Vascular-targeted photodynamic therapy with BF2-chelated Tetraaryl-Azadipyrromethene agents: a multi-modality molecular imaging approach to therapeutic assessment.

BACKGROUND: Photodynamic therapy (PDT) is a treatment modality for a range of diseases including cancer. The BF(2)-chelated tetraaryl-azadipyrromethenes (ADPMs) are an emerging class of non-porphyrin PDT agent, which have previously shown excellent photochemical and photophysical properties for therapeutic application. Herein, in vivo efficacy and mechanism of action studies have been completed for the lead agent, ADMP06. METHODS: A multi-modality imaging approach was employed to assess efficacy of treatment, as well as probe the mechanism of action of ADPM06-mediated PDT. RESULTS: Tumour ablation in 71% of animals bearing mammary tumours was achieved after delivery of 2 mg kg(-1) of ADPM06 followed immediately by light irradiation with 150 J cm(-2). The inherent fluorescence of ADPM06 was utilised to monitor organ biodistribution patterns, with fluorescence reaching baseline levels in all organs within 24 h. Mechanism of action studies were carried out using dynamic positron emission tomography and magnetic resonance imaging techniques, which, when taken together, indicated a decrease in tumour vascular perfusion and concomitant reduction in tumour metabolism over time after treatment. CONCLUSION: The encouraging treatment responses in vivo and vascular-targeting mechanism of action continue to indicate therapeutic benefit for this new class of photosensitiser.

Different forms of Go alpha mRNA arise by alternative splicing of transcripts from a single gene on human chromosome 16.

Go alpha, (gene symbol GNA01), a member of the signal-transducing guanine nucleotide-binding (G) protein family, has been implicated in ion channel regulation. Some tissues contain multiple Go alpha mRNAs of different sizes that differ in the 3' untranslated regions (UTRs). Using sequence-specific 48-base oligonucleotides, two complementary to the different 3' UTRs and one complementary to the coding region, we investigated the origin of the multiple Go alpha transcripts, the organization of the Go alpha gene, the interspecies conservation of 3' UTRs, and the chromosomal localization of Go alpha. Oligonucleotides labeled to high specific activity by using terminal deoxynucleotidyltransferase each hybridized with a single band of restriction enzyme-digested mouse and human DNAs. In three of four digests of human DNA, the two probes specific for the different 3' UTRs hybridized with the same restriction fragment. Thus, these nucleotide sequences are in close proximity in the human genome. The order of the UTRs in the bovine, human, and mouse genomes was confirmed directly by polymerase chain reaction (PCR) amplification and sequencing. Hybridization of bovine oligonucleotide sequence with mouse and human genomic DNA indicated a high degree of interspecies sequence conservation: conservation was confirmed by PCR amplification and sequencing. Bands detected by both UTR probes, as well as the predominant bands detected by a bovine Go alpha cDNA, segregated with human chromosome 16 on Southern blot analysis of human-mouse somatic cell hybrids. We conclude that Go alpha mRNAs with different 3' UTRs arise by alternative splicing of transcripts from a single gene. The UTRs, which exhibit a high degree of interspecies conservation, may play a role in regulation of Go alpha expression during differentiation or in specific tissues. The use of oligonucleotide probes of the type described here represents a new strategy, potentially widely applicable for mapping and elucidating structural features of genes.

Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection.

Solution hybridization is an essential step in sequencing and some point mutation detection methods. In practice, this hybridization is hampered resulting in the need of additional purification of the amplification products. The use of T7 gene 6 exonuclease may lead to efficient production of single-stranded DNA. In this study, the effect of pretreatment with exonuclease on direct cycle sequencing and point mutation detection was analyzed. Exonuclease-treated products were directly cycle sequenced without further purification. This resulted in highly efficient quality improvement for sequencing allowing detection of heterozygotes. Point mutation detection by Point-EXACCT (exonuclease-amplification coupled capture technique) demonstrated detection of one cell containing a mutation in an excess of 75000 wild type cells. Exonuclease-enhanced detection methods offer simple, rapid detection strategies that are easily adaptable for widespread clinical laboratory use. With the use of exonuclease, the detection of heterozygosity using fluorescent cycle sequencing is becoming more reliable. The high sensitivity of Point-EXACCT due to the use of exonuclease makes it a highly promising method for large-scale screening of (pre)malignant changes in patients with a high risk for developing cancer.

Identification and characterization of the human myeloperoxidase promoter.

Myeloperoxidase (MPO) is a microbicidal protein present in the primary granules of myeloid cells. Transcription of the MPO gene is turned on only during the late myeloblast and promyelocyte stages of myeloid maturation. Identification of cis-regulatory elements and transcription factors which regulate the MPO gene should, therefore, shed light on myeloid maturation. We report transfection and in vitro transcription experiments which demonstrate promoter activity in the proximal 5'-flanking region of the human MPO gene. Using a chloramphenicol acetyl transferase (CAT) reporter vector system, and segments of the 5'-flanking MPO DNA, we constructed a series of MPO promoter-CAT expression vectors. By electroporation and lipofectin-mediated transient transfection assays, as well as by in vitro transcription studies, a 594-bp MPO DNA sequence (bp -583 to +11) showed promoter activity in a variety of MPO-expressing and non-MPO-expressing cell lines. Compared with the SV40 early promoter, the MPO promoter had greater relative activity in MPO-expressing than in non-MPO-expressing cell lines, suggesting slight tissue specificity. However, a CAT reporter plasmid containing 1099-bp of 5'-flanking MPO DNA showed greater specificity for MPO expressing cell lines. Analysis of a group of promoter deletion mutants showed that the minimal promoter was contained in a DNA fragment extending from bp-128 to +11. The remainder of the promoter region contained several segments which appeared to enhance the activity of the minimal promoter. One such enhancer sequence was homologous to an enhancer previously described in the human elastase promoter. Activity of the 594-bp MPO promoter in HL-60 was reduced by only approximately 30% following treatment of the cells with chemical inducers of maturation, but the 1099-bp MPO promoter showed 60% reduction in activity after DMSO treatment. A previously described enhancer region in intron 9 of the MPO gene had little or no effect on activity of the 594-bp MPO promoter. The availability of the MPO promoter will facilitate determination of other factors involved in the regulation of this myeloid-specific gene.

Achievements and challenges on policies for allied health professionals who use telehealth in the Canadian Arctic.

We formulated policies and procedures for allied health professionals (AHPs) who provide services using telehealth in Nunavut, Canada's newest Arctic territory. These are a supplement to the clinical policies and procedures already established for Nunavut physicians and nurses. The services were in the areas of audiology, dietetics/nutrition, midwifery, occupational therapy, ophthalmic services, pharmacy, physiotherapy, psychology, respiratory therapy, social work and speech therapy. Documents specific to each of the services were developed, drawing on information from Government of Nunavut data, Nunavut healthcare providers and links made through the Internet. Topics included the scope and limitations of telehealth services, staff responsibilities, training and reporting, professional standards and cultural considerations. We also considered generic policies covering common issues such as jurisdiction, licensing and liability. The policies and procedures for AHPs will enhance and expand the successes already achieved with telehealth in Nunavut. The challenges are to balance the preferred approaches to service provision with the realities of health care and communications in an Arctic setting.

Beta-adrenergic receptor kinase-like activity and beta-arrestin are expressed in osteoblastic cells.

Biologic responses to peptide calciotropic hormones, such as parathyroid hormone (PTH) and calcitonin, exhibit desensitization. As with most hormones, however, the mechanisms of desensitization are not completely understood. For the beta 2-adrenergic receptor (beta 2AR) system, which is coupled to adenylyl cyclase via the stimulatory guanine nucleotide-binding regulatory (G5) protein, homologous desensitization is mediated in part by a receptor-specific kinase (beta ARK) and a soluble cofactor (beta-arrestin). Recently, this system has been reported to be involved in rapid homologous desensitization of the PTH/parathyroid hormone receptor protein (PTHrP) receptor. We have identified the presence of this system in bone using reverse-transcriptase PCR. Nucleotide sequence of PCR fragments from ROS 17/2.8 cells revealed 100% identity with rat brain beta ARK1 and beta-arrestin 1 sequences. Northern analyses with RNA from ROS 17/2.8, UMR 106-H5 cells, and primary cultures of nontransformed neonatal rat calvariae demonstrated two mRNA species of 4 and 2.6 kilobases (kb) for beta ARK and 7.5 kb for beta-arrestin, comparable to those found in bovine brain. beta ARK-like activity was demonstrated in cytosolic extracts of the UMR 106-H5 cells by assessing phosphorylation of the retinal photoreceptor, rhodopsin, by the extracts. Phosphorylation was enhanced with light-activated rhodopsin and by bovine brain G beta gamma subunits; heparin inhibited phosphorylation. These findings are characteristic of beta ARK. Expression of beta-arrestin in the UMR 106-H5 cells was confirmed by immunoblot. Thus, osteoblastic cells express proteins, beta ARK, and beta-arrestin, which may regulate desensitization of calciotropic hormone receptors.

Internal cRNA standards for quantitative northern analysis.

We report a simple method using copy RNA (cRNA) internal standards for quantitative Northern hybridization. This was accomplished by synthesis of a full-length or "half"-length cRNA and mixing these RNA internal standards with samples to be tested for the abundance of a given mRNA. Both full-length and truncated cRNAs are detected in Northern analysis by the nucleic acid detection probe (which can be labeled with 32P or biotin), and the known amount of the truncated cRNA is compared to the homologous mRNA present in the specimen being examined. We demonstrate the usefulness of this method by measuring the expression of renin in rat kidney with ureteral obstruction and angiotensin II receptor (AT1-R) mRNA in kidneys from spontaneously hypertensive rats. It was found that this method of preparing cRNA internal standards successfully controlled for variables (such as differences in loading, transfer and hybridization efficiency) that often frustrate efforts to use Northern analysis as a quantitative tool and enabled direct estimation of absolute mRNA amount present in samples. This technique may have wide applicability and permit a more quantitative use of Northern analysis.

Relation of coronary arterial patency and left ventricular function to electrocardiographic changes after streptokinase treatment during acute myocardial infarction.

One hundred and eighty-eight patients with evolving acute myocardial infarction were treated with intravenous streptokinase. Serial 12-lead electrocardiograms were recorded for 3 hours after treatment and inspected for rapid repolarization changes of the ST segment and T wave. Abrupt electrocardiographic repolarization changes were observed in 106 patients (56%) and were strongly predictive for an open infarct-related coronary artery at a mean of 6 days after treatment (predictive value = 0.92, sensitivity = 0.67). Abrupt electrocardiographic changes were not observed in 82 patients (44%). This absence was not a good predictor of an occluded infarct-related coronary artery (predictive value = 0.4). There was no relation between the presence or absence of abrupt electrocardiographic changes and global or regional left ventricular function after streptokinase treatment. Abrupt repolarization changes after thrombolytic treatment indicate a high probability of an open infarct-related artery. When abrupt repolarization changes do not occur, the patency of the infarct-related coronary artery cannot be predicted with accuracy. Serial electrocardiographic recordings do not provide sufficient information about coronary patency to eliminate the need for coronary arteriography.

Rapid detection of mycobacteria in inflammatory necrotizing granulomas from formalin-fixed, paraffin-embedded tissue by PCR in clinically high-risk patients with acid-fast stain and culture-negative tissue biopsies.

A collection of inflammatory necrotizing granulomas (INGs) negative by acid-fast stain and culture (AFSC) were analyzed by polymerase chain reaction (PCR) for the presence of mycobacteria. Forty-two paraffin-embedded specimens with INGs were collected from patients at high risk for contracting tuberculosis. Twenty biopsies were positive and 22 were negative for mycobacteria by AFSC. Two universal primers specific for all mycobacteria were used to detected a 414 base pair (bp) fragment of 16S rRNA gene. Twenty of 20 biopsies were positive for mycobacteria by both AFSC and PCR (100%), whereas 19 of 22 biopsies negative by AFSC were positive by PCR (86%). Follow-up of patients who were PCR positive but AFSC negative identified nine patients who had subsequent biopsies. Specimens from eight of these nine patients eventually grew Mycobacterium tuberculosis. Our results demonstrate that the detection of mycobacterial DNA by this method should be used in conjunction with AFSC for the initial diagnosis of mycobacterial infection.

Beneficial effects of nafazatrom on ischemic reperfused myocardium.

The effect of nafazatrom, a new antithrombotic agent, was studied in a canine model of regional myocardial ischemia. Nafazatrom was administered 1 mg/kg intravenously every 6 h for 48 h. After 24 h of drug or placebo administration, animals underwent 90 min of occlusion of the proximal left circumflex coronary artery followed by gradual reperfusion over a period of 30 min. Twenty-four hours later, the animals were sacrificed and infarct size was determined by histochemical staining with triphenyltetrazolium chloride. Nafazatrom-treated animals had a significant reduction in infarct size expressed as a percent of the anatomical area at risk for infarction: 21 +/- 5% in the treated group vs. 41 +/- 5% in the control group (X +/- S.E.M., P less than 0.05). Histological examination confirmed the gross results of postmortem histochemical staining. Salvage of ischemically jeopardized tissue appeared to be unrelated to myocardial oxygen demand as there were no hemodynamic differences between groups. The beneficial effects of nafazatrom are presumably related to a limitation of autolytic processes on the heart during and after ischemia as a result of the drug's ability to inhibit lipoxygenase and to prevent the enzymatic degradation of prostacyclin.

Management of acute coronary occlusion during percutaneous transluminal coronary angioplasty: experience of complications in a hospital without on site facilities for cardiac surgery.

OBJECTIVE: To determine whether percutaneous transluminal coronary angioplasty may be safely performed in cardiology centres in the United Kingdom without immediate on site cardiac surgical cover for complications arising at angioplasty. DESIGN: Retrospective review of coronary angioplasties and complications in a hospital without on site cardiac surgical cover. SETTING: All angioplasties were performed in the catheterisation laboratory of the Belfast City Hospital. Revascularisation surgery for complicated coronary angioplasty was performed in the cardiac surgical unit of the Royal Victoria Hospital, 2.4 km away from the catheterisation laboratory. PATIENTS: 540 Coronary angioplasties were performed on 512 patients between late 1982 and November 1988. Indications included stable angina, unstable rest angina, and suitable coronary disease at coronary arteriography after myocardial infarction. MAIN OUTCOME MEASURES: In hospital mortality after complicated coronary angioplasty and delay to surgical revascularisation after acute coronary occlusion at angioplasty. RESULTS: Coronary angioplasty was successful in 444 cases (82%). Acute coronary occlusion occurred in 35 cases (6.5%). Twelve patients required urgent revascularisation surgery and were transferred safely to the surgical unit; none of these patients died. A mean delay of 268 minutes (range 180-390 minutes) occurred before revascularisation compared with 273 minutes (range 108-420 minutes) in the Royal Victoria Hospital, where on site surgical cover was available. The principal cause of delay was the wait for a cardiac operating theatre to become available and not the transfer time between hospitals. Five deaths occurred after coronary angioplasty, a mortality of 0.9%. Three deaths were related to acute coronary occlusion. The absence of immediate surgical help did not influence the outcome in any patient. CONCLUSION: With careful selection of patients coronary angioplasty may be safely performed in a hospital without on site cardiac surgical facilities, provided that these are available at a nearby centre.

The doctor's bag. What do you really need?

BACKGROUND: The doctor's bag should contain essential drugs to treat life threatening emergencies and other serious medical conditions. Practitioners may require these drugs for treating patients in their offices, on home visits and particularly in rural practice for emergency home and roadside calls. OBJECTIVE: To evaluate which medications and equipment need to be included in the doctor's bag. DISCUSSION: The selection of the most appropriate drugs provided by the Pharmaceutical Benefits Scheme (PBS) is discussed. Oral and inhaler preparations for certain specific conditions are also included. Storage and safe keeping of drugs is an important consideration.

Arthralgia: a diagnostic strategy.

For arthritis or arthralgia there is no simple system for diagnostic analysis, but whether it is polyarthritis or monoarthritis, acute or chronic in onset, some general rules apply. Common causes include osteoarthritis (primary and secondary) and viral infection. Drugs should be considered, including those inducing gout. It is still imperative not to miss rheumatic fever, sepsis and tuberculosis in assessment. We may encounter more cases of Lyme disease presenting as arthritis.

Common problems. Tiredness.

When a patient presents with tiredness, the diagnostic model is a useful basis for considering the probable diagnosis. At the same time it is imperative not to overlook the serious, even life threatening, causes such as malignant disease or AIDS. Such an organised approach allows the practitioner to ask the correct questions because, as always, the quality of the history taking is vital in leading to the diagnosis of this difficult symptom.

Ron Elisha. General practitioner and playwright.

Australian Family Physician is privileged to present a regular feature on notable Australian doctors. We are aware that there are hundreds of doctors whose service to their patients and dedication to their vocation make then outstanding people and excellent role models. This feature will present profiles on doctors who have contributed something special in their careers whether it be excellence of service or an additional special skill or achievement other than medical. We hope that readers enjoy learning about their colleagues and invite you to let us know about any members of our profession who would be worthy inclusions in this feature.

Acute sore throat.

The second common problem to be presented in this series is the acute sore throat. The common causes are viral pharyngitis and tonsillitis due to streptococcus pyogenes. Another important cause that warrants attention is Epstein Barr virus (infectious mononucleosis) so that prescribing of penicillins is carefully considered. The sore throat may be the presentation of serious and hidden systemic diseases, such as blood dyscrasias, AIDS and diabetes (due to moniliasis).

The painful arm.

The painful arm often presents a diagnostic challenge. The cause is often obvious, especially with regional pain syndromes including the carpal tunnel and the tennis elbows. However, pain reference syndromes can be confusing and it is advisable to first examine the cervical spine because it is a common and often overlooked cause of arm pain or paraesthesia. It is common for arm pain to cause sleep disturbances and in such cases it is worth considering cervical causes, carpal tunnel syndrome and the thoracic outlet syndromes. The working rule is: patients with thoracic outlet syndrome cannot fall asleep; a carpal tunnel lesion wakes the patient in the middle of the night; and cervical spondylosis wakes the patient with pain and stiffness that persists well into the day.

Diarrhoea.

Diarrhoea is defined as the frequent passage of loose or watery stools. Most patients can easily recognise and accurately define acute diarrhoea as an abrupt change in their bowel habits. Chronic or recurrent diarrhoea is more difficult for the patient to define, since it may mean malabsorption, tenesmus or true diarrhoea. Serious disorders not to be missed include neoplasia, AIDS, various serious infections such as amoebiasis, and inflammatory bowel disease.

Hip pain in children.

Children who present with a limp and pain in the hip, anterior thigh and the knee can have a serious disorder of the hip. The age at presentation tends to provide a guide to the possible problem, such as a slipped upper femoral epiphysis and a stress fracture of the femoral neck in adolescents.

Palpitations.

Palpitations are an unpleasant awareness of beating of the heart. By definition, the condition does not always imply 'racing' of the heart but encompasses any sensation in the chest such as 'pounding', 'flopping', 'skipping', 'jumping', 'thumping' or 'fluttering' of the heart. The problem requires careful attention and reassurance because heart beat is regarded as synonymous with life. To the practitioner it may simply represent anxiety or could be a prelude to a cardiac arrest.