The 5'-CA DNA-sequence preference of daunomycin.

The DNA-sequence specificity of daunomycin was investigated by DNase I footprinting and an E. coli RNA polymerase transcription-inhibition assay. The 5'-CA sequence was identified as being the highest affinity binding site, although other modest affinity (5'-GC, CG, CT, TC, AC) and poor affinity sites (5'-AA, AT, TA) were also observed. The preference of daunomycin for 5'-CA nucleotide sequence suggests that its biological activity may arise from association with the 5'-CA-containing sequences thought to be associated with genetic regulatory elements in eukaryotes.

Biodistribution of haematoporphyrin analogues in a lung carcinoma model.

In an attempt to identify novel compounds useful for the optimization of Photodynamic Therapy (PDT), the tissue localization of new synthetic porphyrins was compared with Photofrin II in nude mice xenografted with a human small cell lung cancer (POVD). Three haematoporphyrin analogues were selected for this study based on prior in vitro photosensitivity screening of a series of 15 such derivatives, as well as on the basis of improved localization in C6 gliomas in mice. Two of the porphyrins yielded better tumour:normal lung ratios than Photofrin II and, of these two, one (P13) is known to exhibit good photosensitization properties both in vitro and in vivo, and is therefore a good candidate as a lead compound for the development of porphyrins suitable for the photodynamic treatment of lung tumours.

Distal arthrogryposis type II: a family with varying congenital abnormalities.

Five family members with distal arthrogryposis in two generations are reported. Cleft lip and palate, micrognathia, ptosis, webbed neck, kyphoscoliosis, and short stature are seen in one or more affected family members. All individuals with distal arthrogryposis also have trismus. This family does not fit any of the recently proposed five subcategories of type II distal arthrogryposis, nor does it fit any other recognized autosomal dominant condition with distal contractures.

A genetic follow-up study of 64 patients with the Pierre Robin complex.

A genetic follow-up study has been performed of 64 infants who were diagnosed as having Pierre Robin complex over a 23-year period in South Australia. Patients and their families were contacted, family history was obtained, and physical examinations were performed with an aim to detect heterogeneity and establish recurrence risks. In 16 deceased patients, detailed autopsy reports allowed the conclusion that 12 (70%) had an underlying syndrome. Twelve of the 47 living patients (26%) were diagnosed as having an underlying syndrome, the most common of which was Stickler syndrome (6 cases). In most cases separation of syndromic cases from the nonsyndromic cases was possible in the neonatal period. In the 34 patients without an underlying syndrome, study of pregnancy and birth details did not reveal any distinctive etiologic factors. There was no recurrence in sibs of this group of patients with nonsyndromic Pierre Robin complex.

Interstitial deletions of the short arm of chromosome 4 in patients with a similar combination of multiple minor anomalies and mental retardation.

Interstitial deletions of chromosome 4 have been described rarely and have had variable presentations. We describe the phenotypic characteristics associated with interstitial deletion of the p14-16 region of chromosome 4 in 7 patients with multiple minor anomalies in common, and with mental retardation. A review of published cases of interstitial deletions of the short arm of chromosome 4 is provided. These deletions present a distinct phenotype which is different from that of Wolf-Hirschhorn syndrome.

Tandem duplication of proximal 22q: a cause of cat-eye syndrome.

A boy with bilateral colobomas, preauricular pits, and developmental delay had a 46,XY,22q+ karyotype. His parents had normal chromosomes. The abnormality of 22q was interpreted as a de novo tandem duplication of 22q11.1----q11.2. Although no anal abnormality was identified, his manifestations are otherwise consistent with those of the cat-eye syndrome. Blood marker results and the levels of galactosidase-2, galactosidase-B and arylsulfatase-A, which are known to be coded on 22q, are normal.

Clinical outcomes of four patients with microdeletion in the long arm of chromosome 2.

We present clinical outcome, through several years of follow-up, of 4 mentally retarded patients, each with a small interstitial deletion in the long arm of chromosome 2, within a region on which clinical reports are infrequent. Our patient 1 was found to have del(2)(q22.3q23.3); patients 2 and 3, del(2)(q23.3q24.2); and patient 4, del(2) (q24.2q31). By comparison of our cases with each other and with those previously published with comparable interstitial deletion, we attempted to identify characteristic clinical findings. Short neck with excessive cervical skin was seen with monosomy of chromosome 2 bands q22.3-q23.3, while hypertrichosis and a peculiar high pitched cry were seen with monosomy of chromosome 2 bands q23.3-q24.2. As suggested by Moller et al. [1984: Hum Genet 68:77-86], a cleft between the first and second toes was seen with monosomy of chromosome 2 bands q24.2-q31. In addition, seizure disorder was present in patients 1 and 4 (with the more proximal and distal deletions, respectively).

Evaluation of porphyrin characteristics required for photodynamic therapy.

The cytotoxicity (in the dark), phototoxicity (red light) and subcellular localization (using confocal laser scanning microscopy) were determined for 15 porphyrins (1-15) in C6 glioma cells. The partition coefficient in 2-octanol was also determined for each porphyrin at pH 7.4. The cytotoxicity increased with pi (log of partition coefficient) up to pi values of +2. The 7 porphyrins with cationic side chains exhibited a classical parabolic correlation between phototoxicity and pi, with maximal activity at a pi value of approximately 1.0. There was also a significant correlation between subcellular localization and degree of phototoxicity, with the three most photosensitive porphyrins all possessing cationic side chains, and all three localizing in mitochondria.

A comparison of the treatment of acute myocardial infarction between St. Petersburg, Russia and Seattle, Washington.

This study compared the baseline demographics, time to treatment and types of treatment of patients presenting with acute myocardial infarction in St. Petersburg, Russia and Seattle, WA. The study included 63 consecutive patients admitted to City Hospital #1 in St. Petersburg in July 1993. Comparative data for Seattle patients was obtained from the multi-year Myocardial Infarction Triage and Intervention project, a registry of all acute myocardial infarction patients hospitalized in the Seattle area. The results show a significantly prolonged time from symptom onset to presentation in Russia (51.8 h vs. 8.0 h; P < 0.001). Aspirin was used slightly more often in Seattle (78.9% vs. 74.6%; P = 0.40) while thrombolysis was used much more often in Seattle (22.5% vs. 6.3%; P = 0.002). There was also much less use of percutaneous transluminal coronary angioplasty (0% vs. 26.9%; P < 0.001), heparin (12.7% vs. 79.1%; P < 0.001), or cardiac catheterization (4.8% vs. 64.3%; P < 0.001). Also, the length of hospitalization was longer in St. Petersburg (23.8 +/- 10.8 vs. 7.5 +/- 5.1 days; P < 0.001). The findings in this study are an impetus for us all in understanding the magnitude of differences currently existing and the challenges for improving health care delivery in Russia.

An unobtrusive measurement method of the horizontal gaze angle.

A horizontal gaze angle measurement device is introduced. By combining a photoelectric viewing device to measure the horizontal eye angle with a similar head angle measurement device, it is possible to measure the horizontal gaze angle without using a headrest. After discussion of circuit diagrams and measurement principles, it is shown that the measurements made with the device yield a reasonable precision. The mean absolute measurement error is below 1 degree. This inexpensive and unobtrusive device covers a visual field of about 20 degrees and can be used in parallel with many tasks. Further, data on the successful application of the device in a driving simulation setting are discussed.

Partial trisomy 3p syndrome.

Two cousins with an unbalanced chromosome translocation (partial trisomy 3p) are described. Both children have a clinically recognizable syndrome of square facies with prominent cheeks, narrow bitemporal regions, psychomotor retardation and congenital heart disease. Extended family studies showed one other individual proven to have partial trisomy 3p karyotype, two retarded individuals with congenital heart disease who probably had it, and 14 balanced carriers of the translocation t(1;3)(q43;p21). This report confirms the characteristic clinical appearance of affected individuals and emphasizes the frequency in which congenital heart disease is the presenting feature of partial trisomy 3p. An additional 22 cases of partial 3p trisomy are reviewed.

Synthesis of polyamine-linked bis-daunomycin hydrazones and their interaction with DNA.

A series of bis-anthracycline hydrazones were prepared, compounds 9, containing either two or three secondary amines in the linker chain as well as the corresponding acetyl (10), or benzoyl (11) protected derivatives. Compounds 9 could not be isolated in pure form as they decomposed during semi-preparative h.p.l.c. Compounds 10a-g and 11b were characterized by f.a.b. mass spectrometry. All compounds bis-intercalated into DNA as judged by viscometric lengthening of DNA and by decreased dissociation kinetics from DNA under conditions of SDS sequestration. The secondary amine containing derivatives (9) exhibited dissociation rates less than 10(-5) that of daunomycin. The apparent affinity of these compounds was so great that the dissociation constants could not be quantitated, and they were only liberated from DNA to monomeric species by a slow hydrolysis process of the hydrazone links. Protection of those derivatives containing two amines in the linker by acetyl (10) and benzoyl (11) groups, decreased the dissociation time constant to 490-2900 s (1400-8000 fold slower than daunomycin) with maximal DNA residence time corresponding to a linker length of 14 atoms (approximately 14 A). Addition of a third protected amine in the linker, compound 10g, enhanced the DNA residence time a further three-fold. In vitro inhibition of transcription analysis showed that all of the bis-anthracyclines exhibited a DNA sequence specificity for 5'-CpA-3' sites, with adjacent intercalation sites decreasing in the order CA greater than AC, TC greater than CT greater than GC, CG, CC, TA, and it is apparent that the presence of the basic amino functions in the bridging chain provides for greatly enhanced formation of the drug-DNA complex.

Elucidation of the DNA sequence preferences of daunomycin.

The sequence specificity of daunomycin was assessed using competition equilibrium dialysis, DNAse I footprinting and an E. coli RNA polymerase transcription inhibition assay; similar studies were performed on adriamycin and a new bis-intercalating daunomycin dimer. The results clearly demonstrate that the highest affinity sites are CA for daunomycin and adriamycin, and CACA for the bis-daunomycin. Other modest affinity (GC, CG, CT, TC, CC, AC) and poor affinity binding sites (AA, AT, TA) were also observed. Our results are in agreement with (a) the observed 5'-pyrimidine-purine-3' sequence preference of intercalating drugs, (b) the reported role played by OH(9) of daunomycin in the stabilization of the drug/DNA intercalation complex, and (c) the thermodynamics of nearest neighbour base-pair unstacking at the intercalation site. The CA specificity of daunomycin and adriamycin suggests that their biological activity may arise from association with the CA containing sequences which are thought to be associated with genetic regulatory elements in eukaryotes. The implications for future anthracycline drug design are presented in this context.

Application of analytical and semi-preparative high-performance liquid chromatography to anthracyclines and bis-anthracycline derivatives.

Efficient high-performance liquid chromatographic (HPLC) methods have been developed for routine analysis of a number of anthracycline derivatives using reversed-phase muBondapak C18 columns and an isocratic methanol-water-ammonium carbonate (or acetate) solvent system. The rates of formation of bis-daunomycin derivatives of alpha, omega-dicarboxylic acid hydrazides have been investigated using the analytical methods developed. It has been demonstrated that the reaction proceeds via an intermediate mono-hydrazone. Both the mono- and bis-hydrazones have been isolated by preparative HPLC.

Bis-daunomycin hydrazones: interactions with DNA.

A series of bis-daunomycin hydrazones were synthesised from diester diamide linking groups derived from alpha,omega-dicarboxylic acids. All members of the series bis-intercalated into DNA, as evidenced by doubling of the lengthening of rod-like DNA compared to daunomycin, and by a 1000-5000 fold slower dissociation from DNA than daunomycin under detergent sequestration conditions. The bis-hydrazones exhibited neighbour exclusion, and occupied 6 bp under saturating conditions of drug. A unique DNA sequence specificity was apparent from transcriptional footprinting of 100 bp of DNA, with the greatest preference for 5'-CACA sites.

The 8p- syndrome.

A boy with severe retardation of growth and development, minor dysmorphic features, severe congenital heart disease, and a 46,XY,8p-karyotype is described. The clinical findings of this boy are compared with those of others reported monosomic for a portion of the short arm of chromosome 8. The red cell glutathione reductase (GSR) level is normal in our patient.

The DNA-association and biological activity of a new bis(14-thiadaunomycin).

A new bis-daunomycin has been synthesized and characterized by 13C-n.m.r. and reversed-phase h.p.l.c. The compound was found to be highly self-associated in aqueous solution and to bis-intercalate into DNA with a residence time about 200-fold greater than the parent compound daunomycin. Although the bis-daunomycin and its parent congener were taken up by V79 Chinese hamster ovarian leukaemia cells to similar extents, the cytotoxicity of the former was considerably lower. The possible in vivo relationship between DNA binding affinity and cytotoxicity is discussed.

Evaluation of a morpholinothiolporphyrin for use in photodynamic therapy.

The photonecrotic effectiveness of a morpholinothiolporphyrin derived from haematoporphyrin was measured in an animal model of cerebral glioma. The dose administered was 20 mg kg-1 and the laser dose varied from 0 to 200 J cm-2. The tumour necrosis was at least as good as that of HpD, and this therapeutic response may be attributed to the targeting of specific 'photopotent' subcellular sites.

Fast atom bombardment mass spectrometry of some anthracycline and bisanthracycline derivatives.

Fast atom bombardment (FAB) mass spectra of daunomycin, four of its derivatives, seven bisanthracyclines and three mixed-functional daunomycin-acridine derivatives are reported. These anthracyclines all exhibited their expected [MH]+ ions and peaks corresponding to the fragmentations which are characteristic of the anthracycline moiety, and in addition the spectra showed enhanced [MH + n]+ (n = 1-4) ions which were attributed to reductive processes occurring in the liquid matrix under FAB conditions. Daunomycin was also observed to form a dimeric cluster ion [M2H]+ together with associated reduced ions under FAB conditions. We have found that FAB mass spectrometry is an ideal method for the qualitative analysis of large, non-volatile derivatives of anthracyclines.

Evaluation of tumour and tissue distribution of porphyrins for use in photodynamic therapy.

A range of pure, monomeric porphyrins were synthesised and their localising capacities compared to HpD and Hp at 6 h and 24 h post injection in the mouse C6 intracerebral glioma model as well as in normal brain, skin, muscle, kidney, spleen, liver, lung and whole blood. The partition coefficients were examined between PBS and 2-octanol over the pH range 7.4-6.6 and pH profiles were established. A parabolic relationship was observed between log (porphyrin tumour concentration) at pH 7.4, with maximal tumour localisation at log (partition coefficient), pi, of approximately zero. Porphyrins with side chains with nett cationic character also exhibited up upward (parabolic) dependence on pi for most tissues studied, with maximal porphyrin localisation at pi of 0-0.5. In contrast, those porphyrins with nett anionic character exhibited a downward (negative) parabolic trend for all eight tissues studied, with minimal porphyrin localisation at pi of approximately zero. Four porphyrins (4, 11, 12, 13) exhibited similar or better tumour localisation than HpD, and two (11 and 12) offer promise as lead compounds for the design of improved porphyrins for use in PDT.