O2-stimulated synthesis of bacteriochlorophyll and carotenoids in marine bacteria

We examined the effects of a limitation of the O₂-supply onthe syntheses of bacteriochlorophyll a_p and carotenoids in isolatesof aerobic marine bacteria, OCh 101 and OCh 114, grown heterotrophically.Whereas they formed these pigments fairly well under high aerationin the dark, a limitation of the O₂-supply resulted in the decreasedsyntheses of bacteriochlorophyll in both strains. Synthesesof carotenoids also were depressed under low aeration but toa lesser extent (especially in OCh 101) than the depressionof bacteriochlorophyll synthesis. Aerobic incubation of a cultureof OCh 101, that previously had been grown semiaerobically,induced the supplementary synthesis of bacteriochlorophyll.This induction was inhibited almost completely by chloram-phenicol.The absorption spectra of suspensions and solvent extracts ofcells grown aerobically or semiaerobically are reported. (Received May 30, 1980; )     

HTHQ (1-O-hexyl-2,3,5-trimethylhydroquinone), an anti-lipid-peroxidative compound: its chemical and biochemical characterizations

Recently, it has become apparent that reactive oxygen species (ROS) play many important roles in biological systems. For example, relationships between many diseases, such as cancer, cardiac infarction and arteriosclerosis, and ROS have been found. It is also well known that anti-oxidative agents scavenge ROS in biological systems, which in turn prevents ROS-related diseases. In our previous efforts to develop effective anti-oxidative compounds, we found that 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), which is a hydroquinone monoalkyl ether, is a potent anti-oxidative agent. Here, the scavenging activities of HTHQ against ROS, such as superoxide anion radicals, hydroxyl radicals, t-butyl peroxyl radicals and singlet oxygens, were examined by the ESR (electron spin resonance)-spin trapping method. Among ROS, HTHQ scavenged t-butyl peroxyl radicals most effectively (IC₅₀=0.31±0.04 mM), showing approximately twice the activity of a well-known lipophilic anti-oxidant, d,l-α-tocopherol (IC₅₀=0.67±0.06 mM), as measured by IC₅₀ values defined as the 50% inhibition concentration of the generated ROS. In addition, a relatively stable ESR spectrum of free radicals due to HTHQ was observed during the reaction of HTHQ and t-butyl peroxyl radicals, indicating a direct reaction of HTHQ and t-butyl peroxyl radicals. The free radicals due to HTHQ were more stable than those derived from d,l-α-tocopherol under the same conditions examined. On the basis of these results, we evaluated anti-lipid-peroxidative activity of HTHQ in three systems involving micelles, liposomes and rat liver microsomes. HTHQ exhibited a similar anti-oxidative activity to that of d,l-α-tocopherol against lipid peroxidation in linolate micelles initiated by addition of Fe²⁺. On the other hand, HTHQ exhibited approximately 4.8-fold higher anti-lipid-peroxidation activity than that of d,l-α-tocopherol against the peroxidation in phosphatidylcholine liposomes initiated by addition of Fe²⁺. Furthermore, HTHQ scavenged the lipid peroxides at a rate approximately 150 times higher than that of d,l-α-tocopherol against Fe³⁺-ADP-induced lipid peroxidation in rat liver microsomes, indicating that the anti-lipid-peroxidation activity of HTHQ might be substantially elevated in biological systems in comparison with that of d,l-α-tocopherol. Based on these results, we suggest that HTHQ reacts directly with peroxyl radicals, such as t-butyl peroxyl radicals and peroxides of linolate micelles, liposomes and microsomes, by scavenging them to form stable free radicals. The resulting free radicals are presumed to be reduced by several reducing mechanisms in biological systems similarly to those of d,l-α-tocopherol, and then the lipid-peroxidation reactions will be terminated. In conclusion, HTHQ was found to be a potent anti-lipid-peroxidative compound and its anti-oxidation activity to be extremely elevated in biological systems, such as that of liver microsomes via the generation of stable free radicals. We propose that HTHQ is a potent anti-oxidative agent for use in future treatments for lipid-peroxide relevant diseases.     

Development and genetic differences of complement activity in rabbits*

The ontogeny of haemolytic complement in rabbit serum and the genetic differences of the activity in five strains of adult rabbits were investigated by single radial haemolysis in gel and a microtiter method with purified complement components and the appropriate haemolytic intermediate cells. The haemolytic complement activity was found as early as the 15th day of foetal life, and increased with age reaching approximately adult level by the 120th day of life. Marked strain differences in both total haemolytic activity and C3 levels of adult rabbit sera were observed. The repeatability of haemolytic activity for an individual serum taken at different times was higher than that for C3 levels and no significant correlation between haemolytic activity and C3 level was observed. An inherited complement deficiency, due to the lack of C6, was found in a strain of Angora rabbits. The genetic studies confirmed that this complement defect was transmitted as an autosomal recessive trait.     

Isolation and characterization of des(Ala-Lys)calmodulin in porcine brain

A calmodulin-like protein -des(Ala-Lys)calmodulin- was isolated from porcine brain extract, and was characterized in comparison to porcine brain calmodulin. Des(Ala-Lys)calmodulin was distinguishable from calmodulin by its slightly faster mobility in 10% polyacrylamide gels without sodium dodecyl sulfate. The protein gave an amino acid composition very similar to calmodulin, and contained one ?-N-trimethyllysyl residue. Comparative peptide mapping of calmodulin and des(Ala-Lys)calmodulin by high performance anion-exchange liquid chromatography, and the subsequent analyses of the isolated peptides, have indicated that des(Ala-Lys)calmodulin lacks the Ala(147)-Lys (148) sequence at the C-terminus of calmodulin. The content of des(Ala-Lys)-calmodulin was about one-tenth of calmodulin.     

Immunohistochemical Studies on the Transneuronal Spread of Virulent Herpes Simplex Virus Type 2 and Its US3 Protein Kinase-Deficient Mutant after Ocular Inoculation

The transneuronal spread of a virulent wild-type herpes simplex virus type 2 (HSV-2) and its US3 protein kinase-deficient (US3 PK^?) mutant was immunohistochemically studied in mice after inoculations into the cornea, anterior chamber, tongue, and masseter muscle. After corneal inoculation, the wild-type virus was demonstrated in various brain stem areas including the trigeminal tract and nucleus, the reticular formation, and cerebellar nucleus group. Viral antigen-positive neurons were strictly confined to the ipsilateral spinal trigeminal nucleus in mice corneally infected with the US3 PK^? mutant. No viral antigens were detected in the central nervous system (CNS) after inoculation with the mutant into the tongue and masseter muscle. However, when mice were immunosuppressed by treatment with cyclophosphamide, both the corneally infected mutant and wild-type virus could invade the CNS. The results suggest that the US3 PK^? mutant principally retains the capacity to spread in the CNS.     

Molecular evidence for human alpha2-HS glycoprotein (AHSG) polymorphism

Alpha2-HS glycoprotein (AHSG) is a human plasma glycoprotein that exhibits genetic polymorphism on isoelectric focusing (IEF). To identify the origin of two common alleles, AHSG*1 and *2, we examined nucleotide exchanges in the gene. AHSG cDNA was obtained by RT-PCR from poly(A) RNA of seven liver tissue samples and subcloned into a plasmid vector. After sequencing, we found six single nucleotide differences in comparison with the originally reported sequence. In particular, the nucleotide substitutions of C to T at amino acid position 230 and C to G at position 238 were common among the samples exhibiting phenotype 2–1 or 2. Since these substitutions might give rise to a NlaIII site and a SacI site, respectively, for the potential AHSG*2, we analyzed these substitutions by PCR-RFLP using genomic DNA of 68 individuals. The result was consistent with the IEF analysis of the corresponding serum, indicating that AHSG*1 was characterized by ACG (Thr) at position 230 in exon 6 and ACC (Thr) at position 238 in exon 7, and that AHSG*2 was characterized by ATG (Met) at position 230 and AGC (Ser) at position 238. Received: 5 March 1996 / Revised: 25 June 1996     

Expression of dystrophin on the cell surface membrane of intrafusal fibers of human skeletal muscle

Summary We examined the morphological expression of dystrophin in the intrafusal muscle fibers in skeletal muscle from normal human and Duchenne muscular dystrophy (DMD) patients, using antisera against the N-terminal and C-terminal regions of dystrophin. The intrafusal fibers of normal muscle express dystrophin on their cell surface membrane, but those of DMD muscle do not.Abbreviation DMD Duchenne muscular dystrophy     

A possible association between basic amino acids of position 13 of DRB1 chains and autoimmune hepatitis

Fifty-one patients with autoimmune hepatitis have been studied for HLA association by conventional serology and also by modified polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) genotyping.HLA-DR4 was significantly associated with autoimmmune hepatitis (46 of 51 patients, 90.2%). DNA typing of the DRB1 gene for 43 DR4-positive patients by using the PCR-RFLP technique revealed that of 43 patients, 33 had DRB1 ^* 0405 (Dw15), five had DRB1 ^* 0406 (DwKT2), four had DRB1 ^* 0403 (Dw13a), two had DRB1 ^* 0401 (Dw4), two of 43 had DRB1 ^* 0407 (Dw13b) and one had DRB1 ^* 0408 (Dw14b). Thus, there was no significant difference in Dw frequencies between DR4-positive patients and DR4-positive healthy subjects. These findings suggest that the DR4-specific sequence (Val 11 and His 13 at amino acid positions 11 and 13, respectively), but not particular Dw-associated DR4 sequence, in the first domain of the DRB1 chain contributes to susceptibility to autoimmune hepatitis among Japanese. Interestingly, all five of the DR4-negative patients had the DR2 specificity (DRB1 ¹⁵⁰² or 1601). Taken together, these results imply that the basic amino acids at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), are most important for determining the predisposition to autoimmune hepatitis. Address correspondence and offprint requests to: M. Ota.