Microexplant isolation from Cactaceae

A method of explant isolation suitable for Cactaceae is described. Small pieces of tissue were removed with a syringe without causing substantial plant injury. Using this method callus cultures were obtained in several Cactaceae species.     

Spectroscopic analysis of [Trp3]-beta-casomorphin analogs. Comparative structure conformation-activity studies

A series of [3-tryptophan]-beta-casomorphin-5([Trp3]-beta-CM-5) analogs were investigated by circular dichroism (CD) and fluorescence spectroscopy to explore their structure-conformation properties in solution. In addition, the comparative opioid activities of these compounds were evaluated using the in vitro guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Specifically, the pentapeptide sequence of [Trp3]-beta-CM-5, H-Tyr-Pro-Trp-Pro-Gly-OH (I) was modified at Pro-2 and Pro-4 by D-Pro substitutions to provide two diastereometric analogs, [Trp3-D-Pro-4]-beta-CM-5 (II) and [D-Pro2,4,Trp3]-beta-CM-5 (III). In the GPI and MVD assays, beta-CM-5 effected IC50 values of 1.3 microM and 8.9 microM, respectively, which confirmed its known mu/delta-selectivity on these two peripheral opioid receptor subtypes. The potencies of compounds I, II, and III were 0.2, 2.0, and less than 0.005 relative to beta-CM-5 on the GPI assay. Compounds I and II exhibited pronounced mu/delta-selectivities (greater than 18.9- and 12.4-fold respectively), whereas compound III was essentially inactive in both the GPI and MVD assays. CD studies of beta-CM-5 and its [Trp3]-beta-CM-5 analogs showed striking differences in their near-UV and far-UV spectra in aqueous or organic solvents. In the far UV CD spectra, weak (20%) alpha-helicity (maximum at 193 nm and minima at 208 and 222 nm) for beta-CM-5 was obtained in trifluoroethanol (TFE); however, none of the [Trp3]-beta-CM-5 analogs showed such CD bands. Of potential relevance to gamma-turn or C7 secondary structure was the observation of a strong negative band at 245 nm for compounds II and III which was not solvent-dependent in H2O or TFE, whereas compound I showed this CD band exclusively in TFE. In the near-UV CD at 275 nm (Trp electronic transition), the relative order of intensities of this band were determined for the [Trp3]-beta-CM-5 compounds to be II greater than I greater than III, which was identical to their relative biological potencies in both the GPI and MVD assays. Fluorescence energy transfer (FET) experiments of compounds I-III provided the intramolecular distances (r) between their Tyr (donor) to Trp (acceptor) side-chains, by the F?rster method, and were as follows: [Trp3]-beta-CM-5, r = 10.6 A; [Trp3, D-Pro4]-beta-CM-5, r = 9.6 A; and [D-Pro2,4,Trp3]-beta-CM-5, r = 11.0 A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physiological fatty liver and hyperlipemia in the fetal guinea pig: chemical and ultrastructural characterization

During its prolonged period of gestation, the fetal guinea pig gradually develops a striking hyperlipemia (plasma triglycerides ca. 500-1500 mg/dl) and fatty liver (hepatic triglycerides ca. 25% of wet weight). The parenchymal cells of the liver contain not only many fat droplets in the cytoplasm, but also large numbers of osmiophilic particles, interpreted as precursors of plasma lipoproteins, within profiles of the cisternae and secretory vesicles of the Golgi apparatus. Similar particles are found in intercellular spaces, in the space of Disse, and in the hepatic sinusoids. Near the end of gestation, these particles enlarge to the size range characteristic of chylomicrons secreted from the intestinal mucosa after ingestion of fat. At the same time, the hyperlipemia increases and is characterized by the accumulation of particles resembling chylomicrons morphologically and chemically. The results are interpreted as evidence of intense hepatic synthesis and secretion of very low density lipoproteins which may be related to the extensive transplacental transport of free fatty acids known to occur in this species. After birth, the hyperlipemia subsides rapidly and the hepatic steatosis more gradually. The blood plasma of the guinea pig fetus also contains moderate amounts of low density and high density lipoproteins. The latter decrease to barely detectable levels during the first 2 wk of postnatal life. Comparably low levels of high density lipoproteins are found in nonpregnant and pregnant adults.     

Fluorescence quenching studies of bovine growth hormone in several conformational states

The use of steady-state fluorescence quenching methods is reported as a probe of the accessibility of the single fluorescent tryptophan residue of bovine growth hormone (bGH, bovine somatotropin, bSt) in four solution-state conformations. Different bGH conformations were prepared by using previous knowledge of the multi-state nature of the equilibrium unfolding pathway for bGH: alterations in denaturant and protein concentration yielded different bGH conformations (native, monomeric intermediate, associated intermediate and unfolded). Because the intramolecular fluorescence quenching which occurs in the native state is reduced when the protein unfolds to any of the other conformations, steady-state fluorescence intensity measurements can be used to monitor bGH unfolding as well as the formation of the associated intermediate. These steady-state intensity changes have been confirmed with fluorescence lifetime measurements for the different conformational states of bGH. Fluorescence quenching results were obtained using the quenchers iodide (ionic), acrylamide (polar) and trichloroethanol (non-polar). Analysis of the results for native-state bGH reveals that the tryptophan environment is slightly non-polar (in agreement with the emission maximum of 335 nm) and the tryptophan is more exposed to acrylamide than most native-state tryptophan residues which have been studied. The tryptophan is most accessible to all quenchers in the unfolded state, because no steric restrictions inhibit quencher interaction with the tryptophan residue. The iodide quenching results indicate that the associated intermediate tryptophan is not accessible to iodide, probably due to negative charges inhibiting iodide penetration. The associated intermediate tryptophan is less accessible to all three quenchers than the monomeric intermediate tryptophan, due to tight packing of molecules in the associated intermediate state.     

Apoptosis in Plants

Apoptosis is a feature of animal cells that explains some aspects of programmed cell death in plants. Differences between plant and animal cell development require that concepts be reexamined to signify how plant cells have evolved the need for cell elimination in the meristematic growth habit, life cycle, and alternation of generations. Central to this theme is the regulation of divisional cycles for mitosis, meiosis, apomeiosis, and their related sexual and asexual reproductive processes. Apoptosis depends on the coordinated expression of genes regulating divisional cycles and apoptotic pathways so that irreversible nuclear and cytoplasmic elimination occurs. Cellular degradation products are salvaged to sustain adaptation, viability, structural function, and ontogeny. The cell wall is usually retained and further differentiated or eliminated. A model of factors predisposing apoptosis and comprising checkpoints in cell divisional cycles is presented for comparisons among plant and animal cells.     

The significance of lipoprotein lipase in rat skeletal muscles.

Lipoprotein lipase was assayed in extracts of acetone-ether powders of rat skeletal muscles. Enzyme activity in soleus had typical characteristics of lipoprotein lipase in other tissues: inhibition by molar NaCl and protamine sulfate and activation by the human apolipoprotein, R-glutamic acid. Activity in muscles with predominantly red fibers (soleus, diaphragm, lateral head of gastrocnemius and anterior band of semitendinosus) was higher than in those with predominantly white fibers (body of gastrocnemius and posterior band of semitendinosus). No effect of a 24 hour fast upon enzyme activity was observed in ten skeletal muscles, but activity decreased substantially in four adipose tissue depots and increased slightly in heart muscle with fasting. Four minutes after intravenous injection of labeled lymph chylomicrons, skeletal muscles with predominantly red fibers incorporated several times more chylomicron triglyceride fatty acids than thos with predominantly white fibers. Estimated lipoprotein lipase activity in total skeletal muscle was about two-thirds that in total adipose tissue of rats fed ad libitum. After a 24 hour fast, total activity in skeletal muscle was about twice that in adipose tissue. These data suggest that a substantial fraction of lipoprotein lipase is in skeletal muscle of rats and that this tissue, especially its red fibers, is an important site of removal of triglycerides from the blood.     

The study of the conditions for the fertilizationin vitro in maize

Suitable conditions for the fertilizationin vitro in maize have been studied. The germinating capacity of pollen in synthetic media was low; it was confirmed that it might be stimulated by supplementing agar with egg yolk. Application of pollen onto styles overhanging from the culture medium of excised ovaries was examined. After 5 days the styles could be cut off the ovaries, for the pollen tubes had already penetrated the embryo sacs. However, better results were obtained when cultivating ovaries along with segments of the maize cob. Solid media were more suitable for the development of kernels. Some of them germinatedin situ and gave rise to normal plants. From nucellar meristems of young kernels a callus could be derived which, on further cultivation, became green and regenerated shoots and roots. The cells of meristems exhibited a varying number of chromosomes.