Intracellular introduction of a fixed quantity of substances by pricking cells using a modified microscope

We report here that pricking cells with a microneedle causes introduction into cells of a fixed quantity of material that is contained in the culture medium. Using fragment A of diphtheria toxin (21 500 D), the volume of injected external medium by a single prick was determined (1.4× 10⁻¹⁵l (μm³)). We also show that immunoglobulin G molecules of 1.6 × 10⁵ D can be introduced into cells by this technique. The speed of the microinjection by this pricking method is fast (by using a modified microscope 2 000–7 000 cells/h).     

Morphological evidence for the innervation of apocrine sweat glands in the general hairy skin of the goat

Summary A histomorphological and histochemical study was made on the nerve supply to the apocrine sweat glands in the general hairy skin of the goat. In harmony with the previous report that the sweat glands in the goat are functionally under the control of sympathetic nervous system, the present study clearly demonstrates cholinesterase-reactive nerve fibers that closely surround the secretory portion of these glands in most of the hairy skin area, though the nerve network is fairly coarse. Analysis with cholinesterase inhibitors indicated that the sudomotor nerves in the goat contain both specific and non-specific cholinesterase.     

New Bradyrhizobium japonicum Strains That Possess High Copy Numbers of the Repeated Sequence RSα

In a survey of DNA fingerprints of indigenous Bradyrhizobium japonicum with the species-specific repeated sequences RSα and RSβ, 21 isolates from three field sites showed numerous RS-specific hybridization bands. The isolates were designated highly reiterated sequence-possessing (HRS) isolates, and their DNA hybridization profiles were easily distinguished from the normal patterns. Some HRS isolates from two field sites possessed extremely high numbers of RSα copies, ranging from 86 to 175 (average, 128), and showed shifts and duplications of nif- and hup-specific hybridization bands. The HRS isolates exhibited slower growth than normal isolates, although no difference in symbiotic properties was detected between the HRS and normal isolates. Nucleotide sequence analysis of 16S rRNA genes showed that HRS isolates were strains of B. japonicum. There was no difference in the spectra of serological and hydrogenase groupings of normal and HRS isolates. Some HRS isolates possessed a tandem repeat RSα dimer that is similar to the structure of (IS30)₂, which was shown to cause a burst of transpositional rearrangements in Escherichia coli. The results suggest that HRS isolates are derived from normal isolates in individual fields by genome rearrangements that may be mediated by insertion sequences such as RSα.     

Generation of Escherichia coli O9a Serotype, a Subtype of E. coli O9, by Transfer of the wb* Gene Cluster of Klebsiella O3 into E. coli via Recombination

Genetic characterization of the wb* gene in a series of Escherichia coli and Klebsiella strains possessing the mannose homopolymer as the O-specific polysaccharide was carried out. The partial nucleotide sequences and PCR-restriction fragment length polymorphism analysis suggested that E. coli serotype O9a, a subtype of E. coli O9, might have been generated by the insertion of the Klebsiella O3 wb* gene into a certain E. coli strain.     

Electron Donation from Cyclic and Respiratory Flows to the Photosynthetic Intersystem Chain is Mediated by Pyridine Nucleotide Dehydrogenase in the Cyanobacterium Synechocystis PCC 6803

The redox kinetics of P700 induced by far-red light and a pulseof strong white light in wild type cells were compared withthose in NAD(P)H dehydrogenase (NDH)-defective mutants of thecyanobacterium Synechocystis PCC 6803. The wild type cells showedthe electron donation from the respiratory donor and the photoreductantgenerated in PS I to P700⁺ through the plastoquinone, whichis mediated by a Hg²⁺-sensitive enzyme. The NDH-defective mutantcells, however, did not show the electron transfer to P700⁺through the plastoquinone from both the photoreductant in PSI and cytosolic electron donors using pyndine nucleotides asan intermediate. Thus, NDH appears to be the site of main entryof electrons into the plastoquinone pool in the NAD(P)H-mediatedcyclic electron flow and the respiratory electron flow in Synechocystis. (Received August 31, 1992; Accepted October 1, 1992)     

Sugars containing a carbon-phosphorus bond : Part V. 5-deoxy-5-(ethylphosphinyl)-D-ribopyranose

The Michaelis-Arbuzov reaction of methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-β-D-ribofuranoside (4) with diethyl ethylphosphonite gave methyl 5-deoxy-5-(ethoxyethylphosphinyl)-2,3-O-isopropylidene-β-D-ribofuranoside (5) which, on treatment with sodium dihydrobis(2-methoxyethoxy)aluminate, afforded methyl-5-deoxy-5-(ethylphosphinyl)-2,3-O-isopropylidene-β-D-ribofuranoside (9). Hydrolysis of 9 with hydrochloric acid yielded a mixture of the anomeric 5-deoxy-5-(ethylphosphinyl)-D-ribopyranoses (10). The hygroscopic, syrupy mixture 10 was converted into a syrup consisting of the two 1,2,3,4-tetra-O-acetyl-5-deoxy-5-(ethylphosphinyl)-D-ribopyranoses (11).     

Foreign protein can be carried into the nucleus of mammalian cell by conjugation with nucleoplasmin

In studies on the specific migration of macromolecules across the nuclear envelope, a karyophilic protein was injected into the cytoplasm of cultured cells and its subsequent location in the cell was examined. Nucleoplasmin of frog nuclear protein was used for this experiment. When [125I]nucleoplasmin was introduced into the cytoplasm of mammalian cells (human and mouse) by red blood cell-mediated microinjection, it rapidly accumulated in the nucleus. When nucleoplasmin conjugated with [125I]IgG against chromosomal protein was introduced similarly, it also accumulated rapidly in the nucleus, and reacted with its antigen inside the nucleus. On the contrary, when IgG alone or IgG conjugated with BSA were introduced, they did not migrate from the cytoplasm into the nucleus. These findings imply that the migration of macromolecules from the cytoplasm to the nucleus does not depend only on their molecular size but also on a specific transport mechanism, and that karyophilic proteins may act as useful carriers in the transfer of exogenous proteins into the nucleus.