Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Chromatin arrangements in intact interphase nuclei examined by laser confocal microscopy

Using a laser confocal microscope, chromatin arrangements in intact interphase nuclei were investigated in four plant species. Chromosomes in these plants have specific segments that can be stained with the fluorescent dye chromomycin A₃ (CMA). We stained centromeres inHordeum vulgare, sub-telomeric regions inSecale cereale, satellites inChrysanthemum multicore, and the satellites and the short arms of chromosomes with satellites inHemerocallis middendorfii. The following points were shown: (1) In mitotic interphase nuclei, the centromere and the telomeres of both arms touched the nuclear membrane and had evident polarity. Some CMA-bodies in sub-telomeric regions do not contact the nuclear membrane. (2) Differentiated nuclei had a non-random construction. Polarity of chromosomes is maintained, however, the chromosomes are far apart from the nuclear membrane. (3) Associations in sub-telomeric regions in the interphase nuclei ofSecale cereale were probably due to the association of heterochromatic regions with identical repeated sequences rather than telomere associlations. (4) In interphase nuclei ofChrysanthemum multicore, satellites fused during interphase.     

Selective elimination of chromosomally unbalanced zygotes at the two-cell stage in the Chinese hamster

The gametic and zygotic selection of genome imbalance was investigated in the Chinese hamster by direct chromosome analyses of spermatocytes and preimplantation embryos from crosses between chromosomally normal females and males heterozygous for a reciprocal translocation, T(2;10)3Idr, abbreviated here as T3. The karyotypes and the frequencies of embryos observed at the first cleavage in the cross +/+female X T3/+male were consistent with those expected from MII scoring in male T3 heterozygotes. Therefore, it was concluded that there was neither gametic selection against genome imbalance nor zygotic selection from fertilization until the first cleavage metaphase. However, 9.1-10.8% of embryos were arrested at the two-cell stage, and karyotypes of these embryos were confirmed as 22(2,10,10,10(2)), 21(2,10,10), and 21(2,10,10(2)). The common abnormality of these embryos was partial monosomy of chromosome 2. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of other segments of chromosomes 2 and 10, had fewer blastomeres than chromosomally balanced embryos. This finding suggests that cleavage of these embryos had been retarded by day 4 of gestation.     

Role of the Photoperiod Preceding a Flower-Inductive Dark Period in Dark-Grown Seedlings of Pharbitis nil Choisy

Flowering responses to a single photoperiod, of various durationsand irradiances, followed by an inductive dark period were investigatedwith dark-grown seedlings of Pharbitis nil Choisy. The numberof flower buds induced in each plant (NFB) increased with theincrease of both duration and irradiance of the photoperiod.Reciprocity did not hold for this photoresponse within the rangeof 0-16 h and 2.5-10 W-m^-2, NFB depending on the duration ratherthan the irradiance. With lengthening of the dark period followinga photoperiod of 8 h or less, two different phases alternatelyappeared so that NFB sharply increased at 20-24 h and 40-43h after the onset of the photoperiod, then gradually decreased.When the photoperiod was longer than 8 h, NFB sharply increasedat 12–16 h after the end of the photoperiod and remainedaround the saturated value with longer dark periods. Far-redlight given immediately after the photoperiod inhibited flowering,the inhibitory effect being stronger the shorter the photoperiod.This far-red effect is mediated by phytochrome and P_FR seemsto be required during the inductive dark period following ashort photoperiod for floral induction. (Received December 23, 1983; Accepted April 12, 1984)     

Clonal propagation of Stevia rebaudiana Bertoni by stem-tip culture

Clonal propagation of Stevia rebaudiana has been established by culturing stem-tips with a few leaf primordia on an agar medium supplemented with a high concentration (10 mg/l) of kinetin. Anatomical examination has suggested that these multiple shoots originate from a number of adventitious buds formed on the margin of the leaf. Innumerable shoots can be obtained by repeating the cycle of multiple-shoot formation from a single stem-tip of Stevia. These shoots produce roots when transferred to a medium containing NAA (0.1 mg/l) without kinetin. The regenerated plantlets can be transplanted to soil.     

Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme.

A series of 16 analogs of 5'-deoxy-5'-adenosylcobalamin (adenosylcobalamin) were examined for their effects on the diol dehydrase system of Klebsiella pneumoniae (Aerobacter Aerogenes). Four analogs, ara-adenosyl-, aristeromycyl-, 3-isoadenosyl-, and nebularylcobalamin, were able to function as coenzymes in the diol dehydrase reaction, coenzyme activity decreasing in that order. Like the native holoenzyme, complexes of the enzyme with these four analogs show a cob(II)alamin-like absorption peak or shoulder in the presence of 1,2-propanediol. Analogs containing hypoxanthine, cytosine, or benzimidazole do not function as coenzymes, but are weak competitive inhibitors in the presence of adenosylcobalamin. Analogs in which the D-ribosyl moiety is replaced by L-ribose or by an alkyl chain of 2 to 6 carbons are inactive as coenzymes, but act as competitive inhibitors with extremely high affinity for the apoenzyme. Complexes with the inactive analogs showed visible spectra similar to those of the corresponding free cobalamins. Upon anaerobic photolysis and subsequent aeration, complexes with the first group of inactive analogs produced unusually stabilized cob(II)alamin, while complexes with the second group of inactive analogs were readily photolyzed to a hydroxocobalamin-enzyme complex. Complexes with adeninylpentyl- and L-adenosylcobalamin were stable to light under the same conditions. These findings suggest that both the ribose and the adenine moiety of the nucleoside participate in enzyme-coenzyme interaction, involving not only the binding to the apoenzyme but also the activation of the carbon-cobalt bond.