Lactococcus strains treated with heat and hen-egg-white lysozyme induce abundant interleukin-12 production by J774.1 macrophages and murine spleen cells.

The IL-12-inducing ability of lactic acid bacteria could be a critical index of immunomodulatory activity, especially in promoting T-helper-1 responses and in suppressing T-helper-2-mediated allergic responses. We aimed to develop a simple method for enhancing the IL-12-inducing ability of bacteria. We examined the in vitro effects of strains of lysozyme-modified Lactococcus (ML-LYS), prepared by heat treatment of the Lactococcus strain in the presence of lysozyme, on the ability of mouse macrophage-like J774.1 cells and spleen cells to produce IL-12. An IL-12-inducing ability greater than that of heat-killed bacteria was shown by 41 of 46 ML-LYS strains in J774.1 cells and by all 46 ML-LYS strains in mouse spleen cells. In contrast, bacteria modified by α-lactalbumin, ß-lactoglobulin, or ovalbumin did not enhance IL-12 production in J774.1 cells. Microscopically, ML-LYS showed stronger resistance to lysozyme and macrophage digestion than did heat-killed bacteria or the other modified bacteria. Addition of chitotriose, a lysozyme inhibitor, enhanced IL-12 production by J774.1 cells stimulated with heat-killed bacteria. Therefore, enhancement of resistance to lysozyme may be a key factor in the strong IL-12-inducing ability of ML-LYS. These findings have important implications for the design of dairy products that have an immunomodulatory effect using the modified bacteria.

Heavy chain variable (VH) region diversity generated by VH gene replacement in the progeny of a single precursor cell transformed with a temperature-sensitive mutant of Abelson murine leukemia virus.

Sequence analysis of a large number of DNA clones containing a functional heavy chain variable, diversity, and joining (VHDJH) complex generated by VH to VHDJH joining (VH gene replacement) in the progeny derived from a common precursor cell transformed with a temperature-sensitive (ts) Abelson murine leukemia virus (A-MuLV) indicates that endogenous VH gene replacement in vitro generates immunoglobulin gene joints distinct from those generated by the usual VH to DJH joining. Such joints keep the pentamer CAAGA at the 3' end of the donor VH segment and lack a recognizable D segment, as can be seen also in vivo. The results suggest that VH gene replacement participates in generating VH region diversity in vivo, as previously postulated. During the joining process, a unique VH gene was selected in all progeny cells, together with a single A nucleotide dominantly added to the junctional boundaries. The basis of these regulatory processes is discussed.

Studies on electrotransfer of DNA into Escherichia coli: effect of molecular form of DNA.

Effects of several molecular forms of DNA were examined on voltage-pulse-mediated transfection or transformation. Among circular DNAs, the single-stranded microvirid DNA was less infective than the double-stranded replicative form, whereas transfectivity of the relaxed or nicked molecule was nearly equal to or slightly lower than that of the supercoiled DNA. The linearized double-stranded DNA derived from phage or plasmid electrotransfects Escherichia coli, albeit at a reduced efficiency. Alkaline denaturation of the linearized DNA resulted in complete loss of the infectivity. Relationship between terminal structure of the linearized DNA and efficiency of the transfection was investigated. Host recombination function did not significantly affect the infectivity of the linearized DNA.

Heterogeneous pathways of Ca2+ metabolism in the triggering of the proliferative process in rat thymocytes.

Concanavalin A induces in rat thymocytes a calcium uptake at 1 h exposure and proliferative response after 24 h exposure. Phosphodiesterase activity parallels the proliferative response (thymidine uptake). Valinomycin, monensin and a small dose of ouabain also induce calcium uptake, but do not lead to thymidine uptake later. The latter treatments reduce, in some instances drastically, the concanavalin A response with respect to thymidine uptake. Trifluoperazine reduces the unstimulated thymidine uptake and the concanavalin A induced thymidine uptake. These results suggest that calcium has a decisive role in inducing proliferation but that some ways of increasing cellular Ca2+ concentration interfere in other steps with the DNA synthesis.

Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M.

A Staphylococcus warneri strain M, newly isolated from processed seafood (smoked Watasenia scintillans), produced an extracellular protease. The protease, designated to as m-PROM (the mature form of PROM), selectively cleaved the carbonyl side of glutamic acid residues in beta-casein. Sequence of N-terminal 27 amino acids of m-PROM, RANVILPNNDRHQINDTTLGHYAPVTF, was found to be similar to those of other glutamyl endopeptidases, V8 protease (Staphylococcus aureus strain V8) and SPase (S. aureus ATCC 12600). To determine the complete primary structure and precursor of PROM, its gene (proM) was cloned and sequenced. The gene proM was found to encode for a protein of 316 amino acids. The amino acid residues from 64 to 90 completely coincided with the N-terminal 27 amino acids of the m-PROM, suggesting that the N-terminal 63 amino acids region of p-PROM (the precursor form of PROM) might be processed posttranslationally. Moreover, the whole amino acid sequence deduced from the primary structure of proM shows significant similarity to those of other glutamyl endopeptidases, V8 protease and SPase. These results suggested that PROM belongs to the glutamyl endopeptidase class. PROM, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.

Loss of the 3p25.3 band is critical in the manifestation of del(3p) syndrome: karyotype-phenotype correlation in cases with deficiency of the distal portion of the short arm of chromosome 3.

Two patients with monosomy for the distal portion of the short arm of chromosome 3 are described. Chromosome analysis on prometaphase cells demonstrated a karyotype of 46,XX,del(3) (p25.3) in one patient and 46,XX,r(3)(p26.1q29) in the other. The former patient showed characteristic clinical manifestations of the 3p- syndrome, including growth failure, mental retardation, microcephaly with a flat occiput, triangular face, synophrys, blepharoptosis, hypertelorism, broad and flat nose, long philtrum, down-turned mouth, micrognathia, apparently lowset and malformed ears, fingers abnormalities, and deafness. The latter patient had a nonspecific phenotype with mental retardation, growth failure and microcephaly. Karyotype-phenotype comparisons in the present cases and 16 previously reported cases with deficiency of the distal portion of 3p suggests that deficiency of the 3p25.3 band is critical to produce the main clinical manifestations of the del(3p) syndrome.

Initial stage of DNA-electrotransfer into E. coli cells.

The mechanism of electrotransfer of DNA into Escherichia coli cells was investigated under conditions optimal for genetic transformation or transfection. Simple mixing in 10% polyethylene glycol 6000 did not cause binding of DNA to the recipient bacteria. When subjected to a high electric field, however, 90-98% of the input plasmid or phage DNAs were complexed with the cells. By application of the electric field, a significant amount of biotin-labeled DNA was bound onto the recipient surface, as detected by fluorescein isothiocyanate coupled avidin. When subjected to a high voltage pulse, DNA molecules were rapidly attracted toward the anode. Concurrently, the electric field induced the orientation of bacterial cells, along the field lines and their movement toward the anode. Since the bacterial movement was relatively slow, a substantial fraction of DNA molecules must strike the cathode-facing end or side of the recipient cells. Irrespective of the high efficiency of DNA transformation, the voltage pulse did not induce release of alkaline phosphate and beta-galactosidase. The electrotransferred DNA first remained sensitive to Tris-EDTA treatment, and became refractory to spheroplasting only after incubation at 37 degrees C. These results indicate that the infecting DNA is electrophoretically plugged to the outer membrane loosened by the voltage pulse.

Effects of cis-diamminedichloroplatinum(II) on Escherichia coli and bacteriophage systems.

The effects of cis-diamminedichloroplatinum(II) (cisplatin) on Escherichia coli cells and bacteriophages were investigated. The bacteriocidal effect of cisplatin was stronger on uvrA or recA mutants than on wild type cells. The drug, like UV, induced prophage development in lysogenic bacteria. Host cell reactivation of alpha3 replicative form (RF) I DNA treated with cisplatin in vitro was more efficient in wild type or recA cells than in uvrA host. When wild type cells were exposed to cisplatin, decay of the host's capacity to sustain the viral multiplication proceeded nearly in parallel with the loss of colony-forming ability, whereas the capacity of uvrA mutant was much more resistant to the drug, as compared with the viability. In the DNA preparation from cisplatin-treated alpha3-infected wild type cells, RF II was deficient, but the RF I molecules extracted from the cells were moderately infective. The microvirid gene A protein, required for RF I-->RF II conversion, was hardly detectable in wild type cells exposed to cisplatin. The possible relationship between uvr+-dependent repair and synthesis of the viral protein is discussed.

The virion proteins encoded by bacteriophage phi K and its host-range mutant phi KhT: host-range determination and DNA binding properties.

The microvirid phage phi K, specific for Escherichia coli K12, contains a circular single-stranded (SS) DNA in the icosahedral virion, which comprises four phage gene products, F (capsid), G (major spike), H (minor spike), and J (core). phi KhT, a host-range mutant of phi K, can grow on E. coli C and B, besides K12, and is more thermosensitive than the parental phage phi K. Sequencing analysis revealed that the genome of phi K and phi KhT consists of 6,089 nucleotides (nt), and codes for eleven genes, whose sequences are similar to those of alpha 3, phi X174, and G4 infective to strain C. In phi KhT, two nt had changed: one is in the gene G, resulting in replacement of the 75th codon Ala with Ser, and the other is at 67th codon of the gene H: Val to Ala. Chemically synthesized gene J protein composed of 23 amino acids (aa) binds to phi K SS DNA more tightly than and preferentially over the host E. coli SS-DNA-binding protein (SSB). These results indicate that the two spike proteins G and H are involved in the determination of phi K host-range, and support a model in which the gene J protein functions in packaging the viral SS DNA into the virion vesicle.

Characteristics of various film cooling jets injected in a conduit.

In the present study, film cooling characteristics by the jets through various easy-to-make straight holes and slots have been investigated. In this experiment, seven kinds of injection geometries were used. They were circular, rectangular, elliptic and oval holes and slots, respectively.

Human cell clones, RSa and UVr-1, differing in their capability for UV-induced virus reactivation and phenotypic mutation.

UVr-1 is a human cell clone established as a variant with increased resistance to cell killing by ultraviolet light (UV, principally 254 nm wavelength) from a UV-sensitive cell clone, RSa. Both cells have been characterized to have much the same capacity of UV-induced DNA repair synthesis in whole cells, and the parent RSa cells were recently found to be hypermutable. In the present study UVr-1 cells were characterized in comparison RSa cells with respect to UV-induced virus reactivation and phenotypic mutation. Survival levels of UV-irradiated vaccinia virus and herpes simplex virus type 1 (HSV-1) were much the same in logarithmically proliferating UVr-1 and RSa cells. Correlated with these host cell reactivation levels, the same extent of UV-induced DNA repair replication synthesis was observed in isolated nuclei of the two cell clones. Enhancement of survival levels of UV-irradiated HSV-1 was detected when proliferating RSa cells were irradiated with UV prior to the virus infection. In contrast, this enhanced virus reactivation (EVR) was not detected in similarly irradiated and infected UVr-1 cells. As for phenotypic mutation frequencies assessed by the cloning efficiency of cells with increased resistance to ouabain cell killing (OuaR), OuaR mutants were not obtained from UVr-1 cells either with or without UV irradiation. When the proliferation of cells was synchronized, both EVR and OuaR mutations were detected in RSa cells irradiated with UV at any cell cycle phase, being greatest in the later half of the G1 phase. However, there was no detectable EVR or mutation in any phase of synchronous UVr-1 cells. The hypomutability of UVr-1 cells and hypermutability of RSa cells in a G1 cell cycle phase was also found even if 4-nitroquinoline 1-oxide was used as a mutagen or mutant cells with increased resistance to 6-thioguanine cell killing were estimated.

Cerebral hemodynamics on near-infrared spectroscopy in hypoxia and ischemia in young animal studies.

Using near-infrared spectroscopy the changes of intracranial oxyhemoglobin, deoxyhemoglobin, total hemoglobin and cytochrome aa3, which show the progression of intracranial oxygenation, hemodynamics and cell metabolism, were recorded during prolonged partial hypoxia induced by carbon dioxide (CO2) and nitrogen (N2), ischemia induced by hyperventilation, and hypoxia during hypoglycemia in neonatal and young rabbits. The reduction of cytochrome aa3 during the terminal stage of CO2-induced prolonged hypoxia, hyperventilation and hypoxia in hypoglycemia suggests that the redox state of cytochrome aa3 will be changed by several combined factors such as oxygen delivery, ATP demand and substrate (glucose) delivery.

The effect of acetazolamide and carbon dioxide on cerebral hemodynamic changes on near-infrared spectroscopy in young rabbits.

The changes of cerebral blood oxyhemoglobin (HbO2), deoxyhemoglobin (HbR), and total hemoglobin (tHb) induced by acetazolamide and CO2 loading on near-infrared spectroscopy (NIRS) were recorded. In anesthetized 2-week-old rabbits, acetazolamide (10 mg/kg i.v.) increased HbO2 and tHb, concomitant with an increase in tissue PCO2, and decreased HbR only at 5 and 10 min. CO2 loading significantly increased HbR and decreased HbO2, and after the termination of CO2 loading, tHb and HbO2 significantly increased and HbR decreased to nearly the baseline value. Thus, NIRS demonstrated cerebral hemodynamic responses as a function of vasomotor reactivity to acetazolamide as well as CO2 loading.

Mumps-induced opsoclonus-myoclonus and ataxia.

A 9-year-old boy manifested acute cerebellar ataxia associated with mumps infection. He developed opsoclonus, myoclonus, tremor, and truncal ataxia 7 days after mumps infection. Lumbar puncture revealed pleocytosis without elevation of protein; ELISA demonstrated an increased IgM titer of cerebrospinal fluid against mumps virus. From these results it was determined that acute cerebellar ataxia was induced by a direct invasion of mumps virus. Electroencephalography demonstrated normal background activity, although alpha-like activity appeared bilaterally in the frontal regions which was induced by eye closure and decreased by eye opening. Polygraphic electroencephalography revealed that the alpha-like activity corresponded to the ocular movement recorded above or lateral to the eyelids by electro-oculography; therefore, the alpha-like activity was considered to be derived from the opsoclonus which was secondary to cerebellar involvement. His neurologic symptoms improved gradually and resolved completely within 3 months after the onset of acute cerebellar ataxia.

Efficient preparation of optically active ketoprofen by Mucor javanicus lipase immobilized on an inorganic support.

Lipase M from Mucor javanicus, one of nine commercially available hydrolytic enzymes, showed good enantioselectivity (E=50) for racemic ketoprofen trifluoroethyl ester in phosphate buffer (pH 7.0) containing 30% acetone. Lipase M immobilized on Toyonite 200-A showed the best selectivity (E=55) and reactivity. Moreover, the lipase could be recycled at least 5 times.

Preparation of optically active trifluoromethylated (3'-indolyl) thiacarboxylic acids, novel plant growth regulators, through lipase-catalyzed enantioselective hydrolysis.

Among a variety of lipases tested, that obtained from Candida antarctica (SP 435) induced enantioselective hydrolysis of trifluoroethyl 5,5,5-trifluoro-4-(3'-indolyl)-3-thiapentanoate (1c). The selectivity could be increased by optimizing the reaction conditions. Thus, good selectivity was achieved (E = 37) in a buffer containing 10% dichloroethane. In order to improve the optical yields, a sequential kinetic resolution was utilized for the preparative-scale enantioselective hydrolysis of 1c using SP 435. Hydrolysis of trifluoroethyl 6,6,6-trifluoro-5-(3'-indolyl)-4-thiahexanoate (2c) with the lipase of Pseudomonas aeruginosa (LIP) in a buffer containing 20% tert-amyl alcohol at 25 degrees C gave excellent selectivity (E=357).

A cytogenetic study of nonpolymalformed patients with mental retardation of clinically undefined etiology: application of a high resolution banding technique.

We performed a cytogenetic study on 140 nonpolymalformed patients with mental retardation of clinically undefined origin, using a high resolution banding technique, to determine how much chromosome abnormalities contribute to the etiology of this condition. A total of 15 patients (10.7%) were found to have autosomal or sex chromosomal abnormalities. Autosomal abnormalities included partial monosomy (5 cases), reciprocal translocation (one case), 13/14 robertsonian translocation (3 cases), unbalanced translocation (one case), inverted duplication of 15q (one case) and mosaic trisomy 21 (one case). Sex chromosomal abnormalities comprised structural rearrangement of the short arm of the X chromosome (one case) and 47, XXY in a pure or mosaic form (two cases). It should be noted that four out of the 5 cases of partial monosomy had subtle interstitial deletions, which might have been unidentified by the conventional G-banding method alone. In one case of the robertsonian translocation 46,XY,t(13;14)/45,XY,t(13;14), a small deletion was thought to have occurred in the cells with a chromosome number of 45. Comparison of clinical features of the 15 chromosomally abnormal patients with those of patients with normal karyotypes did not show any clinical parameter indicative of chromosome imbalance. These results suggest that a subtle chromosomal deletion is specific to mental retardation associated with few malformations. We believe that diagnostic evaluation of mentally retarded patients, even if nonmalformed, should include chromosome analysis using a high resolution banding technique.

Multivariate analysis of dermatoglyphics of severe mental retardates: an application of the constellation graphical method for discriminant analysis.

We studied the dermatoglyphics of 353 severe mental retardates (excluding those with chromosomal abnormalities and major limb malformations), using multivariate analysis, to determine how early intrauterine factors are related to the etiology of mental retardation. First, dermatoglyphics were compared between 140 individuals with undefined prenatal factors and 700 normal controls. After 6 and 9 dermatoglyphic traits were chosen as discriminative variables for males and females, respectively, the data were subjected separately for each sex to the constellation graphical method for discriminant analysis. The same formula as obtained in the idiopathic group was subsequently applied to data from cases in other etiological categories. When the misclassification rate was 0.03, the rates of correct classification of the male patients into the etiological categories of undefined prenatal, defined prenatal, perinatal, postnatal and unknown (no anamnestic data available) categories were 19.7% (13/66), 20.0% (3/15), 8.8% (5/57), 5.0% (1/20) and 7.7% (2/26), while the correct classification rates of females were 24.3% (18/74), 42.1% (8/19), 18.9% (7/37), 5.1% (1/16) and 13.0% (3/23), respectively. The results suggest that early intrauterine factors such as those producing dermatoglyphic deviations may contribute to the pathogenesis of severe mental retardation not only in patients with undefined prenatal etiological factors but also in those with perinatal factors, especially those of the female sex.

Detection of hepatitis B surface antigen subtype adr in an epidemic of papular acrodermatitis of childhood (Gianotti's disease).

Papular acrodermatitis of childhood (PAC) has recently been reported to be associated with hepatitis B surface antigen (HBsAg) subtype ayw. Between September, 1978, and June, 1979, we saw 14 patients with PAC in a small epidemic occurring in Iwakuni City, Japan. HBsAg was detected in sera from all patients. Subtyping of HBsAg in 11 patients showed that 8 had a determinant adr and 3 had no detectable determinant because of low antigen titers. The result suggests that factors other than the specific HBsAg subtype contribute to the development of PAC.

Farber's disease (disseminated lipogranulomatosis): the first case reported in Japan.

We report the first case in Japan, i.e., the first case among oriental subject of Farber's disease. This is a rare disorder of lipid metabolism in infancy subsequent to a genetically-determined defect in ceramide degradation. Main features are characterized clinically by hoarseness, joint swelling, subcutaneous nodules and retarded psychomotor development. Lipid analysis and pathological investigation on the material obtained from a subcutaneous nodule confirmed clearly the presence of ceramide and intracytoplasmic inclusion bodies characteristic for Farber's disease. In this case, we experienced also corneal opacity and striking abnormalities in electroencephalogram, which have apparently not been noticed in the 17 cases hitherto reported.