Biomedical subjects

M Bozzini

Publications and source records attributed to M Bozzini.

5 recordsLinked to original sources

PubMed2000

The alkylated thiohydantoin method for C-terminal sequence analysis.

The alkylated-thiohydantoin method for C-terminal sequencing makes a significant improvement to the thiohydantoin method first described by Schlack and Kumpf. Prior to cleavage from the protein, the C-terminal thiohydantoin is alkylated, making it a better leaving group than the unmodified thiohydantoin. The C-terminal alkylated-thiohydantoin can be cleaved from the protein under conditions that simultaneously form the next thiohydantoin. Combining cleavage and thiohydantoin formation in one step eliminates the need for activating the C-terminal carboxyl group before every sequencing cycle and prevents detection of C-termini formed by random cleavage of peptide bonds in the protein during the sequencing chemistry. The alkylated-thiohydantoin method includes the presequencing modification of cysteine and lysine and the automated modification of aspartic and glutamic acids, serine and threonine. Modifying the reactive side-chain groups improves the ability to sequence through and detect these amino acids. The alkylated-thiohydantoin method can sequence through and detect 19 of the 20 genetically coded amino acids. Sequencing stops at proline residues.

Alkylation↗

PubMed1995

Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme.

Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.

ATP-Binding Cassette Transporters↗

PubMed1992

Sequencing of peptides and proteins from the carboxy terminus.

A new chemical method for carboxy-terminal (C-terminal) protein sequencing has been developed. This approach has been successfully used to sequence 5 residues of standard proteins and 5 to 10 residues of synthetic peptides at low nanomole levels. The sequencing procedure consists of converting the C-terminal amino acid into a thiohydantoin (TH) derivative, followed by transformation of the TH into a good leaving group by alkylation. Next, the alkylated TH is cleaved mildly and efficiently with (N = C V S)- anion, which simultaneously forms a TH on the newly truncated protein or peptide. Thus, after the initial TH derivatization, there is no return to a free carboxyl group at the C-terminus. An additional benefit of this method is that the alkylating moiety can be chosen with a variety of properties allowing for variation in the detection method. This chemistry has been adapted to automated protein sequencers with a cycle time of about 1 h.

Amino Acid Sequence↗

PubMed1991

Radiotherapy of pseudotumors of bone in hemophiliacs with circulating inhibitors to factor VIII.

Pseudotumors of bone in two hemophiliacs with severe factor VIII deficiency and a high level of circulating inhibitors are reported. Both had favorable response to radiation therapy after unsuccessful treatment with factor concentrates and remain free of recurrence at 18-62 months. A review of the literature of cases in which radiotherapy has been used is presented. Radiotherapy in the treatment of pseudotumors of bone in hemophiliacs should be strongly considered, particularly if coagulation factor inhibitors are present.

Adolescent↗

PubMed1975

Laboratory and clinical evaluation of the radiation-potentiating activity of ethyl-N-bis (2,2-dimethylethylamidinophosphoro) carbamate (AB-132).

Ethyl-N-bis (2,2-dimethylethylamidinophosphoro) carbamate (AB-132) has been shown to potentiate the effect of whole-body radiation on inhibition of splenomegaly induced by Friend leukemia virus in ICR/H Swiss mice. The combined effect of AB-132 and radiation does not appear to be related to Friend virus inhibition but seems to act on the proliferating tumor in the spleen. Nine children with advanced cancer were treated with combined administration of local radiation and systemic AB-132. Although regression of tumor was seem, no dramatic effect on survival was apparent. One case of metastatic Ewing's sarcoma showed systemic tumor response to AB-132 in addition to localized response to radiation. Bone marrow depression appeared to be the main side effect of combination therapy.

Animals↗