We have examined trafficking of major histocompatibility complex (MHC) class II molecules in human B cells exposed to concanamycin B, a highly specific inhibitor of the vacuolar H(+)‐ATPases required for acidification of the vacuolar system and for early to late endosomal transport. Neutralization of vacuolar compartments prevents breakdown of the invariant chain (Ii) and blocks conversion of MHC class II molecules to peptide‐loaded, SDS‐stable alpha beta dimers. Ii remains associated with alpha beta and this complex accumulates internally, as ascertained biochemically and by morphological methods. In concanamycin B‐treated cells, a slow increase (> 20‐fold) in surface expression of Ii, mostly complexed with alpha beta, is detected. This surface‐disposed fraction of alpha beta Ii is nevertheless a minor population that reaches the cell surface directly, or is routed via early endosomes as intermediary stations. In inhibitor‐treated cells, the bulk of newly synthesized alpha beta Ii is no longer accessible to fluid phase endocytic markers. It is concluded that the majority of alpha beta Ii is targeted directly from the trans‐Golgi network to the compartment for peptide loading, bypassing the cell surface and early endosomes en route to the endocytic pathway.

All class II major histocompatibility complex genes contain two highly conserved sequences, termed X and Y, within the promoter region(s), which may have a role in regulation of expression. To study trans-acting factors that interact with these sequences, sequence-specific DNA binding activity has been examined by the gel electrophoresis retardation assay using the HLA-DQ213 gene 5' flanking DNA and nuclear extracts derived from various cell types. Several specific protein-binding activities were found using a 45-basepair (bp) HinfI/Sau96I Two highly conserved DNA sequences have been found in all class II a-and ,-chain genes and in a third, related gene, the invariant chain gene, in the region 5' of the transcription start site (9, 10) (see Fig. 1). They are conserved box X [at -100 to -113 base pairs (bp)] and conserved box Y (at -71 to -80 bp). In addition, two regions from -67 to -70 bp and from -81 to t-99 bp are conserved but are characteristic of either a-or A-chain genes. These conserved sequences have been thought to be cis-acting elements involved in regulation of class II genes, although the function of these sequences has not been precisely determined. The analysis by transfection of a series of 5' deletion constructs of the DQ,3 gene has demonstrated the existence of both positive and negative control elements as well as a IFN-y control element in the conserved sequence region (11). To elucidate the interactions of trans-acting regulatory factors with these cis-acting elements, nuclear factors that might bind specifically to them have been sought using the gel electrophoresis DNA-binding assay. MATERIALS AND METHODSCells. Raji and T5-1 are human B-lymphoblastoid cells. RJ 2.2.5 (6) and 6.1.6 (5) are class II-negative variants of Raji and T5-1, respectively. HeLa (human carcinoma) and the human T-lymphocyte cell lines HuT 78 (class II positive), Jurkat and HPB-ALL (both class II negative), were also used. All cells were propagated in RPMI 1640 medium supplemented with 10% fetal calf serum and penicillin/streptomycin/glutamine. DNA Fragment Preparation. The 5' flanking sequences of DQ2,3 gene were from a genomic clone X-42 (12). Subclones MX159, MX128, and MX82 (11) were used for preparation of fragments. End-labeling was carried out with [a-32P]dNTPs and the Klenow fragment of DNA polymerase I, and labeled fragments were isolated by polyacrylamide gel electrophoresis. Chemically synthesized oligonucleotides were used for DNA-binding competition experiments.DNA-Binding Assay. Binding of nuclear factors to DNA probes was analyzed bn polyacrylamide gels as described (13) with minor modifications. Nuclear extracts were prepared as described (14) and incubated with 32P-end-labeled DNA fragments in 10-id, reaction mixtures containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% (vol/vol)

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Dietary supplementation with an essential amino acid L-lysine has been shown to reduce chronic anxiety in humans with low dietary intake of L-lysine. A combination of L-lysine and L-arginine has been documented to normalize hormonal stress responses in humans with high trait anxiety. The present study was carried out in one hundred eight healthy Japanese adults. The aim of study was to find out whether a week-long oral treatment with L-lysine (2.64 g per day) and L-arginine (2.64 g per day) reduces trait and stress-induced state anxiety and basal levels of stress hormones. We confirmed that, without regard to gender, the amino acid treatment significantly reduced both trait anxiety and state anxiety induced by cognitive stress battery. In addition, we found that the treatment with L-lysine and L-arginine decreased the basal levels of salivary cortisol and chromogranin-A (a salivary marker of the sympatho-adrenal system) in male subjects. These results of this double-blind, placebo controlled and randomized study confirm the previous findings in humans and animals and point to a combination of L-lysine and L-arginine as a potentially useful dietary intervention in otherwise healthy humans with high subjective levels of mental stress and anxiety.

A 5-fluorotryptophan-resistant mutant, termed 1041, was isolated from Brevibacterium lactofermentum AJ12036. The anthranilate synthase of 1041 was insensitive to feedback inhibition by tryptophan, and the specific activities of the anthranilate synthase and anthranilate phosphoribosyltransferase of 1041 were 29-and 23-fold higher than those in parental strain AJ12036, respectively. A single-base change (adenine to cytosine) that resulted in a Ser-to-Arg substitution was found in the trpE structural gene of 1041. This substitution was identified as the cause of the desensitization to feedback inhibition by tryptophan of anthranilate synthase in 1041. Another substitution (guanine to adenine) was found at a position in which a mutation would destabilize the p-independent terminator structure within the putative attenuator. The enhanced synthesis of tryptophan enzymes in 1041 could be caused by this substitution in the attenuator.Brevibacterium lactofermentum is an industrially important gram-positive bacterium used for the production of various amino acids. We have focused our efforts on the tryptophan biosynthesis of this microorganism, because tryptophan is an important ingredient in medicines and animal feed as an essential amino acid.Tryptophan biosynthesis in Brevibacterium spp. has been found to be regulated by two mechanisms, feedback inhibition by tryptophan of anthranilate synthase (AS) (6), anthranilate phosphoribosyltransferase (10), and tryptophan synthase (9) and repression by tryptophan of the synthesis of AS and the other tryptophan enzymes (8), as is the case for Escherichia coli and Bacillus subtilis. Furthermore, analysis of the nucleotide sequence of the B. lactofermentum trp operon revealed the presence of operator-and attenuatorlike sequences (3, 5). Thus, we have assumed that repression of the synthesis of tryptophan enzymes in B. lactofermentum is carried out by both the repressor-operator system and the attenuation system, as in E. coli (13). To breed improved tryptophan-producing strains, it is necessary to remove these regulation mechanisms. For this purpose, we have isolated a 5-fluorotryptophan-resistant (4,000 ,g/ml) spontaneous mutant from wild-type B. lactofermentum AJ12036.The spontaneous mutant, termed 1041, was revealed to have an altered AS, which was fully active even in the presence of 10 mM tryptophan, while the activity of the wild-type AS from AJ12036 under the same conditions was less than 1% of that in the absence of tryptophan. Furthermore, the specific activities of AS and anthranilate phosphoribosyltransferase in 1041 were 29-and 23-fold higher than those in parental strain AJ12036, respectively. Determination of the mutation site in 1041 should be useful in clarifying the molecular mechanisms of the regulation of tryptophan biosynthesis in B. lactofermentum.Recombinant plasmids ptrpE97 and ptrpE36 carry the trp operon of 1041 and the 5'-proximal region of the trp operon of AJ12036, respectively (2) (Fig.

Some bacteria belonging to the genera Arthrobacter, Brevibacterium and Corynebac-.terium, so-called glutamic acid-producing bacteria, are highly important as they are used for the industrial fermentation production of various amino acids. The genetic breeding of such amino acid-producing strains has so far been carried out by conventional mutation and selection methods. After a phenol treatment of the supernatant, DNAwas recovered as ethanol precipitation. These DNA samples were analysed by agarose gel electrophoresis with a Tris-acetate buffer system as described by Sharp et al.7) As a result of the screening, two low-molecular-weight plasmids were found. One was found in Brevibacterium lactofermentum ATCC13869 Recentlyand named pAM330.The other was found in Corynebacterium glutamicum ATCC1 3058 and named pHM1519. They were purified.by the CsCl-EtBr equilibrium density gradient centrifugation method, and were confirmed to exist as closed circular molecules. The molecular weights of pAM330 and pHM1519 were determined as 3.0 Md and 1.8 Md, respectively, from electrophoretic mobility in a 0.8% agarose gel. Restriction enzyme analysis was carried out on those plasmids, and the results are shown in Fig. 1. The contents of pAM330 and pHM1519 were roughly analysed by the agarose gel electrophoresis method8'9* as follows: Cells were cultured in 4ml of the CM2Gmediumto the exponential growth phase, and 600 jig/ml of ampicillin was added to the culture medium to ensure the lysozyme digestion. The cells were harvested at 2.5 hr after the addition of ampicillin, and were lysed with lysozyme (lOmg/ml, 37°C 2hr) and SDS (4%). The lysis was completed by heating at 65°C for lOmin, followed by phenol extraction and ethanol precipitation. The DNAsamples were dissolved in TEN buffer and treated with RNase (20/ig/ml, 37°C fir 20min) and pronase (100/^g/ml, 37°C for 20min) sequentially.After phenol extraction and ethanol precipitation, the recovered DNAwas dissolved in TENbuffer, and electrophoresed in a 0.8% agarose gel. The photographs of the gel were taken under irradiation by ultra-violet light, and the negative films of them were analysed by a densitometer (Shimadzu CS-910). As a result, the amounts ofpAM330 and pHM1519 were approximately 1.4% and 9.1% of the total DNA, respectively.To estimate the