A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System

To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained.     

Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.     

Chemotaxis away from thiocyanic and isothiocyanic esters in Pseudomonas aeruginosa

Abstract A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae.     

Lysozyme as a regulator of interleukin-2-activated lymphocyte proliferation

Summary Lysozyme at 1 to 100μg/ml of exposure levels augmented or inhibited proliferative response of human peripheral blood lymphocytes stimulated with interleukin-2 (IL-2). This contradictory effect of lysozyme depended on IL-2 concentration, activating state of lymphocytes, addition time of lysozyme, and serum existence. Lymphocytes increased their IL-2-mediated proliferating ability in response to lysozyme when stimulated with less than suboptimal concentration of IL-2. Lymphocyte activation with anti-CD3 antibody changed the augmented proliferative response into the inhibited response by lysozyme addition whereas elimination of MHC class II molecule-expressing cells augmented the response. Addition of lysozyme within 1 h after IL-2 exposure was most effective in promoting the proliferation whereas additions after 16 to 24 h were ineffective or inhibitory. Addition after longer than 24 h inversely restored the proliferative response. Serum seemed to retard lysozyme action because either sequential serum addition 1 h after exposure of IL-2 and lysozyme to cells or exposure of IL-2 and serum after pretreatment of cells with lysozyme changed the proliferative responsiveness from inhibition into augmentation. Thus lysozyme may regulate lymphocyte proliferation responding to a magnitude of antigenic stimuli and to the progression of cellular events that periodically occur.     

Arrest of cell cycle progression of HeLa cells in the early G1 phase in K+-depleted conditions and its recovery upon addition of insulin and LDL

Cell cycle progression of synchronized HeLa cells was studied by measuring labeling of the nuclei with [³H]thymidine. The progression was arrested in a chemically defined medium in which K⁺ was replaced by Rb⁺ (Rb-CDM) but was restored upon addition of insulin and/or low density lipoprotein (LDL). Cells started DNA synthesis 12 hr after addition of insulin and/or LDL, regardless of the time of arrest, suggesting their arrest early in the G1 phase. After incubation of cells in Rb-CDM containing insulin or LDL singly for 3, 6, or 9 hr, replacement of the medium by that without an addition resulted in marked delay in entry of cells into the S phase, but in its replacement by medium containing both agents, the delay was insignificant. Synthesis of bulk protein, estimated as increase in the cell volume, was not strongly inhibited. From these results we conclude that cell cycle progression of HeLa cells in K^?-depleted CDM is arrested early in the G1 phase and that the arrest is due to lack of some protein(s) required for entry into the S phase that is synthesized in the early G1 phase.     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

High-performance liquid chromatographic determination of hippuric acid in human urine

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean-up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.     

A possible clinical application of multicytokine-producing cytotoxic mononuclear cell (MCCM) therapy

When peripheral blood mononuclear cells (PBMC) were incubated with a streptococcal preparation, OK-432, for 24 h, PBMC acquired cytolytic activity against cultured and fresh human tumor cells. Such PBMC were called OK-432-activated mononuclear cells (OK-MC). OK-MC produce several kinds of cytokines such as interferon (IFN), IFN, and tumor growth inhibitory factor (TGIF) bothin vitro andin vivo. OK-MC-produced cytokines also inhibited the growth of cultured and fresh human tumor cells. The growth inhibition was examined by human tumor clonogenic assay using a double-layer agar technique. The results indicate that two pathways of anti-tumor activity are induced in OK-MC, i.e., cell-mediated and cytokine-mediated.     

Seasonal and areal features of the lagoonal environment in Lake Nakanoumi,a shallow coastal lagoon in Japan

Lake Nakanoumi is a shallow coastal lagoon connected with the Japan Sea by a narrow channel. Over the past decade, land reclamation resulted in a 33% reduction of the lagoon's surface area. The remaining water basin of Lake Nakanoumi is scheduled to be artificially freshened to supply irrigation water for the newly reclaimed lands. This paper deals with the seasonal and areal features of the lagoonal environment prior to the beginning of the artificial desalinization.