Encapsulation of proteins in transparent porous silicate glasses prepared by the sol-gel method.

Novel sol-gel synthetic techniques were used to immobilize copper-zinc superoxide dismutase (CuZnSOD), cytochrome c, and myoglobin (Mb) by encapsulation in stable, optically transparent, porous silica glass matrices under mild conditions such that the biomolecules retained their characteristic reactivities and spectroscopic properties. The resulting glasses allowed transport of small molecules into and out of the glasses at reasonable rates but nevertheless retained the protein molecules within their pores. Chemical reactions of the immobilized proteins could be monitored by means of changes in their visible absorption spectra. Silica glasses containing the immobilized proteins were observed to have similar reactivities and spectroscopic properties to those found for the proteins in solution. For example, encapsulated CuZnSOD was demetallated and remetallated, encapsulated ferricytochrome c was reduced and then reoxidized, and encapsulated met Mb was reduced to deoxy Mb and then reacted either with dioxygen to make oxy Mb or with carbon monoxide to make carbonyl Mb.

Association between Degree of Anaplasia and Degree of Inflammation with the Expression of COX-2 in Feline Injection Site Sarcomas.

Feline injection site sarcomas (FISSs) are mesenchymal neoplasms that develop at the sites of delivery of vaccines or other injectable products. Vaccine adjuvants can trigger an intense and persistent inflammatory response that may lead to neoplastic transformation. The proinflammatory role of cyclo-oxygenase (COX)-2 is well known and its overexpression has prognostic value in multiple neoplastic processes. One hundred and seventeen FISSs were evaluated for the degree of inflammation and anaplasia. Immunohistochemistry was used to determine the expression of COX-2 in these sarcomas. There was a significant association between the degree of inflammation and the expression of COX-2 by neoplastic cells. COX-2 expression was lower in tumours with higher degrees of anaplasia. These findings may be useful in predicting the sensitivity of FISSs to treatment with COX-2 inhibitors. The potential therapeutic use of such agents could then be restricted to tumours with lower degrees of anaplasia.

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1.

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin-Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.

Microvascular lesions and changes in cell proliferation and death, and cytokine expression in the placentas of mice experimentally infected with Equid Herpesvirus 1.

This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.

FKBP-type peptidyl-prolyl cis-trans isomerase from a sulfur-dependent hyperthermophilic archaeon, Thermococcus sp. KS-1.

A gene encoding an FK506 binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase (PPIase) was cloned from a hyperthermophilic archaeon, Thermococcus sp. KS-1, and sequenced. This gene encoded an FKBP with 159 amino-acid residues with a molecular mass of 17.6kDa. Two insertion sequences with 13 and 44 amino acids were found in the regions corresponding to the bulge and flap regions of human FKBP-12, respectively. Comparison with other archaeal FKBP sequences obtained from reported genome sequences revealed that the insertion sequences in the bulge and flap regions were common to archaeal FKBPs. It was also revealed that archaeal FKBPs are classified into two groups: one is approx. 17kDa and the other 27kDa. This Thermococcus FKBP (TcFK) belonged to the smaller archaeal FKBP. In this TcFK, 9 out of 15 amino acid residues forming the FK506 binding pocket of human FKBP12 were found. This gene was expressed in Escherichia coli and the recombinant protein was purified. The purified protein showed PPIase activity and its activity was inhibited by FK506 with an IC50 of 7 microM. This enzyme showed high kinetic stability with a half-life of 40 min at 100 degrees C. Catalytic efficiency of this recombinant PPIase was 1.2-times higher with the substrate N-succinyl-A-L-P-F-p-nitroanilide than with N-succinyl-A-A-P-F-p-nitroanilide.

Guinea-pig liver morphine 6-dehydrogenase as a naloxone reductase.

Elution profiles of guinea-pig liver naloxone reductase and morphine 6-dehydrogenase on Matrex green A, Sephadex G-100 and DEAE-cellulose (DE32) column chromatography used sequentially in the purification procedure were identical. The ratios of the two enzyme activities were almost constant throughout all the purification steps. The two enzymes were similarly more stable at pH 6.0 than at pH 8.0 on storage at 4 degrees. The reversible inactivation of the two enzymes by the removal of 2-mercaptoethanol from the enzyme solution was the same. Inhibitory effects of lithocholic acid, CuSO4, quercitrin, phenylarsine oxide, and prostaglandin E1 on the two enzymes were almost the same. These results indicated that naloxone reductase is identical to morphine 6-dehydrogenase in the guinea-pig liver. For the reduction of naloxone, the enzyme utilized either NADPH or NADH as cofactor, and pH optima were 6.8 with NADPH and 6.2 with NADH. The Km values for NADPH and NADH were 6.5 and 2.2 microM respectively. The Vmax values for naloxone were 1.2 units/mg protein with NADPH and 0.5 unit/mg protein with NADH. The Km values for naloxone were 0.27 mM with NADPH and 0.44 mM with NADH. The reaction product formed by the enzyme was identified as 6 alpha-naloxol by thin-layer and gas-liquid chromatographic analyses. Accordingly, it is clear that the enzyme catalyzes the stereospecific reduction of naloxone to form the 6 alpha-hydroxyl congener.

The primary electron acceptor of green sulfur bacteria, bacteriochlorophyll 663, is chlorophyll a esterified with Delta2,6-phytadienol.

The primary electron acceptor of green sulfur bacteria, bacteriochlorophyll (BChl) 663, was isolated at high purity by an improved purification procedure from a crude reaction center complex, and the molecular structure was determined by fast atom bombardment mass spectroscopy (FAB-mass), (1)H- and (13)C-NMR spectrometry, double quantum filtered correlation spectroscopy (DQF-COSY), heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectral measurements. BChl 663 was 2.0 mass units smaller than plant Chl a. The NMR spectra showed that the macrocycle was identical to that of Chl a. In the esterifying alcohol, a singlet P7(1) signal was observed at the high-field side of the singlet P3(1) signal in BChl 663, while a doublet peak of P7(1) overlapped that of P11(1) in Chl a. A signal of P7-proton, seen in Chl a, was lacking, and the P6-proton appeared as a triplet signal near the triplet P2-proton signal in BChl 663. These results indicate the presence in BChl 663 of a C=C double bond between P6 and P7 in addition to that between P2 and P3. The structure of BChl 663 was hence concluded to be Chl a esterified with 2,6-phytadienol instead of phytol. In addition to BChl 663, two molecules of the 13(2)-epimer of BChl a, BChl a', were found to be present per reaction center, which may constitute the primary electron donor.

Oxy-function promoted hydroboration of conjugated dienes with controlled allylic borane rearrangement.

Hydroboration of conjugated dienes is promoted by the hydroxy and methoxy groups, which also control the rearrangement of the initially produced allylic boranes.

Structure elucidation of glykenin glycosidic antibiotics from Basidiomycetes sp. V. High-performance liquid chromatographic separation of components of glykenin.

The glycosidic antibiotics of the glykenin (GK) family produced by Basidiomycetes sp. were separated into nine components (GK-I-VII and DG) by normal-phase chromatography. It was found that these components differ in the number and location of the acetyl groups in the sugar moiety. Each component (GK-I-VII and DG) was further separated into three isomers (A, B and C), which possess different aglycones, by reversed-phase chromatography on an ODS column with methanol-acetonitrile as eluent. The best composition of the eluent was found to be methanol-acetonitrile-1% trifluoroacetic acid (4:3.5:2.5). The profile analysis of GK-III-VII and DG was also carried out using a modified mobile phase. The combination of normal- and reversed-phase chromatography separated all components of the GK mixture except GK-I and II. The relationship between structure and separation behaviour of GK is discussed.

A new sesquiterpene as an antifouling substance from a palauan marine sponge, dysidea herbacea

Bioassay-guided fractionation of an extract from a marine sponge, Dysidea herbacea, led to the isolation and identification of the new sesquiterpene 1. This compound showed repellent activity against the blue mussel, Mytilus edulis galloprovincialis.

Studies on an antibiotic, albocycline VII. Minor components of albocycline.

As an approach to the search for new potentially useful macrolide antibiotics, we explored the minor components of albocycline (ALB) from the culture broth of Streptomyces bruneogriseus. Eight minor components were isolated and their structures were confirmed as 1 approximately 8. Unexpectedly, they were not glycosidic compounds but only oxidation or reduction products of ALB. Three or four of them will serve as a useful intermediate to introduce amino sugar moiety into ALB skeleton.

Pradimicin S, a new pradimicin analog. III. Application of the frit-FAB LC/MS technique to the elucidation of the pradimicin S biosynthetic pathway.

The biosynthetic pathway of pradimicin S (PRM-S) was investigated by using sinefungin and bioconversion experiments with aglycones of pradimicin A (PRM-A) and Actinomadura spinosa AA0851, a PRM-S producer. Addition of sinefungin to the strain inhibited the formation of 11-O-demethyl-7-O-methylpradinone II (11dM-7M-PNII) as also determined to occur with its addition to the PRM-A producer. In feeding PRM-A aglycone and its analogs to the strain early in PRM-S biosynthesis, good identifications of bioconverted products were obtained by frit-FAB LC/MS as follows: 11-O-demethylpradinone II (11dM-PNII), 11dM-7M-PNII, 11-O-demethylpradinone I (11dM-PNI), 11-O-demethylpradimicinone I (11dM-PMNI) and pradimicinone I (PMNI) were converted to PRM-S. Pradimicin B (PRM-B) and pradimicin L (PRM-L) were converted to PRMs-L and -S and PRM-S, respectively. A biosynthetic pathway for PRM-S is proposed.

Effects of Solanum glaucophyllum toxicity on cell proliferation and apoptosis in the small and large intestine of rabbits.

Vitamin D regulates mineral homeostases and enterocyte proliferation and differentiation. Hypervitaminosis D generates changes in cell proliferation, differentiation and apoptosis in several organs. We analysed morphometric parameters and proliferative and apoptotic indices in the intestinal epithelium of rabbits with hypervitaminosis D induced by the chronic treatment with the calcinogenic plant Solanum glaucophyllum. Rabbits were treated for 15 or 30 days. A group was treated for 15 days and led to possible recovery for 30 days. Another group was nutritionally restricted for 30 days. Morphological, morphometric, proliferative and apoptotic changes were found in the treated animals. Mild atrophy and reduced proliferation was found in the jejunum and ileum. Apoptosis increased in the crypts of the ileum and in the superficial epithelium and crypts of the rectum. Most of the alterations were partially recovered. The possible involvement in these changes of the hypervitaminosis D-like state induced by S. glaucophyllum is discussed.

Restorative effect of intracerebroventricular insulin-like growth factor-I gene therapy on motor performance in aging rats.

Insulin-like growth factor-I (IGF-I) is a powerful neuroprotective molecule in the brain and spinal cord. We have previously shown that intracerebroventricular (i.c.v.) IGF-I gene therapy is an effective strategy to increase IGF-I levels in the cerebrospinal fluid (CSF). Since aging in rats is associated with severe motor function deterioration, we implemented i.c.v. IGF-I gene therapy in very old rats (30-31 months) and assessed the beneficial impact on motor performance. We used recombinant adenovectors (RAds) expressing either green fluorescent protein (GFP) or rat IGF-I. Injection in the lateral or fourth ventricle led to high transgene expression in the ependymal cell layer in the brain and cervical spinal cord. RAd-IGF-I-injected rats but not RAd-GFP-injected controls, showed significantly increased levels of CSF IGF-I. Motor tests showed the expected age-related decline in aged rats. Seventeen-day IGF-I gene therapy induced a significant improvement in motor performance in the aged but not in the young animals. These results show that IGF-I is an effective restorative molecule in the aging brain and spinal cord. The data also reveal that the ependymal route constitutes a promising approach for implementing protective IGF-I gene therapy in the aging CNS.

[Electromyographic (EMG) electrode impedance and EMG activity from anterior temporal muscle and masseter muscle].

The value and change with time of the impedance of surface EMG electrodes and the effects of their difference between the bipolar electrodes on the electromyographic activity from the anterior temporal muscle and the masseter muscle in six adult male subjects with normal occlusion were studied. The results were as follows: 1. In the anterior temporal muscle, if the impedance of the electrode was under 20 k omega it was stable from just after the electrode disc was applied to the skin. In the masseter muscle, if the impedance was under 30 k omega it became stable within two minutes after the electrode was applied. 2. The difference of impedance between the bipolar EMG electrodes did not correlate with EMG activity.

[Frequency analysis of the EMG power spectrum of the anterior temporal and masseter muscles in children and adults].

For the investigation of the functional change of the masticatory muscles along with growth and development, the frequency analysis of the EMG power spectrum was carried out. The subjects were 6 children (5 males and 1 female) with full deciduous dentition (Hellman's dental age IIA) aged 4.5 +/- 0.2 years and 6 adults (4 males and 2 females) with full permanent dentition aged 27.7 +/- 3.8 years. EMG signals were recorded bilaterally by means of bipolar silver surface electrodes from the anterior temporal and masseter muscles when the subjects were chewing chewing gum or performing maximum clenches in the intercuspal position. A fast Fourier transform (FFT) algorithm was used to obtain the power spectrum of the EMG signal. As the total power value from 62.5 to 1000 Hz was 100 per cent, the mean frequencies at 25, 50, 75 and 90 per cent of the cumulative power were calculated. The results were as follows: 1. The mean frequencies at each ratio of the cumulative power were age-dependent and EMG power spectrum patterns significantly shifted to lower frequencies in the muscles of the adults. 2. No statistically significant differences between the chewing and clenching, the anterior temporal and masseter muscle and the left and right side were observed in each group.

[Muscle action potential and masticatory rhythm of anterior temporal and masseter muscles in children and adults].

For the investigation of the functional change of the masticatory muscles along with growth and development, electromyographic evaluation was carried out. The subjects were 6 children (5 males and 1 female) with full deciduous dentition (Hellman's dental age IIA) aged 4.5 +/- 0.2 years and 6 adults (4 males and 2 females) with full permanent dentition aged 27.7 +/- 3.8 years. EMG signals were recorded bilaterally by means of bipolar silver surface electrodes from the anterior temporal and masseter muscles when the subjects were chewing chewing gum or performing maximum clenches in intercuspal position. The cumulative power values from 62.5 to 1000 Hz in the EMG power spectrum during chewing or clenching were calculated as the muscle action potential. The ratio of the action potential of each muscle to the total action potential of four muscles were analyzed. Masticatory rhythm during chewing was analyzed by means of the time parameter (duration, interval and cycle) and their coefficients of variation. The results were as follows: 1. In children the temporal muscles predominated in chewing and clenching, whereas in adults there were three types with Temporal muscles predominating, Masseter muscles predominating and both muscles sharing equally. 2. No statistically significant differences between children and adults were observed in the duration, interval and cycle. 3. In adults the coefficients of variation of the duration, interval and cycle were smaller and the masticatory rhythm was more stable than in children.

Frequency analyses of EMG power spectra of anterior temporal and masseter muscles in children and adults.

To study the functional change of masticatory muscles during growth and development, frequency analyses of surface electromyogram (EMG) power spectra were carried out. The subjects were six children (five males and one female), aged 4.5 +/- 0.2 years, having full deciduous dentition (Hellman's dental age IIA) and six adults (four males and two females), aged 27.7 +/- 3.8 years, having full permanent dentition. EMG signals were recorded bilaterally by using bipolar silver-surface electrodes from the anterior temporal and masseter muscles while the subjects were chewing gum and while performing maximum clenching in the intercuspal position. A fast Fourier transform algorithm was used to obtain the power-spectral density function and the power spectra of the EMG signals. Since the total power value from 62.5 to 1000 Hz was 100 percent, the frequencies at 25, 50, 75, and 90 percent of the cumulative power were calculated. The results showed that the frequencies at every percent of the cumulative power were age-dependent and that the EMG power spectra patterns in adult muscles were shifted to significantly lower frequencies than those in child muscles. The shift was probably caused by differences in the proportion of fiber type and fiber size between muscles of children and adults.

Laser behavior and photostability characteristics of organic dye doped silicate gel materials.

The solgel process is a solution synthesis technique which provides a low temperature chemical route for the preparation of rigid transparent matrix materials. Luminescent organic dye molecules have been incorporated via the solgel method into organically modified silicate (ORMOSIL) polymer host matrices. Optical gain, laser oscillation, and photostability of rhodamine and coumarin dyes doped into ORMOSIL gels are reported. The gel laser materials exhibit peak gain values of 40 cm(-1) and show improved photostability with respect to comparable polymeric host materials.