Compensatory Accumulation Research Articles

Regulation of renal acid-base balance by Epac isoforms in proximal tubule and collecting duct

Proper regulation of acid-base homeostasis has utmost significance for well-being and survival of the living organisms. Up to 50% of emergency room patients have notable disturbances in pH and associated morbific manifestations, which impedes effective treatment. Kidneys play a central role in regulation of acid-base balance by reabsorbing HCO3− via NHE-3 in the proximal tubule (PT) and secreting H+ via V-ATPase pump by the intercalated cells of collecting duct (CD). Exchange proteins directly activated by cAMP (Epac) are most prominently expressed in the proximal tubule and collecting ducts, the sites critical for acid-base transport in the renal tubule. However, the significance and physiological relevance of Epac isoforms (Epac1 and Epac2) for controlling HCO3− reabsorption and H+ secretion and by extension, the systemic pH regulation is not known. Here, we used transgenic mouse models lacking both or individual Epac1 and Epac2 isoforms to examine the systemic adaptation to acid-base insults and to inquire on the perturbed molecular signaling mechanisms and end-effectors in freshly isolated split-opened PTs and CDs using biochemical and fluorescent imaging tools. Deletion of Epac1&2 caused a dramatic decrease in NHE-3 protein levels in an additive manner. Moreover, we detected a marked redistribution of NHE-3 to the base of brush border in proximal tubule cells rendering its inactivation. Consistently, NHE-3 activity was more than 70% decreased upon Epac1&2 deletion, as was assessed by a rate of recovery after intracellular acidification in freshly isolated split-opened PTs. Furthermore, V-ATPase activity was similarly decreased in A-type of intercalated cells in freshly isolated CDs despite compensatory accumulation on the apical side, as monitored with confocal microscopy in cortical and medullary kidney sections of the knockout. In unstressed conditions, Epac1&2 deletion did not affect arterial pH and HCO3− levels, while urinary pH was significantly reduced. Dietary acid load (280 mM NH4Cl in drinking water for 3 days) unmasked severe hyperchloremic metabolic acidosis with pH 7.17±0.01 and HCO3− 14.5±0.6 mM and impaired urinary acidification. Deletion of Epac2 alone recapitulated the phenotype of double knockout, whereas Epac1−/− mice did not demonstrate notable deficiencies in response to dietary acid load. Overall, we concluded that Epac signaling plays a major role in urinary acidification with the dominant contribution of Epac2 isoform by regulating acid-base transport in both proximal tubule (via NHE-3) and the collecting duct (via V-ATPase). Epac1&2 and Epac2 deletion of impairs renal adaptation to the acid load resulting in severe metabolic acidosis. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-23POST1020372 (to K. Pyrshev). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Socio-emotional Skills and the Socioeconomic Achievement Gap

Empirical evidence suggests children’s socio-emotional skills—an important determinant of school achievement—vary according to socioeconomic family background. This study assesses the degree to which differences in socio-emotional skills contribute to the achievement gap between socioeconomically advantaged and disadvantaged children. We used data on 74 countries from the 2018 Programme for International Student Assessment, which contains an extensive set of psychological measures, including growth mindset, self-efficacy, and work mastery. We developed three conceptual scenarios to analyze the role of socio-emotional skills in learning inequality: simple accumulation, multiplicative accumulation, and compensatory accumulation. Our findings are in line with the simple accumulation scenario: Socioeconomically advantaged children have somewhat higher levels of socio-emotional skills than their disadvantaged peers, but the effect of these skills on academic performance is largely similar in both groups. Using a counterfactual decomposition method, we show that the measured socio-emotional skills explain no more than 8.8 percent of the socioeconomic achievement gap. Based on these findings, we argue that initiatives to promote social and emotional learning are unlikely to substantially reduce educational inequality.

Fatty acid composition of quail blood erythrocyte membranes under condition of feeding sodium selenite and nanoselenium

Infectious diseases are a significant problem in poultry farming, because they significantly slow down the growth rate of the industry and lead to losses. For the productive development of the industry and the avoidance of danger to the health of consumers due to the uncontrolled use of these drugs, it is worth using alternative methods of protecting poultry and improving the quality of the obtained products, in particular drugs obtained by nanotechnological means. Nowadays, nanoparticles of various minerals are used in poultry farming, the mineral antagonism of which is reduced in the intestines, compared to preparations of the usual size, which contributes to the modulation of absorption mechanisms, optimization of the immune response, and increased efficiency of digestion. Oxidative stress is a harmful factor for cellular integrity due to the constant release of reactive forms of oxygen. Se is widely used as a supplement to reduce oxidative stress. The element is found in organic and inorganic compounds, replaces Sulfur in protein molecules and is an important part of selenoproteins. Se is known for its antioxidant activity, plays a major role in optimizing redox potential, reproductive processes, hormone metabolism, muscle development, and anticarcinogenesis. Nano-Se results in higher Se retention activity due to smaller size and greater bioavailability. Biogenic selenium nanoparticles (SeNPs), synthesized with the participation of bacteria, have unique physicochemical and biological properties compared to inorganic and organic compounds, and nanoselenium-enriched probiotic bacteria can be effectively used as an alternative to other forms of selenium as food and feed additives. The work compared the composition of fatty acids of lipids extracted from erythrocytes of quail blood, which were injected with sodium selenite and pro-oxidant nano-Se. With the introduction of nanoselenium, a decrease in the level of NFAs was noted, as well as a significant increase in the level of the main PUFAs. The lack of compensatory accumulation of docosapolyene fatty acids in the cell membranes of erythrocytes of birds of the 2nd group can be considered as a factor of the beneficial effect of the administered drug, as well as as a justification for the need to prescribe complex drugs to birds capable of modulating the fatty acid composition of cell membranes. The effectiveness of using a complex nanopreparation consists in normalizing the level of fatty acids and restoring their metabolism at the stage of eicosanoid formation. Key words: fatty acids, quails, peroxide oxidation, selenium, sodium selenite, nanoselenium, nanoparticles, lipids.

Causal Analysis Between Rice Growth and Cadmium Accumulation and Transfer under Arbuscular Mycorrhizal Inoculation

Cadmium (Cd) contamination in rice has been a serious threat to human health. To investigate the effects of arbuscular mycorrhizal fungi (AMF) on the Cd translocation in rice, a controlled pot experiment was conducted. The results indicated that AMF significantly increased rice biomass, with an increase of up to 40.0%, particularly in root biomass by up to 68.4%. Notably, the number of prominent rice individuals also increased, and their plasticity was enhanced following AMF inoculation. AMF led to an increase in the net photosynthetic rate and antioxidant enzyme activity of rice. In the AMF treatment group, the Cd concentration in the rice roots was significantly higher (19.1%‒68.0%) compared with that in the control group. Conversely, the Cd concentration in the rice seeds was lower in the AMF treatment group, indicating that AMF facilitated the sequestration of Cd in rice roots and reduced Cd accumulation in the seeds. Path coefficients varied across different treatments, suggesting that AMF inoculation reduced the direct impact of soil Cd concentration on the total Cd accumulation in seeds. The translocation of Cd was consistently associated with simultaneous growth dilution and compensatory accumulation as a result of mycorrhizal effects. Our study quantitatively analyzed this process through path analysis and clarified the causal relationship between rice growth and Cd transfer under the influence of AMF.

Abstract 1751: BPI-23314: A novel orally bioavailable small molecular BET inhibitor with target protein degradation activity

Abstract The Bromo- and Extra-Terminal (BET) proteins, BRD2, BRD3, and especially BRD4, play important roles in transcriptional regulation of many oncogenes like MYC or BCL2, thus makes it an attractive drug target in multiple pathological settings, particularly in cancer. Earlier BET inhibitors, while effectively bind and inhibit the activity of BRD4, adversely induce compensatory accumulation of the target protein, that may account for their limited preclinical and clinical efficacy. Herein, we report BPI-23314, a potent, selective, and orally available small molecular BET bromodomain inhibitor, which possesses potent target inhibition activity as well as target protein degradation property. In vitro, BPI-23314 induced dose-dependent growth inhibition in a wide range of cancer cell lines, including those from AML, MM, DLBCL, NSCLC, TNBC, and CRPC, etc. Quantitative RT-PCR revealed that BPI-23314 markedly affected the transcription of genes encoding MYC, BCL2, and HEXIM1, thus induced G1 cell cycle arrest and apoptosis in MV-4-11 AML cell line. In vivo, oral administration of BPI-23314 led to tumor growth suppression or regression in multiple xenograft models of AML and MM. Transcriptional modulation of MYC, BCL2 and HEXIM1 were confirmed in BPI-23314-treated xenografts and in normal monkey, supporting their potential as pharmacodynamic biomarkers in clinical trials. Unlike previously described BET inhibitors that induced BRD4 accumulation, BPI-23314 serendipitously decreased BRD4 protein in MV-4-11 cells and in tumors from MV-4-11 xenografts. The feature of target-protein degradation differentiates BPI-23314 from the other BET inhibitors, renders it good in vivo efficacy, and suggests a lower risk of drug resistance in future clinical development. In conclusion, our study establishes BPI-23314 as a novel small molecular BET inhibitor with target BRD4 protein degradation property. BPI-23314 has obtained IND approval from Chinese regulatory authority and is planned to enter Phase 1 clinical development in hematologic malignancies in early 2020. Citation Format: Jing Guo, Yan Xu, Dan Yan, Bo Yu, Weiwei Yu, Haibo Chen, Xiaofeng Xu, Xiangyong Liu, Hong Lan, Lieming Ding, Jiabing Wang. BPI-23314: A novel orally bioavailable small molecular BET inhibitor with target protein degradation activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1751.

Quantitative analysis of normal and pathologic adrenal glands with 18F-FDOPA PET/CT: focus on pheochromocytomas.

Many studies have reported the high performance of 6-fluorine-18-fluorodihydroxyphenilalanine (F-FDOPA) PET/CT in the diagnosis of pheochromocytomas but nobody seems to have investigated physiological and pathological adrenal glands from a quantitative point of view. The purpose of the present study was to assess the quantitative F-FDOPA uptake of normal and pathologic adrenal glands and to establish thresholds to characterize pheochromocytomas. We were especially interested in characterizing the remaining adrenal glands captation after an adrenalectomy. We reviewed 112 F-FDOPA PET/CT scans taken for different indications. A total of 212 adrenal glands, of which 17 were pheochromocytomas, were analyzed on the basis of their functional and morphological features. The final diagnosis was based on histologic proof when available (six pheochromocytomas) or after synthesis of clinical, biological, morphological, and functional results. Maximum standardized uptake value (SUVmax), mediastinum, and liver ratios in case of pheochromocytomas, adenomas, and solitary adrenal glands were determined and compared with those of healthy glands. Receiver operating characteristic curves were determined and areas under the curve were compared for different cutoffs of each index. Pheochromocytomas demonstrated a higher F-FDOPA uptake compared with normal adrenal glands (mean SUVmax: 7.5, SD 4.0, range: 3.5-20.0 vs. mean SUVmax: 2.6, SD: 0.8, range: 1.0-6.9) (P<0.0001). An SUVmax threshold of 4.2 has a sensitivity and specificity of 94 and 98%, respectively. The areas under the curve were 0.988, 0.991, and 0.987 for an SUVmax of 4.2, a mediastinum ratio of 3.0, and a liver ratio of 1.7, respectively. A large number of nonsecreting pheochromocytomas were noticed. On the basis of the SUVmax no statistically significant difference was found between secreting (SUVmax: 8.9, SD: 5.3) and nonsecreting pheochromocytomas (SUVmax: 5.1, SD: 0.9) (P=0.141). After unilateral adrenalectomy, solitary glands presented no increased uptake compared with healthy adrenal glands. An unexpected lower captation was also observed (SUVmax: 2.0, P=0.047). We confirm the high affinity of F-FDOPA for secreting or nonsecreting pheochromocytoma. Indeed within a series of various adrenal glands, only these tumors presented a significant increased uptake compared with normal adrenal glands. Because of a high rate of nonhypersecreting lesions, F-FDOPA can act as a surrogate to biological assays. After an adrenalectomy, the remaining glands did not demonstrate compensatory accumulation of F-FDOPA. To our knowledge this last point has never been addressed.

Rebalance between 7S and 11S globulins in soybean seeds of differing protein content and 11SA4

Protein content and globulin subunit composition of soybean seeds affect the quality of soy foods. In this proteomic study, the protein profile of soybean seeds with high (∼45.5%) or low (∼38.6%) protein content and with or without the glycinin (11S) subunit 11SA4 was examined. 44 unique proteins and their homologues were identified and showed that both protein content and 11SA4 influenced the abundance of a number of proteins. The absence of 11SA4 exerted a greater impact than the protein content, and led to a decreased abundance of glycinin G2/A2B1 and G5/A5A4B3 subunits, which resulted in lower total 11S with a concomitant higher total β-conglycinin (7S). Low protein content was associated with higher glycinin G3/A1aB1b and lower glycinin G4/A5A4B3. Using the proteomic approach, it was demonstrated that 11SA4 deficiency induced compensatory accumulation of 7S globulins and led to a similar total abundance for 7S+11S irrespective of protein content or 11SA4.

BRD4 Degradation By Protacs Represents a More Effective Therapeutic Strategy Than BRD4 Inhibitors in DLBCL

Introduction: BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as an attractive target in multiple oncology indications, particularly hematological malignancies. As a crucial chromatin reader, BRD4 is frequently found to be preferentially localized at super-enhancer regions, which often reside upstream of important oncogenes, including c-Myc, Bcl-xL and Bcl-6. Thus BRD4 inhibitors have gained attention as promising therapeutics in malignancies driven by these key oncogenes, including Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). However, we have recently found that BRD4 inhibitors lead to a compensatory accumulation of BRD4, which results in an incomplete inhibition of c-MYC and lack of apoptosis. These limitations could severely hamper the therapeutic potential of BRD4 inhibitors. To circumvent these limitations, we designed BRD4 degraders using our PROTAC (PROteolysis TArgeting Chimera) platform (Lu et al. Chem Biol. 22:755-763, 2015) and investigated the effects of BRD4 degradation in DLBCL in comparison with inhibitors.Methods and Results: We designed specific BRD4 PROTACs, which contain a BRD4 binding moiety connected to an E3 ubiquitin ligase-recruiting moiety. In all ABC-DLBCL, and GCB-DLBCL cell lines tested, BRD4 PROTACs lead to potent (<10 nM), fast (<4hrs) and robust (>90%) degradation of BRD4. This degradation is accompanied by more pronounced and longer-lasting suppression of downstream signaling events, such as c-MYC, than small molecule inhibitors (JQ1 and OTX015) treatment. We confirmed that BRD4 degradation is E3 ligase-mediated and proteasome-dependent as both excessive E3 ligase binding ligand and proteasome inhibitors (MG132 and carfilzomib) were able to prevent PROTAC-induced BRD4 degradation. Most importantly, BRD4 PROTACs were more effective in inhibiting DLBCL cell proliferation (measured by CellTiter-Glo® Luminescent Cell Viability Assay) and inducing apoptosis (evaluated by caspase3/7 assay and PARP cleavage) compared to BRD4 inhibitors.Conclusion: Hijacking E3 ubiquitin ligases, through PROTAC technology, is an innovative approach to achieve targeted, specific protein degradation. Such targeted degradation can provide important therapeutic advantages. Here, we demonstrated that BRD4 PROTACs lead to robust BRD4 downregulation, which results in better downstream signaling suppression, more pronounced inhibition of proliferation and induction of apoptosis compared to the effects of small molecule inhibitors. The results of on-going efficacy studies using a DLBCL tumor xenograft model will be presented at the meeting. Taken together, BRD4 degradation by PROTACs is a promising therapeutic strategy in DLBCL. DisclosuresLu:Arvinas, LLC: Employment, Equity Ownership. Qian:Arvinas, LLC: Employment, Equity Ownership. Raina:Arvinas, LLC: Employment, Equity Ownership. Altieri:Arvinas, LLC: Employment, Equity Ownership. Dong:Arvinas, LLC: Employment, Equity Ownership. Wang:Arvinas, LLC: Employment, Equity Ownership. Chen:Arvinas, LLC: Employment, Equity Ownership. Crew:Arvinas, LLC: Employment, Equity Ownership. Coleman:Arvinas, LLC: Employment, Equity Ownership. Crews:Arvinas, LLC: Employment, Equity Ownership. Winkler:Arvinas, LLC: Employment, Equity Ownership.

Optogenetic control of phosphoinositide metabolism

Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase(OCRL)), which acts on PI(4,5)P(2) and PI(3,4,5)P(3), was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase(OCRL) to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P(2) and PI(3,4,5)P(3) biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase(OCRL) recruitment and PI(4,5)P(2) dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P(2) biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P(3) synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.

The cathepsin K inhibitor AAE581 induces morphological changes in osteoclasts of treated patients

Inhibitors of Cathepsin K (Cat-K) are recognized as an interesting way to inhibit osteoclast (OC) activity. OCs from patients treated with the anticathepsin-K inhibitor AAE581 (balicatib) were found enlarged. They contained numerous vacuoles filled with tartrate resistant acid phosphatase (TRAcP), an intracellular enzyme that terminates the degradation of collagen internalized in OC transcytotic vesicles. In a phase 2 clinical study, 675 patients with postmenopausal osteoporosis received the Cat-K inhibitor AAE581 at 0, 5, 10, 25, or 50 mg/D during 1 year. Eleven patients had a transiliac bone biopsy, studied undecalcified. Histoenzymatic detection of TRAcP was used to identify and count OC number. The histomorphometrist was not aware of the randomization of patients at the time of analysis. OC were unstained in one patient because of a failure in the fixation protocol, but easily observable in the 10 remaining patients. Whatever the received dose, treated patients exhibited a characteristic aspect of the OC cytoplasm which appeared filled of deeply-stained brown vacuoles, making cells looking like bunches of grape. These round vacuoles, evidenced on TRAcP-stained sections, were due to the accumulation of intracytoplasmic TRAcP. This led to a moderate enlargement of the OC size when compared to a series of control osteoporotic patients. AAE581 did not induce OC apoptosis at any dosage but it modified OC morphology. Cat-K inhibition (inhibiting the extracellular collagen breakdown) is associated with a compensatory accumulation of intracellular TRAcP that could not be used to complete protein degradation. TRAcP is also known to be degraded by Cat-K.

Nucleosome Assembly Depends on the Torsion in the DNA Molecule: A Magnetic Tweezers Study

We have used magnetic tweezers to study nucleosome assembly on topologically constrained DNA molecules. Assembly was achieved using chicken erythrocyte core histones and histone chaperone protein Nap1 under constant low force. We have observed only partial assembly when the DNA was topologically constrained and much more complete assembly on unconstrained (nicked) DNA tethers. To verify our hypothesis that the lack of full nucleosome assembly on topologically constrained tethers was due to compensatory accumulation of positive supercoiling in the rest of the template, we carried out experiments in which we mechanically relieved the positive supercoiling by rotating the external magnetic field at certain time points of the assembly process. Indeed, such rotation did lead to the same nucleosome saturation level as in the case of nicked tethers. We conclude that levels of positive supercoiling in the range of 0.025–0.051 (most probably in the form of twist) stall the nucleosome assembly process.

Suppression of Both ELIP1 and ELIP2 in Arabidopsis Does Not Affect Tolerance to Photoinhibition and Photooxidative Stress

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.

Exact solutions and verification of numerical models for isothermal ice sheets

AbstractComparison of numerically computed solutions to exact (analytical) time-dependent solutions, when possible, is superior to intercomparison as a technique for verification of numerical models. At least two sources of such exact solutions exist for the isothermal shallow ice-sheet equation: similarity solutions and solutions with ‘compensatory accumulation’. In this paper, we derive new similarity solutions with non-zero accumulation. We also derive exact solutions with (i) sinusoidal-in-time accumulation and (ii) basal sliding. A specific test suite based on these solutions is proposed and used to verify a standard explicit finite-difference method. This numerical scheme is shown to reliably track the position of a moving margin while being characterized by relatively large thickness errors near the margin. The difficulty of approximating the margin essentially explains the rate of global convergence of the numerical method. A transformed version of the ice-sheet equation eliminates the singularity of the margin shape and greatly accelerates the convergence. We also use an exact solution to verify an often-used numerical approximation for basal sliding and we discuss improvements of existing benchmarks.

Biological Impact of Elevated Ocean CO2 Concentrations: Lessons from Animal Physiology and Earth History

CO2 currently accumulating in the atmosphere permeates into ocean surface layers, where it may impact on marine animals in addition to effects caused by global warming. At the same time, several countries are developing scenarios for the disposal of anthropogenic CO2 in the worlds' oceans, especially the deep sea. Elevated CO2 partial pressures (hypercapnia) will affect the physiology of water breathing animals, a phenomenon also considered in recent discussions of a role for CO2 in mass extinction events in earth history. Our current knowledge of CO2 effects ranges from effects of hypercapnia on acid-base regulation, calcification and growth to influences on respiration, energy turnover and mode of metabolism. The present paper attempts to evaluate critical processes and the thresholds beyond which these effects may become detrimental. CO2 elicits acidosis not only in the water, but also in tissues and body fluids. Despite compensatory accumulation of bicarbonate, acid-base parameters (pH, bicarbonate and CO2 levels) and ion levels reach new steady-state values, with specific, long-term effects on metabolic functions. Even though such processes may not be detrimental, they are expected to affect long-term growth and reproduction and may thus be harmful at population and species levels. Sensitivity is maximal in ommastrephid squid, which are characterized by a high metabolic rate and extremely pH-sensitive blood oxygen transport. Acute sensitivity is interpreted to be less in fish with intracellular blood pigments and higher capacities to compensate for CO2 induced acid-base disturbances than invertebrates. Virtually nothing is known about the degree to which deep-sea fishes are affected by short or long term hypercapnia. Sensitivity to CO2 is hypothesized to be related to the organizational level of an animal, its energy requirements and mode of life. Long-term effects expected at population and species levels are in line with recent considerations of a detrimental role of CO2 during mass extinctions in the earth's history. Future research is needed in this area to evaluate critical effects of the various CO2 disposal scenarios.

Synaptic pathology of spinal anterior horn cells in amyotrophic lateral sclerosis: An immunohistochemical study

We have applied immunohistochemical techniques to study synaptic alterations of the spinal anterior horn in amyotrophic lateral sclerosis (ALS), and other disorders involving upper or lower motor neurons. A monoclonal antibody to synaptophysin was used. Spinal cord tissues from normal individuals served as controls. As compared to these, a decrease in synaptophysin immunoreactivity was evident in the neuropil in the spinal anterior horn of ALS patients. However, synaptophysin expression in the perikarya and dendrites of remaining normal-appearing neurons in these patients was not decreased and occasionally it was even higher than in control neurons. Similar results were obtained with specimens from patients with lower motor neuron disease. Synaptophysin immunoreactivity in the neuropil and perikarya of the cases with focal spinal cord lesions with bilateral descending tract degeneration was similar to normal controls. Our data suggest that the alterations in synaptophysin expression occurring in ALS are mainly associated with the loss of lower motor neurons, and that the occasional increased perikaryal expression may be due to the neuronal atrophy, compensatory accumulation or abnormal synaptic vesicle degradation.

Cerebral uric acid, xanthine, and hypoxanthine after ischemia: the effect of allopurinol.

The existence of uric acid in mammalian brain was recently reported, but it has not yet become a consensus. The mammalian brain has been thought to lack xanthine oxidase, which catalyzes hypoxanthine to xanthine and xanthine to uric acid as the last steps of ATP degradation in other tissue. Using high-performance liquid chromatography, we performed assays for hypoxanthine, xanthine, and uric acid in rat brain after cerebral ischemia. It was confirmed that all three substances showed significant augmentation in the removed brains and that the chronological order of those increases corresponded to the order in the metabolic pathway. Allopurinol, a specific inhibitor of xanthine oxidase, significantly suppressed the increases in uric acid and xanthine, and a compensatory accumulation of hypoxanthine was observed. From these results, it was concluded that uric acid does exist in the brain, increases after ischemia, and is possibly the end product of purine degradation in the brain. Furthermore, it is suggested that xanthine oxidase exists in the brain and catalyzes the reaction from hypoxanthine to xanthine and then to uric acid. These reactions catalyzed by xanthine oxidase are considered to be a source of free radicals and may play important roles in the pathogenesis of cerebral ischemic injury.

Regulation of neurotransmitter metabolic enzymes and tyrosine hydroxylase mRNA level by nerve growth factor in cultured sympathetic neurones.

The survival of new-born rat sympathetic neurones in culture was increased in a dose-dependent manner by 7S nerve growth factor (NGF). NGF also increased, in a parallel manner, the specific activities of tyrosine hydroxylase (TOH) and choline acetyltransferase (CAT). Total acetylcholinesterase (AcChE) activity increased with NGF concentration, although less distinctly than TOH and CAT. However, NGF caused a large induction of the asymmetric A12 form of AcChE, and to a lesser extent of the globular G1 and G2 forms, whereas the globular G4 form was little affected. This suggests that NGF differentially regulates the synthesis and/or assembly of the various AcChE molecular forms. The levels of TOH mRNA in neurone cultures grown with increasing NGF concentrations were measured by Northern blot analysis with a rat cDNA probe. To correct for variations in the total mass of RNA per neurone, the filters were rehybridized with an 18S rRNA probe. The level of TOH mRNA, measured by the ratio (TOH:18S) of the hybridization signals increased 3.4-fold between 92 and 740 ng ml-1 7S NGF. Increases of TOH specific activity of the same order of magnitude were observed in sister cultures. The deficit in the level of mature TOH mRNA at low NGF concentration was not accompanied by a compensatory accumulation in unprocessed TOH transcripts. As TOH induction is insensitive to RNA polymerase inhibitors, we suggest that NGF regulates the maturation of TOH pre-mRNAs, and that the unprocessed transcripts are rapidly degraded. The long-term regulation of TOH by NGF may thus constitute a case of process-versus-discard control, as defined by J.E. Darnell.

Current concepts on the role of potassium in stomatal movements

A stoma opens when the surrounding guard cell pair increases in turgidity. The increase results from active accumulation of potassium in the guard cell vacuole. The intracellular compartmentation of potassium evokes compensatory accumulation of a yet‐unidentified solute in the guard cell cytoplasm. The source of potassium is other epidermal cells; this indicates that stomatal movements in situ are under control of these cells also. Presumably, guard cell potassium uptake, which is from the apoplast, is mediated by a proton ‐extruding ATPase on the guard cell plasmalemma. The energy source is. oxidative phosphorylation and, to a lesser extent, photosynthetic electron transport. Except for high flux capacity and different responses to applied chemicals, potassium uptake by guard cells is similar to potassium uptake by other plant cells.