MOLECULAR BIOLOGY & HYPERTENSION LABORATORY

Brian J. Morris

  

  

RESEARCH

PERSONNEL in 1994 and 1995

Dr Brian J. Morris Reader (in-charge) University 1978-
Dr Andrew P. Schrader Research Officer NHMRC 1992-
Dr Susan M. Chambers Research Officer NHMRC 1995-
Dr Yi-kun Lou Research Assistant NHF 1988-
Dr Robert Y.L. Zee Research Assistant (0.5) NHMRC 1989-
Dr Han Qin PhD student (0.4; Supervisor, J. Hoh, 0.6) Fac Med Sch'ship 1989-
Li-Hua Ying PhD student NHF Sch'ship 1990-
Amanda Stephen BSc(Hons) student 1994
Dr Lyn R. Griffiths Research Affiliate; Senior Lecturer in
Biochemistry, Griffith Univ. (Gold Coast)
1989-

Current effective full-time personnel = 5.4

  
The research of this Laboratory is directed at finding the molecular genetic basis of cardiovascular disease, particularly hypertension, and determination of the mechanisms of regulation of renin and kallikrein genes.
  

PROJECTS in 1994

Genetic associations for SA gene variant

In a novel approach to hypertension gene identification, mRNAs over-expressed in the kidney of spontaneously hypertensive rats (SHRs) have been cloned, and the locus for one, termed the 'SA' gene, has been found to co-segregate with blood pressure. Sequence data suggest that SA could be a secreted enzyme, but its function is unknown. A polymorphism of the human SA gene which had shown an association with hypertension in Japanese was studied in a severely-affected Caucasian group in Sydney, but no association was detected with hypertension. An association was, however, found with obesity. Furthermore, body mass index (BMI) tracked with the obesity-associated allele. Plasma renin and angiotensinogen were suppressed in subjects with the latter allele, and since plasma angiotensinogen displayed a strong correlation with blood pressure, the putative SA protein could be having a pressor effect.

Low density lipoprotein receptor gene (LDLR) and obesity

Additional confirmation of previous findings by the Laboratory was provided in work by Robert Zee, who showed that a HincII restriction fragment length polymorphism (RFLP) of LDLR was significantly association with obesity in a hypertensive group. Furthermore, BMI tracked with genotypes of the obesity-associated variant. There was also significant linkage disequilibrium between the HincII and the previously-studied ApaLI RFLPs.

A new, common genetic risk factor

After finding that a particular genotype of the angiotensin converting enzyme (ACE) gene further increases the risk of premature death in patients already at high risk of cardiovascular events by having severe hypertension, further research was conducted on subjects with the risk factors 'obesity' and 'high cholesterol'. Death rate appeared to be higher in obese subjects homozygous for the 'death allele' of the ACE gene, but not in those with high cholesterol. Obesity in hypertensive patients did not further increase their risk above that conferred by the latter alone.

Relationship of renin to ACE genotype

The two enzymes required for formation of angiotensin II are renin and ACE. It is now well established that plasma ACE is influenced by ACE genotype. Studies in 1994 showed that there was a reciprocal relationship between plasma renin and ACE across the different ACE genotypes and that the difference was greater in patients with essential hypertension. The reason is probably due to suppression of renin secretion by a negative feedback effect of the higher angiotensin II generated by the genotypes associated with higher plasma ACE concentration.

Insulin receptor gene (INSR) variants and hypertension

A RsaI RFLP, located in intron 9 of INSR was shown previously by the Laboratory to display a strong association with essential hypertension. In 1994 Andrew Schrader found that a NsiI RFLP, located in exon 8 and detected by PCR, also showed an association, albiet weaker, with hypertension. The association was, moreover, stronger in lean and normolipidaemic subjects. The angiotensinogen locus has been linked to hypertension and, interestingly, plasma angiotensinogen was found to track significantly with the major allele of the NsiI RFLP in the hypertensive, but not in the normotensive control group.

Regulation of basal human renin gene (REN) promoter activity

In an extension of previous findings of a functional cyclic AMP response element located at nucleotides -222 to -218 relative to the transcription start site of REN, Li-Hua Ying, in collaboration with Curt Sigmund, Univ. of Iowa, extended these studies to a line of renin-expressing juxtaglomerular cells and identified interactions that may be important in transcriptional control. In addition, expression was studied in different tissues of mice made transgenic for constructs containing the REN promoter coupled to a luciferase reporter gene.

Responses in renin mRNA in rats with renal hypertension

Renin mRNA was accurately quantified in tissues of the rat by Yi-kun Lou using a quantitative reverse-transcriptase PCR technique he had developed previously. In collaboration with Dennis Liu and Judith Whitworth, renin mRNA in different tissues was measured in the two-kidney, one clip (2K1C) model of hypertension. In the kidney with stenosis of the renal artery, renin mRNA was increased more at an early stage (19 days after clipping) than at a later stage (40 days). Renin mRNA was suppressed in the contralateral kidney. These changes were not substantially different in 2K1C rats on a high NaCl intake. Renin mRNA in the adrenal was also increased by renal artery clipping, especially in the early phase of hypertension, suggesting a pathophysiological role. In the hypothalamus, however, it was suppressed at day 19, but this could be alleviated by NaCl loading. In other experiments, the concentration of renin mRNA in response to a low NaCl diet and the ACE inhibitor, enalapril, was determined. Similar directional responses were seen in kidney, adrenal and heart, but suppression was noted in the hypothalamus.

Human kallikrein gene control

Andrew Schrader continued his work on molecular control of a human glandular kallikrein gene expressed in the prostate. This has involved LNCaP prostate metastatic tumour cells for transient expression analyses.

HPV typing of cervical cells with abnormal
IR spectra

Human papillomavirus (HPV) is the cause of most cervical cancer. Cytologically abnormal cells were found to display changes in position and size of bands seen on infrared (IR) spectroscopy and the spectra displayed an approximate match to the grade of cytological abnormality. However, discordance was noted for some Pap smear results. HPV typing revealed the presence of high risk HPV 18 in 3 of 4 samples with severe dysplasia, as well as one sample classed as 'atypia', but having an abnormal IR spectrum. Low-risk HPV 6 or 11 was seen alone in samples with a normal or slightly abnormal IR spectrum, but never in those that showed an IR pattern that was abnormal.
  

RESEARCH PLANNED for 1995

The Laboratory has been involved in collecting large numbers of hypertensive sibships. Following the award of a large grant, 1995 will see the commencement of a national study, involving automated genome scanning using microsatellite markers, in order to identify the causative loci for essential hypertension. In addition, clinical correlates of the INSR-hypertension association will be explored, involving glucose tolerance tests and euglycaemic glucose clamps in collaboration with Richard Donnelly. Furthermore, insulin receptor mRNA will be studied in the SHR. Research will also continue on the molecular control of the human renin and kallikrein genes. Studies will be continued of renin gene expression in tissues of the SHR using quantitative PCR.
  

COLLABORATIONS

Sib-pair linkage studies in hypertension:
Dr Lyn Griffiths, Griffith University (Gold Coast); Prof. Malcolm West and Dr Kim Summers, Prince Charles Hospital; Prof. Nick Martin, Queensland Institute of Medical Research; Dr John Hopper, NHMRC Twin Registry, Univ. of Melbourne; Dr John Whitfield, Royal Prince Alfred Hospital; Prof. W. (Bill) Louis, Austin Hospital; Prof. Trefor O. Morgan, Univ. of Melbourne; Dr Barry McGrath, Monash Medical Centre; Prof. Gary Jennings, Alfred and Baker Medical Unit; Prof. Derek Frewin, Univ. of Adelaide; Prof. John Chalmers, Flinders Medical Centre; Prof. Laurie Beilin, Royal Perth Hosp. (all 1993-present).

Renin gene regulation in renin-synthesizing cells:
Dr Curt Sigmund, Dept of Physiology and Biophysics, Univ. of Iowa (1994-present).

Renin mRNA studies in hypertensive rats:
Prof. Judith A. Whitworth, St George Hospital (1992-present).

Insulin receptor mRNA splice variants in hypertensive rats:
Prof. Judith A. Whitworth, St George Hospital (from 1995).

Insulin resistance vs insulin receptor genotype in hypertensives:
Dr Richard Donnelly, Dept of Pharmacology and Dr John Kelly, Dept of Medicine, St George Hospital (from 1995).

Myosin cDNA and gene cloning:
Dr Joseph F.Y. Hoh (1989-present).

Clinical correlates of plasma ACE vs genotype:
Judith C. Monaghan and Prof. Gordon S. Stokes, Royal North Shore Hospital (1994-present).

HPV typing of cervical cells:
Brian N. Nightingale, Dept of Clinical Biochemistry, Rachel Forster Hospital (1986-1994).
  

FACILITIES

The main laboratory work area is room 203H of the Anderson Stuart Building; other, smaller rooms are adjacent to this, including Dr Morris' office (203F), a room for radioactive labelling and DNA sequencing, a cell culture room (205) and a space for large equipment items (203). The total floor area is ~160 sq. m. Equipment includes the following: Beckman L8-M ultracentrifuge, Beckman J2-21M/E high-speed centrifuge, Applied Biosystems PCR-Mate DNA Synthesizer, DNA sequencing equipment comprising LKB 2010 Macrophore electrophoresis unit, LKB 2297 Macrodrive 5 power supply, LKB 2010-100 Macromould gel casting unit and LKB 2019 Multiheat thermostatic circulator, 2 Perkin-Elmer DNA Thermal Cyclers, Beckman DU 650 spectrophotometer, Berthold Lumat LB 9501 luminometer, Sanyo MDF 391AT ultra low -80°C freezer, Certomat orbital incubator and accessories, Braun HT shaking incubator, Braun HT TM-1 shaker, Polaroid copy camera, UVP transilluminator, Gelman Class II Biosafety cabinet, Gelman CF43S laminar flow cabinet, BioRad Gene Pulser and capacitance extender for electroporation, Dynavac FD-1 freeze dryer, IEC MicroMix microcentrifuge, Eppendorf 5412 and two 5414S microcentrifuges, Beckman microfuge 12, Millipore RO and Milli-Q water filtration system, Tomy ES-315 high-pressure steam sterilizer, LKB and Gilson fraction collectors, peristaltic pumps, Bio-Rad Protean II and Hybaid Electro-4 electrophoresis tanks, Bio-Rad 224 Gel Slab Dryer, LKB 2016 Vacugene vacuum blotting unit and pump, Amicon concentrator, Sartorius 1204 MP balance, LKB Uvicord S flow-through spectrophotometer, LKB 2103 and 2301 power supplies, Grant LTD low temperature water bath, Clayson water bath, Incublock and Paratherm II incubators, National model 5831 vacuum oven, three Labmaster ovens, National microwave oven, Labec and Grant Instruments incubators, two Julabo EM and one Paratherm II water heaters, Clayson Water bath, Hanna pH meter, Chiltern and Dumax stirrers, sterilizer, Gilson automatic pipettes, homogenizers, light-box, Neomedix series 900 geiger counter, radioactivity shields, chromatography columns, SI and SI Genie vortexes, magnetic stirrer, IEC 209-1 magnetic hot plate, Rinnai gas burner, Gared Thermoline refrigerated cabinet, Westinghouse 210 and two Gorenje Pacific chest freezers, Kelvinator 300 freezer, Westinghouse freezamate 312 and 282 refrigerators, rat blood pressure recording equipment, omniscribe chart recorder, one Apple Macintosh Quadra and two Apple Macintosh Classic II PCs, NEC Power Mate/MultiSync II PC, Telecom Commander system with two lines and 5 handsets, assorted glassware, disposable plasticware, other sundry items, enzymes, reagents and chemicals.
  

SCHOLARSHIPS in 1994 and 1995

Australian Postgraduate Research Award, Lou Y-k, 1991-94.

National Heart Foundation Postgraduate Science Research Award, Ying L-H, 1993-present.
  

OTHER RESEARCH ACTIVITIES in 1994

Editorial board of scientific journal

Hypertension Research (1993-present).

Refereeing

Manuscripts: for Journal of Hypertension (4), Hypertension (3), Clinical and Experimental Pharmacology and Physiology (2), American Journal of Physiology (1), American Journal of Hypertension (1), Circulation Research (1), Laboratory and Clinical Medicine (1), Clinical and Experimental Hypertension (1).

Grant applications: for NHMRC (project grants [7], Public Health Research & Development application [1], Research Fellow application [1]), National Heart Foundation (3), ARC, Univ. of N.S.W. Small Grant (1), Merck Sharpe & Dohme Research Foundation (2), Sir Charles Gairdner Hospital Research Advisory Committee (1).

MSc Thesis examined: for Univ. of Melbourne (1).

International rating of conference presentation

Abstract submitted for presentation at 15th International Society of Hypertension Meeting was selected as one of 180 oral presentations, being 2nd highest scoring abstract for Australia, 29th overall out of 1,421 selected for presentation from 2,421 submitted.

Conference attended overseas

16th International Congress of Biochemistry and Molecular Biology, New Delhi, India (Sep).

Seminars

Concord Hospital (Feb).

Anderson Stuart seminar series (Apr).

Fellowship of Jewish Doctors (Oct).

Conference organized

'Genes and Genetics', a satellite symposium of 15th ISH Meeting, Univ. of Sydney (Mar).

Conference organizing committee

National Member, 15th Scientific Meeting of International Society of Hypertension, Melbourne (Mar).

University committees

Member, Biosafety Committee.

Member, Research Report Committee (Faculty of Medicine Representative).

Department co-ordinator:

For submission to Faculty of Medicine Research Review.

For submission to University Research Report.

For preparation of Annual Reports (1989-present) (prior to that 'Research Report' 1988 and 4-5 yearly Research Reports published in 1979, 1983 and 1988).

For calculation of research performance indicators for distribution of a fraction of Faculty funding to Dept.

Annual Report of Sydney Institute for Biomedical Research

Editor for preparation of Annual Reports from inaugural report for 1994.

News media

Press conference

'Hypertension, a disease that affects families', Organized by the National Heart Foundation for Heart Week.

University publications

International Studies Prospectus 1994 (photograph).

'Inventions shown at World Trade fair'. University of Sydney News, 15 Feb 1994, 2.

'New test for cervical cancer'. University of Sydney Gazette, Apr 1994, Vol. 22 (1), 2.

'Researchers discover killer heart gene'. Front page of University of Sydney News, 5 Oct 1994.

Newspaper reports

'Pap smears: Science finds a better way'. Front Page of Sydney Morning Herald, 9 Feb 1994.

Report on cervical cancer test. The Age, 8 Mar 1994.

Report on cervical cancer test. The Advertiser, Adelaide, 10 Feb 1994.

Report on cervical cancer test. The West Australian, Perth, 10 Feb 1994.

'Test developed for cancer virus'. Sunday Age, Melbourne, 27 Mar 1994.

'Heart disease battle plan'. Daily Telegraph Mirror, 3 May 1994.

Report arising from NHF publicity. The Age, 3 May 1994.

Report arising from NHF publicity. Courier Mail, Brisbane, 4 May 1994.

'Found: The heart attack gene'. Front page of Sydney Morning Herald, 6 Oct 1994.

Television

Live interview on cervical cancer test: '11 AM', in Channel 7 Studios, 10 Feb 1994.

Report on cervical cancer test: Channel 10 Evening News, 9 Feb 1994.

Report on gene for heart attack: Channel 10 Evening News, 6 Oct 1994.

Radio interviews

On new cervical cancer test: 2UE, 2WS, 5AD and ABC National, 9 Feb; ABC Brisbane, ABC Adelaide, ABC Perth and 2TN, 10 Feb; 5AA and 5UE, 11 Feb, 1994.

The case for circumcision: 5AA, 14 Feb; FM 1323, Adelaide, Jeremy Cordeau Show, 9 May; ABC Hobart, 27 Jun and 4 Jul; 5MU, 20 Jul, 1994.

On gene for heart death: 5AA, 7 Oct; ABC Perth, 25 Oct 1994.

Magazine reports

'At last - a D-I-Y Pap test'. The New Weekly, 7 Mar 1994, 42-43.

'Simple samples'. Nursing Times, Vol. 90, 30 Mar 1994, 21.

'Test to detect cervical cancer'. Ita, July 1994, 114.

Item on cervical cancer test. New Woman, during 1994.

Newsletter item

'Hypertension. A disease that affects families'. Pulse Beat, National Heart Foundation of Australia (NSW Division), Heart Week, May 1994, 2.

Items in annual reports

'New technique reveals more about renin levels.' Heart Research, NHF, 1993, 3.

Letter to the Editor

'Circumcise now' (title chosen by newspaper). Good Weekend (magazine of Sydney Morning Herald, The Age, Hobart Mercury, etc), 25 June 1994, 8.
  

THESES PASSED in 1994

PhD

Zee RYL (1994) Cross-sectional and linkage analyses of candidate genes in essential hypertension.

Smith DL (1994) Transcriptional regulation of the human renin gene.

BMedSc(Hons)

Stephen AL (1994) The SA gene and essential hypertension. (Result: 2nd Class, Div. 1)
  

5-YEAR RESEARCH PUBLICATIONS

MOST RECENT TOTAL ANNUAL CITATIONS (for 1993): 161

  

FUNDING for 1994 and 1995

NHMRC Renin gene regulation Morris BJ 1994 $44,034
1995 $44,739
1996

NHMRC Human kallikrein gene
regulation
Morris BJ 1994 $44,763
Schrader AP 1995 $45,480
1996

NHMRC Orbital incubator
(equipment)
Morris BJ 1994 $13,278
Hoh JFY
Phillips WD
Mason RS

NHMRC Molecular genetics of essential
hypertension
Morris BJ 1995 $135,291
Griffiths LR 1996
West MJ 1997

NHF Renin gene in hypertension Morris BJ 1993 $27,409
Robinson BG 1994 $30,197
Whitworth JA

NHF Insulin receptor in hypertension Morris BJ 1995 $38,925
Donnelly R 1996
Kelly J

UEG Anderson Stuart Molecular
Facility (equipment)
Morris BJ
and 10 others
1994 $40,000

Ramaciotti Automated facility for genome
scanning in hypertension
(seeding grant)
Morris BJ 1995 $20,000
Griffiths LR

AKF SA gene in hypertension Morris BJ 1995 $7,500

ARC
(Mech C)
Automated DNA sequencing and cell
sorting facility
(*Administered by Univ. of N.S.W.)
Dawes I* 1995 $300,000
Kjelleberg SLA
Chesterman CN
Morris BJ (and 33 others)

Total for 1994: $132,272; plus multi-user equipment grant of $40,000

Total for 1995: $291,934; plus multi-user equipment grant of $300,000 (with Univ. of N.S.W.)

  

  

TEACHING

COURSES TAUGHT

Medical Science 2: Human Life Sciences 2

Acting Course Supervisor: Physiology component of course in second half of year.

Lectures: 6, on endocrinology (introduction, hypothalamus, posterior pituitary, anterior pituitary, adrenal, kidney and cardiac hormones).

Practical classes: 2, each of 3 h, presented 4 times, on endocrinology.

Tutorials: 8: 4 introduction and follow-up sessions associated with practical classes, 2 on lecture material and 1, presented 4 times, to help with preparation for exams.

Examination: Multiple choice (of the 5 alternatives, one correct variety), 'write notes on' and short answer questions.

Medical Science 3/ Science 3: Cardiovascular

Lectures: 11, on hormonal regulation of the cardiovascular system and hypertension.

Practical classes: 1, of 5 h, presented twice (on molecular genetics of cardiovascular disease).

Tutorials: 6, in which 2 students per session discuss an allocated scientific paper.

Examination: Essay-type question; in addition, assessment involved 1 essay written during semester.

Medicine 2

Lectures: 8, on endocrinology (introduction, hypothalamus, posterior pituitary, anterior pituitary, adrenal, kidney and cardiac hormones); 3 Distinction) on genetics of hypertension.

Examination: Mixture of one-word answer, draw diagram or 'write notes on', and multiple true-false questions, plus essay for Distinction course.

Pharmacy 1

Lectures: 11, 8 on endocrinology (introduction, hypothalamus, posterior pituitary, anterior pituitary, adrenal, kidney and cardiac hormones); 3 on reproduction.

Tutorial: 1, on lecture material.

Examination: Multiple choice questions (of the 5 alternatives, 1 correct, variety).

THESIS MARKING

15 h was spent marking theses and essays of students in Honours programme.

STUDENT ADVISER

6 h, Courses and Careers Day and Student Information Day.
  

TEACHING HOURS in 1994

HLS 2 Med 2 Pharm 1 MedSc/Sc3 Total
Lectures 6 11 11 11 39
Tutorials 8 - - 10 (2) 15
Practical classes (no.) 12 (4) - - 10 (2) 22
Student consultations 1 1 1 1 4
Preparing lectures 1 1 1 3 6
Setting exams 1 2 2 1 6
Marking exams 8 15 - 16 39
Marking essays 5 - - 10 15

Total formal contact teaching time = 76 h

Total time = 146 h

  

  

ADMINISTRATION

(see also OTHER RESEARCH ACTIVITIES in 1994)

Faculty of Medicine committee

Member, working party on Endocrinology and on Molecular Medicine for postgraduate medical degree.

Faculty of Science committees

Member, Publicity Committee.

Member, DSc Sub-committee.

Curriculum development

Contribution to discussions concerning a postgraduate medical degree.

Departmental co-ordinator

For Dept submission to Faculty of Science Handbook.

Sydney Association of University Teachers

Departmental representative (1991-present).