ATP-dependent transport for glucuronides in canalicular plasma membrane vesicles

Using rat liver canalicular plasma membrane vesicles, it has been verified that the transport of p-nitrophenyl glucuronide (NPG) across membranes is an ATP-dependent process; the apparent Km for NPG was 20 microM. S-(2,4-dinitrophenyl)-glutathione (DNP-SG) inhibited NPG uptake dose-dependently, and NPG or testosterone glucuronide did ATP-dependent DNP-SG uptake similarly. These results suggest that transport of glucuronide is mediated by an ATP-dependent glutathione S-conjugate carrier.     

A rapid immunogold-silver staining for detection of bromodeoxyuridine in large numbers of plastic sections, using microwave irradiation

Summary A rapid and convenient method for the large scale, immunogold-silver staining (IGSS) of bromodeoxyuridine (BrdU) incorporated by S phase cells, by means of a monoclonal antibody (anti-BrdU) is described. Nineteen slides at a time can be incubated with the antibodies and the protein A-gold (PAG) in staining jars. The antibody and protein A-gold solutions could be used at least five times to incubate new batches of slides. The incubation times with these solutions were shortened by means of microwave irradiation. In this way 200 slides carrying at least 800 sections could be easily processed under the same conditions in one day, using 1.25ml neat antibody solutions of anti-BrdU and rabbit anti-mouse.For light microscopy bothpplastic embedding systems: methylmethacrylate (MMA) and glycolmethacrylate (GMA) can be stained with this technique. The MMA sections, of which the plastic has to be removed before the IGSS, has the advantage of a stronger labelling intensity. The GMA plastic, which contains a cross-linking, agent cannot be removed and consequently for GMA sections it is necessary to incubate the sections with a proteolytic enzyme (trypsin) before the IGSS, to reexpose the antigenic binding sides. However, the GMA sections can be allowed to air dry during the IGSS without negative effects on the morphology. This makes it possible to perform the antibody and the PAG-incubating steps on one day and to finish the IGSS the next day. In this way twice as many GMA slides can be incubated with the same antibody and PAG solutions than with MMA slides.In both plastic embedding systems the intensity of the BrdU labelling was found to be stronger in Carnoy's than in Bouin's fixed sections.     

Ca2+-dependent deimination-induced disassembly of intermediate filaments involves specific modification of the amino-terminal head domain

Peptidylarginine deiminase (proteinarginine iminohydrolase, EC 3.5.3.15) converted some arginine residues to citrulline residues in soluble vimentin, in a micromolar Ca2+-dependent manner and resulted in the loss of polymerization competence of the intermediate filament protein. When about 8 mol of residues/mol of vimentin were deiminated, there was a complete loss of filament forming ability. This enzyme also deiminated vimentin filaments which had been polymerized, and deimination of vimentin filaments resulted in filament disassembly. Similar results were obtained with other intermediate filaments such as desmin and glial filaments. High performance liquid chromatography and amino acid analyses of lysine-specific protease-generated fragments from deiminated vimentin (about 8 mol of citrulline/mol of vimentin) showed a differential deimination of three structural domains. The head domain was predominant. These observations suggest that the head domain strongly influences integrity of the intermediate filament.     

Elements of theS-gene complex VI. Mutations of the self-incompatibility gene,pseudo-compatibility and origin of new self-incompatibility alleles

Spontaneously occurring mutations of theS gene, involving both theS _I and theS _FI classes of alleles, were studied inNicotiana alata. The results showed that while almost all of the irradiation-induced mutants of theS gene requiredS-bearing duplication for their survival, usually in the form of a free fragment, most of the spontaneous mutants in the same species, surprisingly, did not have such a requirement. This difference has been attributed to the greater depth of mutations produced in response to the ionizing radiations, which necessitated complementation for the survival of the mutants. There is a possibility from the data that theS _FI class of alleles may have even less need for the duplication than the S_I class of alleles. Both pollen- and stylar-part mutations of theS gene were obtained, but the majority of the mutations were partial, producing less than half the normal complement of seeds per pod in the mutants. Complementation was observed in the style between a -part mutant alleleS _infF11 ^sup and a normal alleleS _F10, which was the other allele in the parental plant that produced the mutant. No complementation occurred with another normal unrelated alleleS ₂. This observation was similar to that previously recorded in the study of induced mutants inN. alata.In a cross where the two alleles of the pollen parent were both compatible the allele which was also a mutant had an advantage over the other, normal, allele. This suggests that in maize, where the occurrence of mutant forms of theS gene has been demonstrated, the preferential fertilization of ovules by pollen containing the B-chromosomes may be due to the presence of a mutant form of theS gene on the B-chromosome.Besides clear-cutS-gene mutants, there were others, showing mostly irregular, slight compatibility, which did not appear to be directly related to theS-gene mutation. In some of the progeny of certain of these mutants, partial or complete lack of the specificity of one or bothS alleles in the style was observed; in certain others all progeny were normal. This pseudo-compatibility is attributed to cytoplasmic mutations affecting the products of theS gene; however, the possibility of an effect of chance polygenic modifier combinations is not ruled out.Recent literature on theS-gene structure, mutational specificity ofS alleles, and genetic control of pseudo-compatibility is reviewed. The time ofS-gene action is discussed in relation to the mechanism of the generation of new self-incompatibility alleles.     

c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract

In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a netword similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.     

Electron spin echo studies on chloroplasts. Spectral characteristics of electron transport components and light-induced transients

Electron-spin resonance echoes are used to study the complex overlapping ESR spectra of whole chloroplasts. By varying the repetition rate of the microwave pulse sequence, delay time, and pulse width, signals with different longitudinal and transverse relaxation times were extracted. We have identified the echo signals due to plastocyanin and ferredoxins. In addition, we have found a strong signal at g = 4.3, that possibly arises from distorted cytochrome, and weak signals in the region g = 6-9. The strong echo signal at g = 2.0047 (Signal II), is made up of at least three "dark" components that differ in their relaxation times. Upon illumination at 1.2 K several of the echo signals including Signal II show reversible light-induced components. The kinetics of these transients depend on the addition of 3(3,4-dichlorophenyl)-1,1-dimethyl urea. Part of the transients are believed to arise from cyclic electron flow around Photosystem I.     

Characterization of two different factors chemotactic for cancer cells from tumor tissue.

Two different factors chemotactic for cancer cells were extracted in pseudoglobulin fraction of rat ascites hepatoma transplanted tissue. After chromatography on Sephadex G-50 and CM-sephadex, these factors were separated by gel filtration on Sephadex G-100. The factor a was further fractionated by immunoadsorbent chromatography with goat antirat gamma-globulin antibody and then with rabbit antirat hemoglobin antibody; it was a protein with a molecular weight of about 78,000, resembling a chemotactic factor previously reported, and its activity was thermolabile. The previously undescribed factor b was also a protein with a molecular weight of about 14,000 and its activity was thermostable. Intradermal injection of these factors at low concentrations induced an extravascular migration of circulating tumor cells and formation of metastatic secondary tumors; and little difference in the in vivo effect between these factors was observed. It was thus assumed that the combined action of these two factors may be involved in malignant invasion.     

Origin of genetic variation: regulation of genetic recombination in the higher organisms — a theory

Summary Recent studies in the fungi, particularly Neurospora and Schizophyllum, have revealed a number of genetic features which, viewed in conjunction with earlier observations on other organisms, form a pattern, or model, which appears to be basic to the control of recombination in all eukaryotes, including higher organisms. It is assumed that the control is exercised on mechanisms that produce new alleles through recombination, as understood in broad terms and including such a likely phenomenon as gene conversion, which may or may not involve crossing-over, as well as equal and unequal crossing-over. The recombination may thus occur between alleles in either the homozygous or heterozygous condition. In the model, regulatory genes and breeding behaviour are integrated into one self-regulatory system controlling the production of new genetic variation.The model is based on the following five general features, largely substantiated by the results in Neurospora and Schizophyllum: 1) The frequency of recombination in a particular chromosomal region is controlled by specific regulatory genes (rec). 2) There may be a number of such specific, regulatory genes responsible for recombination in a given region. 3) A rec. locus may influence recombination in more than one region. 4) The regulatory genes have no specific physical relationship with the region(s) they control, and are usually located at random in the genome. 5) Of the allelic forms of the regulatory genes it is always the dominant gene which suppresses recombination and the recessive gene which increases recombination. The rec system is epistatic to other genetic elements jointly involved in the overall control of recombination in a specific region. It is suggested that usually the control of recombination in a given region is exercised, cumulatively, by the balance of the dominant and recessive genes of the specific rec loci in the organism. Outbreeding, with the associated high heterozygosity of the regulatory rec loci, virtually switches off recombination, producing few new variations. Inbreeding produces homozygosity of these loci, resulting in certain individuals which will have a considerable number of their regulatory loci in the homozygous recessive condition and in which recombination will be switched on, producing new variation at a high frequency. Inbreeding is thus an integrated, evolutionary system of considerable importance, and is not a degenerate dead end, as many investigators have previously thought.The model has another compensatory function in evolution. In major loci, or in an operon, where there are structural genes and closely linked operator genes, as exemplified by the S locus, there are indications that the present model is concerned with the regulation of both structural and operator genes. The consequences of the model in the two classes of genes, however, are in direct contrast to each other: High heterozygosity which is instrumental in switching off recombination, and which is therefore helpful in maintaining stability in the structural gene, is conducive to functional variation of the operator gene; and high homozygosity, which is instrumental in switching on recombination, and which is therefore helpful in producing variation in the structural gene, is conducive to the stability of the operator gene.This model of the control of genetic variation in a specific chromosomal region is significant in development as well as in evolution, and throws light on a number of hitherto intractable problems peculiar to the higher organisms. For example, the model is helpful in explaining: 1) the origin of new self-incompatibility alleles in the flowering plants; 2) the impressive speciation in the waif flora (and fauna) of the oceanic islands; 3) the presence of high genetic variability in inbreeding species of plants; 4) environmentally-induced heritable variation in certain plants; and 5) the genetic mechanism of antibody diversity in animals.