Loss of the Xeroderma Pigmentosum Group A Gene (XPA) Enhances Apoptosis of Cultured Cerebellar Neurons Induced by UV but Not by Low-K+ Medium

Abstract: To study the involvement of the xeroderma pigmentosum group A gene ( XPA ) in neuronal apoptosis, we cultured cerebellar neurons from mice lacking XPA gene ( XPA ^−/−) and induced apoptosis by exposure to UV irradiation or medium containing a low concentration of potassium (low-K⁺ medium). When cerebellar neurons from postnatal days 15–16 wild-type mice were treated with UV irradiation, apoptotic neuronal death was observed after 24–48 h. About 60% of neurons survived 48 h after UV irradiation at a dose of 5 J/m². On the other hand, neurons from XPA ^−/− mice showed a significantly increased vulnerability to UV irradiation, and >90% of neurons died 48 h after UV irradiation at a dose of 5 J/m². In contrast, low-K⁺ medium induced apoptosis of neurons from mice of each genotype with the same kinetics. These results suggest that the XPA gene is involved in neuronal DNA repair and that it thereby influences apoptosis induced by DNA damage in cultured cerebellar neurons.     

Intracellular Photoreceptive Site for Blue Light-Induced Cell Division in Protonemata of the Fern Adiantum--Further Analyses by Polarized Light Irradiation and Cell Centrifugation

The intracellular localization of the photoreceptive site forblue light-induced cell division in single-celled protonemataof Adiantum capillus-veneris L. was investigated using polarizedlight irradiation and protonemal cell centrifugation. The responseto irradiation with polarized blue light showed no dependenceon the direction of light polarization. However, centrifugationof the protonemata followed by microbeam irradiation showedthat the site of blue light perception could be displaced togetherwith the nucleus. Centrifugal treatment changed the distributionof intracellular organelles at the time of light exposure andbasipetally displaced the nucleus about 90µm. This treatmenthad no effect on the induction of cell division with blue lightif the protonemata were centrifuged again acropetally afterthe light treatment. Microbeam (30x30 µm²) irradiationwith blue light of the apical 45–75 ßm region,the receptive site of blue light in non-centrifuged cell, didnot induce cell division. However, cell division was inducedby irradiation of the nucleus-containing region, indicatingthat the photoreceptive site was displaced together with thenucleus by the centrifugation. These results suggest that theblue light receptor regulating cell division in Adiantum protonematais not likely to be located on the plasma membrane. (Received February 20, 1986; Accepted May 27, 1986)     

Circadian Period Modulation and Masking Effects Induced by Repetitive Light Pulses in Locomotor Rhythms of the Cricket, Gryllus bimaculatus

Effects of 15 min light pulses given at various intervals (every 1, 2, 4, 6, 8 and 12 hr) under constant darkness on the locomotor rhythm were investigated in the adult male cricket, Gryllus bimaculatus. A single pulse per 24 hr induced period modulation in a circadian phase dependent manner, yielding a period modulation curve (PMC): the 15 min light pulse lengthened the period in the early subjective night (CT11-16) and shortened it during the late subjective night to the early subjective day (CT20-5). Frequent light pulses modulated the freerunning period of the rhythm dependent on the interval of the pulses: when compared with the freerunning period in DD (23.74 +/- 0.03 hr) the period was significantly shorter in intervals of 2 and 4 hr, but lengthened when the interval was 1 and 12 hr. Frequent light pulses also resulted in entrainment of the rhythm to run with the period of 24 hr and the ratio of the entrained animals varied from 12% to 72% depending on the interval of the light pulses. The period modulation and the entrainment by the repetitive light pulses could be interpreted according to the PMC. In about 15% of animals, the light pulses induced a rhythm dissociation, suggesting that the bilaterally paired circadian pacemakers have their own sensitivity to the entraining photic information. The light pulse caused a masking effect, i.e., an intense burst of activity. The magnitude of the light induced responses was dependent on the circadian phase. The strongest masking effect was observed in the subjective night. The phase of the prominent period modulation and of the marked masking effects well coincides with the previously reported sensitive phase of the photoreceptive system.     

Regulation of Cell Wall Synthesis by the Clathrin Light Chain Is Essential for Viability in Schizosaccharomyces pombe

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.     

Perithecial Formation in Gelasinospora reticulispora VII: Action Spectra in UV Region for the Photoinduction and the Photoinhibition of Photoinductive Effect Brought by Blue Light

An action spectrum for photoinduction of perithecial formationafter a prior 72 h dark growth period was determined in theUV region with apically growing mycelia of a sordariaceous fungus,Gelasinospora reticulispora. The spectrum exhibited a peak at280 nm. Quantum effectiveness of 280 nm irradiation was ca.1.7 times higher than that of 450 nm light. The number of peritheciainduced by UV radiation was saturated at a lower level as comparedwith blue light. UV radiation having a fluence greater thanthe saturation level decreased the number of induced perithecia.UV radiation that was given after a saturating exposure to inductiveblue light inhibited the inductive effect of blue light. Anaction spectrum for this inhibition exhibited a peak between260 and 270 nm. Monochromatic light beyond 350 nm had no inhibitoryeffect. Inhibitory effects of UV radiation given after inductiveblue light irradiation were observed in the fluence range wherephotoinductive effects of UV radiation became obvious. Therefore,the true height of the UV peak in the photoinduction actionspectrum,when free of distortion from the inhibitory effect, should behigher than the peak obtained in this study. (Received August 20, 1983; Accepted November 4, 1983)     

The Selection of Amber Mutations in Genes Required for Completion of Start, the Controlling Event of the Cell Division Cycle of S. CEREVISIAE

Using a modification of a procedure developed for the isolation of temperature-sensitive mutants defective in the start event of cell division, amber mutations were obtained for two Class-I start genes, cdc28 and cdc37. Genetic analysis demonstrated that co-segregation of an amber suppressor with such alleles was required for viability of spores subsequent to meiosis. These mutations are expected to be useful in the identification of the molecular products of the genes cdc28 and cdc37.     

Temperature-Sensitive Cdc 7 Mutations of Saccharomyces Cerevisiae Are Suppressed by the Dbf4 Gene, Which Is Required for the G(1)/S Cell Cycle Transition

When present on a multicopy plasmid, a gene from a Saccharomyces cerevisiae genomic library suppresses the temperature-sensitive cdc7-1 mutation. The gene was identified as DBF4, which was previously isolated by complementation in dbf4-1 mutant cells and is required for the G1----S phase progression of the cell cycle. DBF4 has an open reading frame encoding 695 amino acid residues and the predicted molecular mass of the gene product is 80 kD. The suppression is allele-specific because a CDC7 deletion is not suppressed by DBF4. Suppression is mitosis-specific and the sporulation defect of cdc7 mutations is not suppressed by DBF4. Conversely, CDC7 on a multicopy plasmid suppresses the dbf4-1, -2, -3 and -4 mutations but not dbf4-5 and DBF4 deletion mutations. Furthermore, cdc7 mutations are incompatible with the temperature-sensitive dbf4 mutations. These results suggest that the CDC7 and DBF4 polypeptides interact directly or indirectly to permit initiation of yeast chromosome replication.     

Transduction of an Ethylene Signal Is Required for Cell Death and Lysis in the Root Cortex of Maize during Aerenchyma Formation Induced by Hypoxia

Ethylene has been implicated in signaling cell death in the lysigenous formation of gas spaces (aerenchyma) in the cortex of adventitious roots of maize (Zea mays) subjected to hypoxia. Various antagonists that are known to modify particular steps in signal transduction in other plant systems were applied at low concentrations to normoxic and hypoxic roots of maize, and the effect on cell death (aerenchyma formation) and the increase in cellulase activity that precedes the appearance of cell degeneration were measured. Both cellulase activity and cell death were inhibited in hypoxic roots in the presence of antagonists of inositol phospholipids, Ca2+- calmodulin, and protein kinases. By contrast, there was a parallel promotion of cellulase activity and cell death in hypoxic and normoxic roots by contact with reagents that activate G-proteins, increase cytosolic Ca2+, or inhibit protein phosphatases. Most of these reagents had no effect on ethylene biosynthesis and did not arrest root extension. These results indicate that the transduction of an ethylene signal leading to an increase in intracellular Ca2+ is necessary for cell death and the resulting aerenchyma development in roots of maize subjected to hypoxia.     

Expression of Lysophosphatidic Acid Receptor 1 and Relation with Cell Proliferation,Apoptosis, and Angiogenesis on Preneoplastic Changes Induced by Cadmium Chloride in the Rat Ventral Prostate

Background

Lysophosphatidic acid (LPA) is a phospholipid growth factor involved in cell proliferation, differentiation, migration, inflammation, angiogenesis, wound healing, cancer invasion, and survival. This study was directed to evaluate the immunoexpression of LPA-1, cell proliferation, apoptosis, and angiogenesis markers in preneoplastic lesions induced with cadmium chloride in rat prostate.

Methods

The following parameters were calculated in ventral prostate of normal rats and rats that received Cd in drinking water during 24 months: percentages of cells immunoreactive to LPA-1 (LI_LPA1), PCNA (LI_PCNA), MCM7 (LI_MCM7), ubiquitin (LI_UBI), apoptotic cells (LI_APO), and p53 (LI_p53); volume fraction of Bcl-2 (V_FBcl-2); and length of microvessels per unit of volume (L_VMV/mm3). Data were analyzed using Student''s t-test and Pearson correlation test.

Results

The LI_LPA1 in dysplastic lesions and normal epithelium of Cd-treated rats was significantly higher than those in the control group. Markers of proliferation were significantly increased in dysplastic lesions, whereas some apoptotic markers were significantly decreased. No significant differences between groups were found in V_FBcl-2. Dysplastic lesions showed a significant increase of LI_p53. The length of microvessels per unit of volume was elevated in dysplastic acini. Statistically significant correlations were found only between LI_LPA1 and LI_UBI.

Conclusions

Our results suggest that LPA-1 might be implicated in dysplastic lesions induced by cadmium chloride development. More studies are needed to confirm its potential contribution to the disease.     

Quantitative Analysis of Sugars in Plant Extracts by Ion-exchange Chromatography,with Special Reference to the Examination of Conditions for Preparing the Sample Sugar Solutions

Critical examinations were made on the conditions for preparing the sugar solutions to be analyzed by ion-exchange chromatography of sugar-borate complexes by the method of Khym and Zill. A procedure was proposed which gave the best recovery of sugars with minimum hydrolysis of sucrose. By means of this procedure, sugar solutions were prepared from potato tubers which had been stored at a high (30°C) or low temperature (6°C). Results of the chromatographic separation and determination of component sugars showed that main sugars present in potato tubers were sucrose, glucose, and fructose. Maltose and pentoses could not be detected. The contents of sucrose, glucose, and especially, fructose were far greater in potatoes stored at a low temperature than in those stored at a high temperature.     

Increased Levels of 3'',5''-Cyclic Adenosine Monophosphate Induced by Cobaltous Ion or 3-Isobutylmethylxanthine in the Incubated Mouse Retina: Evidence Concerning Location and Response to Ions and Light

Dark levels of 3',5'-cyclic adenosine monophosphate (cyclic AMP) of mouse retinas incubated in Earle's medium were elevated by 3-isobutyl-methylxanthine (IBMX) and/or Co2+ or Mn2+, but not by Cd2+, methylverapamil, or excess Mg2+ of Ca2+. Light reduced elevated dark levels of cyclic AMP in the presence of agents known to block the light modulation of post-receptoral neurons (aspartate, Co2+, high Mg2+), a finding consistent with a cyclic AMP metabolism in photoreceptors. Co2+-elevated cyclic AMP levels were not less light-sensitive than cyclic GMP levels. Ouabain substantially increased IBMX-elevated cyclic AMP with a persistent light response, but reduced the dark action of Co2+. IBMX, but not Co2+, also increased cyclic AMP in receptorless (rd/rd) retinas; haloperidol partly reduced this IBMX effect. In normal retinas in Co2+ medium, progressively replacing Na+ by K+ (but not choline+) from 1--50 mM caused a progressive fall in dark, light-sensitive cyclic AMP levels, but from 50 to 100 mM-K+ there appeared haloperidol-preventable increases in both the dark- and light-insensitive levels of cyclic AMP. In IBMX-aspartate medium a haloperidol-preventable, light-insensitive increase in cyclic AMP appeared from 20 mM-K+ upwards. Haloperidol-preventable increases in cyclic AMP as induced by high K+ required Co2+ in normal retinas, but not in receptorless retinas, and 5 nM-Co2+ greatly increased the response to dopamine in receptorless retinas. The post-dopaminergic neurons, which are 4th-order neurons, may have become hypersensitive to dopamine in receptorless retinas consequent to the absent signal from the 1st-order photoreceptors, or directly, as an effect of the same gene underlying the dystrophy.     

Appearance of Mink Cell Focus-Inducing Recombinants during In Vivo Infection by Moloney Murine Leukemia Virus (M-MuLV) or the Mo+PyF101 M-MuLV Enhancer Variant: Implications for Sites of Generation and Roles in Leukemogenesis

One hallmark of murine leukemia virus (MuLV) leukemogenesis in mice is the appearance of env gene recombinants known as mink cell focus-inducing (MCF) viruses. The site(s) of MCF recombinant generation in the animal during Moloney MuLV (M-MuLV) infection is unknown, and the exact roles of MCF viruses in disease induction remain unclear. Previous comparative studies between M-MuLV and an enhancer variant, Mo+PyF101 MuLV, suggested that MCF generation or early propagation might take place in the bone marrow under conditions of efficient leukemogenesis. Moreover, M-MuLV induces disease efficiently following both intraperitoneal (i.p.) and subcutaneous (s.c.) inoculation but leukemogenicity by Mo+PyF101 M-MuLV is efficient following i.p. inoculation but attenuated upon s. c. inoculation. Time course studies of MCF recombinant appearance in the bone marrow, spleen, and thymus of wild-type and Mo+PyF101 M-MuLV i.p.- and s.c.-inoculated mice were carried out by performing focal immunofluorescence assays. Both the route of inoculation and the presence of the PyF101 enhancer sequences affected the patterns of MCF generation or early propagation. The bone marrow was a likely site of MCF recombinant generation and/or early propagation following i.p. inoculation of M-MuLV. On the other hand, when the same virus was inoculated s.c., the primary site of MCF generation appeared to be the thymus. Also, when Mo+PyF101 M-MuLV was inoculated i.p., MCF generation appeared to occur primarily in the thymus. The time course studies indicated that MCF recombinants are not involved in preleukemic changes such as splenic hyperplasia. On the other hand, MCFs were detected in tumors from Mo+PyF101 M-MuLV s. c.-inoculated mice even though they were largely undetectable at preleukemic times. These results support a role for MCF recombinants late in disease induction.     

Quantitation of the niacin metabolites 1-methylnicotinamide and l-methyl-2-pyridone-5-carboxamide in random spot urine samples, by ion-pairing reverse-phase HPLC with UV detection, and the implications for the use of spot urine samples in the assessment of niacin status

A simple ion-pairing reverse-phase HPLC method, with UV diode array detection, was developed and validated for quantitation of the urinary niacin metabolites 1-methylnicotinamide and l-methyl-2-pyridone-5-carboxamide in a single run. Urine samples were purified using a polymer-based mixed mode anion exchange reverse-phase cartridge. Analysis was performed on a reverse-phase C18 column, using a methanol gradient elution system, containing phosphate buffer pH 7.0, 1-heptanesulphonic acid as the ion-pairing agent and trimethylamine as a modifier. The assay was applied to the measurement of the niacin status of two subjects using spot urine samples. The samples were collected over 4 consecutive days and at four time points during 1 day. Status, expressed as the concentration ratios (2-PYR or 1-MN)/creatinine and 2-PYR/l-MN, varied within and between days and was least for fasting samples. This work illustrates the potential of spot urine sampling for niacin status assessment, but highlights the need for further validation prior to its use in field nutritional surveys.     

Neuronal Injury External to the Retina Rapidly Activates Retinal Glia,Followed by Elevation of Markers for Cell Cycle Re-Entry and Death in Retinal Ganglion Cells

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.