Immeasurable Levels Research Articles

Genetic and Agronomic Analysis of Tobacco Genotypes Exhibiting Reduced Nicotine Accumulation Due to Induced Mutations in Berberine Bridge Like (BBL) Genes.

Genetic methodologies for reducing nicotine accumulation in the tobacco plant (Nicotiana tabacum L.) are of interest because of potential future regulations that could mandate lowering of this alkaloid in conventional cigarettes. Inactivation of tobacco genes such as the Berberine Bridge Like (BBL) gene family believed to encode for enzymes involved in one of the latter steps of nicotine biosynthesis could be a viable strategy for producing new tobacco cultivars with ultra-low leaf nicotine accumulation. We introduced deleterious mutations generated via ethyl methanesulfonate treatment of seed or gene editing into six known members of the BBL gene family and assembled them in different combinations to assess their relative contribution to nicotine accumulation. Significant reductions (up to 17-fold) in percent leaf nicotine were observed in genotypes homozygous for combined mutations in BBL-a, BBL-b, and BBL-c. The addition of mutations in BBL-d1, BBL-d2, and BBL-e had no additional significant effect on lowering of nicotine levels in the genetic background studied. Reduced nicotine levels were associated with reductions in cured leaf yields (up to 29%) and cured leaf quality (up to 15%), evidence of physiological complexities within the tobacco plant related to the nicotine biosynthetic pathway. Further nicotine reductions were observed for a BBL mutant line cultivated under a modified production regime in which apical inflorescences were not removed, but at the expense of further yield reductions. Plants in which BBL mutations were combined with naturally occurring recessive alleles at the Nic1 and Nic2 loci exhibited further reductions in percent nicotine, but no plant produced immeasurable levels of this alkaloid. Findings may suggest the existence of a minor, alternative pathway for nicotine biosynthesis in N. tabacum. The described genetic materials may be of value for the manufacture of cigarettes with reduced nicotine levels and for future studies to better understand the molecular biology of alkaloid accumulation in tobacco.

Experimental study of tungsten transport properties in T-10 plasma

First experimental results of tungsten transport investigation in OH and ECRH plasmas in the T-10 tokamak with W-limiter and movable Li-limiter are presented. It is shown that tungsten tends to accumulate (a joint process of cumulation and peaking) near the plasma axis in ohmic regimes. The cumulation of W is enhanced in discharges with high values of the parameter that coincides with accumulation conditions of light and medium impurities in T-10 plasmas. Experiments with Li-limiter show the immeasurable level of Li3+ (0.3–0.5% of ne) of T-10 CXRS diagnostics because of the low inflow of Li with respect to other light impurities. Nevertheless, the strong influence of lithium on inflow of light and tungsten impurities is observed. In discharges with lithized walls, vanishing of light impurities occurs and values of are obtained. It is also shown that the tungsten density in the plasma center decreases by 15 to 20 times while the W inflow reduces only by 2 to 4 times. In lithized discharges with high γ, the flattening of the tungsten density profile occurs and its central concentration decreases up to 10 times during the on-axis ECRH. This effect is observed together with the increase of the W inflow by 3 to 4 times at the ECRH stage.

Stochastic humoral immunity to Bacillus anthracis protective antigen: Identification of anti-peptide IgG correlating with seroconversion to Lethal Toxin neutralization

A substantial fraction of individuals vaccinated against anthrax have low to immeasurable levels of serum Lethal Toxin (LeTx)-neutralizing activity. The only known correlate of protection against Bacillus anthracis in the currently licensed vaccine is magnitude of the IgG response to Protective Antigen (PA); however, some individuals producing high serum levels of anti-PA IgG fail to neutralize LeTx in vitro. This suggests that non-protective humoral responses to PA may be immunodominant in some individuals. Therefore, to better understand why anthrax vaccination elicits heterogeneous levels of protection, this study was designed to elucidate the relationship between anti-PA fine specificity and LeTx neutralization in response to PA vaccination. Inbred mice immunized with recombinant PA produced high levels of anti-PA IgG and neutralized LeTx in vitro and in vivo. Decapeptide binding studies using pooled sera reproducibly identified the same 9 epitopes. Unexpectedly, sera from individual mice revealed substantial heterogeneity in the anti-PA IgG and LeTx neutralization responses, despite relative genetic homogeneity, shared environment and exposure to the same immunogen. This heterogeneity permitted the identification of specificities that correlate with LeTx-neutralizing activity. IgG binding to six decapeptides comprising two PA epitopes, located in domains I and IV, significantly correlate with seroconversion to LeTx neutralization. These results indicate that stochastic variation in humoral immunity is likely to be a major contributor to the general problem of heterogeneity in vaccine responsiveness and suggest that vaccine effectiveness could be improved by approaches that focus the humoral response toward protective epitopes in a greater fraction of vaccinees.

Fractured zone height of longwall mining and its effects on the overburden aquifers

As mining depth becomes deeper and deeper, the possibility of undermining overburden aquifers is increasing. It is very important for coal miners to undertake studies on the height of fractured zone during longwall mining and the effects of longwall mining on the underground water while mining under surface water bodies and underground aquifers. In order to study this problem, piezometers for monitoring underground water levels were installed above the longwall panels in an American coalmine. Large amounts of pre-mining, during mining and post-mining monitoring data were collected. Based on the data, the heights of fractured zones were obtained and the effects of longwall mining on the underground water were studied. The results demonstrate that when the piezometer monitoring wells had an interburden thickness of less than 72.7m, the groundwater level decreased immediately to immeasurable levels and the wells went dry after undermining the face of longwall. The height of the fractured zone is 72.7–85.3m in the geological and mining conditions. The results also show that the calculated values of fractured zones by the empirical formulae used in China are smaller than the actual results. Therefore, it is not always safe to use them for analyses while mining under water bodies.

Methamphetamine‐Induced Early Increase of IL‐6 and TNF‐α mRNA Expression in the Mouse Brain

The mechanisms by which methamphetamine (METH) causes neurotoxicity are not well understood. Recent studies have suggested that METH-induced neuropathology may result from a multicellular response in which glial cells play a prominent role, and so it is plausible to suggest that cytokines may participate in the toxic effects of METH. Therefore, in the present work we evaluated the effect of an acute administration of METH (30 mg/kg in a single intraperitoneal injection) on the interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha mRNA expression levels in the hippocampus, frontal cortex, and striatum of mice. We observed that METH did not induce changes in the IL-1beta mRNA expression levels in both hippocampus and striatum, with immeasurable levels in the frontal cortex. Regarding IL-6, METH induced an increase in the expression levels of this cytokine in the hippocampus and striatum, 1 h and 30 min post injection, respectively. In the frontal cortex, the increase in IL-6 mRNA levels was more significant and remained high even after 2 h. Moreover, the expression levels of TNF-alpha were increased in both hippocampus and frontal cortex 30 min post METH administration, with immeasurable levels in the striatum. We conclude that the pro-inflammatory cytokines IL-6 and TNF-alpha rapidly increase after METH administration, providing a new insight for understanding the effect of this drug of abuse in the brain.

Sucrose phosphate synthase activity and the co-ordination of carbon partitioning during sucrose and amino acid accumulation in desiccation-tolerant leaf material of the C4 resurrection plant Sporobolus stapfianus during dehydration

Both sucrose and amino acids accumulate in desiccation-tolerant leaf material of the C(4) resurrection plant, Sporobolus stapfianus Gandoger (Poaceae). The present investigation was aimed at examining sucrose phosphate synthase (SPS) activity and various metabolic checkpoints involved in the co-ordination of carbon partitioning between these competing pathways during dehydration. In the initial phase of dehydration, photosynthesis and starch content declined to immeasurable levels, whilst significant increases in hexose sugars, sucrose, and amino acids were associated with concomitant significant increases in SPS and pyruvate kinase (PK) activities, and maximal activity levels of phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and NADH-dependent glutamate synthase (NADH-GOGAT). The next phase of dehydration was characterized by changes in metabolism coinciding with net hexose sugar phosphorylation. This phase was characterized by a further significant increase in sucrose accumulation, with increased rates of net sucrose accumulation and maximum rates of SPS activity measured under both saturating and limiting (inhibitory) conditions. SPS protein was also increased. The stronger competitive edge of SPS for carbon entering glycolysis during hexose phosphorylation was also demonstrated by the further decrease in respiration and the simultaneous, significant decline in both PEPCase and PK activities. A decreased anabolic demand for 2-oxoglutarate (2OG), which remained constant, was shown by the co-ordinated decrease in GOGAT. It is proposed that the further increase in amino acids in this phase of dehydration may be in part attributable to the breakdown of insoluble proteins.

Human Breast Cancer Tissue Expresses High Level of Type 1 5′-Deiodinase

Type 1 5'-deiodinase is one of two isoenzymes that participate in conversion of prohormone thyroxine into triiodothyronine (T3). A decrease in type 1 5'-deiodinase expression was observed in renal clear cell carcinoma, thyroid cancer, and lung cancer. The aim of this study was to evaluate type 1 5'-deiodinase activity and mRNA level in breast cancer tissue and non-cancerous surrounding breast tissue. Material was collected from 36 patients undergoing radical mastectomy or local tumor resection. In all non-cancerous breast tissues, type 1 50-deiodinase activity was found to be at a very low or immeasurable level, and type 1 5'-deiodinase mRNA was detected only in 2 out of the 36 samples. By contrast, 20 out of the 36 breast cancer tissues, mainly grades G1 and G2, expressed abundant type 1 5'-deiodinase activity and/or a high mRNA level. Our data demonstrated the presence of type 1 5'-deiodinase in well-differentiated breast cancer tissue. High enzymatic activity of type 1 50-deiodinase can potentially lead to an increase in the production of T3, which may affect target gene transcription, including genes responsible for energy expenditure, growth, differentiation, and proliferation.

Effect of coagulant types on textile wastewater reclamation in a combined coagulation/ultrafiltration system

A combination of coagulation and ultrafiltration (UF) processes for textile wastewater reclamation was investigated using various types of coagulating chemicals such as polyamine, alum, polyaluminum chloride (PACl), and ferric salts. The potential of the combined system was evaluated to replace the existing treatment processes which are composed of a flotation tank (primary step) and a series of filtration beds including sands, granular carbons, and diatomaceous powders. Regardless of the type and dosage of the coagulants, the UF system achieved substantial colloidal particle removal (>97% of turbidity was removed), but membrane fouling was mitigated in a different manner. The degree of fouling reduction was highly dependent upon the type of coagulants used, even though the turbidity and organics removal efficiencies were nearly the same. The polymeric coagulant aggravated membrane fouling, whereas the inorganic coagulants always helped reduce fouling. A residue of the coagulating polymer, which was added in the primary step and its concentration in the effluent was at an immeasurable level, was found to cause serious membrane fouling. The use of polymeric coagulants should be prevented or minimized if UF is considered for textile wastewater reclamation. Polymerized aluminum was found to be the most effective among the coagulants tested, although ferric salt was better than alum in controlling fouling. In particular, it seemed that the characteristics of coagulation chemistry and the coagulated particles had a great impact upon membrane fouling.

Decreased Testosterone in Regularly Menstruating Women with Decreased Libido: A Clinical Observation

Much more information is available concerning decreased libido in postmenopausal than in premenopausal women. Even less is known about androgen deficiency in younger women. We measured total and free testosterone levels in 12 consecutive premenopausal women complaining of decreased libido. Of the 12 women, 8 had low or immeasurable levels of testosterone despite having regular menstrual periods. Androgen precursor hormones, DHEA-S and Androstenedione, were low-normal to high-normal. Treatment with oral DHEA, 50 to 100 mg per day, restored sexual desire in 6 of the 8 women, gave partial improvement in one, and failed in another. Possible significance and etiological mechanism are discussed.

Engineering A23187/EDTA-containing metal-sorbing vesicles for selective extraction of divalent heavy metal ions

Phosphatidylcholine vesicles harboring the lipophilic metal ion carrier A23187 in the bilayer and encapsulating ethylenediamine tetraacetic acid (EDTA) exhibit broadly varied, pH-dependent affinity for Cu 2+, Pb 2+, Cd 2+, and Ca 2+ at micromolar levels. Initial rates of uptake by these metal-sorbing vesicles (MSVs) is strongly dependent on the metal ion affinity of A23187 and the dissociation rate of A23187 complexation with turnover numbers ranging over five orders of magnitude from 27.51 s −1 for Cu 2+ at pH 7.0 to <0.001 s −1 for Ca 2+ at pH 5.5. A modified Briggs–Haldane model fits initial MSV kinetics well and was used to estimate metal ion dissociation rate constants of A23187, which varied from 270 s −1 for Ca 2+ to 18.3 s −1 for Cd 2+. The experimental data support a “pore” type mechanism rather than a membrane-transport-limited model for metal ion uptake in qualitative agreement with the microsecond time-scale expected for carrier–metal ion complex translocation across the lipid bilayer wall of MSVs. The concentration factors achieved by these MSVs, which are controlled by the amount of encapsulated EDTA, varied from 674 for Cu 2+ to 166 for Ca 2+ at pH 7.0 to an immeasurable level for Ca 2+ at pH 5.5. Although several physicochemical properties impact metal ion uptake, MSV selectivity in initial uptake rate or in equilibrium capacity can be described at least semi-quantitatively in terms of the metal ion affinity of the carrier and chelator, as well as the dissociation rate of the metal–carrier complex.

The Evolution of Microstructure in Nonhydrolytic Alumina Xerogels

The effect of drying, aging and thermal treatment of alumina xerogels prepared by the nonhydrolytic route was investigated using SAXS, BET and HR-SEM techniques. The microstructure of the fresh xerogels prepared under different procedures varied drastically, ranging from aerogel-like mass fractals to narrow pore size distribution materials. By variation of the drying conditions the N2-BET surface area was varied from an immeasurable low level up to 600 m2/g. The initial microstructure has a significant influence on the xerogel behaviour during the post-drying heating stage. The ability to produce aerogel-like mass fractal materials from the nonhydrolytic systems is discussed. Finally, a brief theoretical treatment of the drying process of mass fractals is presented as well.

Timescale for producing the geochemical signature of island arc magmas: U-Th-Po and Be-B systematics in recent Papua New Guinea lavas

U-series activities, beryllium isotopes and boron abundances are reported for twenty samples from fourteen Holocene volcanic centers in Papua New Guinea, spanning several tectonic settings. Samples from centers on and behind the volcanic front in the Eastern Bismarck Arc are from a region of normal subduction. The volcanoes overlie a seismic zone 100 to 400 km deep. Samples from the Western Bismarck Arc are from different segments of an eastward-propagating zone of collision between Papua New Guinea and the arc. Measured ratios of ( 230Th) ( 232Th) , 10Be 9Be , and B Be in basalts and andesites from all settings are 1.2–2.9, 2–28, and 8–160, respectively. Rocks from the Eastern Bismarck Arc are characterized by ~ 10% excess uranium and by positively correlated ( 230Th) ( 232Th) , 10Be 9Be , and B Be ratios. The positive correlation of 10Be 9Be with ( 230Th) ( 232Th) rather than with ( 238U) ( 230Th) ratios, and the association of high values with larger fluxes of uranium than thorium, indicate that uranium was added to the arc source and stored there for longer than 10 5 y prior to magma genesis. There was a time lag of hundreds of thousands of years between slab dehydration and melt formation. Lavas from volcanoes up to 230 km above the slab have the same U-series relationships as those at the volcanic front. Beryllium isotope and B Be relationships behind the front differ from those at the front but are still indicative of slab input. The subduction signature is absent only where the slab is 400 km deep. Lavas from the Western Bismarck Arc share most of the characteristics of those at the eastern Bismarck volcanic front except that 10Be concentrations decrease from low to immeasurable levels westward in the direction of earlier collision. The ratio of 10Be (1.5 Ma half-life) to stable boron in the lavas can be used to “date” the collision. Results suggest that suturing began in the westernmost segment in the early Pliocene and within the last million years at 148°E, the site of the present triple junction. Large fluxes of slab-derived elements, including excess 238U which must have developed within the last ~350 ka, are seen in the collision zone lavas even where the slab now beneath the volcanoes may have had a long (>3 Ma) residence time in the mantle. The U-series data from the entire Bismarck volcanic front suggest ~200 Ka between slab dehydration and magma formation and eruption. This is in striking contrast to a similar data set for southern Chile where the elapsed time is inferred to be less than 20 Ka.

Interdiffusion at polyimide interfaces

Interdiffusion at interfaces between deuterated polyamic acid (d-PAA) and polyimide (PI) films was investigated with forward recoil spectrometry, an ion beam analysis technique. The PI films were prepared by spin coating a solution of PAA on a silicon substrate, followed by an anneal at a temperature T i, which produced partial or complete conversion of the PAA to PI. An overlayer of d-PAA was added, in one set of samples by spin coating from solution, and in another set by transferring (in the absence of solvents) a dry d-PAA film onto the PI surface. The bilayer samples were then either annealed at a temperature T d or allowed to stand at room temperature. No interdiffusion occurred in the solventless transfer samples for any combination of temperatures T i or T d from room temperature up to 400°C. Bilayers prepared by spin coating d-PAA from solution directly on partially cured PI films did, however, show significant interdiffusion distances w, which decreased with increasing values of T i to immeasurable levels by T i = 200°C. The decrease in w with increasing T i is thought to be caused by a positive Flory parameter between PAA and PI which increases with the imide fraction in the PI film; the result is an increasing immiscibility between the swollen polymer layers. Subsequent annealing of these spin-coated bilayers at a temperature T d up to 400°C was ineffective in producing any additional interdiffusion. The absence of any thermally activated interdiffusion (even for initially unimidized samples), with either the solventless-transfer or spin-coated preparation methods may be attributed to the rapid increase of the glass transition temperature of the polymer with imidization.

Solvent and Curing Effects on Diffusion at Polyimide Interfaces

AbstractInterdiffusion at interfaces between deuterated polyamic acid (d-PAA) and polyimide (PI) films was investigated with forward recoil spectrometry, an ion beam analysis technique. The PI films were prepared by spin – coating a solution of PAA on a silicon substrate, followed by an anneal at a temperature Ti, which produced partial or complete conversion of the PAA to PI. An overlayer of d-PAA was added in one set of samples by spin – coating from solution and in another set by transferring (in the absence of solvents) a dry d- PAA film onto the PI surface. The bilayer samples were then either annealed at a temperature Td or allowed to stand at room temperature. Bilayers prepared by spin – coating d-PAA from solution directly on partially cured PI films had interdiffusion distances w that decreased with increasing values of Ti to immeasurable levels by Ti = 200 °C. The decrease in w with increasing Ti is thought to be caused by a positive Flory parameter between PAA and PI which increases with the imide fraction in the PI film; the result is an increasing immiscibility between the swollen polymer layers. No interdiffusion occurred in the solventless – transfer samples for any combination of temperatures Ti or Td from room temperature up to 400 °C. Post – annealing of spin – coated bilayers at a temperature Td up to 400 °C was ineffective in producing any additional interdiffusion. Both of these results indicate that thermally activated interdiffusion (even for initially unimidized samples) does not exist in the absence of solvents, an effect attributed to the rapid increase of the glass transition temperature of the polymer with imidization.

Solvent and Curing Effects on Diffusion at Polyimide Interfaces

AbstractInterdiffusion at interfaces between deuterated polyamic acid (d-PAA) and polyimide (PI) films was investigated with forward recoil spectrometry, an ion beam analysis technique. The PI films were prepared by spin - coating a solution of PAA on a silicon substrate, followed by an anneal at a temperature Ti, which produced partial or complete conversion of the PAA to PI. An overlayer of d-PAA was added in one set of samples by spin - coating from solution and in another set by transferring (in the absence of solvents) a dry d-PAA film onto the PI surface. The bilayer samples were then either annealed at a temperature Td or allowed to stand at room temperature. Bilayers prepared by spin - coating d-PAA from solution directly on partially cured PI films had interdiffusion distances w that decreased with increasing values of Ti to immeasurable levels by Ti = 200 °C. The decrease in w with increasing Ti is thought to be caused by a positive Flory parameter between PAA and PI which increases with the imide fraction in the PI film; the result is an increasing immiscibility between the swollen polymer layers. No interdiffusion occurred in the solventless - transfer samples for any combination of temperatures Ti or Td from room temperature up to 400 °C. Post - annealing of spin - coated bilayers at a temperature Td up to 400 °C was ineffective in producing any additional interdiffusion. Both of these results indicate that thermally activated interdiffusion (even for initially unimidized samples) does not exist in the absence of solvents, an effect attributed to the rapid increase of the glass transition temperature of the polymer with imidization.

Frequency and amplitude of gonadotropin-releasing hormone stimulation and gonadotropin secretion in the rhesus monkey.

In adult ovariectomized rhesus monkeys bearing hypothalamic lesions which reduced circulating LH and FSH to undetectable levels, sustained elevated gonadotropin concentrations were reestablished by the intermittent administration of gonadotropin-releasing hormone (GnRH) at the rate of 1 microgram/min for 6 min once every hour. The effects of varying either the frequency or the amplitude of these GnRH pulses on gonadotropin secretion were examined in such animals. Increasing the frequency of GnRH administration from the physiological one pulse per h to two, three, or five pulses h while maintaining a constant infusion rate and pulse duration resulted in gradual declines in plasma gonadotropin concentrations. These declines were most profound at the highest frequencies and the consequence of reduced pituitary responses to individual GnRH pulses. Decreasing the frequency of GnRH pulses from one per h to one every 3 h led to variable declines in plasma LH levels, but circulating FSH invariably rose. Reducing the GnRH infusion rate from 1 to 0.1 mg/min while maintaining constant frequency and pulse duration resulted in abrupt declines in plasma LH and FSH to immeasurable levels, although pulsatile increments in circulating GnRH concentrations without a concomitant reduction in plasma LH concentrations, which remained unchanged. An infusion rate of 0.5 microgram/min resulted in unstable plasma LH and FSH levels. These results demonstrate that changes in the frequency or amplitude of hypophysiotropic stimulation have profound effects on plasma gonadotropin levels as well as on FSH to LH ratios in the circulation. The physiological implications of these observations are discussed.

Actions of gamma-aminobutyric acid on sympathetic ganglion cells.

1. Responses of single ganglion cells in the isolated rat superior cervical ganglion to gamma-aminobutyric acid (GABA) applied via the bathing medium were recorded using intracellular micro-electrodes. 2. GABA produced a large fall in cell input resistance, frequently to immeasurable levels. In thirteen cells showing a modest response to 100 muM GABA, input resistance fell from 50-5 +/-9-5 to 15.9 +/- 3-2 Momega (means +/- S.E. of mean). After correction for resistance leaks introduced by the impaling electrode, the resting membrane resistance Rm and the resistance of the GABA-shunt Rg in these cells were calculated to be 79-3 +/- 16-6 and 35-0 +/- 9-5 Momega respectively. 3. Cells with recorded resting membrane potentials greater than -42 mV were depolarized by GABA; at resting potential less than -42 mV they were hyperpolarized...

Evidence for an Androgen-independent Mechanism Regulating the Levels of Receptor in Target Tissue

Abstract The amount of radioactive dihydrotestosterone (17β-hydroxy-5α-[1,2-3H]androstan-3-one) bound to high molecular weight molecules in the 8 to 10 S fraction of the 100,000 x g supernatant was measured at intervals following castration. As expected, the receptor concentration (and activity per mg of cytosol protein or per µg of DNA) declined steadily to immeasurable levels by the 4th day following castration. However, whereas the size of the prostate and the DNA content per prostate and per mg of tissue continued to fall, the receptor concentration per mg of tissue and per µg of DNA increased to levels similar to those found 1 day following castration. The maximum was reached by the 8th day after castration and was maintained for at least 6 weeks. The results of the sucrose gradient analysis were confirmed by DEAE-cellulose chromatography. The same phenomenon was observed in animals which were either hypophysectomized or adrenalectomized at the time of castration. This finding indicates that restoration of receptor is not mediated by adrenal androgens or pituitary hormones. These observations suggest that there is an androgen-independent mechanism for providing receptor to receive the androgenic stimulus for growth and development as well as for maintenance of the mature prostate.

Partial purification and characterization of peptidyl proline hydroxylase precursor from mouse fibroblasts

A protein, designated CRP, which is immunologically identical to peptidyl proline hydroxylase (involved in collagen biosynthesis), has been isolated from early log phase cultures of L-929 cells. This protein, however, differs from the enzyme in several of its chromatographic characteristics and in being enzymatically inactive. The CRP has a molecular weight between 85,000 and 105,000 compared with peptidyl proline hydroxylase which has a molecular weight between 260,000 and 300,000. The cellular concentration of CRP falls to immeasurable levels in late log phase cultures as peptidyl proline hydroxylase activity becomes maximal. It is concluded that CRP is the inactive precursor of peptidyl proline hydroxylase postulated in earlier studies. It is suggested that CRP is a subunit of the active enzyme and that the functional level of peptidyl proline hydroxylase in this system is regulated through subunit aggregation.

The Physiology of Encystment of Hartmannella castellanii*

SYNOPSIS. Some aspects of the physiology of encystment of the soil amoeba Hartmannella castellanii in a replacement encystment medium consisting of 5 × 10‐2 M MgCl2 have been investigated. It is suggested that measurement of the cellulose produced during encystment in the synthesis of the cyst wall is a more reliable measure of the process than other methods tried.The degree of encystment was dependent on the physiologic state of the amoebae and the composition of the growth medium, but the initial pH of the encystment medium (C. 4.0‐8.5) had little effect on the process. The requirement for Mg during encystment was probably not due to its deficiency during growth. Encystment was inhibited to varying extents by inhibitors of protein synthesis, tetracycline and chloramphenicol and also by arsenate, arsenite and iodoacetate; sodium fluoride, malonate and 2, 4‐dinitrophenol were without marked effect.Addition of glucose and α‐ketoglutarate to the replacement medium led to improvement in the encystment response. The presence of glutamate and histidine during encystment led to cell death. Other carbon and nitrogen sources had no effect.During encystment there was an increase in the metabolic activity of the amoebae, as measured by their oxygen consumption. This was accompanied by a decrease of about 40% in cellular dry weight and protein content. Of the other chemical components, there were marked initial increases in the levels of total carbohydrates and pentose which were followed by their depletion during cellulose synthesis.Encystment was completed after about 64 hr when the synthesis of cellulose was complete and the oxygen uptake of the amoebae fell to an immeasurable level.