Steroidogenic enzyme histochemistry in the testis of Sprague Dawley rats following the administration the water extracts from Carica papaya seed.

Water extracts from pawpaw seed have been reported to reversibly decrease the testicular weight and to suppress spermatogenesis, and fertility of Wistar rats. The reversible changes become evident, 30 - 45 days after the withdrawal of the extract. The possible effect of this extract on the activities of steroidogenic enzymes of the testis has not been investigated. Water extract of papaya seeds was administered to male Sprague Dawley rats ad libitum for 84 days. Following the discontinuation of the extracts, ten rats each were sacrificed on days 0, 10, 20 and 30 after the withdrawal. Their testes were quickly dissected out and frozen. Cryostat sections, 10µm thick were cut. These sections were used for immunohistochemical stains for side chain cleavage enzyme and aromatase, and for histochemical stains for 17-ß Hydroxysteroid dehydrogenase, 3-ß Hydroxysteroid dehydrogenase. We conclude that the water extract of papaya seed suppresses the activities of steroidogenic enzymes in the testis of Sprague Dawley rats, and that this may contribute to reversible suppression of spermatogenesis, a property that gives a possible male contraceptive potential.

Evaluation of methylthio-, methylsulfinyl-, and methylsulfonyl-analogs of alkanes and alkanoic acids as cardiac inotropic and antifungal agents.

A group of alkane and alkanoic acid compounds of general formula MeS(O)m(CH2)nR [m = 0-2; n = 1, 5, 13; R = Me, CO2H(Na)] were synthesized for evaluation as cardiac inotropic and antifungal agents. Inotropic activity was determined as the ability of the test compound to modulate in vitro guinea pig atrium contractility. The oxidation state of the S-atom was an important determinant of inotropic modulation since the thio (m = 0) analogs exhibited a positive inotropic effect. In contrast, the sulfinyl (m = 1) and sulfonyl (m = 2) analogs exhibited a negative inotropic effect. A pentyl spacer (n = 5) provided the largest positive or negative inotropic effect. The relative positive, and negative, inotropic potency orders with respect to the R-substituent were Me > or = CO2H, and CO2Na > or = Me, respectively. The most potent positive inotrope MeS(CH2)5Me (EC50 = 4.49 x 10(-6) M) could serve as a useful lead-compound for the design of a new class of positive inotropic agents. In a broad spectrum antifungal screen, the minimal inhibitory concentration (MIC) range for the five most active compounds was MeSO2(CH2)5Me (0.46-1.83 mM), MeS(CH2)13Me (0.31-1.23 mM), MeSO(CH2)13Me (< 0.009-1.87 mM), MeSO2(CH2)13Me (0.27-1.09 mM), and MeS(CH2)13CO2H (0.27-1.09 mM), relative to the reference drug Ampotericin B (< 0.0002-0.002 mM). The most active antifungal agent MeSO(CH2)13Me was selective against C. guillermondi, C. neoformans, S. cerevisiae, and A. fumigatus (strain TIMM 1776).

Sample preparation for high throughput accurate mass analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

An automated sample preparation for high throughput accurate mass determinations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been developed. Sample preparation was performed with an automated workstation and automated mass analyses were performed with a commercial MALDI-TOF mass spectrometer. The method was tested with a 41-sample library. MALDI-TOFMS was found to give the needed sensitivity, accurate mass measurement, and soft ionization necessary for structure confirmation, even of mixtures. A mass accuracy of 5 ppm or less was obtained in over 80% of known compound measurements. A mass accuracy better than 10 ppm was obtained for all measurements of known compounds. Analyses of parallel synthesis products resulted in 77% of the measurements with a mass accuracy of 5 ppm or better.

Double blind, randomised, placebo controlled, crossover, multicentre study to determine the efficacy of a 0.1% (w/v) sodium hyaluronate solution (Fermavisc) in the treatment of dry eye syndrome.

BACKGROUND/AIMS: Hyaluronan (sodium hyaluronate) has been shown to confer objective and subjective improvement in patients with dry eye syndrome. This study compared the efficacy and safety of a 0.1% solution of hyaluronan with 0.9% saline, when administered topically to the eye, in the treatment of symptoms of severe dry eye syndrome. METHODS: A randomised, double blind, crossover clinical trial in which subjects were randomised to receive either hyaluronan or saline, applied as one or two drops to the eye, three or four times a day or as required. After 28 days' treatment, subjects crossed over to the other study medication for a further 28 days' treatment. RESULTS: 70 subjects were included in the analyses of efficacy and significant improvements in Schirmer's score (p=0.0006) and rose bengal staining score (p=0.0001) were observed during treatment with hyaluronan. In a subjective assessment of the effectiveness of two treatments, a majority of subjects felt that hyaluronan was more effective than saline in alleviating the symptoms of burning and grittiness (p<0.001). No adverse events attributable to hyaluronan treatment were reported. CONCLUSION: The study demonstrates a clear benefit of hyaluronan over saline, in both subjective and objective assessments of dry eye syndrome. Hyaluronan was shown to be well tolerated.

Determining affinity-selected ligands and estimating binding affinities by online size exclusion chromatography/liquid chromatography-mass spectrometry.

Size exclusion chromatography (SEC) isolation of affinity-selected ligands combined with reverse phase liquid chromatography-mass spectrometry (LC-MS) is an effective means for identifying members of mixtures which form tightly bound noncovalent complexes with target proteins. A potential liability of the approach is that the SEC isolation is carried out under nonequilibrium conditions favoring protein/ligand complex dissociation. At long SEC isolation times and/or for complexes with fast off-rates the extent of dissociation can jeopardize the ability to detect the affinity-selected components. Additionally, equilibrium binding affinities cannot be exactly determined from the measured distribution of isolated ligands. We present here an online SEC/LC-MS system for determining affinity-selected members of active mixtures which reduces this liability. A kinetic model of the SEC isolation process is developed to determine the practical limits for the application of the method and to extrapolate equilibrium binding affinities from the nonequilibrium data. The utility of online SEC/LC-MS for identifying affinity-selected ligands and for estimating binding affinities is demonstrated for a small molecule mixture of compounds with known binding affinities and for a simple combinatorial mixture.

Synthesis, rotamer orientation, and calcium channel modulation activities of alkyl and 2-phenethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(3- or 6-substituted-2-pyridyl)-5-pyridinecarboxylates.

A group of racemic alkyl and 2-phenethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(3- or 6-substituted-2-pyridyl)-5-pyridinecarboxylates (13a-q) was prepared using a modified Hantzsch reaction that involved the condensation of a 3- or 6-substituted-2-pyridinecarboxaldehyde (7a-j) with an alkyl or 2-phenethyl 3-aminocrotonate (11a-d) and nitroacetone (12). Nuclear Overhauser (NOE) studies indicated there is a significant rotamer fraction in solution where the pyridyl nitrogen is oriented above the 1,4-dihydropyridine ring, irrespective of whether a substituent is located at the 3- or 6-position. A potential H-bonding interaction between the pyridyl nitrogen free electron pair and the suitably positioned 1,4-dihydropyridine NH moiety may stablize this rotamer orientation. In vitro calcium channel antagonist and agonist activities were determined using guinea pig ileum longitudinal smooth muscle (GPILSM) and guinea pig left atrium (GPLA) assays, respectively. Compounds having an i-Pr ester substituent acted as dual cardioselective calcium channel agonists (GPLA)/smooth muscle-selective calcium channel antagonists (GPILSM), except for the C-4 3-nitro-2-pyridyl compound which exhibited an antagonist effect on both GPLA and GPILSM. In contrast, the compounds with a phenethyl ester group, which exhibited antagonist activity (IC50 = 10(-5)-10(-7) M range) on GPILSM, were devoid of cardiac agonist activity on GPLA. Structure-activity relationships showing the effect of a substituent (Me, CF3, Cl, NO2, Ph) at the 3- or 6-position of a C-4 2-pyridyl moiety and a variety of ester substituents (Me, Et, i-Pr, PhCH2CH2-) upon calcium channel modulation are described. Compounds possessing a 3- or 6-substituted-2-pyridyl moiety, in conjuction with an i-Pr ester substituent, are novel 1,4-dihydropyridine calcium channel modulators that offer a new drug design approach directed to the treatment of congestive heart failure and may also be useful as probes to study the structure-function relationships of calcium channels.

New polyazaporphine chemistry for the origin of life.

Molecular orbital spectral predictions suggest that 2,5,7,10,12,15,17,20-octaaza-21H, 23H-porphine has a visible spectral range closely matching that of chlorophyll-a. Since the octaazaporphine is, in its core, a simple derivative of an (HCN)12 oligomer, this fact, together with its spectral properties, would suggest that it occupies a high rank as a primordial porphinic solar energy transducer for photochemistry essential to life's formation. The demonstration that the mass 324 hexahydrooctaazaporphine is formed in protic media by the cyclotetramerization of imidazol-4-aminohydroxonium ion or the derived nitrenium ion, and that a mass 318 species consonant with that of the Huckel aromatic octaazaporphine is observed in the course of these studies, strongly supports the proposed octaazaporphine synthesis in a prebiotic hydrocyanic acid milieu.

Fundamentals of the application of matrix-assisted laser desorption-ionization mass spectrometry to low mass poly(methylmethacrylate) polymers.

Matrix-assisted laser desorption-ionization (MALDI) time of flight is shown to give a molar peak area response for isolated methylmethacrylate oligomers that have 25 and 50 repeat units when run on three different instruments in reflectron or linear mode and using three different matrix materials. In addition, fragmentation was not observed in any of the three different matrices or at higher laser power. No spectral differences were observed for syndiotactic and isotactic methylmethacrylate oligomers. These results suggest that the low most probable peak values observed for narrow distribution poly(methylmethacrylate) standards by MALDI mass spectrometry are not the result of mass discrimination or fragmentation.

The fundamentals of applying electrospray ionization mass spectrometry to low mass poly(methyl methacrylate) polymers.

Electrospray ionization (ESI) is capable of ionizing many soluble polymers. The ESI spectra are complex because of overlap of the multiply charged ions of the oligomer distribution, causing current computer transform programs to fail. However, it is possible to determine the origin of the multiply charged ions, making it feasible to write a program designed to transform ESI polymer spectra. To assess the value of such a program for polymer analysis, isolated monodisperse methyl methacrylate (MMA) oligomers (25 and 50 repeat units) were used to determine molar signal response and propensity for fragmentation.The sum of the peak areas for the multiply charged MMA 50-mer was found to be only about 66% of the summed peak areas for the 25-mer for the same molar concentration. However, conversion of the multiply charged peak areas to the singly charged representations, with peak area compression taken into account, gave equal signal responses for the 25-and 50-mers. Signal response variations due to the tacticity of the MMA oligomers were not observed. Fragmentation of the MMA oligomers also was shown not to occur under normal ESI conditions. Therefore, transformation of the polymer spectra to the singly charged molecular ion distribution should allow accurate calculation of average molecular weights, polydispersity, end group mass, and repeat unit mass.

Clinical correlations of osteoarthritis associated with a single-base mutation (arginine519 to cysteine) in type II procollagen gene. A newly defined pathogenesis.

OBJECTIVE: To investigate the occurrence and clinical correlation of the arg519-to-cys mutation in the type II procollagen gene in patients with osteoarthritis (OA). METHODS: Sixty-six subjects from 7 families with a strong family history of generalized OA and 13 patients with erosive OA were evaluated clinically and radiologically. Blood samples from 58 subjects in the familial OA group and from all 13 patients with erosive OA were obtained for DNA analysis. Exon 31 of COL2A1, which spans residue 519, was amplified by polymerase chain reaction. RESULTS: The arg519-to-cys mutation was detected in 2 of the 7 families with generalized OA. In these 2 families, the mutation was present in the 2 probands and in 19 other clinically affected family members, as well as in 3 (so-far) clinically unaffected family members (ages 25, 14, and 11 years). It was absent in 18 clinically unaffected members tested. The mutation was associated with a distinctive pattern of early-onset, aggressive, generalized OA with a mild spinal chondrodysplasia. Inheritance was autosomal dominant. No mutation was found in any of the patients with erosive OA. CONCLUSION: The arg519-to-cys mutation defines a new pathogenic factor in generalized OA with characteristic clinical and radiologic features. The demonstration of a mutation in 3 of 8 families with OA studied thus far suggests a significant incidence of genetically related clinical OA.

Enantioselective syntheses and calcium channel modulating effects of (+)- and (-)-3-isopropyl 5-(4-methylphenethyll) 1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)-3,5-pyridinedicarboxylates.

The (+)- and (-)-enantiomers of 3-isopropyl 5-(4-methylphenethyl) 1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)-3,5-pyridinedicarboxylate were synthesized using an efficient highly enantioselective (ee > or = to 96%) variant of the Hantzsch dihydropyridine synthesis. The key step in this procedure involved the asymmetric Michael addition of a metalated chiral aminocrotonate, derived from D-valine or L-valine, respectively, to the Knoevenagel acceptor (Z)-2-isopropoxycarbonyl-1-(2-pyridyl)-but-1-en-3-one. Both enantiomers exhibited a dual cardioselective partial calcium channel agonist (positive inotropic)/smooth muscle selective calcium channel antagonist effect. The relative in vitro smooth muscle calcium channel antagonist activities of the (-):(+) enantiomers was 26:1. In contrast, the (+)-enantiomer exhibited a greater in vitro positive inotropic effect on guinea pig left atrium where the contractile force was maximally increased by 14.8% at a concentration of 1.63 x 10(-8)M.

Production of perfluoroalkylated nanospheres from buckminsterfullerene.

Perfluoroalkylated nanospheres have been prepared by reaction of fullerenes with a variety of fluoroalkyl radicals. The latter are generated by thermal or photochemical decomposition of fluoroalkyl iodides or fluorodiacyl peroxides. Up to 16 radicals add to C(60) to afford easily isolable fluoroalkylated derivatives. The monosubstituted radical adducts were detected by electron spin resonance in the early stages of the fluoroalkylation reactions. These spheroidal molecules are thermally quite stable, soluble in fluoroorganic solvents, chemically resistant to corrosive aqueous solutions, and more volatile than the parent fullerenes. Films of the sublimed material display properties typical for a perfluoroalkylated material.

Sensitivity of NCI-H292 human lung mucoepidermoid cells for respiratory and other human viruses.

NCI-H292 mucoepidermoid carcinoma cells from human lungs were shown in an earlier report to be a fully adequate substitute for primary rhesus monkey kidney (MK) cells for the isolation and propagation of the human paramyxoviruses. Although sensitivity for ortho- and paramyxoviruses was the principal reason for using MK cells, the cells were also sensitive to many other viruses, which constituted another important value of MK cells. That MK cells supported the initial isolation and growth of so many respiratory viruses made it a mandatory cell type for any clinical laboratory. We therefore felt it was imperative to evaluate the virus spectrum of NCI-H292 cells, which are being used as a substitute for MK cells in many laboratories. In the present report, we show that NCI-H292 cells are sensitive for vaccinia virus, herpes simplex virus, adenoviruses, BK polyomavirus, reoviruses, measles virus, respiratory syncytial virus, some strains of influenza virus type A, most enteroviruses, and rhinoviruses, in addition to the parainfluenza and mumps viruses originally reported. Furthermore, these viruses replicate in NCI-H292 cells to the same virus and antigen titers and at the same speed of replication as they do in their usually preferred cells. The NCI-H292 cells are therefore an excellent substitute for MK cells in terms of laboratory safety, ease of availability, paramyxovirus isolation, and broad virus spectrum but cannot substitute for MK cells for the isolation of influenza viruses.

An electrospray ion source for magnetic sector mass spectrometers.

Electro spray mass spectra of carbonic anhydrase (MW ∼ 29000) and ovalbumin (MW ∼ 45000) were obtained on a double focusing magnetic secter mass spectrometer by using a single stage of mechanical pumping in the interface between atmospheric pressure and high vacuum. Full scan spectra of lysozyme were recorded on 15 fmoles consumed. In addition, accurate mass measurement was demonstrated for peptides and proteins, and resolution in excess of 10,000 (m /△m, 10% valley) was observed. These results clearly show that high performance magnetic sector mass spectrometers can be advantageously interfaced to an atmospheric pressure electrospray ion source.

Multicenter evaluation of synovectomy in the treatment of rheumatoid arthritis. Report of results at the end of five years.

A controlled multicenter evaluation of open surgical synovectomy in the treatment of rheumatoid arthritis (RA) was carried out over a period of 5 years after the operation. There was evidence of benefit in some knees but none in finger joints. Synovectomy performed at a stage of minimal damage did not result in fewer recurrences of disease activity than when done after greater damage had occurred. The results showed little longterm value of surgical synovectomy in the general treatment of RA or in the prevention of recurrences of disease activity or progressive articular damage. These results emphasize the need for controls in trials of this type.

Intralesional recombinant alpha-2 interferon for the treatment of patients with condyloma acuminatum or verruca plantaris.

We conducted a multicenter double-blind study comparing human recombinant intralesional alpha-2 interferon (IFN) and placebo in 237 patients with the clinical diagnosis of condyloma acuminatum or verruca plantaris. A single wart on each patient was injected with 0.1 mL containing 10(6) IU of IFN, 10(5) IU of IFN, or placebo three times weekly for three weeks, and the response to treatment was followed up for 12 weeks. Among 91 of the 114 patients with condyloma acuminatum who completed the study, complete clearing of the treated wart occurred in 16 (53%) of 30 patients receiving 10(6) IU of IFN compared with six (19%) of 32 receiving 10(5) IU of IFN and four (14%) of 29 receiving placebo. In the group of 100 patients with plantar warts, there was no apparent benefit associated with interferon administration. Seven patients (3%) had treatment discontinued due to adverse reactions. Intralesional alpha-2 IFN is of benefit in the treatment of a single condyloma; its role in the treatment of multiple lesions remains to be clarified. Its role in the treatment of verruca plantaris, where no response was seen, also remains to be clarified.

Salivary gland aplasia: an ectodermal disorder?

A case of a patient presenting with the symptoms of lifelong xerostomia is discussed. Clinical investigation demonstrated aplasia of all the major salivary glands, lacrimal gland hypoplasia and enamel hypoplasia. The management and aetiology of the case is discussed.