Comparison between recombinant baculo- and adenoviral-vectors as transfer system in cardiovascular cells.

The development of effective gene-therapeutic applications for cardiovascular disorders is in part limited by the lack of appropriate delivery systems. In an attempt to overcome this deficiency, we investigated the ability of baculoviral vectors to transduce human cardiovascular cells, for which data are missing in literature. Additionally, baculovirus ability to transduce target cells was compared to that of an adenoviral vector, a well characterized and widely used viral vector. Transduction experiments, performed using baculo/adenoviral vectors expressing the enhanced green fluorescence protein, revealed that, under the experimental condition considered, baculoviruses but not adenoviruses efficiently transduce human coronary smooth muscle cells (hCSMC); an opposite behavior was noticed for human coronary endothelial cells (hCEC). Thus, baculoviral vectors are potentially indicated as transfer system in the treatment of coronary restenosis, where growth inhibitory genes should reach hCSMC but not hCEC. When used to transduce human cardiomyocytes and fibroblasts, both vectors behaved similarly. Finally, studies on cellular DNA replication revealed a more prolonged and pronounced negative effect on cells transduced by adenoviral compared to baculoviral vectors. Our data indicate that baculoviruses represent an attractive alternative to adenoviruses as transfer vectors in cardiovascular cells and that baculovirus have the potential to be used as gene transfer system in cardiovascular diseases such as restenosis.

Expression of somatostatin receptors in inflammatory lesions and diagnostic value of somatostatin receptor scintigraphy in patients with ANCA-associated small vessel vasculitis.

OBJECTIVE: To assess the usefulness of somatostatin receptor (SSTR) scintigraphy for the evaluation of disease activity in the upper and lower respiratory tract in ANCA-associated vasculitis (AASV). METHODS: Thirty-two consecutive patients with AASV were subjected to SSTR scintigraphy as part of their initial diagnostic evaluation and follow-up. The presence of SSTRs in inflammatory lesions was evaluated with immunohistochemistry in selected cases. RESULTS: In AASV, specificity of SSTR scintigraphy for active vs non-active disease was 96% for pulmonary disease and 100% for ear, nose and throat (ENT) involvement, while sensitivity was 86% and 68%, respectively. Absence of previously present tracer accumulation characterized treatment responders, and treatment resistance was reflected by repeated positive scintigraphy. We could demonstrate the expression of SSTRs in lung and mucosal biopsies obtained from patients with active Wegener's granulomatosis and with microscopic polyangiitis. CONCLUSION: SSTR scintigraphy is useful for the assessment of AASV, indicating disease activity, disease extent and treatment efficacy. SSTRs are expressed in both granulomatous as well as non-granulomatous AASV.

[New design concept to improve medical application software]. / Neues Design-Konzept zur Verbesserung der medizinischen Anwendersoftware.

Up to now, there has been no software model available that adequately addresses the growing importance of flexibility in using different information tools. Based on Java, Jini technology provides distributed and therefore robust software architecture. In case of an application crash, Jini is able to minimize the damage by adding functions that reverse the effects of the crash. Owing to increasing user mobility, it is necessary to be able to receive location-independent information. The growing use of powerful telecommunications suggests the application of 'wireless application protocol' (WAP) mobile telephones also for medical purposes. This paper presents the application of these new software trends (Jini and wireless application protocol).

Sentinel lymph node detection with large human serum albumin colloid particles in breast cancer.

Detection of metastatic involvement of lymph nodes is essential for management and prognostic evaluation in breast cancer patients. The success of lymphatic mapping depends on identifying the sentinel lymph node(s) draining the primary tumour. However, when mapping is performed with a radiocolloidal agent, the number of hot lymph nodes varies with the agent and its size, among other factors. In this study, we evaluated prospectively the detection rate of sentinel lymph nodes in breast cancer when injecting large particles (100-600 nm) of human serum albumin colloids (Senti-Scint). In 128 consecutive breast cancer patients without palpable lymph nodes, pre-operative static lymphoscintigraphic mapping of the breast was performed after subcutaneous injection of 15 MBq of the radiocolloid. Lymphoscintigrahic results were compared with intra-operative surgical gamma detection probe and blue dye mapping data. Pre-operative lymphoscintigraphy and surgical gamma detection probe both correctly detected 203 sentinel lymph nodes in 122/128 patients (95%), while blue dye mapping showed only 183 sentinel lymph nodes in 82% of the patients. Only one or two sentinel lymph nodes were identified in each patient, which allowed the surgeon easily to find the sentinel lymph node(s) intra-operatively. In conclusion, lymphoscintigraphy with large particles of human serum albumin colloids is a helpful and reliable procedure for the surgical management of breast cancer.

The inefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent.

BACKGROUND: Ecteinascidin 743 (Et 743), a natural product derived from a marine tunicate, is a potent antitumor agent presently in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine via a unique mechanism involving catalytic activation. The sequence selectivity of Et 743 is governed by different patterns of hydrogen-bonding to DNA, which results in differential reversibility of the covalent adducts. As determined by nuclear magnetic resonance spectroscopy, the preferred sequences 5'-PuGC and 5'-PyGG are stabilized by a hydrogen-bonding network, while the non-preferred sequences 5'-NG(A/T) are much less stabilized due to the lack of a key hydrogen bond to the GC base pair on the 3'-side of the alkylated guanine. RESULTS: Mammalian cell lines (XPB, XPD, XPF, XPG, and ERCC1) deficient in the nucleotide excision repair (NER) gene products show resistance to Et 743. The recognition and subsequent incision of Et 743-DNA adducts by the bacterial multisubunit endonuclease UvrABC were used to evaluate DNA repair-mediated toxicity as a rationale for the resistance of NER-defective cell lines and the antitumor activity of Et 743. The Et 743-DNA adducts are indeed recognized and incised by the UvrABC repair proteins; however, the pattern of incision indicated that the non-preferred, and less stable, sequences (i.e. 5'-NG(A/T)) modified with Et 743 are generally incised at a much higher efficiency than the preferred, more stable sequences (i.e. 5'-PuGC or 5'-PyGG). In addition, within the same Et 743 recognition sequence, the level of incision varies, indicating that flanking regions also contribute to the differential incision frequency. CONCLUSIONS: The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity.

[Determination of therapy management in rectal carcinoma by staging with 18-FDG-PET]. / Bestimmung des Therapiemanagements beim Rectumcarcinom durch Staging mittels 18-FDG-PET.

The curative treatment of carcinoma of the rectum in the early stage of the disease is radical local surgery. If there is a solitary liver metastasis, resection is also a curative treatment. This report describes a female patient with rectal carcinoma, in whom a solitary liver metastasis in the left lobe was diagnosed only by FDG-PET and verified at surgery. This case report demonstrates the potential role of FDG-PET even for primary staging in detecting occult hepatic and extrahepatic metastases, thus significantly influencing the therapeutic management and prognosis of these patients.

Synthesis, DNA cross-linking activity, and cytotoxicity of dimeric mitomycins.

Dimeric DNA cross-linking compounds have emerged as important new antitumor agents. We report the synthesis and biochemical evaluation of a select set of dimeric mitomycins in which the two mitomycin units are tethered at either the mitomycin C(7) amino or the aziridine N(1a) positions. Significantly, mitomycin C (1) itself is the prototypical bioreductive DNA cross-linking agent. DNA cross-linking experiments using a denaturing-gel-electrophoresis-based assay showed that the extent of DNA cross-linking for select dimeric mitomycins can exceed that of the parent compound, mitomycin C, and that the reaction proceeds, in part, at the two distal C(1) sites in the mitomycins. The efficiency of DNA cross-linking depended on the nature of the linker and the position of linker unit's attachment. When we compared the efficiency of DNA cross-linking for the dimeric mitomycins with their in vitro cytotoxicities in cultured human tumor cells, we observed a poor correlation. The mitomycins that gave the highest levels of DNA cross-linked adducts displayed the weakest cytotoxicities. These findings determined that the denaturing-gel-electrophoresis-based assay was a poor predictor of cytotoxic activity.

Functionalized amino acid anticonvulsants: synthesis and pharmacological evaluation of conformationally restricted analogues.

Proven conformationally restricted analogues of anticonvulsant functionalized amino acids (FAAs) were prepared using short-range cyclizations and evaluated in pharmacological assays providing new information concerning the structural requirements for FAA function.

Regional cerebral blood flow in patients suffering from post-traumatic stress disorder.

OBJECTIVE: The aim of the study was to determine whether regional cerebral blood flow in survivors of torture suffering from post-traumatic stress disorder (PTSD) differed significantly from that in healthy controls. METHOD: We examined the cerebral regional distribution of 99m-technetium-hexamethylpropyleneamineoxime (HMPAO) using single photon emission computed tomography (SPECT) in 8 patients and in 8 healthy controls. A semi-quantitative analysis was performed in which symmetrical regions of interest (ROI) were drawn in all subjects. RESULTS: Regional blood flow was markedly more heterogeneous in patients suffering from PTSD than in healthy controls. The differences are significant. CONCLUSION: Severe psychological trauma induced by torture can cause neurobiologic alterations that may contribute, even years after the original trauma, to a number of complaints commonly expressed by patients suffering from PTSD.

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles.

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug DNA modification was accomplished with I and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) condilions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda-exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.

C(5)-C(5a)-modified bicyclomycins: synthesis, structure, and biochemical and biological properties.

Bicyclomycin (1) is a novel antibiotic that targets rho transcription termination factor in Escherichia coli. We have demonstrated that retention of the C(5)-C(5a) exomethylene unit in 1 is not essential for inhibition. In a recent paper we proposed a working model for 1 and rho function and suggested that 1 binds in a cleft with the C(5)-C(5a) exomethylene unit directed toward the dimeric interface of two rho monomers. This report examines the bicyclomycin C(5)-C(5a) structural constraints necessary for retention of rho inhibitory activity. Three classes of C(5)-C(5a)-modified bicyclomycins have been prepared and their inhibitory activities evaluated in the poly C-dependent ATPase and filter disk antimicrobial assays. The first series consisted of 12 analogues (8-19) that contained a C(5a)-unsaturated substituent and possessed C(5E)-geometry. The second set were a pair of C(5a)-substituted C(5E)- and C(5Z)-geometrical isomers (21 and 23). The final group of compounds consisted of six C(5)-C(5a)-dihydrobicyclomycins (24-28, 34) where the terminal substituent was systematically varied. We find that extending the C(5)-C(5a) double bond with unsaturated substituents provides bicyclomycin derivatives with excellent inhibitory activities in the biochemical assay, and that enhanced inhibitory activity is observed for the C(5E) geometrical isomer compared with its C(5Z) counterpart. Finally, C(5a)-substituted dihydrobicyclomycin inhibitory activity appears to be tightly regulated by the nature and spatial placement of the C(5a)-terminal substituent with respect to the [4.2.2]-bicyclic ring system. The observed biochemical activities for the C(5a)-extended conjugated bicyclomycin derivatives and the (5E) and (5Z) isomers were correlated with a structural model for the 1-rho complex.

Synthesis and structural studies of aza analogues of functionalized amino acids: new anticonvulsant agents.

We have reported that functionalized amino acids 1 display potent anticonvulsant activities in mice and rats, and that the activity resides primarily in the D-isomer. In this study we investigated whether selectively replacing the C(2) tetrahedral atom with a trivalent nitrogen provides compounds with comparable activity. Six functionalized N(2)-substituted semicarbazides (3) were prepared. X-ray crystallographic analysis of 1-acetyl-4-benzyl-2-(thiazol-2-yl)semicarbazide (13) showed that it lost asymmetry and adopted a configuration midway between the corresponding D- and L-amino acid derivatives. Evaluation of 3 in both mice (ip) and rats (po) showed that the compounds exhibited significant anticonvulsant activities but in most cases at levels lower than their amino acid counterparts. One of the semicarbazides, 13, displayed excellent activity in mice and rats that compared favorably to that of phenytoin.