Porphyrin-related photosensitizers for cancer imaging and therapeutic applications.

A photosensitizer is defined as a chemical entity, which upon absorption of light induces a chemical or physical alteration of another chemical entity. Some photosensitizers are utilized therapeutically such as in photodynamic therapy (PDT) and for diagnosis of cancer (fluorescence diagnosis, FD). PDT is approved for several cancer indications and FD has recently been approved for diagnosis of bladder cancer. The photosensitizers used are in most cases based on the porphyrin structure. These photosensitizers generally accumulate in cancer tissues to a higher extent than in the surrounding tissues and their fluorescing properties may be utilized for cancer detection. The photosensitizers may be chemically synthesized or induced endogenously by an intermediate in heme synthesis, 5-aminolevulinic acid (5-ALA) or 5-ALA esters. The therapeutic effect is based on the formation of reactive oxygen species (ROS) upon activation of the photosensitizer by light. Singlet oxygen is assumed to be the most important ROS for the therapeutic outcome. The fluorescing properties of the photosensitizers can be used to evaluate their intracellular localization and treatment effects. Some photosensitizers localize intracellularly in endocytic vesicles and upon light exposure induce a release of the contents of these vesicles, including externally added macromolecules, into the cytosol. This is the basis for a novel method for macromolecule activation, named photochemical internalization (PCI). PCI has been shown to potentiate the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins, immunotoxins, gene-encoding plasmids, adenovirus, peptide-nucleic acids and the chemotherapeutic drug bleomycin. The background and present status of PDT, FD and PCI are reviewed.

Global analysis of transcription kinetics during competence development in Streptococcus pneumoniae using high density DNA arrays.

The kinetics of global changes in transcription patterns during competence development in Streptococcus pneumoniae was analysed with high-density arrays. Four thousand three hundred and one clones of a S. pneumoniae library, covering almost the entire genome, were amplified by PCR and gridded at high density onto nylon membranes. Competence was induced by the addition of CSP (competence stimulating peptide) to S. pneumoniae cultures grown to the early exponential phase. RNA was extracted from samples at 5 min intervals (for a period of 30 min) after the addition of CSP. Radiolabelled cDNA was generated from isolated total RNA by random priming and the probes were hybridized to identical high density arrays. Genes whose transcription was induced or repressed during competence were identified. Most of the genes previously known to be competence induced were detected together with several novel genes that all displayed the characteristic transient kinetics of competence-induced genes. Among the newly identified genes many have suggested functions compatible with roles in genetic transformation. Some of them may represent new members of the early or late competence regulons showing competence specific consensus sequences in their promoter regions. Northern experiments and mutational analysis were used to confirm some of the results.

Transient gene asymmetry during sporulation and establishment of cell specificity in Bacillus subtilis.

Sporulation in Bacillus subtilis is initiated by an asymmetric division generating two cells of different size and fate. During a short interval, the smaller forespore harbors only 30% of the chromosome until the remaining part is translocated across the septum. We demonstrate that moving the gene for sigmaF, the forespore-specific transcription factor, in the trapped region of the chromosome is sufficient to produce spores in the absence of the essential activators SpoIIAA and SpoIIE. We propose that transient genetic asymmetry is the device that releases SpoIIE phosphatase activity in the forespore and establishes cell specificity.

Using a double blind controlled clinical trial to evaluate the function of a Diabetes Advisory System: a feasible approach?

This paper assesses the feasibility of using a double blind controlled clinical trial to evaluate the function of a decision support system by applying such a design to the evaluation of a Diabetes Advisory System (DIAS). DIAS is based on a model of the human carbohydrate metabolism and is designed an interactive clinical tool, which can be used to predict the effects of changes in insulin dose or food intake on the blood glucose concentration in patients with insulin dependent diabetes. It can also be used to identify risk periods for hypoglycaemia. and to provide advice on insulin dose. The latter feature was evaluated in the present study. We believe double blind controlled clinical trials are prerequisites for clinical application of many decision support systems, and conclude that the present double blind controlled clinical trial is a suitable evaluation method for the function of DIAS.

Risk of haemorrhage from transurethral prostatectomy in acetylsalicylic acid and NSAID-treated patients.

Postoperative bleeding in patients who regularly ingest acetylsalicylic acid (ASA) has been reported after several types of surgery. However, data on the influence of ASA on the risk of haemorrhage from transurethral prostatectomy (TUR-P) have been conflicting. We have studied retrospectively the unselected clinical records of all patients undergoing TUR-P in the Department of Urology at Hvidovre Hospital (during 1992-1994) with special focus on the use of ASA and non-steroidal anti-inflammatory drugs (NSAIDs). In total, 457 records were examined: 99 patients on ASA/NSAID received 42 units of blood, while 358 patients free from such medication received 68 units of blood, a significantly smaller amount (p = 0.0390). We conclude that ASA and NSAIDs increase the risk of bleeding during and after TUR-P, and we recommend the withdrawal of these drugs for one week before TUR-P.

Plasmids for ectopic integration in Bacillus subtilis.

Plasmids have been constructed that allow integration by a double recombination event at the thrC locus of the Bacillus subtilis (Bs) chromosome. These plasmids can be used either for construction of merodiploid strains and complementation analysis, or for construction of transcriptional fusions to the Escherichia coli lacZ gene. The plasmids contain an antibiotic (An) marker selectable in Bs, as well as an additional An marker outside of the region that can recombine into the chromosome. When used in conjunction with recipient strains containing a third An marker at their thrC locus, these plasmids allow easy identification of transformants issued from a marker exchange event without additional Campbell-type integration. The existing plasmids used for ectopic integration at the amyE locus have been modified similarly.

Time course of platelet alpha granule release in acute myocardial infarction treated with streptokinase.

OBJECTIVE: To determine the time course of platelet alpha granule release in patients with acute myocardial infarction treated with streptokinase. DESIGN: A prospective study. SETTING: Coronary care unit. PATIENTS: Nine with myocardial infarction treated with both streptokinase and aspirin, and nine with acute chest pain but without myocardial infarction, who were treated with aspirin only. METHODS: All patients received 250 mg aspirin on admission and 150 mg once daily thereafter. All patients who fulfilled the indications for streptokinase received 1.5 megaunits, in a single infusion. After the initial medication, serial measurements of plasma beta thromboglobulin and plasma platelet factor 4 were performed at fixed intervals after the onset of chest pain. The primary endpoint sought was the peak value of beta thromboglobulin and platelet factor 4 in each individual. RESULTS: The median peak plasma beta thromboglobulin in the infarction group was substantially higher than in those without infarction, at 37 (range 12 to 210) v 15 (9 to 36) mg/litre, P < 0.01. The corresponding values for plasma platelet factor 4 were 4.6 (2.4 to 60.0) v 2.2 (< 2 to 8.5) mg/litre, P < 0.01. Increased values were seen only within the first 12 h after onset of chest pain, and after 12 h there was no difference between the patients with myocardial infarction and those without. Aspirin treatment did not abolish alpha granule release. CONCLUSIONS: In patients with acute myocardial infarction treated with streptokinase the content of the alpha granules is released within the first 12 h after the onset of chest pain. Aspirin apparently does not abolish this release.

Antibiotic-resistance cassettes for Bacillus subtilis.

The genes encoding resistance to four different antibiotics (erythromycin, kanamycin, tetracycline and spectinomycin) were cloned in the polylinker of various Escherichia coli plasmid vectors. These cassettes can be inserted into cloned Bacillus subtilis (Bs) genes and used to create tagged chromosomal disruptions after recombination into Bs and selection in the presence of the appropriate antibiotic.

Cell-type specificity during development in Bacillus subtilis: the molecular and morphological requirements for sigma E activation.

Development in Bacillus subtilis involves the formation of two cell types with activation of the transcription factors sigma F in the forespore and sigma E in the mother cell. Activation of sigma E is due to the processing of the inactive precursor pro-sigma E, which requires the putative protease SpoIIGA and the presence of active sigma F. We have introduced missense mutations altering the promoter recognition properties of sigma F. These mutations abolish pro-sigma E processing, suggesting that sigma F is involved through its transcriptional activity and that the processing machinery responds to a signal generated by the product(s) of some unidentified gene(s) transcribed in the forespore. The role of the septum in transducing this signal was investigated. Induction of sigma F during exponential growth in cells producing SpoIIGA and pro-sigma E led to a high level of processing and sigma E activity. Moreover, pro-sigma E was efficiently processed in a mutant strain blocked prior to septation and synthesizing sigma F in active form at the onset of sporulation. Therefore, the sporulation septum is not required for induction of pro-sigma E processing and pro-sigma E can be processed in the same cell in which sigma F is active. These results suggest that some unknown mechanism must exist to prevent sigma E from becoming active in the forespore.

Identification and characterization of the Bacillus subtilis spoIIP locus.

We have identified an additional sporulation gene, named spoIIP, in the region of the Bacillus subtilis chromosome located immediately downstream of the gpr gene (227 degrees on the genetic map). A null mutation of spoIIP arrests sporulation at an early stage of engulfment (stage IIii), a phenotype similar to that already described for spoIID and spoIIM mutants. This gene encodes a 401-residue polypeptide, which is predicted to be anchored in the membrane, most of the protein being localized outside the cytoplasm. The spoIIP gene is transcribed from a promoter located in the interval between the gpr and the spoIIP reading frames. This promoter has the structural and genetic characteristics of a sigma E-dependent promoter. Transcription of spoIIP is abolished by a mutation in spoIIGB, the gene encoding sigma E, and can be induced during exponential growth in cells engineered to produce an active form of sigma E. Plasmid integration-excision experiments leading to the formation of genetic mosaics during sporulation indicate that as with SpoIID and SpoIIM, SpoIIP is required only in the mother cell. Disruption of spoIIP had little or no effect on the expression of sigma F- and sigma E-controlled regulons but inhibited transcription from sigma G-dependent promoters and abolished transcription from promoters under the control of sigma K. We propose that, together with SpoIID and SpoIIM, the SpoIIP protein is involved in the dissolution of the peptidoglycan located in the sporulation septum.

Excess histidine enzymes cause AICAR-independent filamentation in Escherichia coli.

High-level expression of the hisHAFI genes in Escherichia coli, cloned under the control of an IPTG-inducible promoter, caused filamentation, as previously reported in Salmonella typhimurium. We speculated that this filamentation might be produced by an action of the HisH and HisF enzymes on their product AICAR (amino-imidazole carboxamide riboside 5'-phosphate), a histidine by-product and normal purine precursor, possibly by favouring the formation of ZTP, the triphosphate derivative of AICAR. However, filamentation occurred even in the absence of carbon flow through the histidine and purine pathways, as observed in a hisG purF strain lacking the first enzyme in each pathway. Filamentation thus does not require either the normal substrate or products of the overproduced histidine enzymes and must reflect another activity.

AICAR is not an endogenous mutagen in Escherichia coli.

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5'-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur+ his+ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac+ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.

[Magnesium and ischemic heart disease]. / Magnesium og iskaemisk hjertesygdom.

Our knowledge about magnesium in health and disease has increased during the past ten years. Many authors have demonstrated possible magnesium depletion in the population as a whole and particularly among cardiac patients receiving diuretics. Evidence suggests that this magnesium depletion may play a role in the development of arteriosclerosis. It has been demonstrated that long term supplementation of the diet with magnesium reduces the frequency of heart disease. However, the matter is still not proven. It is postulated further that magnesium depletion aggravates the outcome of acute myocardial infarct and has a tendency to provoke arrhythmia. Finally, it has been shown that the potassium depletion frequently observed among cardiac patients may actually arise from magnesium depletion. On this basis, many authors have employed magnesium therapy in various cardiac diseases. Some authors have demonstrated that magnesium therapy reduces the mortality in AMI patients. These studies are, however, too few and too limited to be conclusive. In order to investigate this question, an international multicentre trial (ISIS-4) will be conducted to investigate the influence of magnesium therapy on the mortality after acute myocardial infarction.

[Basedow's disease and thyroid stimulating hormone producing pituitary adenoma in a patient]. / Mb. Basedowi og thyreoideastimulerende hormonproducerende hypofyseadenom hos samme patient.

TSH-producing adenomas of the pituitary gland are very rare. Synchronous combinations of TSH-producing adenomas with other causes of hyperthyroidism are certainly extremely rare. We present the second known case, reported in the literature, consisting of observations for 12 years in a woman aged 43 years, who presented with active Graves' disease and an apparently inactive pituitary macro-adenoma. However, after normalisation of serum T3 and serum T4 levels by antithyroid medication for one year, the serum TSH rose inappropriately and continued to rise for the following 11 years. Insidious growth of the adenoma also occurred. After one year of medical treatment, a huge goitre was resected (210 g) leaving the patient euthyroid, clinically and biochemically, for four years. Hereafter, hyperthyroidism developed again this time without Graves' disease. We conclude that the patient experienced hyperthyroidism on two occasions, the first caused by Graves' disease and then caused by a TSH-producing pituitary adenoma.

[Immunosuppression with cyclosporin induces clinical remission and improved beta cell function in patients with newly diagnosed insulin-dependent diabetes. A national and international multicenter study]. / Immunosuppression med ciklosporin inducerer klinisk remission og forbedret beta-cellefunktion hos patienter med nykonstateret insulinkraevende diabetes. En national og international multicenterundersøgelse. The Canadian-European Randomized Control Trial Group.

A prospective, randomized, double-blind, placebo-controlled international multicenter trial including 188 newly diagnosed insulin-dependent diabetic (IDDM) patients was undertaken with the aim of investigating whether immunosuppression for one year with ciklosporin (Cs) could induce and maintain clinical remission and improvement of beta-cell function. The relative odds for non-insulin-requiring remission at one year were increased approximately five times in the Cs-treated group. After three months Cs-treated patients achieved more than a doubling of beta-cell function compared to baseline than did placebo-treated patients, and the Cs-treated group maintained this improvement in beta-cell function for 12 months, whereas the placebo-group lost beta-cell function during the same period. Short duration of disease (less than or equal to six weeks of symptoms, less than or equal to two weeks of insulin treatment) was associated positively with remission, as was an elevated proinsulin/C-peptide ratio, especially in patients with the tissue-type HLA-DR 3,4; 4,X and X,X. Cs-treatment inhibited the formation of antibodies against insulin and islet cell components, but islet cell antibody status at entry was not predictive of remission. Cs-treatment caused a reversible decrement of kidney function as measured with serum creatinine and the calculated creatinine clearance, but studies of renal physiology and kidney biopsies performed on a limited subset of patients indicated that Cs treatment in IDDM patients for one year induced a slight chronic nephropathy in some of these.(ABSTRACT TRUNCATED AT 250 WORDS)

Fast, stable method for harvesting leukocytes for measuring intracellular magnesium.

A new method for isolating blood leukocytes and measuring intracellular leukocyte magnesium is based on three successive centrifugations (1300 x g, 5 min, 22 degrees C), with no use of Ficoll, followed by two washes. We express the leukocyte magnesium concentration in micromoles of magnesium per gram of protein. Magnesium is measured by atomic absorption spectrophotometry, protein by the classic Lowry method. With the method 8% to 28% of the leukocytes are isolated and 0% to 0.025% of the erythrocytes. Of the isolated leukocytes 86% to 93% are viable. The magnesium concentration is constant at centrifugal forces greater than 1000 x g, at a centrifugation time of 5 to 15 min, and with use of two or three washes. However, the cells lose 1% of their magnesium content per hour when samples are left at 22 degrees C. The temperature influences this. In blood samples from 98 volunteer blood donors, mean leukocyte magnesium concentrations were 26.4 (SD 3.9) mumol/g. There was no significant correlation between the magnesium concentration and the relative amount of neutrophils.

Nephrotoxicity of cyclosporin A in humans: effects on glomerular filtration and tubular reabsorption rates.

The contention that cyclosporin A (CyA) nephrotoxicity may be due to renal afferent arteriolar constriction was inferred from rat studies showing CyA to increase renal vascular resistance, to reduce glomerular filtration rate (GFR) and delivery of tubular fluid from the end of the proximal tubule to the loop of Henle (Vprox), and to increase proximal fractional reabsorption. In order to test whether the mechanism of human CyA nephrotoxicity is similar to its rat analogue, and whether CyA treatment causes prolonged renal malfunction after drug withdrawal, renal function was investigated with clearance techniques including lithium clearance (CLi) as a measure of Vprox. The subjects were patients (n = 11) with previously normal renal function, given CyA in the treatment of ocular manifestation of extrarenal disease, or bone-marrow transplant recipients. Nine out of these eleven patients were investigated before and during CyA treatment: GFR (P less than 0.05) and Vprox (P less than 0.005) decreased while proximal fractional reabsorption increased (P less than 0.01). In six patients investigated before CyA was given, and re-examined a mean of 273 days (range 84-384 days) after CyA withdrawal, CLi was reduced (P less than 0.05) while mean GFR was not significantly lowered (0.5 greater than P greater than 0.2). In one of these six patients GFR was reduced to a subnormal value of 26 ml min-1 (1.73 m2 body surface)-1. In conclusion, human and rat CyA nephrotoxicity have the same pattern of renal functional deterioration. Cyclosporin A nephrotoxicity was evident in patients investigated a mean of 9 months after CyA withdrawal.