Microbial metabolism of 1-aminoanthracene by Beauveria bassiana

Article abstract of DOI:10.1016/j.bmc.2008.03.030

The carcinogen and mutagen, 1-aminoanthracene, was efficiently metabolized by the fungal strain Beauveria bassiana ATCC 7159 to yield three new metabolites identified as 1-acetamido-5-[(4′-O-methyl-β-d-glucopyranosyl)oxy]anthracene, 1-acetamido-8-[(4′-O-methyl-β-d-glucopyranosyl)oxy]anthraquinone, and 1-acetamido-6-[(4′-O-methyl-β-d-glucopyranosyl)oxy]anthraquinone, together with 1-acetamidoanthracene and 1-acetamidoanthraquinone. Formation of these metabolites suggests that the metabolic pathways of 1-aminoanthracene in B. bassiana ATCC 7159 involve acetylation, oxidation, hydroxylation, and O-methylglucosylation.

Full text of DOI:10.1016/j.bmc.2008.03.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5085–5089
Microbial metabolism of 1-aminoanthracene by Beauveria bassiana
Jixun Zhan and A. A. Leslie Gunatilaka*
Southwest Center for Natural Products Research and Commercialization, Office of Arid Lands Studies,
College of Agriculture and Life Sciences, The University of Arizona, 250 East Valencia Road, Tucson, AZ 85706-6800, USA
Received 16 January 2008; revised 5 March 2008; accepted 10 March 2008
Available online 14 March 2008
Abstract—The carcinogen and mutagen, 1-aminoanthracene, was efficiently metabolized by the fungal strain Beauveria bassiana
ATCC 7159 to yield three new metabolites identified as 1-acetamido-5-[(40-O-methyl-b-D-glucopyranosyl)oxy]anthracene, 1-acetam-
ido-8-[(40-O-methyl-b-D-glucopyranosyl)oxy]anthraquinone, and 1-acetamido-6-[(40-O-methyl-b-D-glucopyranosyl)oxy]anthraqui-
none, together with 1-acetamidoanthracene and 1-acetamidoanthraquinone. Formation of these metabolites suggests that
the metabolic pathways of 1-aminoanthracene in B. bassiana ATCC 7159 involve acetylation, oxidation, hydroxylation, and
O-methylglucosylation.
ꢀ 2008 Published by Elsevier Ltd.
1. Introduction
mation of 1-aminoanthracene resulted in five
metabolites of which three were new compounds. Struc-
tural analysis of these metabolites suggested that
biotransformation pathway of this substrate by B. bassi-
ana involved acetylation, oxidation, hydroxylation, and
O-methylglucosylation.
Aromatic amines are widely used in industry and many
aniline derivatives are synthetic precursors to a variety
of colorants routinely employed in the textile, paper,
leather, plastic, cosmetic, pharmaceutical, and food
industries.1,2 Most aromatic amines are known to be
mutagenic and carcinogenic especially after their meta-
bolic activation.3 The aromatic amine, 1-aminoanthra-
cene (1), has been reported to be mutagenic3 and to
possess genotoxic potency in Drosophila melanogaster.4
This compound could also be transformed into a di-
rect-acting mutagen by the reaction with nitrites5 fre-
quently used in the preservation of meat products.
Although bacterial biodegradation and biotransforma-
tion of polycyclic aromatic hydrocarbons (PAHs) such
as anthracene and phenanthrene are known,6–8 no bio-
transformation studies of 1-aminoanthracene have thus
far been reported. In continuing our interest in biotrans-
formation of aromatic compounds,9–11 we have investi-
gated the metabolism of 1-aminoanthracene by the
fungal strain, Beauveria bassiana ATCC 7159. This
strain has previously been used in the biotransformation
of more than 300 different substrates12 and has dis-
played a variety of enzymatic activities.13–16 Biotransfor-
A probable sequence of these enzymatic reactions is
proposed.
2. Results and discussion
Five metabolites of 1-aminoanthracene (1) were isolated
from the biotransformation broth and freeze–dried
mycelia of B. bassiana ATCC 7159. Two of the metabo-
lites were identified as 1-acetamidoanthracene (2) and 1-
acetamidoanthraquinone (3) on the basis of their mass
and NMR spectroscopic data, and by comparison with
synthetic samples obtained by acetylation of 1-aminoan-
thracene and 1-aminoanthraquinone. The third metabo-
lite (4) was determined to have the molecular formula
C23H25NO7 by high-resolution ESI-MS and its NMR
spectra indicated structural similarities to 2. Compared
to 1-acetamidoanthracene (2) it had an additional
C7H13O6 which was consistent with the presence of a
4-O-methylglucose moiety, a group which we have pre-
viously found B. bassiana ATCC 7159 was capable of
introducing to aromatic substrates.11 The 13C NMR
spectrum of this metabolite analyzed with the help of
its DEPT spectrum showed the presence of 13 methines,
one oxygenated methylene, two methyls, and seven
Keywords: 1-Aminoanthracene; Biotransformaion; Beauveria bassi-
ana; Microbial metabolism; Acetylation; Oxidation; Hydroxylation;
O-Methylglucosylation.
*
Corresponding author. Tel.: +1 520 741 1691; fax: +1 520 741
1468; e-mail: leslieg@ag.arizona.edu
0968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.03.030

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J. Zhan, A. A. Leslie Gunatilaka / Bioorg. Med. Chem. 16 (2008) 5085–5089
Table 2. 13C NMR (125 MHz) spectroscopic data for metabolites 4–6
(in DMSO-d6)
quarternary carbon signals. It had an aromatic methine
less and a quarternary carbon (d 152.7) more than 2 (Ta-
ble 2) suggesting that it was substituted by another
group at an aromatic position. The presence of a 4-O-
methylglucose moiety in this metabolite was confirmed
by the occurrence of five methine carbon (CH) signals
at d 100.5, 73.6, 75.7, 79.0, and 76.1, one methylene car-
bon (CH2) signal at d 60.3 and one OCH3 signal at d
59.7 in its 13C NMR spectrum. Two singlets at d 8.70
Position
d, multiplicities
4
5
6
1
133.4, qC
120.2, CH
125.0, CH
125.5, CH
152.7, qC
106.8, CH
125.8, CH
121.6, CH
120.3, CH
131.7, qC
126.4, qC
121.4, CH
124.3, qC
131.3, qC
168.9, qC
13.7, CH3
100.5, CH
73.6, CH
75.7, CH
79.0, CH
76.1, CH
60.3, CH2
59.7, CH3
141.6, qC
125.2, CH
136.7, CH
122.5, CH
123.1, CH
136.3, CH
121.6, CH
158.2, qC
187.1, qC
115.1, qC
118.0, qC
181.8, qC
136.4, qC
136.4, qC
170.3, qC
26.2, CH3
101.2, CH
74.4, CH
77.2, CH
79.7, CH
76.7, CH
61.1, CH2
60.6, CH3
141.3, qC
125.4, CH
135.4, CH
121.7, CH
112.6, CH
161.9, qC
122.1, CH
129.7, CH
185.5, qC
127.9, qC
117.4, qC
181.8, qC
134.2, qC
133.9, qC
169.4, qC
25.3, CH3
99.7, CH
73.3, CH
75.9, CH
78.7, CH
75.7, CH
60.0, CH2
59.6, CH3
2
3
4
5
1
and d 8.97 in the H NMR spectrum of this metabolite
were assigned to H-9 and H-10, respectively. The
6
7
1
remaining signals in its H NMR spectrum (Table 1)
8
9
9a
analyzed with the help of COSY showed two similar
spin-systems of three aromatic protons with coupling
constants suggesting C-5 or C-8 to be the likely positions
to which the 4-O-methylglucose moiety was introduced.
The high J value of the anomeric proton H-10 suggested
the presence of a b-glycosidic linkage in 4. The correla-
tions of proton H-10 (d 8.97) with C-4 (d 125.5) and C-5
(d 152.7) in the HMBC spectrum indicated that the 4-O-
methylglucose is attached to C-5 through an oxygen.
Based on the foregoing evidence, this metabolite was
identified as 1-acetamido-5-[(40-O-methyl-b-D-glucopyr-
anosyl)oxy]anthracene (4).
9b
10
10a
10b
11
12
10
20
30
40
50
60
40-OCH3
Metabolite 5 exhibited a peak due to [M+Na]+ at m/z
480.1270 in its high-resolution ESI-MS, suggesting a
molecular formula of C23H23NO9. Spectroscopic data
for this compound were very similar to those of 1-acet-
amidoanthraquinone (3) and suggested that a 4-O-meth-
ylglucose moiety was introduced during the
biotransformation. This was further confirmed by the
presence of sugar signals at d 101.2, 74.4, 77.2, 79.7,
76.7, 61.1, and 60.6 in its 13C NMR spectrum (Table
2). The two carbonyl group signals at d 187.1 and d
181.8 confirmed that it is an anthraquinone. The HMBC
correlations of H-6 to C-8 and C-10a as well as H-5 to
C-10 and C-7 suggested that the newly introduced 4-
O-methylglucose moiety is linked to the anthraquinone
moiety at C-8 through an oxygen atom. Based on the de-
tailed analysis of COSY, HSQC, and HMBC spectra,
this biotransformation product was identified as 1-acet-
amido-8-[(40-O-methyl-b-D-glucopyranosyl)oxy]anthra-
quinone (5).
Metabolite 6 had the same molecular formula as 5. In its
13C NMR spectrum (Table 2) two carbonyl signals at d
185.5 and d 181.8, the signals for NHCOCH3 moiety at
C-1, and the signals for the 4-O-methylglucose moiety
Table 1. 1H NMR (500 MHz) spectroscopic data for metabolites 4–6
(in DMSO-d6)
Position
d (multiplicities, J)
1
were observed. In its H and COSY NMR spectra, the
4
5
6
spin-system for H-2, H-3, and H-4 was the same as that
of 5, while the second spin-system showed a different
splitting pattern (d, dd, and d) with coupling constants
of 8.8 and 2.4 Hz, from which it could be deduced that
the 4-O-methylglucosyloxy moiety is located at C-6 or
C-7. The HMBC correlations of H-8 to C-6 and C-10a
confirmed the attachment of the sugar moiety to C-6
of 6. Thus, this metabolite was identified as 1-acetam-
ido-6-[(40-O-methyl-b-D-glucopyranosyl)oxy]anthraqui-
none (6).
1-NH
10.4 (s)
7.75*
11.9 (s)
8.87 (d, 6.1)
7.86*
12.1 (s)
2
3
4
5
6
7
8.96 (d, 8.1)
7.87 (t, 8.0)
7.93 (d, 7.0)
7.66 (d, 2.4)
7.48 (t, 7.8)
7.91 (d, 8.4)
7.82*
7.67 (d, 8.3)
7.84*
7.91 (d, 7.7)
7.12 (d, 7.4)
7.43 (t, 8.0)
7.54 (dd,
8.8, 2.4)
8.21 (d, 8.8)
8
7.75*
8.70 (s)
9
10
12
10
20
30
40
50
60
8.97 (s)
2.25 (s)
2.25 (s)
2.26 (s)
Biotransformation of 1-aminoanthracene (1) by B. bas-
siana ATCC 7159 resulted in three major (4–6) and
two minor (2 and 3) metabolites. As the chemical acety-
lation of 1-aminoanthraquinone required more vigorous
conditions than those required for 1-aminoanthracene
(see Section 3, 3.8 and 3.9), it is probable that during
the microbial biotransformation, the acetylation reac-
tion of 1-aminoanthracene (1) to provide 1-acetamido-
anthracene (2) would have preceded the oxidation of it
to 1-aminoanthraquinone (3). Feeding B. bassiana
5.13 (d, 7.1)
3.53 (m)
3.48 (m)
3.13 (t, 9.0)
3.52 (m)
5.15 (d, 6.9)
3.43 (m)
3.45 (m)
5.18 (d, 7.7)
3.32 (m)
3.48 (m)
3.09 (t, 9.0)
3.48 (m)
3.08 (t, 9.2)
3.51 (m)
3.69 (dd, 11.2, 4.8) 3.64 (brd, 11.6) 3.64 (dd,
10.0, 5.5)
3.56 (m)
40-OCH3 3.49 (s)
3.53 (m)
3.47 (s)
3.54 (m)
3.46 (s)
* Overlapping signals.

J. Zhan, A. A. Leslie Gunatilaka / Bioorg. Med. Chem. 16 (2008) 5085–5089
5087
ATCC 7159 with synthetic 1-acetamidoanthracene (2)
resulted in the isolation of metabolites 3–6 whereas feed-
ing with synthetic 1-acetamidoanthraquinone (3) re-
sulted in only metabolites 5 and 6 (Fig. 1). The above
findings together with the difference in rates observed
for the chemical acetylation of 1-aminoanthracene (1)
and 1-aminoanthraquinone (2) suggested that the sub-
strate, 1-aminoanthracene, was first acetylated giving
1-acetamidoanthracene which would then undergo fur-
ther biotransformation by two different pathways
(Fig. 1). The 4-O-methylglucose moiety can then be
introduced to 1-acetamidoanthracene to form 1-acetam-
ido-5-[(40-O-methyl-b-D-glucopyranosyl)oxy]anthracene
(4), involving hydroxylation of the aromatic carbon
C-5 followed by 4-O-methylglucosylation of the newly
introduced hydroxyl group. On the other hand, 1-ace-
tamidoanthracene can be first oxidized to 1-acet-
amidoanthraquinone (3) followed by hydroxylation at
C-6 or C-8 and 4-O-methylglucosylation of the newly
introduced hydroxyl groups affording metabolites 5
and 6 (Fig. 1). The overall metabolism of 1-aminoan-
thracene by B. bassiana therefore appears to involve
four different enzymes, acetylase, oxidase, hydroxylase,
and 4-O-methyglucosyltransferase. This constitutes the
first report of the microbial metabolism of 1-aminoan-
thracene (1).
acetamido groups may possess decreased mutagenicity
compared with the substrate, 1-aminoanthracene (1),
and that the metabolism of 1 by B. bassiana may be car-
ried out for the detoxification of this mutagen.
3. Experimental
3.1. General methods
Melting points were determined with a Gallenkamp
micromelting point apparatus and are uncorrected.
Optical rotations were measured with a Jasco DIP-370
digital polarimeter. Infrared (IR) spectra were recorded
in KBr disks with a Shimadzu FTIR-8300 spectrometer.
UV spectra were recorded in CH3OH using a Shimadzu
UV-1601 spectrometer. 1D (1H and 13C) and 2D
(DEPT, COSY, HSQC, and HMBC) NMR spectra were
recorded on a Bruker DRX-500 instrument (at 500 MHz
for 1H NMR and at 125 MHz for 13C NMR). The
chemical shift (d) values are given in parts per million
(ppm) relative to TMS at 0 ppm. The coupling constants
(J values) are reported in Hertz (Hz). Standard pulse se-
quences were used for distortionless enhancement by
polarization transfer (DEPT), heteronuclear single
quantum coherence (HSQC), and heteronuclear multi-
ple bond coherence (HMBC) experiments. High-resolu-
tion electrospray ionization mass spectra (ESI-MS) were
obtained with a JEOL HX110A mass spectrometer.
Although in this study, we have not evaluated the muta-
genicity of 1-aminoanthracene (1) and its biotransfor-
mation products 2–6, Trieff et al. have previously
reported that the mutagenicity of aromatic amines was
enhanced with increasing lipophilicity and was reduced
when the amino group was located ortho to the ring
junction or present as an acetamido moiety.17 Thus, it
is possible that the biotransformation products 2–6 con-
taining polar O-methylglucosyl moieties and carrying
3.2. Substrate, fungus, and the medium
The substrate, 1-aminoanthracene, purchased from Sig-
ma–Aldrich Inc. (Milwaukee, WI, USA) was found to
be ca. 90% pure. It was therefore purified by reversed-
phase thin-layer chromatography (RP-TLC) by eluting
H
COCH3
11 12
N
9
8
1
9a
9b
10b
10a
5
10
4
6'
CH2OH
O
4'
O
H3CO
1'
HO
4
OH
iii
H
COCH3
NH2
N
9
1
8
5
i
H
COCH3
O
N
ii
10
4
2
1
H3CO
HO
O
CH2OH
O
iii
iii
3
H
COCH3
O
N
HO
H
COCH3
O
O
N
CH2OH
O
O
H3CO
HO
O
OH
O
5
6
Figure 1. Microbial metabolism of 1-aminoanthracene by B. bassiana ATCC 7159. Reagents: (i) Acetylation; (ii) oxidation; (iii) hydroxylation
followed by O-methylglucosylation.

5088
J. Zhan, A. A. Leslie Gunatilaka / Bioorg. Med. Chem. 16 (2008) 5085–5089
with MeOH/H2O (9:1) to obtain material of >99% pur-
ity as determined by HPLC analysis. The fungus, B. bas-
siana ATCC 7159 (ATCC, Manassas, USA), was
cultured in potato dextrose broth (PDB, Difco, Plym-
outh, MN, USA) medium.
which a dark yellow extract (63.1 mg) was obtained after
the evaporation of the solvent. This extract was subjected
to Sephadex LH-20 (3.0 g) size-exclusion chromatogra-
phy eluting sequentially with CH2Cl2 (30 mL), 5% MeOH
in CH2Cl2 (30 mL), 10% MeOH in CH2Cl2 (20 mL), 20%
MeOH in CH2Cl2 (10 mL), and MeOH (20 mL). Twenty-
three fractions (5 mL each) were collected and combined
into eight fractions on the basis of their TLC profiles to
provide fractions F (5.2 mg), G (11.3 mg), H (1.8 mg), I
(0.5 mg), J (6.0 mg), K (6.6 mg), L (6.8 mg), and M
(19.8 mg). Fractions G and H were separated by re-
versed-phase TLC (MeOH/H2O, 85:15), leading to the
isolation of an additional amount of 1-acetamidoanthra-
cene (2) (0.4 mg). Fractions I, J, and K were subjected to
normal phase TLC (MeOH/CH2Cl2, 13:87) followed by
purification on reversed-phase TLC (MeOH/H2O,
30:70), yielding further quantities of 1-acetamidoanthr-
aquinone (3) (7.6 mg), 1-acetamido-8-[(40-O-methyl-b-
D-glucopyranosyl)oxy] anthraquinone (5) (3.4 mg), and
1-acetamido-6-[(40-O-methyl-b-D-glucopyranosyl)oxy]-
anthraquinone (6) (3.6 mg).
3.3. Culture and biotransformation procedures
A small scale metabolism experiment of 1-aminoanthra-
cene (1) with B. bassiana ATCC 7159 was carried out in
a 125 mL Erlenmeyer flask containing 50 mL of the
PDB medium. The flask was placed in a rotary shaker at
150 rpm and 25 ꢁC. After 3 days, when the fermentation
broth turned red, 3.0 mg of 1-aminoanthracene (300 lL
of a solution of 10 mg/mL in acetone) was added and
the flask was maintained under the same conditions for
additional 7 days. Two controls were used. Culture con-
trol consisted of fermentation broth of B. bassiana ATCC
7159 with the same volume of acetone but without the
substrate, and the substrate control consisted of sterile
PDB medium with the same amount of a solution of
1-aminoanthracene in acetone but without the microor-
ganism. Both controls were incubated under the same
conditions. The preparative scale experiment was carried
out in 3· 250 mL flasks, each containing 100 mL of PDB
under the same conditions as for the small scale experi-
ment. A total of 40 mg of 1-aminoanthracene were used
(13.3 mg/flask).
3.4.1. 1-Acetamido-5-[(40-O-methyl-b-D-glucopyranosyl)
25
D
oxy]anthracene (4). White powder; mp 250–251 ꢁC; ½aꢀ
ꢁ69.5 (c 0.05, MeOH); UV (MeOH) kmax (loge) 208.0
(5.29), 246.5 (5.47), 357.0 (4.76), 375.0 (4.88), 394.5
(4.78) nm; IR (KBr) mmax 3373, 3246, 3053, 2928, 2889,
1653, 1543, 1462, 1313, 1263, 1215, 1109, 1042, 991
cmꢁ1; HRESIMS m/z 428.1712 [M+1]+ (calcd for C23H26
1
3.4. Extraction, isolation, and identification of the
metabolites
NO7, 428.1709). H and 13C NMR data, see Tables 1
and 2.
The cultures were filtered to separate mycelia, and the
combined culture filtrate (300 mL; pH 2.69) was neutral-
ized with 1.0 N aqueous NaOH and extracted withEtOAc
(3· 300 mL). Evaporation of EtOAc under reduced pres-
sure yielded a dark yellow solid (57.5 mg), a portion
(53.1 mg) of which was subjected to size-exclusion chro-
matography on a column of Sephadex LH-20 (3.0 g)
and eluted with hexane/acetone (3:2) (30 mL), hexane/
acetone (1:1) (30 mL), hexane/acetone (1:2) (30 mL), hex-
ane/acetone (1:3) (30 mL), acetone (20 mL), and finally
with MeOH (10 mL). Five fractions (30 mL each) were
collected, and evaporation of the solvents yielded frac-
tions A (4.0 mg), B (14.8 mg), C (16.2 mg), D (6.4 mg),
and E (4.6 mg). Except for fraction A, all other fractions
contained similar spots as judged by both normal phase
(hexane/acetone; 1:2) and reversed-phase preparative
TLC (CH3OH/H2O; 70:30). Fraction A was separated
by reversed-phase TLC (MeOH/H2O; 85:15), yielding
1-acetamidoanthracene (2) (0.1 mg) and 1-acetamidoanthr-
aquinone (3) (0.3 mg). Fractions B, C, D, and E were
separated on normal phase preparative TLC (CH2Cl2/
MeOH; 87:13) followed by purification on reversed-phase
preparative TLC (MeOH/H2O; 70:30), resulting in the
isolation of 1-acetamido-5-[(40-O-methyl-b-D-glucopyr-
anosyl)oxy]anthracene (4) (10.6 mg), 1-acetamido-8-[(40-
O-methyl-b-D-glucopyranosyl)oxy] anthraquinone (5)
(8.4 mg) and 1-acetamido-6-[(40-O-methyl-b-D-glucopyr-
anosyl)oxy]anthraquinone (6) (8.4 mg).
3.4.2. 1-Acetamido-8-[(40-O-methyl-b-D-glucopyranosyl)
oxy]anthraquinone (5). Yellow powder; mp 248 ꢁC
25
(decomposed); ½aꢀ ꢁ26.5 (c 0.03, MeOH); UV kmax
D
(MeOH) (loge) 223.5 (4.19), 261.5 (4.07) nm, 395.0
(3.51); IR (KBr) mmax 3499, 3275, 2926, 2885, 1699,
1663, 1593, 1531, 1458, 1340, 1288, 1267, 1111, 1094,
1067, 995 cmꢁ1; HRESIMS m/z 480.1268 [M+Na]+
(calcd for C23H23NO9Na, 480.1270). 1H and 13C
NMR data, see Tables 1 and 2.
3.4.3. 1-Acetamido-6-[(40-O-methyl-b-D-glucopyranosyl)
oxy]anthraquinone (6). Yellow powder; mp 251–252 ꢁC;
25
½aꢀ ꢁ100.0 (c 0.01, MeOH); UV (MeOH) kmax(loge)
D
216.5 (4.45), 268.5 (4.48), 397.5 (3.77) nm; IR (KBr) mmax
3479, 3425, 2966, 2920, 2878, 1705, 1641, 1597, 1522,
1342, 1302, 1273, 1258, 1163, 1082, 1013 cmꢁ1; HRE-
SIMS m/z 458.1451 [M+1]+ (calcd for C23H24NO9,
1
458.1442). H and 13C NMR data, see Tables 1 and 2.
3.5. Acetylation of 1-aminoanthracene
Ac2O (100 lL) was added with stirring to a solution of 1-
aminoanthracene (7.3 mg) in pyridine (300 lL). The mix-
ture was allowed to stand at 25 ꢁC for 1 h, and then dried
under nitrogen to afford 1-acetamidoanthracene (2)
(8.5 mg, 96%).
3.6. Acetylation of 1-aminoanthraquinone
The freeze–dried mycelia of B. bassiana from the above
experiment were extracted with 3· 50 mL of MeOH, from
Ac2O (200 lL) was added with stirring to a solution of
1-aminoanthraquinone (7.3 mg; Sigma–Aldrich, 97%

J. Zhan, A. A. Leslie Gunatilaka / Bioorg. Med. Chem. 16 (2008) 5085–5089
5089
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stand at 80 ꢁC for 2 h, and then dried under nitrogen to
afford 1-acetamidoanthraquinone (3) (8.3 mg, 95%).
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Acknowledgments
Financial support from the NCI/NIH (Grant R01 CA
90265) and Arizona Biomedical Research Commission
is gratefully acknowledged. We thank Mr. C. J. Seliga
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