[The assessment and course of development of cognitive abilities in the elderly]. / Diagnostik und Verlauf von kognitiven Fähigkeiten bei älteren Menschen.

BACKGROUND: The assessment of the appropriate level of development in children belongs to the standard duties of physicians in the public health system. Due to a steady increase of dementia in Germany the assessment of cognitive abilities of the elderly is becoming more and more the focus of future activities. Such an assessment of cognitive functioning reveals whether the respective person is aging normally or whether the impaired cognitive functioning is probably based on a pathological process. OBJECTIVE: The aim of the present study is to present cognitive changes in the aged and 2 psychometric tests for the assessment of cognitive functioning: the Wechsler Adult Intelligence Scale (WAIS-IV) and the Short Cognitive Performance Test (SKT), a test for the assessment of memory impairments and impairment of attention. In addition, similarities and dissimilarities are presented. METHODS: As part of a multi-centre study in German-speaking countries the data of 504 cognitively healthy persons between the age of 60 and 90 were tested with the WAIS-IV and the SKT. RESULTS AND CONCLUSION: The results revealed a significant cognitive decline in the fluid and crystal intelligence depending on age. Only 2 subtests of the WAIS-IV (General Information and Block Design) showed no significant variation due to age. The SKT scores of memory and attention correlated significantly with almost all subtests of the WAIS-IV. The highest correlations were between the SKT attention score and the WAIS-IV subtests for processing speed, perceptual reasoning and working memory. The decline in cognitive abilities is mainly due to reduced capacities in speed of information processing and working memory.

Suspect novel adverse drug reactions to trimethoprim-sulphonamide combinations in horses: a case series.

Adverse drug reactions to trimethoprim-sulphonamide combinations are common in many species, manifesting as gastrointestinal tract disorders, dermatopathies and blood dyscrasias. In this case series, neurological abnormalities in 4 horses being treated with trimethoprim-sulphonamide combinations at normal dosages and in one foal that received an overdose are described. The horses developed hypermetric gait, agitation and erratic behaviour. All signs resolved once medication was withdrawn, and no horse had residual deficits. No other cause for observed neurological deficits could be determined. These clinical signs appear to represent a novel adverse drug reaction to some commonly used antimicrobial combinations.

[From "schizophrenia" to "disturbance of the integrity of the self": causes and consequences of renaming schizophrenia in Japan in 2002]. / Von der "Schizophrenie" zur "Störung der Einheit des Selbst" : Ursachen und Folgen der Umbenennung der Schizophrenie in Japan im Jahre 2002.

In August 2002 the Japanese Society of Psychiatry and Neurology decided to rename the Japanese expression for schizophrenia from Sêshin Bunretsu Byô to Tôgô Shicchô Shô. Currently the psychiatric classification systems ICD-10 and DSM-IV are under revision. Against this background the Japanese process of renaming a psychiatric disorder is of high interest as far as the clinical, social and cultural implications of the new name are concerned.The authors give an overview of the Japanese process of renaming schizophrenia. Its background and realization are explained and the expectations of Japanese physicians, patients and their families related to the new name are analysed. Furthermore, its effects are evaluated. The aim of the paper is to clarify in how far the Japanese example may serve as a model for evaluating the possible implications that a renaming or nosological redefinition of schizophrenia might have in the course of the revision process of ICD 10 and DSM IV.

[The situation of the "small disciplines" using the example of the history, theory, and ethics of medicine]. / Die Situation der "kleinen Fächer" am Beispiel der Geschichte, Theorie und Ethik der Medizin.

There is no clearly defined concept of the "small disciplines" (i.e., smaller fields of study). They have minor facilities and therefore they do not take center stage in a medical school with regard to research and education. Thus, in times of tight resources, the necessity of those disciplines is questioned. In particular, disciplines which cover the field of social medicine in a broader sense and which are not based on molecular medicine, respectively, do not belong to a clinical major discipline may be threatened with "extinction". This is probably not the case with "History, Theory, Ethics of Medicine" (German abbreviation: GTE) which was introduced by the Approbationsordnung für Arzte (license to practise medicine order) in 2002. It is now established in almost all of the German medical schools. The paper describes the contemporary, sometimes controversially discussed situation of GTE stressing its importance for medical education and the scientific self-conception of medicine. The medical humanities are an essential location for the critical self-reflection of medicine - indispensable for medical schools and in the end also medical care.

Association between hyperinsulinaemia and laminitis severity at the time of pituitary pars intermedia dysfunction diagnosis.

BACKGROUND: Hyperinsulinaemia is the suspected component of insulin dysregulation having the strongest association with laminitis and occurs variably in equids with pituitary pars intermedia dysfunction (PPID). OBJECTIVES: We hypothesised that magnitude of hyperinsulinaemia correlates with laminitis severity in PPID-affected equids. Furthermore, we hypothesised that owners can be unaware of chronic endocrinopathic laminitis. STUDY DESIGN: Cross-sectional study. METHODS: Serum insulin concentrations, owner-reported laminitis history and radiographic evidence of laminitis were determined in 38 client-owned horses and ponies with confirmed PPID. Laminitis severity was classified into four categories (normal [nonlaminitic], mild, moderate or severe laminitis) based on degree of distal phalangeal rotation. Animals were also categorised as normoinsulinaemic (<20 µU/ml), mildly hyperinsulinaemic (20-50 µU/ml) and severely hyperinsulinaemic (>50 µU/ml). One-way ANOVA, t tests and Fisher's exact tests were performed. RESULTS: While owners reported laminitis in 37% of animals, 76% were laminitic based on study criteria (P = 0.01). Owners reported laminitis more frequently in hyperinsulinaemic vs. normoinsulinaemic animals; recognition increased with severity of hyperinsulinaemia (P = 0.03). Mean insulin concentrations were higher in equids with moderate to severe radiographic laminitis (geometric mean 74.1, 95% confidence interval (CI) 38.4-143.1 uU/ml) vs. those classified radiographically as normal to mild (31.9, 95% CI 21.1-48.1 uU/ml P = 0.03). MAIN LIMITATIONS: Dynamic insulin testing was not performed; some normoinsulinaemic animals might have had subtle insulin dysregulation. CONCLUSIONS: Although radiographic abnormalities were present in most animals at the time of PPID diagnosis, chronic laminitis remained unrecognised by many owners. Owner awareness of laminitis increased with severity of hyperinsulinaemia and higher insulin concentrations were detected in association with more severe radiographic changes. The Summary is available in Chinese - See Supporting Information.

Brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona and act as intermediate hosts.

We tested the hypothesis that brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona, the agent of equine protozoal myeloencephalitis (EPM), and act as intermediate hosts for this parasite. In summer 1999, wild caught brown-headed cowbirds were collected and necropsied to determine infection rate with Sarcocystis spp. by macroscopic inspection. Seven of 381 (1.8%) birds had grossly visible sarcocysts in leg muscles with none in breast muscles. Histopathology revealed two classes of sarcocysts in leg muscles, thin-walled and thick-walled suggesting two species. Electron microscopy showed that thick-walled cysts had characteristics of S. falcatula and thin-walled cysts had characteristics of S. neurona. Thereafter, several experiments were conducted to confirm that cowbirds had viable S. neurona that could be transmitted to an intermediate host and cause disease. Specific-pathogen-free opossums fed cowbird leg muscle that was enriched for muscle either with or without visible sarcocysts all shed high numbers of sporocysts by 4 weeks after infection, while the control opossum fed cowbird breast muscle was negative. These sporocysts were apparently of two size classes, 11.4+/-0.7 microm by 7.6+/-0.4 microm (n=25) and 12.6+/-0.6 microm by 8.0+/-0 microm (n=25). When these sporocysts were excysted and introduced into equine dermal cell tissue culture, schizogony occurred, most merozoites survived and replicated long term and merozoites sampled from the cultures with long-term growth were indistinguishable from known S. neurona isolates. A cowbird Sarcocystis isolate, Michigan Cowbird 1 (MICB1), derived from thin-walled sarcocysts from cowbirds that was passaged in SPF opossums and tissue culture went on to produce neurological disease in IFNgamma knockout mice indistinguishable from that of the positive control inoculated with S. neurona. This, together with the knowledge that S. falcatula does not cause lesions in IFNgamma knockout mice, showed that cowbird leg muscles had a Sarcocystis that fulfills the first aim of Koch's postulates to produce disease similar to S. neurona. Two molecular assays provided further support that both S. neurona and S. falcatula were present in cowbird leg muscles. In a blinded study, PCR-RFLP of RAPD-derived DNA designed to discriminate between S. neurona and S. falcatula showed that fresh sporocysts from the opossum feeding trial had both Sarcocystis species. Visible, thick-walled sarcocysts from cowbird leg muscle were positive for S. falcatula but not S. neurona; thin-walled sarcocysts typed as S. neurona. In 1999, DNA was extracted from leg muscles of 100 wild caught cowbirds and subjected to a PCR targeting an S. neurona specific sequence of the small subunit ribosomal RNA (SSU rRNA) gene. In control spiking experiments, this assay detected DNA from 10 S. neurona merozoites in 0.5g of muscle. In the 1999 experiment, 23 of 79 (29.1%) individual cowbird leg muscle samples were positive by this S. neurona-specific PCR. Finally, in June of 2000, 265 cowbird leg muscle samples were tested by histopathology for the presence of thick- and thin-walled sarcocysts. Seven percent (18/265) had only thick-walled sarcocysts, 0.8% (2/265) had only thin-walled sarcocysts and 1.9% (5/265) had both. The other half of these leg muscles when tested by PCR-RFLP of RAPD-derived DNA and SSU rRNA PCR showed a good correlation with histopathological results and the two molecular typing methods concurred; 9.8% (26/265) of cowbirds had sarcocysts in muscle, 7.9% (21/265) had S. falcatula sarcocysts, 1.1% (3/265) had S. neurona sarcocysts, and 0.8% (2/265) had both. These results show that some cowbirds have S. neurona as well as S. falcatula in their leg muscles and can act as intermediate hosts for both parasites.

Effects of age and diet on glucose and insulin dynamics in the horse.

BACKGROUND: Age and diet may affect insulin sensitivity (SI) but these factors have received limited investigation in horses. OBJECTIVES: To measure minimal model parameters during an insulin-modified frequently sampled intravenous glucose tolerance test (FSIGTT) after adaptation to a forage only diet (HAY) or forage supplemented with either starch/sugar-rich (SS) or oil/fibre-rich (FF) concentrate feeds; and to assess glucose and insulin responses to a standardised meal challenge (SMC, 4 g/kg BW of SS) after diet adaptation in adult and aged mares. STUDY DESIGN: Latin square design with eight adult (5-12 years) and nine aged (>19 years) healthy mares. METHODS: Diets were fed for 6 weeks, and the FSIGTT and SMC were performed after 31-32 and 41 days on each diet respectively. Data were analysed by a mixed ANOVA for repeated measures. RESULTS: Acute insulin response to glucose (AIRg) was greater and SI was lower in aged horses, compared with adults, regardless of diet. Both AIRg and SI were greater in aged mares after adaptation to SS, as compared with HAY. Similar trends, although not statistically significant, were observed after adaptation to SS in adult mares. Peak insulin concentration and area under the insulin vs. time curve during the SMC were greater in aged than adult mares with all diets. Furthermore, area under the glucose vs. time curve was lower after adaptation to SS, when compared with other diets, in both groups. MAIN LIMITATIONS: Transient weight loss occurred at the beginning of the study and only one sex was included. Incomplete ingestion of the SMC by four mares was another limitation. CONCLUSIONS: Insulin responses to i.v. and enteral nonstructural carbohydrate challenge increase with age in healthy horses, regardless of diet fed.

In vitro and in vivo antileukemic effect of novel dimers consisting of 5-fluorodeoxyuridine and arabinofuranosylcytosine.

Various amphiphilic heterodinucleoside phosphates containing 1-beta-D-arabinofuranosylcytosine (ara-C) and 5- fluorodeoxyuridine (5-FdUrd) have recently been synthesized in order to increase the efficacy of ara-C and 5-FdUrd. Employing growth inhibition and growth recovery assays, we evaluated the in vitro effects of four of these dimers (No. 2, 2A, 3, 10) in L1210 and P388D1 murine leukemia cells. Although ara-C and 5-FdUrd appeared equimolar in all dimers, their contribution to the cytotoxicity of these agents was different. Thus, the liberation of ara-C and 5-FdUrd from their dimeric origin and their subsequent metabolic activation had a different course. In another set of experiments, we examined the in vivo effects of these agents in mice. The dimer with the highest cytotoxicity in vitro exerted the lowest acute toxicity and yielded the lowest therapeutic effect in vivo. The obtained data indicate that dimers with slower liberation of ara-C and 5-FdUrd were less cytotoxic, but prolonged liberation of both antimetabolites protected them from inactivation and extended the time period of therapeutic action. Some of the dimers exceeded the synergistic effects yielded by simultaneous application of both ara-C and 5-FdUrd. The significantly higher therapeutic potential of these new antitumor agents indicates that further studies are warranted.

Response to nasopharyngeal oxygen administration in horses with lung disease.

REASONS FOR PERFORMING STUDY: Guidelines for administration of oxygen to standing horses are unavailable because previous investigations of the efficacy of oxygen administration to increase arterial oxygenation in standing horses have produced equivocal results. OBJECTIVE: To determine the effect of nasal oxygen supplementation on inspired and arterial blood gas tensions in control horses and those with moderate to severe recurrent airway obstruction (RAO). METHODS: Normal horses (n = 6) and horses during an attack of RAO induced by stabling (n = 6) were studied. Oxygen was administered through either one or 2 cannulae, passed via the nares into the nasopharynx to the level of the medial canthus of each eye. Intratracheal inspired oxygen and carbon dioxide concentration and arterial blood gas tensions were measured at baseline and during delivery of 5, 10, 15, 20 and 30 l/min oxygen. RESULTS: Nasal cannulae and all but the highest oxygen flow rates were well tolerated. Fractional inspired oxygen concentration (F(I)O2) increased with flow but was significantly lower at all flow rates in horses with RAO compared with controls. Arterial oxygen tension (PaO2) was significantly increased (P < 0.001) by all flow rates, but was always lower in RAO-affected animals. At 30 l/min, PaO2 increased to 319 +/- 31 mmHg in control horses and 264 +/- 69 mmHg in horses with RAO. Additionally, a large arterial to end-tidal gradient for CO2 in RAO-affected horses was observed, indicating increased alveolar deadspace ventilation in these animals. CONCLUSIONS: The use of nasal cannulae to deliver oxygen effectively increases both F(I)O2 and PaO2 in horses with moderate to severe RAO. Oxygen flow rates up to 20 l/min are well tolerated, but flow rates of 30 l/min produce occasional coughing or gagging. POTENTIAL RELEVANCE: Oxygen therapy delivered by means of an intranasal cannula is a highly effective means of increasing arterial oxygen tension in horses with respiratory disease. Generally, flows of 10-20 l/min should be effective. If higher flows (20-30 l/min) are necessary, they should be delivered by means of 2 cannulae.

Conformation affinity of cross-linked polyacrylic gels towards nucleobases, nucleosides and nucleotides.

By radical copolymerisation of monofunctional acrylic derivatives with 1,4-tetramethylene dimethacrylate, cross-linked polyacrylic gels which show a high affinity towards nucleic acid residues have been synthesised. Using these polyacrlic gels, mixtures of nucleobases, nucleosides and nucleotides can be chromatographically separated to differing extents, in some cases quantitatively. The elution of nucleobases and nucleosides from the gel in the order Cyt, Gua, Thy, Ade (or dC, dG, dT, dA and C, G, U, A, respectively) shows that Ade (dA, A) is retarded to the highest degree from the gel matrix, Cyt (dC, C) to the lowest degree. Further, the results of the separations prove that the affinity of the polyacrylic gels is much stronger towards deoxyribonucleosides than towards ribonucleosides and nucleobases. The affinity of the polyacrylic gels towards nucleobases and nucleosides depends mainly upon their tertiary structure whereas the functional side groups of the polyacrylate matrix do not contribute significantly to the interaction of the gels.

Investigation of peptide-nucleotide interactions using template chromatography. The specific recognition of peptide sequences by oligodeoxy adenylic acids.

Polyvinylalcohol has been substituted with oligodeoxyadenylic acid and the resulting polyanion polyvinyl (pA)n irreversibly attached to DEAE-cellulose. Peptide-oligonucleotide interactions have been studied using a column chromatographic technique with the polyvinyl (pA)n-DEAE-cellulose as a stationary phase.

Labelling of liposomes with intercalating perylene fluorescent dyes.

The high fluorescent potential and the exceptional photostability of lipophilic derivatives of perylene-3,4:9,10-bis(dicarboximides) are utilized for the fluorescence-labelling of liposomes. The preparation of the liposomes is effected by supersonic starting from a lipid mixture consisting of the matrix lipids soy lecithin, cholesterol, alpha-tocopherol and the perylene dyes. From a multitude of perylene derivatives investigated only those are optimally incorporated into the bilayer membrane of unilamellar liposomes which are substituted at both nitrogen atoms by one or two linear hydrocarbon groups. In order to attain an optimal fluorescent quantum yield, about 200 to 300 dye molecules can be incorporated per liposome. The liposomes thus obtained have a diameter of about 70 to 80 nm, are homogeneous and may be stored for more than seven months. Neither the fluorescent properties nor the stability of these liposomes are influenced by the additional incorporation of various ara C-derivatives and lipophilic anchor groups which subsequently enable the coupling of antibodies to the liposomes. As the water-insoluble perylene dyes are incorporated into the bilayer membrane, the aqueous inner volume of the liposomes remains available for a further utilization.

Palmitoyl derivatives of L-cysteine, cysteamine, L-cystine, cystamine and their incorporation into the bilayers of unilamellar liposomes.

The amino groups of the amino acids L-cysteine and L-cystine as well as their biogene amines cysteamine and cystamine were derivatized with palmitoyl residues. The obtained lipophilic R-SH and R-S-S-R components were incorporated into the bilayers of unilamellar liposomes. The resulting liposomes carrying about 2000 functional groups each remained stable and homogeneous during 60 days after incorporation of N-palmitoyl cysteamine and N,N'-dipalmitoyl cystamine. The incorporation of the lipophilic amino acid derivatives, however, destabilized the resulting liposomes. Via the thiol residues of the functionalized liposomes activated molecules can be linked to the liposomal surface by disulfide bonds.

Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy.

The two coupling agents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) and SATA (N-succinimidyl-S-acetylthioacetate) were compared in their efficiency and feasibility to couple monoclonal antibodies (Abs) via thioether linkage to liposomes functionalized by various lipophilic maleimide compounds like N-(3-maleimidopropionyl)-N2-palmitoyl-L-lysine methyl ester (MP-PL), N-(3-maleimidopropionyl)phosphatidylethanolamide (MP-PE), N6-(6-maleimidocaproyl)-N2-palmitoyl-L-lysine methyl ester (EMC-PL), and N-(6-maleimidocaproyl)phosphatidylethanolamine (EMC-PE). The composition of the liposomes was soy phosphatidylcholine (SPC), cholesterol, maleimide compounds and alpha-tocopherol (1:0.2:0.02:0.01, mol parts), plus N4-oleylcytosine arabinoside (NOAC) as cytostatic prodrug (0.2 mol parts) and a new, lipophilic and highly fluorescent dye N,N'-bis(1-hexylhfetyl)-3,4:9,10-perylenebis(dicarboximid ) (BHPD, 0.006 mol parts). From the maleimide derivatives MP-PL was the most effective in terms of preservation of the coupling activity in dependence of liposome storage. The coupling of the monoclonal A B8-24.3 (mouse IgG2b, MHC class I, anti H-2kb) and IB16-6 (rat IgG2a, anti B16 mouse melanoma) to the drug carrying liposomes was more effective and easier to accomplish with SATA as compared to SPDP. Coupling rates of 60-65% were obtained with SATA at molar ratios of 12 SATA:1 Ab:40 maleimide spacer groups on the surface of one liposome. The highest coupling rates with SPDP were obtained at the ratio of 24 SPDP:1 Ab:40 liposomal maleimide groups, with an Ab binding efficiency of only 20-25%. The optimal in vitro binding conditions to specific target cells (EL4 for B8-24.3-liposomes and B16-F10 for IB16-6-liposomes) were determined by cytofluorometric measurement of the liposomal BHPD fluorescence with SATA linked Abs. Optimal immunoliposome binding to specific epitopes on the target cells was achieved with 1-2 Ab molecules coupled to one liposome, with immunoliposome concentrations of 20-130 nM and with a small incubation volume of 0.3-0.4 ml. The specificity of the binding of B8-24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy. Antibody mediated endocytic uptake of immunoliposomes could be demonstrated by transmission electron microscopy.

Interleukin-2- and interferon alfa-2a-based immunochemotherapy in advanced renal cell carcinoma: a Prospectively Randomized Trial of the German Cooperative Renal Carcinoma Chemoimmunotherapy Group (DGCIN).

PURPOSE: We conducted a prospectively randomized clinical trial to compare the efficacy of three outpatient therapy regimens in 341 patients with progressive metastatic renal cell carcinoma. PATIENTS AND METHODS: Patients were stratified according to known clinical predictors and were subsequently randomly assigned. Treatment arms were: arm A (n = 132), subcutaneous interferon alfa-2a (sc-IFN-alpha-2a), subcutaneous interleukin-2 (sc-IL-2), and intravenous (IV) fluorouracil; arm B (n = 146): arm A treatment combined with per oral 13-cis-retinoic acid; and arm C (n = 63), sc-IFN-alpha-2a and IV vinblastine. RESULTS: Treatment (according to the standard 8-week Hannover Atzpodien regimen) arms A, B, and C yielded objective response rates of 31%, 26%, and 20%, respectively. Arm B, but not arm A, showed a significantly improved progression-free survival (PFS) compared with arm C (P =.0248). Both arm A (median overall survival, 25 months; P =.0440) and arm B (median overall survival, 27 months; P =.0227) led to significantly improved overall survival (OS) compared with arm C (median OS, 16 months). All three sc-IFN-alpha-2a-based therapies were moderately or well tolerated. CONCLUSION: Our results established the safety and improved long-term therapeutic efficacy of sc-IL-2 plus sc-INF-alpha-2a-based outpatient immunochemotherapies, compared with sc-INF-alpha-2a/IV vinblastine.

Parasitemia in an immunocompetent horse experimentally challenged with Sarcocystis neurona sporocysts.

Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in Americans. Most cases are attributed to infection of the central nervous system with Sarcocystis neurona. Parasitemia has not been demonstrated in immunocompetent horses, but has been documented in one immunocompromised foal. The objective of this study was to isolate viable S. neurona from the blood of immunocompetent horses. Horses used in this study received orally administered S. neurona sporocysts (strain SN 37-R) daily for 112 days at the following doses: 100/day for 28 days, followed by 500/day for 28 days, followed by 1000/day for 56 days. On day 98 of the study, six yearling colts were selected for attempted culture of S. neurona from blood, two testing positive, two testing suspect and two testing negative for antibodies against S. neurona on day 84 of the study. Two 10 ml tubes with EDTA were filled from each horse by jugular venipuncture and the plasma fraction rich in mononuclear cells was pipetted onto confluent equine dermal cell cultures. The cultures were monitored weekly for parasite growth for 12 weeks. Merozoites grown from cultures were harvested and tested using S. neurona-specific PCR with RFLP to confirm species identity. PCR products were sequenced and compared to known strains of S. neurona. After 38 days of in vitro incubation, one cell culture from a horse testing positive for antibodies against S. neurona was positive for parasite growth while the five remaining cultures remained negative for parasite growth for all 12 weeks. The Sarcocystis isolate recovered from cell culture was confirmed to be S. neurona by PCR with RFLP. Gene sequence analysis revealed that the isolate was identical to the challenge strain SN-37R and differed from two known strains UCD1 and MIH1. To our knowledge this is the first report of parasitemia with S. neurona in an immunocompetent horse.

Litomosoides carinii microfilarial sheaths: partial amino acid sequences of several major polypeptide constituents.

Isolated sheaths from Litomosoides carinii microfilariae were disintegrated by reduction with dithiothreitol and were 14C-carboxymethylated. Five major sheath proteins thus solubilized were purified by size exclusion chromatography and reversed-phase HPLC (rpHPLC). Proteolytic fragments of complete sheaths and of the single sheath proteins were isolated by rpHPLC and were N-terminally sequenced. A library of 27 partial sheath polypeptide sequences was thus established, 21 of which could be assigned to three L. carinii sheath structural genes (shp1,2, and 3/3a) isolated on the basis of this and of previous amino acid sequence information. The remaining peptides document the presence of at least one additional major sheath constituent.

[3H]Dopamine release by d-amphetamine from striatal synaptosomes of reserpinized rats.

The injection of reserpine, 5 mg/kg i.p. (ipRes), the regimen employed by a majority of investigators, results in synaptosomal and vesicular preparations which are incompletely reserpinized as determined by [3H]dopamine ([3H]DA) accumulation. Reserpine administered by the subcutaneous route, 5 mg/kg (scRes), appears to produce complete reserpinization. Release of [3H]DA by d-amphetamine (Amph) was observed from striatal synaptosomes prepared both from normal rats and those pretreated with reserpine intraperitoneally but not from those injected subcutaneously. In the more completely reserpinized scRes synaptosomes, so little [3H]DA had accumulated that release by Amph was not measurable, indicating that if a labile, reserpine-resistant, extravesicular DA storage pool releasable by Amph is present under these conditions, it must be extremely small. In scRes monoamine oxidase (MAO)-inhibited preparations, Amph released preloaded [3H]DA located in the cytosol in the absence of functional vesicles. Although chromatographic analysis of the superfusate from ipRes striatal synaptosomes showed that significant amounts of preloaded [3H]DA were released by Amph, the level of dihydroxyphenylacetic acid was not increased over controls, indicating that Amph releases only DA and not its metabolite and is also acting as a MAO inhibitor. No [3H]DA could be released by Amph from superfused hyposmotically shocked normal or ipRes synaptosomes, suggesting that an intact membrane is required for Amph-induced release.

Induction of cell cycle-dependent cytotoxicity and apoptosis by new heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine in PC-3 human prostate cancer cells.

Fluorodeoxyuridine (5-FdUrd) is an antineoplastic agent with clinical activity against different types of solid tumours. To enhance the effectiveness of this drug, we have synthesised new heterodinucleoside phosphate dimers of 5-FdUrd. These dimers were compared to 5-FdUrd for their cytotoxic effect and the cell cycle dependence of cytotoxicity, as well as for their capacity to induce apoptosis and inhibit thymidylate synthetase (TS) in androgen-independent human PC-3 prostate tumour cells. Incubation of the cells with the dimers N(4)-palmitoyl-2'-deoxycytidylyl-(3'-->5')-5-fluoro-2'-deoxyuri din e (dCpam-5-FdUrd) and 2'-deoxy-5-flourouridylyl-(3'-->5')-2'-deoxy-5-fluoro-N(4)-octa decylc ytidine (5-FdUrd-5-FdC18) resulted in a marked cytotoxicity with IC(50) values of 4 microM, similar to 5-FdUrd. In contrast to 5-FdUrd, 100% toxicity was achieved with concentrations of 100-200 microM 5-FdUrd-5-FdC18. Flow cytometric analysis revealed an increase in the cell population in S-phase after treatment with 5-FdUrd, 5-FdUrd-5-FdC18, and dCpam-5-FdUrd from 36 to 63%, 50%, and 77%, respectively. dCpam-5-FdUrd was more potent than 5-FdUrd in arresting the cell cycle. Significant S-phase arrest was indicated by a decreased proportion of cells in G1- and G2/M-phases. Cell cycle arrest and inhibition of cell proliferation were followed by apoptosis, as shown by a 6- to 8-fold increased binding of Apo2.7 antibody, a 9- to 11-fold increase in caspase-3 activity, DNA fragmentation, and by cell morphology showing the appearance of apoptotic bodies. Importantly, 5-FdUrd-5-FdC18 increased the number of apoptotic cells to 160% compared to 5-FdUrd under the same conditions. As with 5-FdUrd, the two dimers also inhibited TS in a time- and concentration-dependent manner, although requiring 100-fold higher concentrations. In conclusion, dCpam-5-FdUrd and 5-FdUrd-5-FdC18 exert stronger cytotoxicity and induce more S-phase arrest and apoptosis than does 5-FdUrd in PC-3 cells, suggesting their potential role in the treatment of human prostate cancer.

New lipophilic alkyl/acyl dinucleoside phosphates as derivatives of 3'-azido-3'-deoxythymidine: inhibition of HIV-1 replication in vitro and antiviral activity against Rauscher leukemia virus infected mice with delayed treatment regimens.

The antiretroviral activity of two new lipophilic derivatives of azidothymidine (AZT), N4-hexadecyl-2'-deoxyribocytidylyl-(3',5')-3'-azido-2',3'-deoxythy midine (N4-hexadecyldC-AZT) and N4-palmitoyl-2'-deoxyribocytidylyl-(3',5')-3'-azido-2',3'-deoxythy midine (N4-palmitoyldC-AZT) was evaluated in comparison to AZT. In vitro the drugs were tested in human immunodeficiency virus 1 (HIV-1) infected CD4+ HeLa and H9 cells. The in vivo antiviral effect of these derivatives was analysed in Rauscher leukemia virus (RLV) infected mice. The derivatives were incorporated into small liposomes. In vitro both derivatives inhibited virus proliferation in both HIV-1 infected cell lines in a similar dose-responsive manner as AZT. In a plaque reduction assay, using HeLa cells, the IC50 values were 0.035 microM for AZT, 0.5 microM for N4-hexadecyldC-AZT and 4.5 microM for N4-palmitoyldC-AZT, whereas p24 antigen analysis on H9 cells gave IC50 values of 0.005 microM, 0.05 microM and 0.05 microM, respectively. RLV infected mice were treated with intermittent schedules i.p. or i.v. on days 1, 6, 11, and days 16 or 0, 3, 7, and 11 after infection. Regimens with further delayed drug application were on days 3, 7, and 11 and 7 and 11 only. While i.p. treatment with total doses of 380-1140 mg/kg free AZT resulted in 10-30% inhibition of RLV induced splenomegaly, the derivatives gave inhibitions of 37-94%. Late onset of treatment with the derivatives was significantly more effective as compared to free AZT. Intravenous treatment with N4-hexadecyldC-AZT was effective, but with AZT was inactive. The discrepancy in antiviral activity of the AZT derivatives found between the in vitro and in vivo test systems emphasizes the importance of investigating the activity of drug derivatives in vivo.