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Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif psiKXE/D, where psi represents a large hydrophobic amino acid, and X represents any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process.

Using the technique of integrative mapping with three vectors carrying chromosomal rDNA sequences, one of two rRNA operons of loofah witches' broom (LfWB) phytoplasma was constructed. This is the first complete rRNA operon of a phytoplasma to be reported. The operon has a context of 5'-16S-23S-5S-3' with a tRNA(Ile) gene in the ITS and tRNA(Val) and tRNA(Asn) genes downstream from the 5S rRNA gene. Although the other operon has not been cloned, the DNA sequence of a PCR-amplified product shows that it has no tRNA(Ile) gene in the ITS region. The complete nucleotide sequences of 16S, 23S, and 5S rDNA are 1538, 2864, and 113 bp, respectively. Five -10-like sequences, but no -35 sequences, were found within a 494-bp leader region. There was a TG dinucleotide two nucleotides upstream from each -10-like sequence. The existence of a TG dinucleotide at this position has been reported to enhance the efficiency of a promoter without a -35 region. The regions immediately flanking the 5' and 3' ends of 16S and 23S rDNA can form long basepaired stems that contain sites for processing by RNase III. No obvious sequence for a rho-dependent or rho-independent termination site was found downstream from the tRNA(Asn) gene. The transcription may stop within a pyrimidine-rich region, as has been reported for several polypeptide-encoding genes and rRNA operons of archaeobacteria. The presence of the tRNA genes downstream from the 5S rRNA gene in the rRNA operon of LfWB phytoplasma further supports the hypothesis that phytoplasmas are phylogenetically closer to acholeplasmas than to mycoplasmas. The phylogenetic relatedness of LfWB phytoplasma to other phytoplasmas is discussed on the basis of the nucleotide sequence of rRNA genes and ITS.

The recombinant clone expressing a 60 kDa (P60) antigen was isolated from Escherichia coli by screening a lambda EMBL3 genomic library using rabbit produced antiserum against Mycoplasma hyopneumoniae. Sequence analysis revealed that an interrupted (by a UGA codon) open reading frame coding for a 72 kDa protein (P72) may contain the P60 antigen gene. Western blot analysis with an anti-P60 monospecific antibody confirmed the presence of a P72 antigen from the total protein of M. hyopneumoniae, and a 72 kDa protein was also expressed in E. coli after changing the codon (UGA to UGG) by site-directed mutagenesis. BLAST (Basic Local Alignment Search Tool) comparison showed that the amino acid sequences of P72 share approximately 70% homology with the phosphotransferase enzyme I (PTSI) of bacteria and other mycoplasma species. The biological function of the P72 cytosolic protein was further confirmed by complementation using an E. coli ptsI mutant. The bacterial phosphoenolpyruvate-sugar phosphotransferase system (PTS) is known to mediate the uptake and phosphorylation of carbohydrates and to be involved in signal transduction. The immune responses of specific pathogen free (SPF) pigs and farm animals toward this unique antigen were observed. The transcription start positions of the PTSI gene were determined in M. hyopneumoniae and E. coli by primer extension experiments and the promoter site was also predicted.

The recombinant clone expressing the 42 kDa protein (P42) of Mycoplasma hyopneumoniae in Escherichia coli was analyzed. The 4.4 kb HindIII-Xmal DNA fragment expressing the p42 gene product encodes three ORFs: p42 and p16 in the forwarding strand, p24 in the reverse strand. Sequence comparisons revealed that p42 could be part of a p65 gene, and has 62% identities with Mycoplasma genitalium HSP70 gene and 56% identities with Bacillus subtilis dnaK gene; p16 and p24 genes share 73% and 47% identities with Erysipelothrix rhusiopathiae dnaJ gene and Pseudomonas fluorescens uvrC gene, respectively. Further analysis demonstrated that P42 is indeed a heat shock protein and the monospecific antibodies against P42 can block the growth of Mycoplasma hyopneumoniae.

There were very few cases of mushroom poisoning every year in Taiwan and yet no fatal incident have been reported. A species named Amanita phalloides had high lethality rate (22-33%). Physicians should be aware of it's toxic property and be able to treat the patient in case of mushroom poisoning. However, mycologist sometimes might not distinguish edible from toxic mushroom so that educating people not to ingest wild mushroom is very important. We present 4 children with gastrointestinal syndrome after ingesting wild mushroom. We also introduce some basic approaches in treating mushroom poisoning in this paper.

We collected 1984 to 1993's health statistics to calculate the incidence and trend of SIDS in Taiwan. Deaths of SIDS were listed in the International Classification of Diseases (ICD) code 798.0. All infants under 1 year of age died of SIDS were included in our study. We found Taiwan was a low incidence area of SIDS. The incidences of SIDS from 1984 to 1993 were increased from 0.25 to 0.56 per 1000 live births. We also found the SIDS had inclining trend from 1984 to 1993. Whether the reason of inclining trend of SIDS in Taiwan is due to westernization of living style needs further exploration.