Synthesis and characterization of high-molecular mass polyethylene glycol-conjugated oligonucleotides

PURPOSE: Evaluation of skin layer-specific prednicarbate (PC) biotransformation, possibly explaining the improved benefit/risk ratio of this topical corticosteroid in atopic dermatitis (1,2). METHODS: Metabolism of PC in keratinocyte and fibroblast monolayers derived from human juvenile foreskin was evaluated. Drug concentration was determined by HPLC/UV-absorption. Accompanying cell viability tests (MTT-tests) were performed to exclude toxic drug effects. RESULTS: Keratinocytes hydrolyzed the double ester PC (2.5 x 10(-5) M) at position 21 to the monoester prednisolone 17-ethylcarbonate (P17EC) which nonenzymatically transformed to prednisolone 21-ethylcarbonate (P21EC). This metabolite was enzymatically cleaved to prednisolone (PD), the main biotransformation product at 24 hours. Fibroblasts, however, showed a distinctively lower enzyme activity. Both, PC and P17EC (or rather P21EC) were hydrolyzed to a minor extent only. The biotransformation pathway, however, was the same. When P17EC was added separately, it transformed to P21EC and again was cleaved by keratinocytes to a much higher extent. Despite of the rather high glucocorticoid concentration MTT-tests proved a non-disturbed cell viability and proliferation rate. CONCLUSIONS: Extrapolating our results to the in-vivo situation, topically applied PC may be metabolized by epidermal cells during skin penetration. A complex mixture of compounds reaches the dermis, whose fibroblasts are barely able to metabolize the steroids. Since skin atrophy is less pronounced with PC as compared to conventional halogenated glucocorticoids, less potent PC metabolites appear to be the dominant species in the dermis.     

Barley lipid-transfer protein complexed with palmitoyl CoA: the structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands

The expression of cadherin-8 was mapped by in situ hybridization in the embryonic and postnatal mouse central nervous system (CNS). From embryonic day 18 (E18) to postnatal day 6 (P6), cadherin-8 expression is restricted to a subset of developing brain nuclei and cortical areas in all major subdivisions of the CNS. The anlagen of some of the cadherin-8-positive structures also express this molecule at earlier developmental stages (E12.5-E16). The cadherin-8-positive neuroanatomical structures are parts of several functional systems in the brain. In the limbic system, cadherin-8-positive regions are found in the septal region, habenular nuclei, amygdala, interpeduncular nucleus, raphe nuclei, and hippocampus. Cerebral cortex shows expression in several limbic areas at P6. In the basal ganglia and related nuclei, cadherin-8 is expressed by parts of the striatum, globus pallidus, substantia nigra, entopeduncular nucleus, subthalamic nucleus, zona incerta, and pedunculopontine nuclei. A third group of cadherin-8-positive gray matter structures has functional connections with the cerebellum (superior colliculus, anterior pretectal nucleus, red nucleus, nucleus of posterior commissure, inferior olive, pontine, pontine reticular, and vestibular nuclei). The cerebellum itself shows parasagittal stripes of cadherin-8 expression in the Purkinje cell layer. In the hindbrain, cadherin-8 is expressed by several cranial nerve nuclei. Results from this study show that cadherin-8 expression in the embryonic and postnatal mouse brain is restricted to specific developing gray matter structures. These data support the idea that cadherins are a family of molecules whose expression provides a molecular code for the regionalization of the developing vertebrate brain.     

The effects of hormone replacement therapy (HRT) on lipid metabolism in the menopause

Immunohistochemical localizations of carbonic anhydrase isozymes (CA-I, CA-II and CA-III) in bovine male reproductive tracts were studied. In bulls, no immunoreaction was seen after treatment with antibodies to CA-I, -II and CA-III in the testis. Specific staining for CA-III, however, was evident in peritubular cells in interstitial tissue of the testis, epididymis. CA-II activity could be detected in epithelium of the epididymis, ductus deferentis and ampulla ductus deferentis. Especially, a strong reaction for CA-II was seen in apical in epithelium of the epididymis in the initial and middle segment. CA-I activity was only founded in ductus deferentis and ampulla ductus deferentis. No or a weak reaction for CA-I, CA-II and CA-III were seen in the three accessory reproductive glands. Specific immunostaining for CA-II and CA-I could be observed in the organ, suggesting the bicarbonate in bovine semen to derive primarily from the genital tract and not accessory reproductive organs. CA-III-positive peritubular cells in interstitial tissue were also stained for alpha smooth muscle actin, and were very similar to contractile myofibroblast cells (Wrobel et al., 1979).     

Non-covalent complexes between DNA-binding drugs and double-stranded deoxyoligonucleotides: a study by ionspray mass spectrometry

The non-covalent complexes between some DNA-binding drugs and duplex oligodeoxynucleotides were studied by ionspray mass spectrometry, with the aim of evaluating the suitability of this technique to screen rapidly a series of drugs exerting their activity through non-covalent binding to specific base sequences of DNA. Two classes of drugs were considered, distamycins (which show affinity for the minor groove of DNA) and anthracyclines (which interact through intercalation between bases). For the former, d(CGCGAATTCGCG)2 was chosen as the model oligodeoxynucleotide. Following optimization of sample preparation and instrumental conditions, the complexes of different distamycins were observed; depending on the ligand considered, 1:1 or 2:1 complexes were formed preferentially. A semi-quantitative evaluation of the relative affinities was made by measuring the ratio of the complexes signals to those of the duplex, and also by competitive binding with equimolar amounts of distamycin. For anthracyclines, the daunorubicin-d(CGATCG)2 complex was chosen as the model for a preliminary mass spectrometric study; however, the signals of the duplex and the complex were very low compared with the monomer signal. Since the complex was known to be stable in solution, this was ascribed to gas-phase instability, probably caused by electrostatic repulsion between negatively charged phosphate groups.     

Endogenous serine protease inhibitor modulates epileptic activity and hippocampal long-term potentiation

Protease nexin-1 (PN-1), a member of the serpin superfamily, controls the activity of extracellular serine proteases and is expressed in the brain. Mutant mice overexpressing PN-1 in brain under the control of the Thy-1 promoter (Thy 1/PN-1) or lacking PN-1 (PN-1-/-) were found to develop epileptic activity in vivo and in vitro. Theta burst-induced long-term potentiation (LTP) and NMDA receptor-mediated synaptic transmission in the CA1 field of hippocampal slices were augmented in Thy 1/PN-1 mice and reduced in PN-1-/- mice. Compensatory changes in GABA-mediated inhibition in Thy 1/PN-1 mice suggest that altered brain PN-1 levels lead to an imbalance between excitatory and inhibitory synaptic transmission.     

Stress myocardial gammatomography in the diagnosis of multivessel coronary disease

OBJECTIVE: The aim of the study was to evaluate the diagnostic yield of 99m-Technetium-methoxy-isobutyl-isonitrile (MIBI) SPET for identification of individual coronary artery disease and in the prediction of multivessel involvement. METHODS: Stress/rest myocardial SPET and coronary arteriography were evaluated in 231 consecutive patients (age 58 +/- 10 years, 26% women) without prior myocardial infarction. 149 patients had coronary narrowing > 50%: 104 with multivessel disease and 45 with one vessel disease. Tomographic stress defect score was obtained by semiquantitative analysis (maximal score 65). Univariate and multivariate analysis was performed to identify discriminant parameters between one vessel and multivessel patients. RESULTS: The sensitivity and specificity for the identification of individual artery disease was 74% and 85% for left anterior descending artery, 79% and 85% for right coronary and 45% and 96% for circumflex artery. In the prediction of multivessel involvement the sensitivity was 65%, specificity 87%, positive predictive value 81% and negative predictive value 76%. In the bivariate analysis, four parameters differed significantly between one vessel and multivessel disease patients: ST downslope > 1 mm (p = 0.01), ST downslope/heart rate corrected (p = 0.005), reversible defects in two or more regions (p = 0.009) and SPET score (p = 0.002). In the multivariate analysis the probability of multivessel disease was 90% when ST depression > 1 mm and SPET score > 20 were associated and the probability was lowered to 16% when these criteria were not present. CONCLUSION: Myocardial SPET with MIBI offers an accurate localization of individual coronary artery disease, mainly in left anterior descending artery and right coronary artery lesions. Combined evaluation of ST depression and extension of myocardial stress defects improved prediction of multivessel involvement.     

Surgery in hemopathies: experience in ambulatory care and day surgery

The one day surgery experience of our team developed in many years of a Special Surgical Service in Haematologic Diseases (within the III Surgical Department directed by Prof. G. Di Matteo of the University of Rome "La Sapienza") is herein reported. From 1989 to December 1995, 2,126 haematologic patients (1127 M, 999 F) were operated in day-surgery regimen. Five-hundred-fifty-six patients were over 65 years old. In most cases surgery was required for diagnostic purposes to ascertain the type, the stadiation or re-stadiation of the haematologic disease. Five-hundred-eighty-three operations were carried on the axillary region, 825 on the cervical region, 202 on the supraclavicular region and 163 on the groin region. In 729 patients a diagnosis of non-Hodgkin lymphoma was obtained, while 308 patients resulted affected by Hodgkin lymphoma. In 124 patients metastases from solid tumors (pulmonary, mammary, thyroidal adenocarcinoma, etc.) were found at histologic examination of the specimen. Furthermore, other types of pathologies such as lateral neck cysts, salivary gland adenomas, schwannomas, groin and crural hernias were identified. Outpatient surgery and one day surgery represent a valid procedure for the early diagnosis of haemotologic diseases also taking into account the low cost and the minimal invasiveness.     

Binding of actinomycin D to DNA oligomers of CXG trinucleotide repeats

Actinomycin D (ACTD) binding propensities of DNA with CXG trinucleotide repeats were investigated using oligomers of the form d[AT(CXG)n = 2-4AT] and their corresponding heteroduplexes, where X = A, C, G, or T. These oligonucleotides contain -CXGCXG-, -CXGCXGCXG-, and -CXGCXGCXGCXG- units that can form homoduplexes containing one, two, and three GpC binding sites, respectively, with flanking X/X mismatches. The corresponding heteroduplexes contain these same sites with flanking Watson-Crick base pairs. It was found that oligomers with X = G exhibit weak ACTD affinities whereas those with X not equal to G and n = 3 exhibit unusually strong ACTD binding affinities with binding constants ranging from 2.3 x 10(7) to 3.3 x 10(7) M-1 and binding densities of approximately 1 drug molecule/strand (or 2/duplex). These binding affinities are considerably higher than those of their shorter and longer counterparts and are about 2- and 10-fold stronger than the corresponding CAG.CTG and CGG.CCG heteroduplexes, respectively. The CTG-containing oligomer d[AT(CTG)3AT] stands out as unique in having its ACTD dissociation kinetics being dominated by a strikingly slow process with a characteristic time of 205 min at 20 degrees C, which is 100-fold slower than d[AT(CAG)3AT], nearly 10-fold slower than the corresponding heteroduplex, and considerably slower than d[AT(CTG)2AT] (63 min) and d[AT(CTG)4AT] (16 min). The faster dissociation rate of the n = 4 oligomer compared to its n = 2 counterpart is in apparent contrast with the observed 10-fold stronger ACTD binding affinity of the former. It was also found that d[AT(CCG)3AT] exhibits the slowest dissociation rate of the CGG/CCG series, being more than an order of magnitude slower than that of its heteroduplex (tau slow of 43 vs 2 min). The finding that a homoduplex d[AT-CXG-CXG-CXG-AT]2 can bind two ACTD molecules tightly is significant since it was thought unlikely for two consecutive GpC sites separated by a single T/T mismatch to do so.     

Chronic recurrent multifocal osteomyelitis

On the basis of a preliminary study of antimycobacterial activity of thiobenzanilides a series of eight thiosalicylanilides have been prepared. Synthetized compounds have been examined in vitro against Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium avium and Mycobacterium fortuitum. All compounds have been found very active. The values of minimal inhibitory concentrations are summarized in Table 1. 3',4'-Salicylanilide was selected for the following research. The compound have been found inactive in vivo (on experimental murine tuberculosis).     

Determinants of the kinetics of very low-density lipoprotein apolipoprotein B-100 in non-obese men

1. Apolipoprotein B-100 (ApoB) is the principal structural and functional protein of the pro-atherogenic lipoproteins. Elevated plasma apoB is an independent risk factor for coronary artery disease. In the present study we aimed to assess the factors that determine the kinetics of apoB in the very low-density lipoprotein (VLDL) in healthy men. 2. We studied 17 non-obese men who were consuming an ad libitum diet and had the following characteristics: mean (+/-SD) age 45.5 +/- 9.7 years, body mass index (BMI) 25.1 +/- 1.4 kg/m2, waist:hip ratio 0.91 +/- 0.04, serum cholesterol 5.2 +/- 0.6 mmol/L, triglycerides 1.08 +/- 0.53 mmol/L and high-density lipoprotein-cholesterol 1.24 +/- 0.31 mmol/L. Daily dietary intake was as follows: total fat 76 +/- 26 g, carbohydrate 238 +/- 67 g, protein 103 +/- 33 g and alcohol 20 +/- 16 g. 3. The kinetics of VLDL ApoB were studied using a primed, constant infusion (1 mg/kg per h) of 1-[13C]-leucine over 8 h with measurement of isotopic enrichment of ApoB using gas chromatography/mass spectrometry. The fractional turnover rate of VLDL ApoB was estimated using a monoexponential function. The mean (+/-SD) absolute hepatic secretion rate (ASR) of ApoB was 8.5 +/- 4.6 mg/kg per day and the fractional catabolic rate (FCR) was 7.9 +/- 5.6 pools/day. The ASR was significantly correlated with the waist:hip ratio (r = 0.60; P = 0.04), but not with age, BMI, weight or nutrient intake. The FCR was significantly and inversely correlated with plasma triglycerides (r = -0.53; P = 0.03) and alcohol intake (r = -0.48; P = 0.05). 4. In conclusion, the hepatic secretion of VLDL ApoB in nonobese, healthy men is primarily determined by the waist:hip ratio, a measure of visceral fat. This is consistent with the hypothesis that the rate of lipid substrate supply in the liver regulates the output of ApoB. The fractional catabolism of VLDL ApoB may, however, be inversely related to alcohol intake and appears to determine the plasma concentration of triglycerides.