Kluyveromyces lactis sexual pheromones. Gene structures and cellular responses to alpha-factor

The Kluyveromyces lactis genes for sexual pheromones have been analyzed. The alpha-factor gene encodes a predicted polypeptide of 187 amino acid residues containing four tridecapeptide repeats (WSWITLRPGQPIF). A nucleotide blast search of the entire K. lactis genome sequence allowed the identification of the nonannotated putative a-pheromone gene that encodes a predicted protein of 33 residues containing one copy of the dodecapeptide a-factor (WIIPGFVWVPQC). The role of the K. lactis structural genes KlMFalpha1 and KlMFA1 in mating has been investigated by the construction of disruption mutations that totally eliminate gene functions. Mutants of both alleles showed sex-dependent sterility, indicating that these are single-copy genes and essential for mating. MATalpha, Klsst2 mutants, which, by analogy to Saccharomyces cerevisiae, are defective in Galpha-GTPase activity, showed increased sensitivity to synthetic alpha-factor and increased capacity to mate. Additionally, Klbar1 mutants (putatively defective in alpha-pheromone proteolysis) showed delay in mating but sensitivity to alpha-pheromone. From these results, it can be deduced that the K. lactis MATa cell produces the homolog of the S. cerevisiaealpha-pheromone, whereas the MATalpha cell produces the a-pheromone.     

Increased runs of homozygosity in the autosomal genome of Brazilian individuals with neurodevelopmental delay/intellectual disability and/or multiple congenital anomalies investigated by chromosomal microarray analysis

Runs of homozygosity (ROH) in the human genome may be clinically relevant. The aim of this study was to report the frequency of increased ROH of the autosomal genome in individuals with neurodevelopmental delay/intellectual disability and/or multiple congenital anomalies, and to compare these data with a control group. Data consisted of calls of homozygosity from 265 patients and 289 controls. In total, 7.2% (19/265) of the patients showed multiple ROH exceeding 1% of autosomal genome, compared to 1.4% (4/289) in the control group (p=0.0006). Homozygosity ranged from 1.38% to 22.12% among patients, and from 1.53 to 2.40% in the control group. In turn, 1.9% (5/265) of patients presented ROH ≥10Mb in a single chromosome, compared to 0.3% (1/289) of individuals from the control group (p=0.0801). By excluding cases with reported consanguineous parents (15/24), the frequency of increased ROH was 3.4% (9/250) among patients and 1.7% (5/289) in the control group, considering multiple ROH exceeding 1% of the autosome genome and ROH ≥10Mb in a single chromosome together, although not statistically significant (p=0.1873). These results reinforce the importance of investigating ROH, which with complementary diagnostic tests can improve the diagnostic yield for patients with such conditions.     

Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The k_cat/K_m values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.     

Identification of active regions for neurite outgrowth activity of neurocrescin

We previously identified and cloned a neurite outgrowth promoting protein, Neurocrescin (NC), from the extract of the chick denervated leg muscles. In this study, we explored the active region of NC for neurite outgrowth. Using the deletion mutants of NC, we tested their neurite outgrowth activity in the cultured telencephalic neurons of E5 chick embryos. We found three regions which independently had significant neurite outgrowth activity comparable with that of the extract of the chick denervated leg muscles. These regions were not homologous to any well-known active sites such as the laminin active region, IKVAV. In parallel, searching the endogenous deletion mutants of NC in the rat brain, we cloned a mutant in which the region including the larger part of one of the three active regions was deleted. The neurite outgrowth activity of the mutant was significantly lower than that of normal NC. These results suggest the physiological significance of these active regions.     

Isolation and characterization of the Schizosaccharomyces pombe cDNA encoding the mitochondrial endonuclease(1)

We isolated the cDNA of the fission yeast mitochondrial endonuclease SpNUC1, which consists of 322 amino acids and has a significant homology with the budding yeast NUC1 and mammalian endonuclease G. Comparison of the cDNA sequence with the genomic sequence showed that the gene consists of three exons and two introns and spans 1.31 kb. The enzyme localization in mitochondria was demonstrated by expressing the SpNUC1-green fluorescent protein fusion in the yeast. The endonuclease was activated by truncation of the amino-terminal region of the protein, indicating that the enzyme is encoded as an inactive precursor. The active enzyme degraded single-stranded DNA and RNA, the activity being dependent on Mg(2+) (Mn(2+)).     

Cutting edge: Gln22 of mouse MD-2 is essential for species-specific lipopolysaccharide mimetic action of taxol

MD-2 associates with the extracellular domain of Toll-like receptor 4 (TLR4) and greatly enhances LPS signaling via TLR4. Taxol, which mimics the action of LPS on murine macrophages, induces signals via mouse TLR4-MD-2, but not via human TLR4-MD-2. Here we investigated the molecular basis for this species-specific action of Taxol. Expression of mouse MD-2 conferred both LPS and Taxol responsiveness on human embryonic kidney 293 cells expressing mouse TLR4, whereas expression of human MD-2 conferred LPS responsiveness alone, suggesting that MD-2 is responsible for the species-specificity as to Taxol responsiveness. Furthermore, mouse MD-2 mutants, in which Gln(22) was changed to other amino acids, showed dramatically reduced ability to confer Taxol responsiveness, although their ability to confer LPS responsiveness was not affected. These results indicated that Gln(22) of mouse MD-2 is essential for Taxol signaling but not for LPS signaling.     

Intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice

Thymus- and activation-regulated chemokine (TARC; CCL17) is a lymphocyte-directed CC chemokine that specifically chemoattracts CC chemokine receptor 4-positive (CCR4(+)) Th2 cells. To establish the pathophysiological roles of TARC in vivo, we investigated here whether an mAb against TARC could inhibit the induction of asthmatic reaction in mice elicited by OVA. TARC was constitutively expressed in the lung and was up-regulated in allergic inflammation. The specific Ab against TARC attenuated OVA-induced airway eosinophilia and diminished the degree of airway hyperresponsiveness with a concomitant decrease in Th2 cytokine levels. Our results for the first time indicate that TARC is a pivotal chemokine for the development of Th2-dominated experimental allergen-induced asthma with eosinophilia and AHR. This study also represents the first success in controlling Th2 cytokine production in vivo by targeting a chemokine.     

Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels

The effects of hyaluronic acid (HA) on the proliferation and chondroitin sulfate (CS) synthesis of chondrocytes embedded in collagen gels were examined. Articular cartilage was isolated from the humerus, femur, and tibia of 21 10-week-old Japanese white rabbits. Chondrocytes isolated by collagenase digestion were embedded in type I collagen gels and cultured in Dulbecco's modified Eagle's medium (DMEM) with various doses of HA for 4 weeks. Histological and biochemical evaluations were performed at postculture weeks 1, 2, 3, and 4. For biochemical evaluations, isomers such as chondroitin 6-sulfate (delta(di)-6S) and chondroitin 4-sulfate (delta(di)-4S) synthesized by cultured chondrocytes were determined by high performance liquid chromatography (HPLC) combined with fluorometry. Morphological and histological studies demonstrated that HA-treated chondrocytes in collagen gel proliferated profusely while maintaining their phenotype. At postculture week 4, 0.1 mg/ml of HA induced an eightfold increase in cell counts compared with HA pretreatment values, or 1.5-fold more than control group. Synthesis of delta(di)-6S (delta(di)-6S content/cell) in groups treated with 0.01 and 0.1 mg/ml of HA significantly increased, while gel accumulation rates in groups treated with 0.1 and 1.0 mg/ml of HA scored significantly higher values than other groups. In collagen gel culture, HA enhanced the proliferation and delta(di)-6S synthesis of chondrocytes while maintaining their phenotype. In clinical application, since the supply of autologous chondrocytes for transplantation is not unlimited, the HA-treated culture method may be useful for increasing the number of chondrocytes and thus improving the quality of implants.     

Molecular cloning and expression of a mouse thiamin pyrophosphokinase cDNA

Thiamin pyrophosphokinase (EC 2.7.6.2) catalyzes the pyrophosphorylation of thiamin with adenosine 5'-triphosphate to form thiamin pyrophosphate. A mouse thiamin pyrophosphokinase cDNA clone (mTPK1) was isolated using a combination of mouse expressed sequence tag database analysis, a two-step polymerase chain reaction procedure, and functional complementation screening with a Saccharomyces cerevisiae thiamin pyrophosphokinase-deficient mutant (thi80). The predicted protein contained 243 amino acid residues with a calculated molecular weight of 27,068. When the intact mTPK1 open reading frame was expressed as a glutathione S-transferase fusion protein in Escherichia coli lacking thiamin pyrophosphokinase, marked enzyme activity was detected in the bacterial cells. The corresponding 2.5-kilobase pair mRNA was expressed in a tissue-dependent manner and was found at relatively high levels in the kidney and liver, indicating that the mode of expression of mTPK1 genes differs with cell type. The expression of mTPK1 genes in cultured mouse neuroblastoma and normal liver cells was unaffected by the thiamin concentration in the medium (10 microM versus 3.0 nM). This is the first report on identification of the primary sequence for mammalian thiamin pyrophosphokinase.