Synthesis of the human proinsulin sequence 71-86, I: Synthesis of sequence 71-86 as a monomeric cyclic biscystine peptide derivative and as its tetra-S-trityl derivative (author's transl)]

The synthesis of sequence 71-86 of human proinsulin (sequence 6-21 of human insulin A-chain) via its monomeric cyclic dicystine peptide derivative (XXII) and the corresponding tetra-S-trityl-peptide derivative (XVIII) is described. The good solubility of the derivatives (XXII, XVIII) in orgranic solvents makes it possible to purify both the derivatives in their protected form. The S-trityl derivative (XVIII) enables the synthesis of the fragment 71-86 in a scale needed for the planned synthesis of human proinsulin.     

Synthesis of human proinsulin sequence 71-86, II: Improved synthesis of the fragment.

An improved synthesis of a fully protected hexadecapeptide Bpoc-Cys(Trt)-Cys-(Trt)-Thr(But)-Ser(But)-Ile-Cys(Trt)-Ser(But)-Leu-Tyr(But)-Gln-Leu-Glu(OBut)-Asn-Tyr(But)-Cys(Trt)-Asn-OBut, which corresponds to the amino acid sequence 71-86 of human proinsulin, is described. The final product was obtained by the condensation of three fragments 71-74, 75-77 and 78-86.     

Dp71Δ78‐79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71Δ_78‐79 dystrophin mutant is stably expressed in PC12‐C11 cells. To investigate the effect of Dp71Δ_78‐79 overexpression on the protein profile of PC12‐C11 cells, we compared the expression profiles of undifferentiated and NGF‐differentiated PC12‐C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein was downregulated, and five were upregulated. Dp71Δ_78‐79 overexpression had a greater effect on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein with the highest upregulation was HspB1. Changes in HspB1 expression were validated by Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12‐C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process. Our results show that Dp71Δ_78‐79 affects the expression level of some proteins and that the stimulated neurite outgrowth produced by this mutant is mainly through upregulation and phosphorylation of HspB1.     

Synthesis and antifungal activity of oxygenated cholesterol derivatives

A series of oxygenated cholesterol derivatives were prepared from new synthetic methods and evaluated for their in vitro antimicrobial properties against human pathogens. The activity was highly dependent on the structure of the different compounds involved. The best results were obtained with hydroxy ketones 2, 4 and 5 and diketone 7 exhibiting activities against S. cerevisiae (ATCC 28383) and Candida albicans (CIP 1663-86). For example, compound 2 exhibited high activities against C. albicans (CIP 1663-86) and Amphotericine B and miconazole resistant strain C. albicans (CIP 1180-79) at a concentration of 1.5 microg/mL.     

Synthesis and Biological Activity of the Functional Derivatives of 3- and 4-(Dimethyl-aminomethyl)-1,2-dithiolanes

A variety of derivatives of 3- and 4-(dimethylaminomethyl)-1,2-dithiolanes were prepared as their biological equivalents in an analogous way to that for the nereistoxin derivatives. Most of them showed insecticidal and acaricidal activity. High potency against Chilo suppressalis was displayed by S,S′-[2-(dimethylaminomethyl)trimethylene] bis(thiobenzoate), and a broad spectrum of activity was observed for 5-(dimethylaminomethyl)-1,2,3-trithiane. The stereochemistry of the 1,3-dithianes is also discussed.     

Well Differentiated Follicular Neoplasms of the Thyroid: Reproducibility and Validity of A''Decision Tree''Classification Based On Nucleolar and Karyometric Features

This study was conducted on fine-needle aspirates of well differentiated follicular neoplasms of the thyroid. A 'decision tree' classification based on the percentage of nucleolated nuclei, percentage of nuclei with two or more nucleoli and mean major nuclear diameter was adopted. We observed that the reproducibility and the validity of the follicular adenoma vs follicular carcinoma discrimination are greater than in the subjective evaluation. Moreover, similar classification results were obtained when measurements were performed either with a fully automated image analysis system or with semiautomatic or manual instrumentation. As for reproducibility of the inter-instrument comparisons, the k statistic values ranged from 0.85 to 1.00 (mean value 0.90, that is, an 'almost perfect' degree of agreement); in the subjective evaluations, the inter-observer comparisons showed values ranging from 0.20 to 0.56 (mean value 0.37, that is, 'fair'). In the decision tree classification, feature value thresholds were selected in order to have specificity of 100% and the predictive value of a positive result (carcinoma) of 100%; accuracy was 87% (range 86-89%), sensitivity 74% (71-79%), the predictive value of a negative result (adenoma) 79% (78-82%). In the subjective evaluation the values were as follows: accuracy 67% (64-71%), sensitivity 57% (50-64%), specificity 77% (71-79%), predictive value of a negative result (adenoma) 64% (61-69%), predictive value of a positive result (carcinoma) 71% (67-75%). The conclusion is that, by using a routine microscope equipped with a micrometer, the preoperative diagnosis of follicular carcinoma from smears can be formulated with a high degree of certainty.     

Alternative splicing regulates the nuclear or cytoplasmic localization of dystrophin Dp71

The subcellular distribution of Dp71 isoforms alternatively spliced for exon 71 and/or 78 was examined. The cDNA sequence of each variant was fused to the C-terminus of the green fluorescent protein and the constructs were transfected transiently in the cell lines HeLa, C2C12 and N1E-115. The subcellular distribution of the fused proteins was determined by confocal microscope analysis. The Dp71 isoform lacking the amino acids encoded by exons 71 and 78 was found exclusively in the cytoplasm whereas the variants containing the amino acids encoded by exon 71 and/or exon 78 show a predominant nuclear localization. The nuclear localization of Dp71 provides a new clue towards the establishment of its cellular function.     

Characterization of Dp71Δ(78-79), a novel dystrophin mutant that stimulates PC12 cell differentiation

Dp71 has an important role in the central nervous system. To better understand the function of Dp71 domains in neuronal differentiation, PC12 cells were stably transfected with a dystrophin mutant, Dp71Δ(78-79) , which lacks exons 78 and 79. Based on the percentage of cells bearing neurites and neurite length analyses, we found that cells stably expressing Dp71Δ(78-79) (PC12-C11) differentiate more efficiently than non-transfected cells. While wild-type cells reach their maximum differentiation 9-12 days after initiating the differentiation process, the PC12-C11 cells reach differentiation in 4-6 days. Protein expression analysis showed a down-regulation of Dp71a and an up-regulation of Dp71ab and/or Up71, β-dystroglycan and neuron-specific enolase in undifferentiated and in neural growth factor differentiated PC12-C11 cells. No change was observed in the expression of Grb2 and Up400. The subcellular localization of Dp71Δ(78-79) was in the cell periphery, and there was no change in localization during the differentiation process, which was also observed throughout the neurite extensions.     

Genomic cloning of a heat shock cognate 71-1 gene (HSC71-1) from the hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae).

The self-fertilizing fish Rivulus marmoratus (R. marmoratus) heat shock cognate 71 (HSC71) gene was cloned and characterized recently (Park et al., 2001). Here, we report the isolation of a homologue of the R. marmoratus HSC71 gene via screening of an R. marmoratus genomic DNA library. A 12,591 bp genomic fragment was sequenced and found to contain a 2844 bp open reading frame that consisted of 8 exons and showed high similarity to the previously reported R. marmoratus HSC71 gene. The two genes differed slightly at exons 5 and 8, and intron 3. On a deduced amino acid sequence level, the two R. marmoratus HSC71 genes were highly similar (89.3% in amino acid residues). In this paper, the author presented a homologous gene (R. marmoratus HSC71-1) similar to R. marmoratus HSC71 gene.     

Three-dimensional structure of murine anti-p-azophenylarsonate Fab 36-71. 2. Structural basis of hapten binding and idiotypy

Comparison between the structures and solvent-accessible surfaces of the antigen-binding fragments of two murine anti-p-azophenylarsonate monoclonal antibodies, one bearing a major cross-reactive idiotype of A/J strain mice (36-71) and one lacking the idiotype (R19.9; Lascombe et al., 1989), highlight the structural basis for the determination of hapten affinity and idiotypy. Since the sequence of R 19.9 is identical with the germline-encoded sequence at 16 positions in both heavy-chain and light-chain variable regions where somatic mutations and junctional differences have occurred to produce the 36-71 sequence, the structure of R 19.9 can be used to model the structure of the germline-encoded antibody (36-65) in the regions around these sites. These 16 sequence differences exclude the third heavy-chain complementarity-determining region because R 19.9 utilizes a D gene segment not associated with the predominant idiotype, which is 4 residues longer than the canonical D gene segment utilized in the sequences of 36-71 and 36-65. This difference between the structures of R 19.9 and 36-71 does not affect the validity of using the structure of R 19.9 to model the structure of 36-65 since the third heavy-chain complementarity-determining region is highly solvent-exposed in both 36-71 and R 19.9, and does not interact with any of these 16 sites. Comparing the structures of 36-71 and R 19.9 suggests that only three of the differences in the heavy-chain sequences, and three of the differences in the light-chain sequences of 36-71 and 36-65, increase the affinity for hapten.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of the a subunit of human factor XIII

Factor XIII is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The complete amino acid sequence of the a subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gtll cDNA library prepared from human placenta mRNA was screened with an affinity-purified antibody against the a subunit of human factor XIII and then with a synthetic oligonucleotide probe that coded for a portion of the amino acid sequence present in the activation peptide of the a subunit. Six positive clones were identified and shown to code for the a subunit of factor XIII by DNA sequence analysis. A total of 3831 base pairs was determined by sequencing six overlapping cDNA clones. This DNA sequence contains a 5' noncoding region or a region coding for a portion of a pro-piece or leader sequence, the mature protein (731 amino acids), a stop codon (TGA), a 3' noncoding region (1535 nucleotides), and a poly(A) tail (10 nucleotides). When the a subunit of human factor XIII was digested with cyanogen bromide, 11 peptides were isolated by gel filtration and reverse-phase HPLC. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 363 amino acid residues were identified. These amino acid sequences were in complete agreement with those predicted from the cDNA. The a subunit of factor XIII contained the active site sequence of Tyr-Gly-Gln-Cys-Trp, which is identical with that of tissue transglutaminase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane.     

Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments

Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII.     

Oxidation of 3-C-(2-amino-2-deoxy-D-glucopyranosyl)-1-propene compounds and the structure of 3-C-(2-amino-2-deoxy-D-glucopyranosyl)-1,2-propanediol derivatives for a synthesis of 2,3-didehydro-2,7-dideoxy-N-acetylneuraminic acid

Oxidation of 5-acetamido-4,8-anhydro-1,2,3,5-tetradeoxy-D-glycero-D-ido-non-1-enitol [3-C-(2-amino-2-deoxy-beta-D-glucopyranosyl)-1-propene] was studied to search for preparative routes to aminodeoxy didehydro nonulosonic acid derivatives. Since only moderate chiral induction was observed with osmium tetroxide dihydroxylation as well as with peracid epoxidation, the catalytic asymmetric dihydroxylation conditions were applied to give the stereocontrolled formation of 1,2-propanediol derivatives. The structures of these diastereoisomeric 1,2-propanediol derivatives were determined by X-ray crystallographic analyses. The formation of diastereoisomeric 1,2-propanediols also varied with the nature of 2-substituent on the aminodoexy glycosyl moiety. Thus 5-acetamido-4,8-anhydro-3,5-dideoxy-D-erythro-L-ido-nonitol [(2S)-3-C-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-1,2-propanediol] was obtained predominantly up to 70% from 3-C-(2-acetamido-2-deoxyglycosyl)-1-propene by the use of ADmixbeta reagent. The (2S)-propanediol derivative was transformed in a five-step reaction sequence to 2,3-didehydro-2,7-dideoxy-N-acetylneuraminic acid.     

Identification of a human SCARB2 region that is important for enterovirus 71 binding and infection

We previously identified human scavenger receptor class B, member 2 (SCARB2), as a cellular receptor for enterovirus 71 (EV71). Expression of human SCARB2 (hSCARB2) permitted mouse L929 cells to efficiently bind to virions and to produce both viral proteins and progeny viruses upon EV71 infection. Mouse Scarb2 (mScarb2) exhibited 85.8% amino acid identity and 99.9% similarity to hSCARB2. The expression of mScarb2 in L929 cells conferred partial susceptibility. Very few virions bound to mScarb2-expressing cells. The viral titer in L929 cells expressing mScarb2 was approximately 40- to 100-fold lower than that in L929 cells expressing hSCARB2. Using hSCARB2-mScarb2 chimeric mutants, we attempted to map the region that was important for efficient EV71 infection. L929 cells expressing chimeras that carried amino acids 142 to 204 from the human sequence were susceptible to EV71, while chimeras that carried the mouse sequence in this region were not. Moreover, this region was also critical for binding to virions. The determination of this region in hSCARB2 that is important for EV71 binding and infection greatly contributes to the understanding of virus-receptor interactions. Further studies will clarify the early steps of EV71 infection.     

An 86Y-labeled mirror-image oligonucleotide: influence of Y-DOTA isomers on the biodistribution in rats

A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.