Quantitative structure-activity relationship of cytokinin-active adenine and urea derivatives

The substituent effect of N⁶-alkyl and -aralkyl adenines on the promotion of the growth of tobacco callus was analysed quantitatively using physico-chemical substituent parameters and regression analysis. The results indicated an optimum steric condition for activity in terms of the maximum width of the N⁶-substituents from the bond-axis connecting the N⁶-atom with its α carbon atom. The electron withdrawing effect of the N⁶-substituent enhances the activity. The substituent effect on the cytokinin activity of phenyl- and diphenyl- urea derivatives determined by Bruce and Zwar using the tobacco pith-block assay was also analysed. The results suggest that position-specific steric and hydrophobic effects of aromatic substituents participate in the variation in activity rationalizing the general trend of the activity; meta >para >ortho derivatives, for both series of compounds. The electronic effect is significant for the activity of diphenylureas but not for that of phenylureas which show somewhat different modes of interaction between the two series at the site of action. Based on inferences made from the correlations, hypothetical maps for the mode of interaction of these three sets of compounds at the site of action have been proposed.     

Failure to verify the presence of pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase by gas chromatography/mass spectrometry

The presence of covalently bound pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase (BPAO) was examined by the use of gas chromatography/mass spectrometry. The enzyme was subjected to proteolysis with proteinase in the presence of [U-13C]PQQ as an internal standard. After isolation and derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ, respectively, by selected ion monitoring (SIM). In the SIM profile, although the sample extract obtained from BPAO treated with proteinase clearly showed the peak at m/z 462 for the internal standard, there were no peaks detectable at m/z 448, showing the absence of PQQ in the proteolysis digest of BPAO. Thus, our results do not support the claim that BPAO contains covalently bound PQQ in its structure.     

Decreased levels of steroid 21-hydroxylase [P450(c21)] and its mRNA in an adrenocortical adenoma associated with 21-hydroxylase deficiency

Adrenocortical adenoma incidentally found in a 37-yr-old female patient, with simple virilizing form of 21-hydroxylase deficiency, was studied. Cultured adenoma cells revealed excessive secretion of 17 alpha-hydroxyprogesterone in response to 10(-8) M ACTH, compared with those of 11-deoxycortisol and cortisol, which indicated impaired activity of the 21-hydroxylase. To elucidate the molecular mechanisms of this defective 21-hydroxylase in the adenoma, we analyzed the gene encoding specific cytochrome P450 (P450c21) for steroid 21-hydroxylation and its expression. DNA and RNA were extracted from the adrenal adenoma and were hybridized with a probe of human P450c21 gene, by Southern and Northern blot analysis. In Southern blot analysis with Taq I, Bgl II or Bam HI, there was no difference between the pattern of restriction fragments in DNA from the adenoma and normal peripheral leucocytes. Northern blot analysis of the adenoma showed the same size of P450c21 mRNA as in the normal adrenal gland, but the amount was low--about a half that of the normal adrenal. In Western blot analysis with polyclonal antibody to P450c21, only a small amount of P450c21 protein was detected in the adenoma, although it was found to be of the same molecular weight as that in the normal adrenal gland. In view of these findings it is conceivable as one of possibilities that a mild and small mutation in the structural or promotor region of the P450c21 gene may cause the decreased 21-hydroxylase activity in this adenoma.     

Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry.

A detailed procedure for the assay of free pyrroloquinoline quinone (PQQ) in human and rat samples by gas chromatography/mass spectrometry (GC/MS) has been established with stable-isotopic PQQ as internal standard. PQQ was extracted from the samples, after addition of the internal standard, with butanol under acid conditions and with Sep-Pak C18 cartridges. After derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ by selected ion monitoring, respectively. Trace amounts of free PQQ were detected in eight organs, plasma and urine of the human, and in three organs of the rat. The PQQ level was highest in the human spleen (5.9 +/- 3.4 ng/g tissue, followed by the pancreas and lung, and it was below detection limits for human brain and heart. Trace levels of PQQ were also found in rat small intestine, liver and testis. Our data are far below those measured by the redox cycling method of Gallop's group for human plasma, adrenal and urine.     

Assimilation and transport of nitrogen in rice I. 15N-labelled ammonium nitrogen

The assimilation and transport of ¹⁵N-labelled ammonium nitrogenin rice plants (Oryza sativa L.) was studied. Plants assimilatedlarge amounts of nitrogen from labelled ammonium into theiramides and amino acids, particularly in the roots and stem,at the end of a 4-day ¹⁵N feeding and 10 days later in the upperleaves, especially in the blades. Although the incorporationof ¹⁵N into all the nitrogen fractions of the newly emergedpanicle was evident, it was particularly pronounced in the amidesand amino acids of the soluble fractions. The upper leaves hada greater ¹⁵N incorporation in their organic N-fractions thandid the lower ones. Amides and amino acids are considered tobe the main forms of nitrogen transported to the shoot fromthe ammonium assimilated in the roots. The transport of theorganic forms of nitrogen was possibly greater to the upperleaves than to the lower ones. The nitrite fraction had more ¹⁵N than did the nitrate fractionin all parts of the plant, particularly in the upper leaf blades.It appeared that some of the ammonia might have been oxidizedto nitrite, then to nitrate in some parts of the plant; probablyin the upper leaves. The synthesis of protein and nucleic acid occurred rapidly inthe upper leaves, especially in the blades, also in the rootsas evidenced by the considerable incorporation of ¹⁵N in theinsoluble fractions of these parts. The variation in ¹⁵N-distribution,during the 10 days, in the different plant parts suggests thatthe nitrogen incorporated during protein synthesis in the rootsand tillers was remobilized and transported to the upper partsof the shoot. A concept for the transport of organic nitrogenouscompounds from the roots to shoot through the phloem and xylemof the rice plant stem is discussed. (Received May 11, 1974; )     

Modulation of mouse anti-trinitrophenyl plaque-forming cell affinity by adjuvants or lectins.

The efffects of several kinds of adjuvants or lectins, such as Corynebacterium parvum, dextran, poly AU, poly IC, dibutyryl cAMP, concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. The numbers of anti-TNP PFC in the spleens of mice which had been injected with C. parvum 7 days in advance were greater than those in controls after immunization with TNP-coupled heterologous erythrocytes, while the affinity of antibodies released by these PFC. Copolymers of nucleotides, poly AU and poly IC, were capable of enhancing splenic anti-TNP PFC responses, but showed almost no altering of PFC affinity. Dibutyryl cAMP did not have any effect on this system. Con A had potencies to both augment the number of anti-TNP PFC and heighten the PFC affinity, while PHA seemed to lack these potencies. Injection of PWM in the presence of antigen increased the number of anti-TNP PFC and heightened slightly the PFC affinity. These results indicate that the heightening of the affinity at the cellular level is regulated in ways different from the augmenting effects on the number of anti-TNP PFC by adjuvants or lectins. These results are discussed in the light of the mode of action of the substances used.     

Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.     

Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.     

Biological effects of Er:YAG laser irradiation on the proliferation of primary human gingival fibroblasts

We investigated the biological effects of Er:YAG laser (2940‐nm; DELight, HOYA ConBio, Fremont, California) irradiation at fluences of 3.6, 4.2, 4.9, 6.3, 8.1 or 9.7 J cm^?2 at 20 or 30 Hz for 20 or 30 seconds on primary human gingival fibroblasts (HGFs). Irradiation at 6.3 J cm^?2 promoted maximal cell proliferation, determined by WST‐8 assay and crystal violet staining, but was accompanied by lactate dehydrogenase release, on day 3 post‐irradiation. Elevation of ATP level, Ki67 staining, and cyclin‐A2 mRNA expression confirmed that Er:YAG affected the cell cycle and increased the number of proliferating cells. Transmission electron microscopy showed alterations of mitochondria and ribosomal endoplasmic reticulum (ER) at 3 hours post‐irradiation at 6.3 J cm^?2, and the changes subsided after 24 hours, suggesting transient cellular injury. Microarray analysis revealed up‐regulation of 21 genes involved in heat‐related biological responses and ER‐associated degradation. The mRNA expression of heat shock protein 70 family was increased, as validated by Real‐time PCR. Surface temperature measurement confirmed that 6.3 J cm^?2 generated heat (40.9°C post‐irradiation). Treatment with 40°C‐warmed medium increased proliferation. Laser‐induced proliferation was suppressed by inhibition of thermosensory transient receptor potential channels. Thus, despite causing transient cellular damage, Er:YAG laser irradiation at 6.3 J cm^?2 strongly potentiated HGF proliferation via photo‐thermal stress, suggesting potential wound‐healing benefit.