A facile method for detection of the genuine sapogenin from a saponin extract

We present a simple, efficient system for preparative polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate. The instrument features a novel sample collection assembly which is fitted directly onto the bottom of the gel cylinder. Buffer pumped uniformly upward through a porous plastic disk from the lower tray sweeps the emerging sample into a collection system with high efficiency. Measures which prevent the destruction of tryptophan residues and the blockage of α-amino groups observed in electrophoresed polypeptides are described.     

Specific in vivo binding of 77Br-p-bromospiroperidol in rat brain: A potential tool for gamma ray imaging

The binding of the gamma labeled neuroleptic, ⁷⁷Br-$\text{p}$-bromosprioperidol, in the rat brain was examined $\text{in vivo}$. This binding parallels the binding of ³H-spiroperidol, in that binding is especially high in dopaminergically innervated areas, is saturable, and is displaced by high doses of unlabeled spiroperidol (1–5). Thus, ⁷⁷Br-$\text{p}$-bromospiroperidol is a suitable ligand for use in gamma ray imaging techniques for $\text{in vivo}$ monitoring of receptor binding.     

Epstein-Barr virus RNA in Burkitt tumor tissue.

Analysis of the viral RNA in four Burkitt tumor biopsies indicates that tumor tissue contains RNA homologous to at least 3–6% of the DNA of Epstein-Barr virus (EBV). Most of these RNA species accumulate in the polyadenylated RNA fraction of Burkitt tumor tissue. Two approaches have been used to determine the location within the EBV genome of the DNA sequences which encode stable RNA in two Burkitt tumor biopsies, F and S, which contain 6–10 copies per cell of at least 80% of the EBV genome. With the first approach, ³²P-EBV DNA homologous to polyadenylated or nonpolyadenylated RNAs from the F, S or R tumors was hybridized to blots of fragments of EBV DNA. With the second approach, polyadenylated or nonpolyadenylated RNAs from the F or S tumors were hybridized to separated, labeled fragments of EBV DNA in solution. The results indicate that first, most of the viral RNA in Burkitt tumor tissue is encoded by approximately 20% of the Hsu I D fragment, 20% of the Eco RI A/Hsu I A double-cut fragment and 3% of the Hsu I B fragment of EBV DNA; second, an abundant RNA species in tumor tissue is homologous to the “additional DNA” present in the W91 and Jijoye/HR-I Burkitt tumor isolates of EBV and absent in the B95-8 virus, an isolate of EBV from outside the Burkitt endemic region; and third, there is little or no homology to other regions of the EBV genome.     

New reagents for the introduction of the thiomethyl group at sulfhydryl residues of proteins with concomitant spectrophotometric titration of the sulfhydryls: Methyl 3-nitro-2-pyridyl disulfide and methyl 2-pyridyl disulfide

Two new reagents for the titration of sulfhydryl groups in peptides and proteins and for their temporary blocking with the thiomethyl group have been developed. The sulfhydryl groups in cysteine, glutathione, and papain react quantiatively under mild conditions with these reagents, methyl 3-nitro-2-pyridyl disulfide and methyl 2-pyridyl disulfide, with concomitant methanethiolation and without the need to employ a large excess of reagent. Because of the chromophoric properties of the 3-nitro-2-thiopyridone and 2-thiopyridone products, spectrophotometric titration of the sulfhydryl groups can be carried out, accompanying their methanethiolation. The modification of the sulfhydryl groups in peptides and proteins with thiomethyl is rapidly and completely reversible upon addition of thiols such as l-cysteine.     

Age-related changes in in vitro immunoglobulin secretion: Comparison of responses to T-dependent and T-independent polyclonal activators

In vitro Ig secretion by peripheral blood mononuclear cells (MNCs) from old and young donors, in response to T-dependent (TD) [pokeweed mitogen (PWM)] and T-independent (TI) [Salmonella paratyphii B (SPB)] activation were compared. In older donors, the IgG and IgA responses to PWM were comparable to those of young donors; the IgM response was reduced in the elderly. With SPB activation, IgA response was again preserved, whereas IgG response was reduced and IgM secretion was markedly decreased. These data indicate class-specific changes in Ig responsiveness to both TD and TI cell activators with age. The reduction in TI-induced IgG and IgM responses in the elderly suggest that changes in B cells themselves have occurred. The preservation of the TD IgG response in concert with reduced TI response indicates that a decline in T-suppressor influences over B cells in the elderly coupled with reduced B-cell synthesizing capacity can result in apparent “preservation” of the final Ig response. In keeping with the above postulate, analysis of individual elderly donors' responses indicated that some of the old donors responded to PWM, but not SPB; none of the old donors responded to SPB and not PWM. In contrast, some young donors did respond to SPB, but not PWM. These results also suggest that nonresponse to PWM in young donors relates to an override of functionally intact B cells by T-regulator influences.     

The activity of adenosyl-d-methionine and adenosyl-2-methylmethionine in transmethylations

The d-methionine- and 2-methyl-dl-methionine analogs of the enzymatic methyl donor, (?)S-adenosyl-l-methionine, were synthesized by methylation of S-adenosyl-d-homocysteine and S-adenosyl-2-methyl-dl-homocysteine with methyl iodide. By chromatographic purification, S-adenosyl-d-methionine and S-adenosyl-2-methyl-dl-methionine were obtained. The structure of the latter was ascertained by hydrolysis to 2-methylmethionine in strong acid, and to 5′-methylthioadenosine and 2-methylhomoserine at pH 4. Reference material of the latter compound was obtained by alkaline hydrolysis of 2-methylmethionine methylsulfonium iodide. The sulfonium compounds were tested as methyl donors with N-acetylserotonin O-methyltransferase, l-homocysteine S-methyltransferase, histamine N-methyltransferase, and guanidinoacetate N-methyltransferase. In most instances, methyl donor activity was observed.     

Transdetermination of imaginal wing disc fragments of Drosophila melanogaster

Fragments of the imaginal wing disc of Drosophila melanogaster were cultured in adult hosts before transfer to larvae for metamorphosis. Transdetermination occurred only after at least 2 weeks of culture in vivo, producing structures of the leg, antenna, head, and thoracic spiracle. Details of the transdetermined structures and their locations with respect to normal wing disc structures are reported. We present evidence suggesting that regulation can occur between the wing and the second leg imaginal discs, and we propose that many transdeterminations which involve neighboring discs may result from such interdisc regulation.     

Specifically induced suppression of T cell and NK-like cytolytic activity by ingested soluble antigens

The effect of feeding xenoserum (xs) on cytolytic cell activity induced by parenteral injection was examined in C3H/N mice. Spleen cells were cultured with xs and then assayed for cytolytic activity against a panel of 51Cr-labeled YAC-1, AKR-A, or P815 target cells. Prior feeding resulted in significant suppression of responses stimulated by injection and culture. The induction of these responses was antigen specific for xs whereas the effector stage represented polyclonal activation of cytolytic cells. Some effector cells were lysed by either anti-Lyt 2 or anti-NK- 1.2 and complement and some were blocked by anti-Lyt 2 or anti-T200 in the cytotoxicity assay. Thus, both cytolytic T and NK-like cells were suppressed by antigen feeding. Activity of TH cell-derived factors which enhance cytolytic activity ("promoter" factor, interferon, and interleukin 2) also was diminished in culture supernatants of cells from mice fed soluble antigens. The conclusion that polyclonal cytolytic responses induced by soluble antigen can be regulated by prior enteric stimulation is made.     

A non-peptide morphine-like compound from brain

A non-peptide morphine-like compound was extracted from calf brain and partially purified by immunoabsorption to antimorphine antibodies. The material was able to depress electrically stimulated contractions of the myenteric plexus-longitudinal muscle of the isolated guinea pig ileum. This effect could be prevented and reversed by naloxone and by antimorphine antibodies. Biological activity and morphine-like immunoreactivity were not affected by treatment with proteolytic enzymes. These results represent the first independent confirmation of the non-peptide morphine-like compound reported by Gintzler $\text{et al}$.     

Biosynthesis of adenosyl-d-methionine and adenosyl-2-methylmethionine by Candida utilis

Supplementation of the culture medium of Candida utilis with d-methionine or 2-methyl-dl-methionine leads to intracellular synthesis of S-adenosyl-d-methionine and S-adenosyl-2-methylmethionine. The identity of the sulfonium compounds was established by tracer technique, chromatography, acid hydrolysis, and examination of the released methionine and 2-methylmethionine. In addition to the expected sulfur amino acid component, both adenosine sulfonium fractions contained S-adenosyl-l-methionine. This is explained by transmethylation of S-adenosyl-d-methionine and of S-adenosyl-2-methyl-methionine with endogenous l-homocysteine; the resulting l-methionine reacts with ATP to form S-adenosyl-l-methionine. Experiments with purified cell-free preparations of S-adenosylmethionine synthetase (EC 2.5.1.6) from C. utilis confirmed the reaction of ATP with d-methionine or 2-methyl-dl-methionine.     

芝加哥天蓝法在甲屑真菌检测中效果的评估

目的探讨芝加哥天蓝染色法对甲真菌病患者标本检测效果。方法收集皮肤科门诊确诊的甲真菌感染患者标本160例,同时用KOH湿片法、荧光染色法和芝加哥天蓝染色法对真菌进行镜下形态学观察,并比较真菌检出率。结果芝加哥天蓝染色法和荧光染色法对真菌的形态结构清晰易辨。芝加哥天蓝染色法、荧光染色法和KOH湿片法的检出率分别为60.6%、68.5%、47.5%;荧光染色法与芝加哥天蓝染色法的检出率结果比较差异无统计学意义(χ~2=0.176 2,P=0.674 0),芝加哥天蓝染色法和荧光染色法与KOH湿片法的结果比较差异有统计学意义(χ~2=102.012 9,P0.000 1;χ~2=57.010 3,P0.000 1)。结论如实验室没有荧光显微镜,芝加哥天蓝染色法检测甲真菌病患者标本效果较好。     

Localization of chemotactic peptide receptors on rabbit neutrophils

The chemotaxis of blood leukocytes is initiated by the binding of a chemoattractant to specific receptors on the leukocyte cell surface. Although a great deal is known about the biochemical and morphological events accompanying chemotactic activation, there is very little morphological information about the chemoattractant receptors themselves. This latter information is needed so that we may understand the mechanism by which these inflammatory cells detect and respond to chemical gradients. One class of chemotactic factors extensively used to characterize the complex behavioral responses following leukocyte activation are the synthetic formylmethionyl peptides. These peptides, now known to be the analogs of the naturally occurring N-terminal peptides produced by bacteria, are released into culture medium and are believed to be responsible, at least in part, for the accumulation of leukocytes at the sites of bacterial infection. We have localized the receptors for the chemotactic hexapeptide N-formylnorleucyl-leucyl-phenylalanine-norleucyl-[125I]tyrosyl-lys ine [N-fNle-Leu-Phe-Nle-[125I]Tyr-Lys] on whole rabbit peritoneal neutrophils (PMN) using light microscope autoradiography. By this method, the inherent formylpeptide receptor distribution on cells incubated at 4 degrees C appears to be uniform over the surface of both rounded and structurally polarized PMN. Following a short 37 degrees C incubation, cells retain a large proportion of labelled hexapeptide at or near the cell surface and, in addition, polarized PMN redistribute the hexapeptide anteriorly away from the cell uropod.     

Interaction of iodinated enkephalin analogues with opiate receptors.

Radioiodinated derivatives of the metabolically stable enkephalin analogues, [DAla²,Leu⁵]- and [DAla²,DLeu⁵]-enkephalin, have been prepared. Such derivatives show sterospecific binding to receptors in brain homogenates and some neuroblastoma cell lines such as NG108-15 and N4TG1. The relative effects of levorphanol and dextrorphan and Na⁺ and Mn⁺⁺ ions on enkephalin binding in brain and cells indicate that the iodinated derivatives are interacting with opiate receptors. Levorphanol is considerably more potent in displacing specifically bound enkephalin than dextrorphan. Sodium ions at physiological concentrations decrease enkephalin binding whereas manganese ions enhance it. Unlabelled monoiodo derivatives retain high potency in the guinea-pig ileum, mouse vas deferens and receptor binding assays. Unlabelled diiodo derivatives show far lower potency in these assays. It is concluded that radio-iodinated derivatives containing one iodine per molecule retain high affinity for the opiate receptor but diiodo derivatives do not.     

Embryogenesis of peripheral nerve pathways in grasshopper legs. I. The initial nerve pathway to the CNS

The founding of the first nerve path of the grasshopper metathoracic leg was examined at the level of identified neurons, using intracellular dye fills, immunohistochemistry, Nomarski optics, and scanning and transmission electron microscopy. The embryonic nerve is established by the axonal trajectory of a pair of afferent pioneer neurons, the tibial 1 (Ti1) cells. Following a period of profuse filopodial sprouting, the Ti1 axonal growth cones, possessing 75- to 100-microns-long filopodia, navigate a stereotyped path across the limb bud epithelium to the base of the appendage and into the CNS. The Ti1 axons grow from cell to cell along a chain of preaxonogenesis neurons spaced at intervals along the pathway, forming dye-passing junctions with them. The contacted neurons subsequently undergo axonogenesis and follow the pioneer axons into the CNS. Later arising neurons project their axons onto the cell bodies of the chain, thereby establishing the principal branch points of the nerve. Among the later arising afferents are the sensory neurons of the femoral chordotonal and subgenual organs. The morphology of the adult nerve appears to be determined by the stereotyped positioning of neurons in the differentiating limb bud and by the resultant axonal trajectories established during the first 10% of peripheral neurogenesis.     

Embryogenesis of peripheral nerve pathways in grasshopper legs. III. Development without pioneer neurons

We have examined the consequence of deleting the first pathfinding neurons to differentiate in the metathoracic leg, cell pair tibial 1 (Ti1) (C. M. Bate, 1976, Nature (London) 260, 54-56; H. Keshishian, 1980, Dev. Biol. 80, 388-397) on the development of two uniquely identifiable follower sensory neurons, and upon the subsequent development of nerve 5B1 in the leg. Following the equivalent of 10-15% of embryonic development in culture the follower sensory neurons were found to have formed topologically normal axonal trajectories in the leg, and to have established contacts with later differentiating sensory and motor axons in an essentially normal fashion. The results show that followers can navigate the route normally taken by the pioneers, and suggest that the pioneers do not have unusual pathfinding capabilities.     

Embryogenesis of peripheral nerve pathways in grasshopper legs. II. The major nerve routes

In the preceding paper (H. Keshishian and D. Bentley, 1983a, Dev. Biol. 96, 89-102) the events leading to the morphogenesis of nerve 5B1 in the grasshopper embryonic metathoracic leg were presented. Here the role of later differentiating peripheral neurons in establishing the other major nerves of the leg is examined. In addition to the (tibial 1) (Ti1) pioneer neuron cell pairs that establish nerve 5B1 in the tibia femur, and coxa-trochanter, six later differentiating cells and/or cell pairs were identified and examined with respect to their role in peripheral nerve ontogeny. Nerve path pioneering was observed in two cell pairs of the distal tarsus (Ta1 and Ta2), by neurons of the posterior proximal tibia (Ti2), the posterior midfemur (neurons F3 and F4), and by an additional cell pair in the anterior coxal-trochanteral region of the limb bud (cell pair, CT2). In addition, efferent projections onto limb and epithelia played an important role in establishing nerve branches. In two nerves the axonal trajectory from the periphery to the CNS is established by afferent and efferent pathfinding axons meeting halfway and overgrowing each other's established projections. For each nerve branch examined it was found that axons projected initially to the cell bodies of previously arising neurons along the trajectory. The location along the limb bud ectoderm where neurons arise, and hence their ultimate cell body positions, played an important role in organizing the fasciculation of follower axons and establishing branch points.     

Analysis of population cytokinetics of chick myocardial cells in tissue culture

The growth of embryonic chick cardiac myocytes and fibroblasts in tissue culture was evaluated by the kinetics of nuclear labeling during continuous exposure to [3H]thymidine. The fraction of mitotically active cells, the mean intermitotic period and the population doubling times were determined in each cell type during 3 weeks in culture. After 24 hr in culture, 90% of the muscle cells were mitotically active with minimal population doubling times of 65 hr. By 17 days in culture only 5% of the myocytes continued to divide with population doubling times greater than 3000 hr. Primarily, the lengthening of doubling times was due to a withdrawal of cells from the mitotic cycle and much less to a lengthening of the intermitotic period. Growth of cardiac muscle cells from embryonic hearts from 4 to 10 days of development was also compared. Muscle cells from younger hearts displayed greater mitotic activity than those from older hearts at equivalent times in culture.     

Turnover of chloroplast and cytoplasmic ribosomes during gametogenesis in Chlamydomonas reinhardi

A number of novel observations on ribosomal metabolism were made during gametic differentiation of Chlamydomonas reinhardi. Throughout the gametogenic process the amount of chloroplast and cytoplasmic ribosomes decreased steadily. The kinetics and extent of such decreases were different for each of the two ribosomal species. Comparable rRNA degradation accompanied this ribosome degradation. Concurrent with the substantial ribosome degradation was the synthesis of rRNA, ribosomal proteins and the assembly of new chloroplast and cytoplasmic ribosomes throughout gametogenesis. The newly synthesized chloroplast ribosomes exhibited distinctively faster turnover than their cytoplasmic counterpart. Cytoplasmic ribosomes, pulse-labeled in early gametogenic stages, retained label until differentiation was nearly complete even though a net decrease in the level of cytoplasmic ribosomes continued, indicating that the newly synthesized cytoplasmic ribosomes were preferentially retained during differentiation. Hence the regulation of ribosome metabolism during gametogenesis contrasts with the conservation of ribosomes obtained during vegetative growth of C. reinhardi and other organisms. This unique pattern of ribosome metabolism suggests that new ribosome synthesis is necessary during gametogenesis and that some specific structural or functional difference relating to the development stage of the life cycle might exist between degraded and newly synthesized ribosomes.     

Intact B-cell activity is essential for complete expression of experimental allergic encephalomyelitis in Lewis rats

The mechanisms of the inhibitory effect of the serum thymic factor (FTS) on allograft immunity have been studied on both cellular and humoral immune responses of skin allografted mice. FTS-induced suppression of allogeneic skin graft rejection was correlated with a transient diminution of in vivo alloreactive CTL production in the spleen, whereas the generation of allo-anti-H-2 antibodies was not affected. The involvement of suppressor cells in the effect of FTS was supported by the observation that irradiated spleen cells from FTS-treated recipients (bearing a 9-day allograft) suppressed the in vitro CTL generation.