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133)11-1-410 

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A-83-22 

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NAJUR l*VO L. 236 MARCH 10 1972 iVC ^341 T/o 

Where the sandstone has remained buried and natural 

weathering has not yet reached into it, the sulphur content 

decreases from several tenths of a per cent near the coal to less 

than one tenth of a per cent near the top of the sandstone. 

There is no region similar to the highly leached weathered zone 

observed in the same sandstone when it contacts the surface. 

The pattern is shown in Fig. 1 h. 

A few of the rock columns sampled to 16 m show no highsulphur zone and have chromas higher than 2. Mild geological 

folding at these locations has resulted in rock fracture with 

subsequent weathering reaching greater depths. In such cases, 

a study of the fresh high walls resulting from mining operations 

verifies suspected lateral penetration of weathering from 

fractures, indicated by iron oxide chromas enclosing an 

interior of massive, low-chroma sandstone at depths below the 

6 m weathered zone indicated in Fig. 1. This lateral penetration of weathering, extending inward several centimetres from 

the vertical fissures, vividly illustrates the "weathering front" 

(ref. 6). Evaluation of chromas in such bicoloured specimens 

consistently distinguishes between high sulphur and low sulphur 

portions of the fragments (Table 1). 

Table 1 SulDhur in Relation to Chroma in Six Natural Bicoloured 

Fragments of Lower Mahoning Sandstone * 

Fragment Chroma 

1 

1 

2 

2 

3 

3 

4 

4 

5 

5 

6 

6 

4 

0 

3 

0 

6 

0 

6 

0 

4 

0 

6 

2 

Position in fragment 

Near surface 

Interior 

Near surface 

Interior 

Near surface 

Interior 

Near surface 

Interior 

Near surface 

Interior 

Near surface 

Interior 

Sulphur (%) 

< 0.001 

0.175 

0.048 

0.288 

0.060 

0.198 

< 0.001 

0.035 

0.032 

0.140 

0.004 

0.010 

* Ground to <250 \im; colour evaluated on dry sample. 

Higher chromas penetrate several centimetres from bedding or 

fracture planes, low chromas typify interiors. 

"Although the presence of sulphides, other reduced chemical 

species, and low chroma (grey) coloration might indicate the 

effects of the water table, t!ie 6 m depth from the present land 

surface is well above the present permanent water table. The 

evidence for oxidation to greater depths on fracture planes 

confirms that a permanent water table is not involved. Prolonged saturation of the sandstone is a likely condition within 

the rock that has retained its low chroma colour and pyrite. 

This condition is related to rock porosity, position in the 

Mcction, recharge by meteoric water, and rock chemistry. 

Details of these influences have not been studied, but their 

resultant interactions evidently have maintained an oxidation 

potential low enough to preserve pyrite in the low chroma rock. 

The inherent low fertility of soil developed from Lower 

Mahoning sandstone1 is readily understood in view of the 

.iroperties of the pre-weathered parent material. The solubilization and subsequent leaching of all but the most acidinsoluble compounds by the sulphuric acid formed by oxidation 

if the pyrite in the weathering sandstone have left rock which 

is almost barren of plant nutrients. Measurements of available 

plant nutrient cations throughout the depths studied indicate 

mense removal of essential plant nutrients from the 6 m 

oxidized zone. This strong acid leaching during the early 

>tages of soil development accounts for low base saturation 

levels and modern classification of these soils as Dystrochrepts 

or Udults1. 

The usual method of spoil disposal in this area is backfilling 

iind covering with material favourable for planned land use, 

usually afforestation, forage growth, or specialty uses. The 

otal sulphur and chroma profiles in near-surface Lower 

Masoning sandstone indicate that there are adequate volumes 

of pyrite-free rock material to place in the oxidation zone of 

spoil heaps to avoid acid pollution. But nutrients must be 

added before vigorous' vegetation can be established. On the 

other hand, the bulk of potentially toxic low chroma material 

below a depth of 6 m constitutes a formidable barrier to 

revegetation and a likely prolonged source of acid water 

pollution unless protected from oxidation by burial or other 

means. Studies are under way to determine weathering of 

spoil materials, rate of plant nutrient release, and the progress 

of soil property genesis, which are expected to proceed 

differently in fractured spoil materials than in stratified sediments in situ. 

This work was supported by the Environmental Protection 

Agency, Water Quality Office. 

WALTER E. GRUBE, jun. 

RICHARD M. SMITH 

EVERETT _M. JENCKS 

RABIND, 

College of Agriculture and Forestry, 

West Virginia University, 

Morgantoyvn, West Virginia 26506 

Received July 23; revised November 16,1971. 

TUSli8-.! 

m I 3 1892 lU 

1

 White. I. C, Bull. US Geol. Surv., No. 65 (189] 

3

 Grimsley, G. P., Iron Ores, Salt and Sandsh 

Geol. Surv., 4 (1909). 

* Jackson, M. L., Soil Chemical Analysis (Prentice-Hall, 1958). 

* Munsell Soil Color Charts (Munsell Color Co., Inc., Baltimore, 

Maryland). 

5

 Temple, K. L, and Koehler, W. A., Drainage from Bituminous 

Coal Mines, West Virginia Eng. Exp. Sta. Res. Bull. No. 25 

(1954). 

4

 Oilier, C, in Weathering (American Elsevier, New York, 1969). 

1

 Soil Survey Staff, Placement of Series—Northeast Region, USDASCS (Northeast Regional Technical Service Center, Upper 

Darby, Pennsylvania, 1969). _ 

DOCKET 

VJ*r 

BIOLOGICAL SCIENCES 

Defective Synthesis of cc and (3 Globin 

Chains in Lead Poisoning 

THE cells of many tissues, including bone, red cells, liver, brain 

and nerves, absorb lead1 which accumulates chiefly on the 

mitochondrial membrane. Here it disturbs mitochondrial 

metabolism and produces the swelling and disruption revealed 

by electron microscopy2. The most obvious metabolic defect 

is the inhibition of the synthesis of haem (and porphyrin). 

Lead inactivates at least three mitochondrial enzymes in the 

haem pathway (8 aminolevulinic acid synthetase3, 5-aminolaevulinic dehydrase3

, and haem synthetase*). This aspect of 

lead toxicity (reviewed in ref. 5) reduces haem production and 

leads to the accumulation and excessive excretion of several 

porphyrin intermediates. Lead seems to affect red cells 

chiefly by causing increased destruction and interfering with 

haemoglobin synthesis, as judged by the presence of ring 

sideroblasts in the bone marrow and hypochromia of the red 

cells. In normal red cell precursors ihe synthesis of haem and 

globin is finely balanced, but in diseases such as sideroblastic 

anaemia7 and iron deficiency anaemia (R. Barr, A. V. Hoffbrand and J. M. W., unpublished) a defect in haem synthesis 

leads to a disturbance of globin synthesis. A previous report 

indicated that globin synthesis was defective in lead poisoning8, 

but no reports have dealt with the synthesis and assembly of 

a and B chains, which we have now studied. 

Reticulocyte-rich blood samples from 3 yr old twins, who 

had developed lead poisoning after eating old paint, were 

prepared and incubated for 60 min with

 3H-leucine and the 

cells weic divided into two equal aliquots. From one aliquot 

w prep Med "imour**" "lobi;-! bx' 2'J'I!"" the c)!s <1!rcc'.!y to 

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60 SO 100 120 I JO 

Fraction No. 

Fig. 1 Pattern of radioactivity of protein fractions o f lysate o f 

twin 1 separated by 'G-100 Sephadex' chromatography. , 

Absorbance -280;nm; •—•, radioactivity (d.p.m. ml.-'). 

Note that the radioactivity associated with the dextran blue 

peak (arrow) is nearly as great as that associated with haemoglobin peak. 

acid acetone. From the other aliquot we made "pure" globin 

by separating the haemoglobin from other proteins by 

chromatography of a lysate of washed cells on 'G-100 Sephadex'. The radioactivity and absorbance of the protein fractions 

of the lysate eluted from the column were measured. Finally, 

the pure haemoglobin fraction was concentrated by ultrafiltration and the globin precipitated by addition of acid 

acetone. The a and fJ chains from impure and pure globins 

were separated by chromatography on CMC-23 using 8 M 

urea-phosphate buffers'. The radioactivity associated with 

the a and f3 chain protein peaks was measured, and then the 

total radioactivity of each peak was determined (Table 1). 

The synthesis of a and P chains of their reticulocytes was 

measured in vitro, at the time the patients first presented and 

6 and 12 weeks later while they were recovering after treatment 

with penicillamine. 

When first studied the patients' haemoglobin levels were 

7.0 and 7.6 g 100 ml.-1 and blood lead levels 98 ug 100 ml.-1. 

The radioactivity of the protein fractions of the cells separated 

by chromatography on a 'G-100 Sephadex* is shown in 

Fig. I. (Only the results of one twin are shown: the other 

twin's results are identical.) Two important features are shown. 

First, the radioactivity of the void column peak is nearly as 

great as the radioactivity of the pure haemoglobin fraction. 

(This peak contains ribosomes and apoferritin and its radioactivity is normally only one-twentieth to one-fifteenth of the 

radioactivity associated with the haemoglobin fraction.) 

800 

r 

I 20 0 .10 

Fraction No. 

Fig. 2 First experiment. Pattern of radioactivity as associated 

with the a and P chains of twin 1, separated on CMC-23. , 

Absorbance 280 nm; •—•, "impure" globin (c.p.m. ml.-'); 

O O. "pure" globin (c.p.m. ml.-'). Note: (I) in "impure" 

globin large amount of "y" peak radioactivity; P chain radioactivity >a chain radioactivity; (2) in "pure" globin "y" 

peak has disappeared; fall in P chain radioactivity but all a 

chain radioactivity recovered. 

P.3 

NATURE VOL. 236 MARCH 10 19 

I 

Table 1 Laborat 

Hb(g 100 ml."') 

MCHC (%) 

Reticulocytes (%) 

Blood lead (ng 100 ml." 

UbF(% ) 

HbA,(% ) 

Mb electrophoresis 

ory Invc 

') 

istigations on 

Twin I 

7.3 

28.0 

10.0 

98.0 

3.2 

1.3 

A 

Patients 

Twin 2 k 

7.6 

27.5 

11.5 

98.0 

3.0 

1.8 

A 

Second, the amount of radioactivity associated with haemo 

globin fraction (3,000 d.p.m. ml.-1) is very low for a reticulocyii 

count of 10%. Together these findings indicate indirectly tha 

the synthesis of the haemoglobin tetramer is abnormal. Direc 

evidence for this was obtained from studies of the synthesi: 

of the isolated a and fi. chains. 

o 

•6 

o a: 

hraclion No. 

Fig. 3 Second experiment. Pattern of radioactivity of a and P 

chainsof (win I. Note: (1) in "impure" globin fall of "y" peak 

activity and relative increase in P chain and a chain radioactivity; (2) most of the p and a chain radioactivity recovered 

from "pure" globin. 

The radioactivity associated with the a and (3 chains isolated 

from the impure globin preparations is shown in Fig. 2. Most 

of the radioactivity was found in a peak in front of the P chain 

peak in the region corresponding to the y chain. This peak 

is called "y". The radioactivity of the P chain was less than 

this initial peak but significantly greater than that associated 

with the a chain peak. The ratios of the radioactivity of the 

a: P + "Y" chains were 0.23 and 0.25 in twin 1 and twin 2, 

respectively, and the ratios of the radioactivity of the a:p 

chains alone, 0.60 and 0.61. The pattern of radioactivity 

associated with the a and P chains isolated from pure globin 

preparations showed several differences. The "y" peak of 

radioactivity had disappeared and there was a fall in the radioactivity of the P chains. Virtually all the radioactivity associated with the a chain in the impure globin sample, however, 

was recovered in the o chain isolated from the pure globin. 

Six weeks later the pattern of radioactivity obtained after 

elution of the impure globin preparation shows a fall in radioactivity associated with the "y" peak and a relative increase 

in the radioactivity associated with the P an.d a chains (Fig. 3). 

The pattern of radioactivity of the chains isolated from the 

pure globin preparation was very similar except that the "y" 

peak was missing and the specific activity of the P chain was 

reduced. The ratios of radioactivity of a : P + "y" peaks made 

from the impure globin were 0.65 and 0.68 and the o: P ratios 

0.83 and 0.87. The findings of the final experiments, 12 weeks 

later, are shown in Fig. 4. The radioactivity of the "y" peak 

had disappeared and the radioactivity of the a and P chain 

peaks from both the pure and impure globin preparations was 

very similar. The ratio of the radioactivity of a:P + "y" 

chains was 0.89 and 0.90 and the a: p ratio 0.93 and 0.96. 

Fig. 5 shows the ratios of radioactivity of the a:P + "Y" 

chains made from impure globin preparations from each twin 

UPM «iMman.B,n.wiJM-tfmmJHUgw "mmrtmSTJyWii CT^.^».».fiSiB 
P.4 

NATURE }/0L 236 MARCH 10 1972 

.0.8 

0.7 

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g 0.5 

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0.3 

0.2 

0.1 

.Gradient 

- 4r 

10 

j 

-

I \ / v 

i \ i \ J L ^ V 

20 30 40 50 

Fraction No. 

800 

700 1 

E 

600 g 

500- S" iviiy oacl 

200 « 

100 

Fig. 4 Third experiment. Pattern of radioactivity of a and P 

chains, twin I. Note: absence of "y" radioactivity and nearly 

equal amounts of p and a chain activity, most of which is 

recovered from the "pure" globin. 

plotted against the haemoglobin values. As the haemoglobin 

levels rise, and the blood lead levels fall, the a: P + "y" ratio 

becomes near to 1.0. 

These findings indicate that in the reticulocytes of the -

patients globin synthesis is disordered. In the initial experiments, the P chain peak of the impure globin preparation was 

more radioactive than the a chain peak. Because the chains 

were isolated from whole cells this finding indicates that the 

total synthesis of p chains was greater than that of a chains. 

But radioactivity of the a chain peak was virtually the same 

in the impure and pure globin preparations, which indicates 

that nearly all the a chains synthesized combine with P chains 

and haem to form haemoglobin. The finding that the radioactivity of the P chain peak was less in the pure globin preparations again suggests that there was a relative excess of P chains 

which existed as a free (uncombined) pool. 

The nature of the "y" peak is uncertain. A similar peak of 

activity has been found in preparations made from marrow 

cells cultured without haem'0,11. The peak was originally 

thought to be due to the synthesis of true y chains10, but 

recent analysis has shown it to be a protein of a larger molecular weight with a different peptide sequence11

. An alternative suggestion is that it is apoferritin11

. Our data support 

this as we were unable to demonstrate this peak in the pure 

preparations and also found a large amount of radioactivity 

associated with the apoferritin peak after 'G-100 Sephadex' 

Chromatography. Moreover, the level of Hb F (a2Pj) in the 

two patients was never greater than 3% and higher levels 

would have been expected if this peak was true of y chain. 

A similar although lesser degree of defective synthesis is 

seen in two other conditions due to haem deficiency, namely 

sideroblastic anaemia7 and iron deficiency anaemia (R. Barr, 

A. V. Hoffbrand and J. M. W., unpublished). It is tempting 

?•:• 

!•. 

V 

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V 

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73 dio 

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o 

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o 

0.9 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 Blood level 

(/'g 100ml.-') 98 45 20 

;,. 7.0 8-0 9.0 10.0 11.0 

V Hb values <g 100ml.'1) 

- Fig. 5 Correlation between ratios of total radioactivity of the 

«***»: P+ "y" chains wiih haemoglobin values of both twins. Blood 

--••V lead levels are also indicated. S.Tv.ir. 3; C.twir. T 

73 

to postulate that lead, in addition to its undoubted effect 

on haem synthesis, directly affects the synthesis and/or assembly 

of the globin chains. In support of this conclusion, when 

reticulocytes are incubated with lead in vitro polysomal disaggregation occurs12

. This has also been observed in reticulocytes with haem (iron) deficiency, however, and the 

disordered synthesis in lead poisoning may be wholly due to 

haem deficiency. 

It is uncertain whether protein synthesis is disturbed in other 

cells but in reticulocytes the abnormal synthesis of globin 

chains seems to be a factor in the pathogenesis of the anaemia 

of lead poisoning. 

J. M. WHITE 

MR.C Group on Haemolytic Mechanisms, 

Royal Postgraduate Medical School 

D. R. HARVEY 

Department of Child Health, 

Hammersmith Hospital, 

Ducane Road, London W\2 

Received July 13; revised September 20, 1971. 

* Barry, P. S. I., and Mossman, D. B., Brit. J. lndust. Med.,11, 339 

(1970). 

2

 Bessis, M., and Breton-Gorius, J., Blood, 19, 635 (1962). 

1

 Dresel, E. I. B., and Falk, J. E., Biochem. J., 63, 80 (1965). 

4

 Goldberg, A., Ashenbracker, H., Cartwright, G. E., and Wintrobe, M. M., Blood, 11, 821 (1956). 

5

 Goldberg, A., Seminars Haematol., 5, 424 (1968). 

6

 Waldron, H. A., Brit. H. lndust. Med., 23, 83 (1964)._ 

7

 White, J. M., Brain, M. C.and Ali, M. A. M., Brit. J. Haematol., 

20,263(1971). 

• Kasenaar, A., Morel!, H., and London, I. M.,J. Biol. Chem.,229, 

423(1957). 

9

 Clegg, J. B., Naughton, M. A , and Weatherall, D. J., J. Mol. 

JB/O/.,19,91(1966). 

10

 Gabuzda, T. G., Melvin, M., Silver, R. K., and Sachs, C, Proc. 

Thirteenth Congr. Intern. Soc. Haematol., 8 (1970). 

11

 Wood, N., Whittacker, J. A., Clegg, J. B., and Weatherall, D. J., 

Absl. Brit. Soc. Haematol., 19 (1971). 

11

 Trakatellis, A. C, Heinle, E., Montjar, M., Axelrod, A. E., and 

Jensen, W. K, Arch. Biochem. Biophys., 112, 89 (1965). 

Transmission of Scrapie to the 

Cynomolgus Monkey (Macaca 

fascicularis) 

SCRAPIE has been transmitted to the cynomolgus, or crabeating, monkey (Macaca fascicularis) with an incubation 

period of more than 5 yr from the time of intracerebral 

inoculation of scrapie-infected mouse brain. The animal 

developed a chronic central nervous system degeneration, with 

ataxia, tremor and myoclonus with associated severe scrapielike pathology of intensive astroglial hypertrophy and proliferation, neuronal vacuolation and status spongiosus of grey 

matter. The strain of scrapie virus used was the eighth 

passage in Swiss mice (NIH) of a Compton strain of scrapie 

obtained as ninth intracerebral passage of the agent in goat 

brain, from Dr R. L. Chandler (ARC, Compton, Berkshire). 

The monkey, a juvenile cynomolgus, was inoculated on 

April 18, 1966, with a 10% suspension of the scrapie-infected 

mouse brain in phosphate buffered physiological saline, pH 

7.4, which had been clarified by centrifugation at 3,500 r.p.m. 

for 30 min. The animal received 0.2 ml. of such suspension by 

each of five routes: intracerebral, intravenous, intraperitoneal, 

intramuscular and subcutaneous. This suspension had an 

LD50 infectivity titre of 10~7 S ml."1 by intracerebral 

inoculation in mice. 

Five years and five months after inoculation, the animal 

developed slight ataxia and tremors of the head and extremities. 

These signs became progressively more severe with generalized 

body tremors more prominent during feeding and on agitation. 

Uy C sucks afici onset liieic v>a:> pionounccu locomotor ataxia 

and cl^msinc^ ir. handling food and myoclonic jerks wci