Studies on cytochrome c. Part II. Synthesis of the protected heptapeptide (sequence 17–23) of Baker's yeast iso-1-cytochrome c

Synthesis is described for the N-o-nitrophenylsulfenylheptapeptide tert-butoxycarbonylhydrazide corresponding to positions 17–23 of the amino acid sequence of baker's yeast iso-1-cytochrome c. Moreover a new method of selective removal of the S-acetamidomethyl group for analytical and preparative purpose is reported.     

Aerobiological monitoring of the 'Sistine Chapel': airborne bacteria and microfungi trends

The present paper reports the results of a bacteriological and mycological monitoring carried out on the airborne microflora of the Sistine Chapel. The general aim of the study was to evaluate the impact of the flow of visitors, as well seasonal effects, on the qualitative and quantitative variations of microorganisms. Two sampling campaigns were carried out in May and November 1997. A Surface Air System (SAS) sampler (active system) and a sedimentation based sampler (passive system), supported by an original plinth, were used. Temperature, humidity and carbon dioxide were detected. VITEK SYSTEMS jr. for Staphylococcus spp. and microscopic observation for microfungi were the identification methods. In spite of the conditioning and filtration air system, initial results with both samplers, show a positive correlation between the airborne microorganisms and presence and number of visitors. The SAS samples showed higher microbial load, for both bacteria and fungi, than the passive ones, but the epidemiological meaning of the differently collected data varies. The increase during visiting hours of human Staphylococcus spp. is stronger than the airborne bacterial load increase. The microfungi most frequently isolated were Cladosporium spp. and Penicillium spp. These preliminary data underline the significance of the survey for the protection of such a precious environment, and encourage the Authors to continue the ongoing monitoring. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on cytochrome c. Part I. Synthesis of the protected hexadecapeptide (sequence 1–16) of Baker's Yeast iso-1-cytochrome c

The general strategy for the synthesis by conventional procedure of N- and C-terminal fragments and of the entire sequence of baker's yeast iso-1-cytochrome c is discussed. The synthesis of the N-benzyloxycarbonylhexadecapeptide tert-butoxycarbonylhydrazide corresponding to the sequence 1–16 of the apoprotein is described in detail.     

Further evidence of a duplication in 17p11.2 in families with recurrence of HMSN Ia (Charcot-Marie-Tooth neuropathy type Ia)

Five Italian families with recurrence of cases of Charcot-Marie-Tooth disease (type Ia) were analysed using three closely linked DNA probes that detect polymorphisms in the region 17p11.2. The probe pVAW409R3 detected the presence of a duplication in all the affected subjects, but not in the subjects with normal electromyographic (EMG) findings. This observation confirms previous data indicating the association of the duplication with the disease, suggesting that, at least in populations of European origin, the duplication might be the molecular feature diagnostic of the pathological trait.     

Partial trisomy 4q: Two cases with a familial translocation t(4;18)(q27;q23)

Clinical and cytogenetic data of two related patients, both trisomic for the segment 4q27 to qter, are reported. Familial studies determined that the mothers of the two probands were carriers of the same balanced translocation between chromosomes 4 and 18. Altogether, two partial trisomies 4q, five balanced karyotypes, and one 45,X0 karyotype were found in the family. The 18 cases reported to date are reviewed with respect to the karyotype-phenotype correlation.     

Polystyrene Embedding and Spreading of Sections at Lower Temperature

Embedments of histological material for the light microscope using a polystyrene solution (Frangioni and Borgioli Polystyrene Embedding Medium, Polysciences Inc., Catalog no. 15858) has several advantages (Frangioni and Borgioli 1979) including: ease of use, consistency of results, possibility of obtaining sections of any thickness using even a steel knife microtome, possibility of using all staining methods applicable to paraffin embedments, and superior quality of the mounts. Excellent preservation of morphology has been confirmed by pictures taken under the electron microscope of osmium fixed material embedded in polystyrene (Frangioni and Borgioli 1979).     

The effect of S-adenosyl-l-methionine on ischemia-induced disturbances of brain phospholipid in the gerbil

Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries with reported procedures. Changes in the energy status of brain demonstrated that carotid ligation was effective. At different time intervals from ligation, groups of gerbils were given either saline of S-Adenosyl-L-methionine (SAMe) by the intraventricular (i.v.) route (1.6 mg/Kg body wt. twice, at each 10 min interval), or by the intraperitoneal (i.p.) administration (200 mg/Kg body wt.) or subcutaneously (s.c.) with 40 mg/Kg body wt, daily, for two weeks. Control animals, with and without SAMe, together with the ischemic groups, were decapitated directly into liquid nitrogen, 10 min after ligation. Brain neutral and polar lipid, together with free fatty acids, which were all labeled in vivo by the intraventricular injection of [1-14C]arachidonic acid 2 hr prior to ligation, were extracted, purified and separated by conventional procedures. SAMe when injected i.v. or i.p. noticeably corrected the changes in polar lipid by reversing the decrease of brain phosphatidylcholine and choline plasmalogen, as well as of their labeling, which was due to ischemia. Concurrently with this action, SAMe treatment (i.v. and i.p.) also provided to some extent to re-establish the normal level of labeling of ethanolamine lipids. When SAMe was given s.c., no effect was present. SAMe had no effect on the increase of free fatty acid and diglyceride due to ischemia. The prevention by SAMe of the changes of choline lipids suggests that a stimulation of the methyltransferase reaction may occur in the ischemic brain, due to increased substrate (SAMe) availability. This effect may be important for cell survival, since membrane phospholipid derangements alter the properties of the membrane.     

A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the α2,6-sialyltransferase (α2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both α2,6- and α2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing α2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the α2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-γ and the tissue inhibitor of metalloproteinases-1. Interferon-γ purified from the universal host carried 40.4% α2,6- and 59.6% α2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-γ produced by normal CHO cells.