Determination of the number of active GroES subunits in the fused heptamer GroES required for interactions with GroEL

A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.     

Mg2+-, Ca2+-dependent unwinding of DNA by poly-L-glutamic acid

In order to examine a possibility that the high acidic amino acid region in the nonhistone protein HMG(1+2) is concerned with the Mg2+-, or Ca2+-dependent unwinding of DNA by the HMG(1+2) (1,2), poly-L-glutamic acid was employed as an acidic model peptide for thermal melting temperature analysis. The poly-L-glutamic acid bound to DNA either in the presence or absence of Mg2+. The poly-L-glutamic acid unwound DNA double-helix to a similar extent to HMG(1+2) in the presence of Mg2+ or Ca2+, but not in the absence of them. These results may suggest that the high acidic amino acid region in HMG(1+2) participates in Mg2+-, Ca2+-dependent unwinding of DNA double-helix.     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.     

The Om(1e) Mutation in Drosophila Ananassae Causes Compound Eye Overgrowth Due to Tom Retrotransposon-Driven Overexpression of a Novel Gene

Optic morphology (Om) mutations in Drosophila ananassae are a group of retrotransposon (tom)-induced gain-of-function mutations that map to at least 22 independent loci and exclusively affect the compound eye morphology. In marked contrast to other Om mutations, which are characterized by fewer-than-normal and disorganized ommatidia, the Om(1E) mutation exhibits a peculiar phenotype as enlarged eyes with regularly arrayed normal ommatidia. To characterize the Om(1E) mutation, we have carried out molecular analyses. A putative Om(1E) locus cloned by tom tagging and chromosome walking contained two transcribed regions in the vicinity of tom insertion sites of the Om(1E) mutant alleles, and one of these regions was shown to be the Om(1E) gene by P element-mediated transformation experiments with D. melanogaster. The Om(1E) gene encodes a novel protein having potential transmembrane domain(s). In situ hybridization analyses demonstrated that the Om(1E) gene is expressed ubiquitously in embryonic cells, imaginal discs, and the cortex of the central nervous system of third instar larvae, and specifically in lamina precursor cells. Artificially induced ubiquitous overexpression of Om(1E) affected morphogenesis of wing imaginal disc derivatives or large bristle formation. These findings suggest that the Om(1E) gene is involved in a variety of developmental processes.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-α, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-α mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-α protein by DLD-1 and CoLo320DM cells was confirmed by TGF-α specific monoclonal antibody binding assay. The spontaneous³H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-α monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-α produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

Construction of New Shuttle Vectors for Acetbacter

Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0 kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.     

Blood vascular architecture of the rat lingual papillae with special reference to their relations to the connective tissue papillae and surface structures: a light and scanning electron microscope study

Subepithelial blood vessels of the rat lingual papillae and their spatial relations to the connective tissue papillae and surface structures were demonstrated by light and scanning electron microscopy. In the rat, four types of papillae were distinguished on the dorsal surface of the tongue, i.e. the filiform, fungiform, foliate and circumvallate papillae. Vascular beds of various appearance were found in all four types of lingual papillae: a simple or twisted capillary loop in the filiform papilla; a basket- or petal-like network in the fungiform papilla; a ring-like network in the foliate papilla, and a conglomerated network surrounded by double heart-shaped capillary networks in the circumvallate papilla. These characteristic vascular beds corresponded to the shape of the connective tissue papillae and surface structures. The vascular bed beneath the gustatory epithelium in the fungiform, foliate and circumvallate papilla consisted of fine capillary networks next to the taste buds.     

Discriminant analysis of hematological and serum biochemical values in cynomolgus monkey (Macaca fascicularis) bred and reared under the indoor individually-caged conditions

The data on hematological and serum biochemical properties of laboratory-bred cynomolgus monkeys (Macaca fascicularis) at different ages were analyzed by discriminant analysis. All the animals had been bred and reared under uniform environmental conditions at Tsukuba Primate Center for Medical Science, N.I.H., Japan. The items used were as follows: red blood cell count (RBC), hematocrit value (Ht), hemoglobin concentration (Hb), mean corpuscular volume (MCV), white blood cell count (WBC), glutamic oxaloacetic transaminase activity (GOT), glutamic pyruvic transaminase activity (GPT), total protein concentration (TP), albumin concentration (ALB), albumin-globulin ratio (A/G), blood urea nitrogen (BUN), glucose concentration (GLU), total cholesterol concentration (TCHO), free cholesterol concentration (FCHO), triglyceride concentration (TG) and alkaline phosphatase activity (ALP). In total, 1086 animals in 10 age groups were examined. Data analyses were done with respect to the difference of sex. Discrimination was possible by Mahalanobis' generalized distance between centroids of groups. In canonical discriminant analysis (discriminant analysis with reduction of dimensionality), age was highly correlated to the value of the first canonical variate. From the approximate relative value of the eigenvector of the first canonical variate, the most discriminant variables are WBC, TP, ALB, A/G, TCHO, FCHO, TG, and ALP. It can be concluded that periodic measurement of these 8 parameters is necessary and sufficient to monitor the physiological conditions of growing monkeys.