Viomycin resistance: alterations of either ribosomal subunit affect the binding of the antibiotic to the pair subunit and the entire ribosome becomes resistant to the drug.

Subcellular localization of $\text{[}{}^3\text{H]1α,24(R)-dihydroxyvitamin}$ D₃ and $\text{[}{}^3\text{H]1α,24(S)-dihydroxyvitamin}$ D₃ in rat intestinal mucosa was investigated in comparison with the $\text{[}{}^3\text{H]1α-hydroxyvitamin}$ D₃. The 24(R) and 24(S) isomers of 1α,24-dihydroxyvitamin D₃ were gradually transformed to 1α,24(R)25-trihydroxyvitamin D₃ and 1α,24(S)25-trihydroxyvitamin D₃, and the plasma concentrations of these metabolites were 10.30 and 1.36 pmol/ml, respectively. The major portions of the administered compounds distributed in the nuclear fraction of the intestinal mucosa remained unchanged, and the amounts of 1α,24(R)-dihydroxyvitamin D₃ and 1α,24(S)-dihydroxyvitamin D₃ were 4.25 and 0.306 pmol/g intestinal mucosa, respectively. No detectable amount of the metabolites, 1α,24(R)25-trihydroxyvitamin D₃ and 1α,24(S)25-trihydroxyvitamin D₃ were found in the same nuclear fractions. In the case with the $\text{[}{}^3\text{H]1α-hydroxyvitamin}$ D₃, however, the compound was rapidly metabolized to 1α,25-dihydroxyvitamin D₃.The metabolite, 1α,25-dihydroxyvitamin D₃, was seen in the nuclear fraction of the intestinal mucosa at a concentration of 2.44 pmol/g intestinal mucosa.     

Molecular cloning and sequencing of the cDNA of rat alpha 1-protease inhibitor and its expression in COS-1 cells

A cDNA clone for alpha 1-protease inhibitor (pc alpha 1P1212) was isolated from a lambda ZAP rat liver cDNA library. The 1.4 kb cDNA insert of pc alpha 1P1212 contained an open reading frame that encodes a 411-residue polypeptide (46,125 Da), in which a signal peptide of 24 residues was identified by comparison with the NH2-terminal sequence of the purified protein. Three potential sites for N-linked glycosylation were found in the molecule, accounting for the difference in molecular mass between the predicted form and the purified protein (56 kDa). The deduced primary structure of rat alpha 1-protease inhibitor showed 68.5% homology to that of the human inhibitor. We then constructed the expression plasmid pSV2 alpha 1PI from pSV2-gpt and pc alpha 1P1212, and transfected it into COS-1 cells. The transfected cells synthesized a molecule which was precipitated with anti-(rat alpha 1-protease inhibitor)-IgG and had the same molecular size as that of the inhibitor produced by rat hepatocytes.     

Cloning of the yeast SFL2 gene: its disruption results in pleiotropic phenotypes characteristic for tup1 mutants

We have identified a yeast gene, SFL2 (suppressor gene for flocculation), which complemented a newly isolated sfl2 mutant. This mutation causes asexual cell aggregation. The strain bearing the SFL2 gene disruption exhibited pleiotropic phenotypes characteristic for tup1 mutants. Physical mapping and complementation analysis suggested that the cloned SFL2 gene is identical to the TUP1 gene. The SFL2 gene encodes a 669-amino acid protein which has domains rich in glutamine, as does the SSN6 protein.     

Intracellular processing of complement pro-C3 and proalbumin is inhibited by rat alpha 1-protease inhibitor variant (Met352----Arg) in transfected cells

Complement C3, when its cDNA was transfected into COS-1 cells, was synthesized as a precursor, pro-C3, which was intracellularly processed into the alpha and beta subunits, although not completely. A cDNA for rat alpha 1-protease inhibitor (alpha 1-PI) was mutated in vitro to encode its variant with the modified active site (Met352----Arg). In cells co-transfected with the mutant alpha 1-PI cDNA and the C3 cDNA, pro-C3 expressed was secreted without being processed into the subunits. Co-transfection of the mutant alpha 1-PI cDNA and the albumin cDNA also resulted in the inhibition of intracellular conversion of proalbumin into serum-type albumin. No inhibition of the processing of each preform was observed in cells co-transfected with the normal alpha 1-PI cDNA. Taken together, the results indicate that the alpha 1-PI variant (Met352----Arg) expressed inhibits specifically an intracellular enzyme which is involved in the proteolytic processing of both pro-C3 and proalbumin.     

Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface.

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).     

Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406. Structural importance of the crown domain

Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP.     

Triple immunofluorescent labeling of FtsZ, dynamin, and EF-Tu reveals a loose association between the inner and outer membrane mitochondrial division machinery in the red alga Cyanidioschyzon merolae.

In the mitochondria of primitive eukaryotes, FtsZ and dynamin are part of the machinery involved in division of the inner and outer membranes, respectively. These genes also commonly function in the same manner during chloroplast division. In this study, a relationship between the localization of the inner and outer division machinery was directly shown for the first time. Triple immunofluorescent labeling was performed in the red alga Cyanidioschyzon merolae by a device using narrow bandpass filter sets and bright photostable dyes. FtsZ (CmFtsZ1) and dynamin (CmDnm1) localizations were examined simultaneously throughout the mitochondrial division cycle with an alternative mitochondrial marker protein, the mitochondrial translation elongation factor EF-Tu, whose localization was also shown to be identical to the mitochondrial matrix. FtsZ and dynamin did not necessarily co-localize when both were recruited to the mitochondrial constriction site, indicating that inner and outer dividing machineries are not in tight association during the late stage of division.     

Structural requirements for the stability of novel cephalosporins to AmpC beta-lactamase based on 3D-structure

AmpC beta-lactamase is one of the leading causes of Pseudomonas aeruginosa (P. aeruginosa) resistance to cephalosporins. FR259647 is a cephalosporin having a novel pyrazolium substituent at the 3-position and exhibits excellent activity (MIC=1 microg/mL) against the AmpC beta-lactamase overproducing P. aeruginosa FP1380 strain in comparison with the third-generation cephalosporins FK518 [Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21-24, 1990, Abs. 454; Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21-24, 1990, Abs. 455; Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21-24, 1990, Abs. 456; Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21-24, 1990, Abs. 457] (MIC=16 microg/mL) and ceftazidime (CAZ) (MIC=128 microg/mL). The stability of FR259647 and FK518 to AmpC beta-lactamase was evaluated using MIC assays against both the P. aeruginosa PAO1 strain and a PAO1 mutant strain overproducing AmpC beta-lactamase as a differential assay, which indicates that the main difference derives from their stability to AmpC beta-lactamase. A structural analysis using computer simulations indicated that the difference in stability may be due to steric hindrance of the 3-position substituents causing differential affinity. This steric hindrance may disturb entry of the cephalosporins into the binding pocket. We predicted the possibility of inhibition of entry as a potential means of enhancing stability by conformational analysis. In order to validate this speculation, novel FR259647 derivatives 4-9 were designed, calculated, synthesized, and evaluated. As a result, we demonstrated that their probability of entry correlated with the MIC ratio of the mutant strain to the parent strain and supports the validity of our model.     

Nonhuman primate intestinal villous M-like cells: an effective poliovirus entry site

Humans and some Old World monkeys, chimpanzees, and cynomolgus macaques, are susceptible to oral poliovirus (PV) infection. Interestingly, rhesus macaques, although sensitive to injected PV, are not susceptible to gut infection. Not much is known about the initial event of gut infection by PV in rhesus macaques so far. Here, we show that PV can efficiently enter the lamina propria (LP) by penetrating across intestinal villous M-like cells in rhesus macaques. We found by immunofluorescence analysis that PV effectively invades LP rather than germinal centers (GCs) in rhesus macaques despite expressing PV receptor CD155 on cells within GCs and LP. Furthermore, energy dispersive X-ray spectroscopy demonstrated that gold-labeled PV is spatiotemporally internalized into villous M-like cells and engulfed by macrophage-like cells in LP. These results suggest that rhesus macaques may be resistant to productive gut PV infection owing to a defective translocation of PV to GCs.