the portal vein One physiological function of liver alcohol dehydrogenase may be to metabolize these products Structural studies have established that mammalian alcohol dehydrogenases have a distant evolutionary link to both the yeast and bacterial enzymes Ingested alcohol is metabolized to acetaldehyde mainly by the action of liver alcohol dehydrogenase

656 citations

Journal ArticleDOI
TL;DR: Variant isoenzymes produced at the two polymorphic alcohol dehydrogenase loci account for the differences in enzyme electrophoretic patterns observed among individuals, and this may accounts for the 2‐ to 3‐fold variation in alcohol elimination rate among individuals.

562 citations

Journal ArticleDOI
TL;DR: Many of the phase I drug-metabolizing enzyme exhibit dynamic perinatal expression changes that are regulated primarily by mechanisms linked to birth and secondarily to maturity, suggesting that birth is necessary but not sufficient for the onset of expression.
Abstract: Although some patterns are beginning to emerge, our knowledge of human phase I drug-metabolizing enzyme developmental expression remains far from complete. Expression has been observed as early as organogenesis, but this appears restricted to a few enzymes. At least two of the enzyme families that are expressed in the fetal liver exhibit a temporal switch in the immediate perinatal period (e.g., CYP3A7 to CYP3A4/3A5 and FMO1 to FMO3), whereas others show a progressive change in isoform expression through gestation (e.g., the class I alcohol dehydrogenases). Many of the phase I drug-metabolizing enzyme exhibit dynamic perinatal expression changes that are regulated primarily by mechanisms linked to birth and secondarily to maturity. A few of these enzymes are not detectable until well after birth, suggesting that birth is necessary but not sufficient for the onset of expression (e.g., CYP1A2). Tissue-specific expression adds to the complexity during ontogeny. For example, CYP3A7 expression is restricted to the fetal liver. However, with few exceptions, complete temporal relationship information during development is not known. Furthermore, most studies have concentrated on hepatic expression and much less is known about extrahepatic developmental events.

350 citations


Cites background from "Developmental changes and polymorph..."

  • ...The seminal and pioneering work of Smith et al. (1971) provided definitive evidence for the progressive expression of the three class 1 enzymes, ADH1A (ADH ), ADH1B (ADH ), and ADH1C (ADH ), during development....

    [...]

  • ...Differing from the earlier report by Smith et al. (1971), ADH1A transcripts dominated in the lung, whereas in the fetal kidney, only transcripts for either or both ADH1B and ADH1C were present....

    [...]

Journal ArticleDOI
TL;DR: An overview of DME developmental expression patterns is provided and some implications of the data with regards to drug therapy are discussed and common themes emerging from the current knowledge also will be discussed.

345 citations

Journal ArticleDOI
W. Kalow1
TL;DR: Interethnic differences in drug-metabolising capacity may be substantial, and they are sufficiently frequent to warrant attention, and new efforts are needed to adapt pharmacokinetic methods to make them suitable for population studies.
Abstract: Interethnic differences in drug-metabolising capacity may be substantial, and they are sufficiently frequent to warrant attention. Such differences may consist of different mean values of quantitative traits in separate populations, or of different frequency distributions as produced by the occurrence of genetic enzyme variants. The collection of population data requires the investigation of substantial numbers of subjects. This may be no problem if drug-metabolising enzymes occur in blood or are sufficiently stable in their tissues to allow investigation in vitro. However, if investigations require the use of probe drugs, new efforts are needed to adapt pharmacokinetic methods to make them suitable for population studies. This development of methods is further called for because genetic variants seem to be more easily detected through the assessment of particular metabolites than through the determination of pharmacokinetic parameters of the parent drug. Many studies with probe drugs comparing different populations have given results that are equivocal in terms of the nature-nurture interplay. However, a set of data with antipyrine has pointed to environmental factors as the principal determinant of differences in metabolising capacity, while data with debrisoquine have indicated monogenically controlled variation of one facet of the cytochrome P-450 system. In several instances, statistically significant differences between population means have been established by testing small numbers of subjects, numbers insufficient to establish distribution patterns that would allow the recognition of genetic polymorphism. The populations studied range from Greenlanders to South African Blacks, but most comparisons pertain to Caucasians and Orientals.

238 citations

References
More filters
Journal ArticleDOI
TL;DR: The results presented here suggest that variants may be found in alcohol dehydrogenase during development, and studies which make use of purified enzyme and isoenzyme preparations are needed.
Abstract: Extract: In the present study, alcohol dehydrogenase activity (ADH) has been measured in human liver tissue during development, and a comparison made between certain kinetic properties of crude enzyme preparations from fetal and adult liver. The fetal livers were obtained from legal abortions. The liver tissue from children and adults was acquired during surgery of the abdomen in cases where no macroscopic abnormality of the livers was observable. The liver tissue was frozen immediately after excision. Studies indicated that the enzyme activity was stable at −20° for up to 6 days. All the determinations were made within 24 hours of the liver being sampled. A 10% liver homogenate was prepared in ice-cold 0.25 M sucrose containing 1 % Triton X-100. After centrifugation of the homogenate for 10 minutes at 5000 × g at 3°C, samples were taken from the supernatant for protein determination and for enzyme assays. Table I presents the enzyme activity levels in human liver during the development from a fetus to an adult organism. The results are expressed as milliunits per g liver wet weight and per 100 mg soluble liver protein. ADH activity is detectable in 2-month-old fetuses, although it amounts to no more than 3–4: % of adult activity. Activities of adult range are found after 5 years of age. Considerable varation exists in the activity of adult livers. The relationship between pH and the rate of reaction appears in figure 1. The final pH figures of the reaction mixtures are given; these were determined with a Radiometer pH meter. Adult human liver ADH has a pH optimum of about 10.4, and the pH optimum for the fetal enzyme preparations is 10.0. Lineweaver-Burk analyses which illustrate the relationship between the concentration of ethanol and NAD, and the ADH activity in adult and fetal liver are presented in figures 2 and 3. The results presented are typical of three cases studied in each group. Close agreement was found for all constants in the different enzyme preparations examined. The apparent Km values for ethanol were 3400 μM. and 1100 μM, and for NAD 70 μM and 150 μM in fetal and adult enzyme preparations respectively. Speculation: These results do not constitute a foundation for a conclusion whether the fetal and the adult enzyme preparations are composed of a similar isoenzyme pattern. Studies which make use of purified enzyme and isoenzyme preparations are needed. Nevertheless, the results presented here suggest that variants may be found in alcohol dehydrogenase during development.

187 citations

Journal ArticleDOI
TL;DR: A simple screening test is described to differentiate between atypical and normal alcohol dehydrogenase in liver homogenate.
Abstract: An atypical alcohol dehydrogenase was found in two human livers. The anomalous enzyme, which has been purified, differs from the normal regarding (a) pH rate profile, (b) substrate specificity, and (c) sensitivity to metal binding agents. Thiourea inhibits the variant enzyme, but activates normal human liver alcohol dehydrogenase. A simple screening test is described to differentiate between atypical and normal alcohol dehydrogenase in liver homogenate. Total alcohol dehydrogenase activity was considerably higher in livers containing the atypical enzyme.

181 citations