1170

pulmonary vascular resistance, suggesting that penta-zocine has a pulmonary vasoconstrictor effect.

Scott and Adgey 13 found rises in cardiac outputand systemic arterial pressure and a fall in peripheralvascular resistance after administration of pentazocinein myocardial infarction. Jewitt and co-workers 2,3found a much greater rise in systemic arterial pressurewith no change in cardiac output. Peripheral vascularresistance, therefore, rose. They attributed the risein L.V.E.D.P. which they observed to the increasedafterload imposed on a depressed left ventricle by thisincreased resistance. The rise in pulmonary-arterypressure reflected the rise in L.V.E.D.P. We have beenunable to confirm the increase in peripheral vascularresistance or the rise in L.v.E.D.P. In animals theadministration of pentazocine is followed by improvedleft ventricular function with increased myocardialcontractility and without an increase in L.V.E.D.P.

or in myocardial oxygen consumption. There was atransient fall in peripheral vascular resistance. 11These findings and our own indicate no significantimpairment of left ventricular function. Previousconcern about the use of pentazocine in acute myo-cardial infarction may well have been exaggerated.Mild respiratory depression develops after 30 mg.

pentazocine,8 but it is insufficient to cause the rise inpulmonary-artery pressure. We did not demonstratea statistically significant fall in PaO2 although othershave done so. However, Paco:! increased significantlyby nearly 6 mm. Hg, indicating a reduction in alveolarventilation. Neither the fall in Pao2 nor the rise inPaCO2 were sufficient to produce any significantcirculatory effect.10, 12

Since pulmonary-artery diastolic pressure correlatesclosely with L.V.E.D.P. when pulmonary vascularresistance is low,7 its use as an indirect measure ofL.V.E.D.P. has gained wide acceptance. 6 Our findingsunderline the importance of considering changes inpulmonary vascular resistance when interpretingalterations in pulmonary-artery diastolic pressure infuture investigations using this technique.We thank Miss Ann Colthart for skilled technical assistance

and Prof. K. W. Donald for provision of laboratory facilities.We wish to acknowledge financial support from WinthropLaboratories.

Requests for reprints should be addressed to D. G. J., De-partment of Cardiology, Royal Infirmary, Edinburgh EH3 9YW.

REFERENCES

1. Scott, M. E., Orr, R. Lancet, 1969, i, 1065.2. Jewitt, D. E., Maurer, B. J., Hubner, P. J. B. Br. med. J. 1970,

i, 795.3. Jewitt, D. E., Maurer, B. J., Hubner, P. J. B, Shillingford, J. P.

Br. Heart J. 1971, 33, 145.4. Rickards, A. F., Smithen, C. S., Sowton, E. Ann. clin. Res. 1971, 3,

199.5. George, M., Lassers, B. W., Muir, A. L., Julian, D. G. Cardiovasc.

Res. 1970, 4, 127.6. Rapaport, E., Scheinmann, M. M. Mod. Conc. cardiovasc. Dis.

1969, 38, 55.7. Jenkins, B. S., Bradley, R. D., Branthwaite, M. A. Circulation, 1970,

42, 75.8. Lal, S., Savidge, R. S., Chhabra, G. P. Lancet, 1969, i, 379.9. Kaltman, A. J., Herbert, W. H., Conroy, R. J., Kossman, C. E.

Circulation, 1969, 34, 377.10. Prys-Roberts, C., Kelman, G. R., Greenbaum, R., Robinson, R. H.

Br. J. Anœsth. 1967, 39, 533.11. Levitsky, S., Mullin, E. M., Sloane, R. E., Morrow, A. G. Am.

Heart J. 1971, 81, 381.12. Fishman, A. P., McClement, J., Himmelstein, A., Coumand, A.

J. clin. Invest. 1952, 31, 771.13. Scott, M. E., Adgey, A. A. J. Curr. ther. Res. 1971, 13, 81.

HÆMOGLOBIN SOUTHAMPTON, &bgr;106 (G8)Leu →Pro: AN UNSTABLE VARIANT

PRODUCING SEVERE HÆMOLYSIS

R. D. HYDE M. D. HALL

Department of Hœmatology, General Hospital, SouthamptonUniversity Hospital Group, Southampton SO9 4XY

B. G. WILTSHIRE* H. LEHMANN

Medical Research Council Abnormal Hœmoglobin Unit,University Department of Biochemistry,

Cambridge CB2 1QW

Summary A new mutation has produced a

hemoglobin variant, Hb Southampton.The nature and position of the aminoacid substitutioncomplicated its identification, but the resultant severehæmolytic anæmia correlates with the type of substitu-tion and the associated instability of the molecule.

Introduction

EXPERIMENTS of Nature occasionally producechanges which could be closely predicted from a

knowledge of the effect of a mutation on D.N.A. tran-scription. When such a mutation affects a part of thehaemoglobin molecule which determines the molecularconformation or the function of haem, the haemoglobinmay be permanently oxidised (haemoglobin M diseases)or become unstable. Instability of haemoglobin isthe commonest cause of congenital Heinz bodyanæmia,1 and recent developments in protein analysishave resulted in the identification of approximately 25haemoglobin variants in such patients. 2 Most geneticstudies have shown the anomaly to be inherited, butoccasional new mutations have been described.We describe here a new mutation in a child with

severe hæmolytic anaemia.

Case-reportA girl, born in 1960, was noticed to be pale at the age

of 3 years. Intermittently the urine was very dark, andwhen seen in 1965 there was icterus and a palpable spleen.Chronic intravascular hasmolysis and unconjugated hyper-bilirubinaemia (serum-bilirubin 2-2-3-4 mg. per 100 ml.)were demonstrated, but extensive investigation of knowncauses of haemolysis, of red-blood-cell metabolism, and ofglutathione and A.T.P. concentrations gave results whichwere consistent with the observed reticulocytosis of30-55% of red blood-cells except for the followingobservations:

(1) The red blood-cells were hypochromic and included3% of contracted cells, 2% microspherocytes, and4% showed coarse basophilic stippling, but therewere no Heinz bodies nor methxmoglobin.

(2) After incubation of sterile blood for 48 hours at

37 °C, many red-blood-cell inclusions appeared in60% of cells, an excess of methxmoglobin formed,and spontaneous haemolysis was increased to 20%but did not fall when blood was incubated with0-05M glucose or 0°025M A.T.P.

(3) Alkali-resistant haemoglobin amounted to 5—11%and 8 per 1000 red blood-cells contained non-elutedHbF in a Kleihauer acid-elution test.

(4) Although levels of HbA were normal and no

hæmoglobin variants could be detected on electro-

* Present address: Roche Products Ltd., Welwyn Garden City, Herts.

1171

phoresis, it was noted that attempts to prepare a

hsemolysate by chloroform extraction 3 producedspontaneous gelation.

(5) A heat-unstable haemoglobin, amounting to 15% oftotal globin prepared by hydrochloric-acid/acetoneprecipitation 4 was detected when hæmolysatesprepared with carbon tetrachloride were heated at50°C.

Further investigation between 1965 and 1970 confirmedthe presence of an unstable haemoglobin, but no abnormalitycould be detected by peptide fingerprint analysis.

Family StudyThe propositus is the sixth born and only female child

of unrelated English parents with no family history ofhxmolytic anaemia and no known relationship to other

English families with unstable-haemoglobin diseases.Detailed investigation of both parents and all seven brothersshowed no evidence of haemolysis nor of any unstablehaemoglobin. Blood-grouping studies revealed no evidenceof irregular parentage.

Clinical ProgressThe child maintained normal activity with haemoglobin

concentrations of 7-0-8-6 g. per 100 ml., reticulocytosis of30-49% of red blood-cells, and required only occasionalblood-transfusion. Development had been normal withheight and weight in the 25th centile for normal Britishchildren.

The development of newer methods of separationof unstable haemoglobin variants 5 and of aminoacidsequence analysis by aminoethylation (AE) followedby Edman degradation enabled an aminoacidsubstitution to be detected.The unstable hxmoglobin was precipitated from the

haemolysate by isopropanolftrisfhydrocMoric-acid.5 Itwas dissolved in 0.1N hydrochloric acid and the

globin was precipitated using acidic acetone. The aand &bgr; chains were separated on a Whatman CM23.column, using a sodium-ion gradient of 0.01M to0.06M and a pH of 6-7.* s The &bgr;-chain was amino-ethylated,’ gel-filtered, and freeze-dried. It was then

digested with trypsin and fingerprints were prepared.These showed that a new divalent-sulphur-containingspot had appeared below the peptide Tp &bgr;2, which wasanalysed and found to consist of Tp &bgr;12a (residues&bgr;105—112) with one of the three leucyl residues beingreplaced by one of proline (see table). The sequence ofthis peptide is leu-leu-gly-asn-val-leu-val-AE cys. Todecide which of the three possible leucine residues hadbeen replaced, the peptide was submitted to two runsof Edman degradation,8 and on each occasion resub-mitted to aminoacid analysis. On the first Edman

degradation one residue of leucine was lost, but on the

AMINOACID COMPOSITION OF PEPTIDE &bgr;XIa &bgr;105—112) OF THEAMINOETHYLATED &bgr;-CHAIN OF THE UNSTABLE HÆMOGLOBIN

25-3 nmoles per residue.* AE Cys is partially destroyed during the hydrolysis of the peptide

preceding aminoacid analysis.

second occasion one of proline was lost, indicatingthat the proline was in position 2 (R106) of this peptide.To confirm this, the peptide after removal of its firstresidue was submitted to dansyl Edman degradation, 8and there again the result suggested that the N terminalresidue of this peptide (&bgr;106—112) was one of proline.The dansyl peptide obtained after one round ofEdman degradation had been hydrolysed with 6Nhydrochloric acid for only 6 hours rather than the usual16 hours, as proline was expected in the second position.Later in the course of this work it was found that

when the &bgr;-chain was digested with trypsin for 3hours, rather than the usual 2, a better yield of themutant peptide was obtained. It seems that the

proline in position &bgr;106 may inhibit the action oftrypsin in hydrolysing the bond between Tp &bgr;11 and12a.

The propositus is thus a heterozygote for HbA anda new unstable haemoglobin, Hb Southampton, &bgr;106(G8) Leu—Pro.

Discussion

Severe haemolysis, associated with the appearanceof red-blood-cell inclusions after splenectomy, has beendescribed as congenital Heinz body anaemia during thepast 20 years, and in approximately twenty familiesdifferent unstable haemoglobin variants have beenidentified. Where genetic information has been

recorded, the unstable haemoglobin has usually beeninherited as an autosomal dominant, although therehave been four variants which have occurred as

sporadic cases.A new variant, Hb Southampton, has now been

identified in only one of eight children of hxmatologi-cally normal parents. It is presumed to have arisen inthe propositus from a single point mutation at position2 of the triplet coding for &bgr;106 where the observedsubstitution of proline for leucine could arise from atransition mutation of a uracil to a cytosine (U-C)in R.N.A. With stable hxmoglobin variants transversionmutations occur, as expected, twice as often as

transitions, but there is a relative lack of transversionmutations producing viable unstable hxmoglobins.9 HbSouthampton is a further example of a non-transversionmutation producing an unstable hxmoglobin withsevere hxmolysis.The substitution of the iminoacid proline for leucine

in position G8 of the -chain would interrupt thehelical sequence and severely distort the tertiarystructure of the molecule at a point where there isdirect contact with hæm.10

In common with the four previously describedsubstitutions by proline at other helical positions inthe molecule 11—14 Hb Southampton is unstable andproduces severe haemolysis. In-vitro instability to heatand isopropanol is accompanied by in-vivo precipita-tion of haemoglobin within red blood-cells, with severeshortening of red-blood-cell survival. The spleen canremove some of the precipitated haemoglobin by its" pitting " action, leaving hypochromic and contractedred blood-cells without visible precipitate in vivo.When splenectomy has been performed in other

patients with uncompensated hæmolytic anaemia, manyHeinz bodies appear, but there has been little clinical

1172

improvement in those with variants producing themost severe haemolysis. -

We thank Dr. M. J. Simpkiss for referring the patient forinvestigation and the Wessex Regional Hospital Board ResearchCommittee for support.

Reprint requests should be addressed to R. D. H.

REFERENCES

1. Dacie, J. V., Grimes, A. J., Meisler, A., Steingold, L., Hemsted,E. H., Beaven, G. H., White, J. C. Br. J. Hœmat. 1964, 10, 388.

2. Carrell, R. W., Lehmann, H. Semin. Hemat. 1969, 6, 116.3. Lehmann, H., Huntsman, R. G. Man’s Haemoglobins; p. 268.

Amsterdam, 1966.4. Anson, M. L., Mirsky, A. E. J. gen. Physiol. 1929, 13, 469.5. Carrell, R. W., Kay, R. Br. J. Hœmat. 1972, 23, 615.6. Clegg, J. B., Naughton, M. A., Weatherall, D. J. J. mol. Biol.

1966, 19, 91.7. Jones, R. T. Cold Spring Harbor Symp. quant. Biol. 1964, 29, 297.8. Gray, W. R. Methods Enzymol. 1967, 11, 469.9. Lehmann, H., Huntsman, R. G. in The Metabolic Basis of In-

herited Disease (edited by J. B. Stanbury, J. B. Wyngaarden,and D. S. Fredrickson); p. 1398. New York, 1972.

10. Perutz, M. F. Nature, 1970, 228, 726.11. Sansone, G., Carrell, R. W., Lehmann, H. ibid. 1967, 214, 877.12. Opfell, R. W., Lorkin, P. A., Lehmann, H. J. med. Genet. 1968, 5,

292.13. Schneider, R. A., Satoshi, U., Alperin, J. B., Brimhill, B., Jones,

R. T. New Engl. J. Med. 1969, 280, 739.14. Kleihauer, E. F., Reynolds, C. A., Dozy, C. A., Wilson, A. M.,

Moores, R. R., Berenson, M. P., Wright, C. S., Huisman, T. H.J. Biochim. biophys. Acta, 1968, 154, 220.

ISOLATION OF INFECTIOUS MEASLES

VIRUS IN MEASLES ENCEPHALITIS

V. TER MEULEN

Y. KÄCKELL

D. MÜLLER

M. KATZ

R. MEYERMANN

Department of Clinical Virology, Institute of Virology,University of Würzburg, Federal Republic of Germany;Virus Laboratory, Department of Pœdiatrics, and Depart-ment of Neuropathology, University of Göttingen, FederalRepublic of Germany; and School of Public Health, Collegeof Physicians and Surgeons, Columbia University, New

York, U.S.A.

Summary A 40-year-old man developed encepha-litis 3 days after onset of acute measles.

He died 6 weeks later. Histopathological findings wereconsistent with late type of post-infectious measlesencephalitis. Direct inoculation of a homogenate offresh brain tissue obtained at necropsy into susceptibletissue cultures produced no infectious virus. However,co-cultivation of cells derived from this brain tissuewith those of a continuous line of African greenmonkey kidney tissue culture yielded an infectiousvirus that was identified as measles.

Introduction

NEUROLOGICAL complications develop in between1 in 400 and 1 in 1000 cases of acute measles.’, 2 The

encephalopathy (or encephalitis as it is more commonlycalled) usually appears at the time of recovery frommeasles, often when the temperature has returned tonormal. However, there have been cases in which theneurological symptoms appeared even before theonset of the rash.3 Indeed, involvement of the centralnervous system (C.N.s.) may be much more common

than is the clinical expression of it, because the electro-encephalogram (E.E.G.) of patients with acute measleshas been reported as abnormal in more than half ofthe cases .4,5The mechanism of this neurological complication

has not been elucidated. An obvious explanationwould be a direct involvement of the brain by themeasles virus. Such a concept received some supportin two publications. One claimed transmission ofmeasles infection to rhesus monkeys by inoculationof brain tissue from a patient with measles encephalitis,but the virus itself was not isolated; the basis for its" recovery " was seroconversion of the animals.However, since rhesus monkeys in captivity invariablyseroconvert

" spontaneously ", the report was dis-counted. The other paper described isolation ofinfectious measles virus from cerebrospinal fluid

(c.s.F.) of one patient considered to have measlesencephalitis. However, no details about the isolatedagent were provided.’ 7We have isolated measles virus from the brain ob-

tained at necropsy of a patient who died from measlesencephalitis.

Case-reportThe patient was a 40-year-old German male, who

presented with fever and respiratory distress. The followingday he developed a typical measles exanthem and wasnoted to have Koplik spots. 2 days later his fever abated,but respiratory distress increased and he was admitted toa rural hospital. There he was noted to be restless anddisoriented; he was slightly hypertensive (blood-pressure160/100 mm. Hg), had right bronchopneumonia, deter-mined by X-ray, and his c.s.F. showed pleocytosis with151 cells per 0- 1 ml., all of them mononuclear; c.s.F. sugar was80 mg. per 100 ml. and Pandy test was+ +. 2 weeks beforethe onset of the patient’s symptoms his son had hadclinically typical measles. The past history of the patientwas unremarkable. He had not had measles and had notbeen given measles vaccine.The patient was given supportive respiratory care,

steroids, digitalis, and was treated with tetracycline andampicillin for his pneumonia. Because his conditionbecame worse, he was transferred to the intensive-careunit at the university hospital in Gottingen. By that timehe was comatose and had no deep tendon reflexes. Hisc.s.F. continued to show mononuclear cells and a positivePandy test. Chest X-ray showed bilateral broncho-pneumonia. His E.E.G. was diffusely slow. Supportivetreatment with antibiotics and steroids and respiratory carewere continued. In addition, measures were taken to

reduce cerebral oedema by infusing hypertonic solutions.The patient remained comatose during his entire stay inthe hospital and died as a result of respiratory arrest 6weeks after the onset of his illness.

Additional studies, carried out at the university hospitalin Gottingen, revealed C.S.F. protein ranging between48 and 156 mg. per 100 ml., colloidal gold curve of theparetic type, and raised c.s.F. gamma-globulin with valuesranging from 7-7 to 25-6 mg. per 100 ml. Clinical diagnosiswas measles encephalitis.

Materials and Methods

NecropsyThe brain was examined under aseptic conditions 2

hours after the patient’s death. Several representativetissue blocks were obtained from all regions of the brainand prepared for histology, immunofluorescence, virus

isolation, and cell culture.