Ketoconazole susceptibility of yeasts by the FCST method

The flow cytofluorometric susceptibility test (FCST) was extended fromCandida albicans to additional clinically relevant yeasts.Candida tropicalis, C. parapsilosis, C. lusitaniae, C. krusei, C. guilliermondii, Torulopsis glabrata, andRhodotorula rubra were also amenable to the FCST. Of the most frequently encountered yeasts,C. albicans andC. tropicalis exhibited the E_max response, which is believed to be an in vitro indicator of ketoconazole susceptibility with potential clinical relevance.Torulopsis glabrata, for which ketoconazole therapy is less effective, did not exhibit the E_max response; rather it exhibited an MIC effect, but usually at ketoconazole concentrations greater than those therapeutically achievable.     

Susceptibility of clinical isolates of fungi to saperconazole

The in vitro activity of saperconazole against 228 strains of mycotic agents belonging to 48 species was investigated. Susceptibility testing was performed using a microtiter broth dilution method. Isolates of Candida albicans, C. tropicalis and Torulopsis glabrata showed distinct intra-species variation of susceptibility with MIC values ranging from 0.045 to 100 mg l^–1. The drug was inhibitory for the dermatophytes at a relatively narrow range of concentrations, most isolates being inhibited at MIC 0.78 mg l^–1. The strongest antifungal potency of saperconazole was exerted against clinical isolates of the genus Aspergillus (MIC 90% = 0.19 mg l^–1). Concentrations up to 100 mg l^–1 had no macroscopically recognizable effect on the growth of zygomycetous fungi (Mucor, Rhizopus, Syncephalastrum). Species of the genus Absidia with their good sensitivity are an exception. Justification of in vitro susceptibility testing of triazoles is discussed. In the author's opinion, MIC values can serve as an informative parameter showing the range of indications of these antifungals for treatment. It is concluded that saperconazole exhibits a very good activity against a broad spectrum of medically important fungi in vitro and can be considered a promising antifungal drug.     

Contrasting acclimation responses to elevated CO2 and warming between an evergreen and a deciduous boreal conifer

Rising atmospheric carbon dioxide (CO₂) concentrations may warm northern latitudes up to 8°C by the end of the century. Boreal forests play a large role in the global carbon cycle, and the responses of northern trees to climate change will thus impact the trajectory of future CO₂ increases. We grew two North American boreal tree species at a range of future climate conditions to assess how growth and carbon fluxes were altered by high CO₂ and warming. Black spruce (Picea mariana, an evergreen conifer) and tamarack (Larix laricina, a deciduous conifer) were grown under ambient (407 ppm) or elevated CO₂ (750 ppm) and either ambient temperatures, a 4°C warming, or an 8°C warming. In both species, the thermal optimum of net photosynthesis (T_optA) increased and maximum photosynthetic rates declined in warm‐grown seedlings, but the strength of these changes varied between species. Photosynthetic capacity (maximum rates of Rubisco carboxylation, V_cmax, and of electron transport, J_max) was reduced in warm‐grown seedlings, correlating with reductions in leaf N and chlorophyll concentrations. Warming increased the activation energy for V_cmax and J_max (E_aV and E_aJ, respectively) and the thermal optimum for J_max. In both species, the T_optA was positively correlated with both E_aV and E_aJ, but negatively correlated with the ratio of J_max/V_cmax. Respiration acclimated to elevated temperatures, but there were no treatment effects on the Q₁₀ of respiration (the increase in respiration for a 10°C increase in leaf temperature). A warming of 4°C increased biomass in tamarack, while warming reduced biomass in spruce. We show that climate change is likely to negatively affect photosynthesis and growth in black spruce more than in tamarack, and that parameters used to model photosynthesis in dynamic global vegetation models (E_aV and E_aJ) show no response to elevated CO₂.     

Standardization of hyphal growth inhibition rate as a means of evaluating Microsporum spp. in vitro susceptibility to terbinafine, griseofulvin, and ciclopiroxolamine

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.     

Short-and long-term effects of cadmium on transmembrane electric potential (E m) in maize roots

In this study, the effects of Cd on root growth, respiration, and transmembrane electric potential (E _m) of the outer cortical cells in maize roots treated with various Cd concentrations (from 1 μM to 1 mM) for several hours to one week were studied. The E _m values of root cells ranged between −120 and −140 mV and after addition of Cd they were depolarized immediately. The depolarization was concentration-dependent reaching the value of diffusion potential (E _D) when the Cd concentration exceeded 100 μM. The values of E _D ranged between −65 to −68 mV (−66 ± 1.42 mV). The maximum depolarization of E _m was registered approx. 2.5 h after addition of Cd to the perfusion solution and in some cases, partial (Cd > 100 μM) or complete repolarization (Cd < 100 μM) was observed within 8–10 h of Cd treatment. In the time-dependent experiments (0 to 168 h) shortly after the maximum repolarization of E _m a continuous concentration-dependent decrease of E _m followed at all Cd concentrations. Depolarization of E _m was accompanied by both increased electrolyte leakage and inhibition of respiration, especially in the range of 50 μM to 1 mM Cd, with the exception of root cells treated with 1 and 10 μM Cd for 24 and 48 h. Time course analysis of Cd impact on root respiration revealed that at higher Cd concentrations (> 50 μM) the respiration gradually declined (∼ 6 h) and then remained at this lowest level for up to 24 h. All the Cd concentrations used in this experiment induced significant inhibition of root elongation and concentrations higher than 100 μM stopped the root growth within the first day of Cd treatment. Our results suggest that Cd does not cause irreversible changes in the electrogenic plasma membrane H⁺ ATPase because fusicoccin, an H⁺ ATPase activator diminished the depolarizing effect of Cd on the E _m. The depolarization of E _m in the outer cortical cells of maize roots was the result of a cumulative effect of Cd on ATP supply, plasmalemma permeability, and activity of H⁺ ATPase.     

Physiological characteristics of chemostatically grownCitrobacter freundii as a function of the specific growth rate and type of nutrient limitation

Citrobacter freundii was grown aerobically in a chemostat on a mineral medium witn galactose or glucose as carbon and energy sources under limitation by carbon or nitrogen source respectively. At various specific growth rates ranging from 7 to 95% μ_max the culture in steady state was analysed and growth yield, specific metabolic rate of substrate utilization, intracellular concentration of pyruvate, ATP, ADP, AMP and energy charge were determined and plotted as functions of dilution rate. In all four types of experiments the physiological state of cells remained practically independent of dilution rate up toD = 0.6 μ_max, and at a given specific growth rate nearly independent on μ_max and type of limitation. At approximatelyD = 0.6 μ_max, which is close to the maximum output dilution rateD _m, the physiological state of the cells changed: growth yields decreased and intr cellular pyruvate and adenylates concentrations increased. Consequently, in a given medium two dilution rates exist at which growth rate dx/dt is the same but the physiology of the population is quite different.     

Effect of cell membrane-active antimicrobial agents on membrane potential and viability of mycoplasmas

The membrane potential ofMycoplasma mycoides subsp.capri has been determined to beE _M=−48 mV±10%, inside negative. In this study we investigated the influence of cell membrane-active antimicrobial agents, viz., valinomycin, gramicidin, polymyxin, and clotrimazole, on membrane potential and viability ofM. mycoides subsp.capri. Valinomycin, an ionophore with extreme potassium selectivity, induced a membrane hyperpolarization,E _M=−110 mV. Valinomycin was not cidal, but static to mycoplasmas. Obviously the potassium drain induced by valinomycin can be compensated for by the organisms. Gramicidin is an antibiotic forming cation conduction channels across membranes. It induced a rapid depolarization,E _M=+23 mV, of mycoplasma membranes. At low concentrations, gramicidin had a static effect, whereas at high concentrations it was cidal to mycoplasmas. The rapid permeation of cations through the stationary ion channels formed by gramicidin obviously exerts an inhibitory or even lethal effect on mycoplasma metabolism and growth. Polymyxin B induced a depolarization,E _M=−35 mV, of mycoplasma membranes only when the organisms had been pretreated and hyperpolarized with valinomycin. After treatment with both valinomycin and polymyxin B, a slight inhibition of mycoplasma growth was observed. Clotrimazole, a synthetic imidazole antimycotic, hyperpolarized mycoplasma membranes (E _M=−80 mV). At high concentrations clotrimazole was cidal, whereas at low concentrations it was static to mycoplasmas.     

Dihydrotestosterone and estradiol-17β mutually neutralize their inhibitory effects on human vascular smooth muscle cell growth in vitro

We reported previously that high concentrations of either estradiol-17β (E₂) or dihydrotestosterone (DHT) inhibit growth of human cultured vascular smooth muscle cells (VSMC), mediated by cell membrane receptors and MAP-kinase–kinase activity (MEK). We now tested whether the presence of the opposite gender's dominant sex hormone modifies these effects. We incubated VSMC with various concentrations of E₂ and DHT or protein bound hormones (E₂–BSA or T–BSA), alone or in various combinations. High concentration of E₂ or E₂–BSA inhibited VSMC growth and stimulated MEK. In the presence of 3 nM DHT, high concentration of E₂ no longer inhibited ³[H] thymidine incorporation or increased MEK. Moreover, when high DHT concentration (300 nM) was added to VSMC exposed to high E₂, VSMC growth actually increased without change in MEK. DHT at 300 nM suppressed VSMC growth and increased MEK while 0.3 nM E₂ had only marginal effect on this interaction, and 30 nM E₂ reversed the inhibitory effect of DHT on cell growth. The inhibitory effects of both E₂ and DHT on VSMC cell growth and the stimulation of MEK was apparently mediated by cell membrane receptors, as it persisted when bovine serum albumin (BSA)-bound hormones were used. Further, inhibition of VSMC growth induced by E₂–BSA was reversed in the presence of T–BSA and vice versa. These results suggest that while female and male sex hormones affect VSMC growth similarly, they interfere in a dose-, hormone- and MEK-dependent manner with each other's effect.     

MOUSE NEUROBLASTOMA CELL ADENYLATE CYCLASE: REGULATION BY 2-CHLOROADENOSINE, PROSTAGLANDIN E1 AND THE CATIONS Mg2+, Ca2+ AND Mn

—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg²⁺ or Mn²⁺ in addition to ATP. In the presence of Mg²⁺, the K_m for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non-cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn²⁺ cause inhibition. Prostaglandin E₁ and 2-chloroadenosine activate the enzyme. The K_m of the cyclase for 2-chloroadenosine is 6 μm . Activation by 2-chloroadenosine leads to an increase in V_max but does not effect the K_m for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E₁ and 2-chloroadenosine is inversely related to the Mg²⁺ concentration. Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a K_i of 5 ± 10^?4m . Ca²⁺ interaction with the enzyme in the absence or presence of either 2-chloroadenosine or prostaglandin E₁ appears cooperative (i.e. Hill coefficients of ?2). Ca²⁺ inhibition is non-competitive with respect to either ATP or 2-chloroadenosine but is progressively diminished by increasing Mn²⁺ concentrations. Divalent cation effects and activation by 2-chloroadenosine and prostaglandin E₁ of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non-neuronal tissue.     

In vitro antifungal effects of potassium bicarbonate on <Emphasis Type="Italic">Trichoderma</Emphasis> sp. and <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Bicarbonates are often utilized in the food industry to avoid fermentation and to improve pH, flavor, and texture. In the same manner, bicarbonates have been demonstrated to control postharvest phytopathogens; however, there are no reports describing the effects of these chemical compounds either on soil-borne pathogens such as Sclerotinia sclerotiorum or on antagonist fungi such as Trichoderma species. This study evaluated the antifungal effect of increasing concentrations (0, 2, 4, 6, 8, 10, 25, and 50 mM) of potassium bicarbonate (KHCO₃) on the growth of Trichoderma sp. strain R39 and S. sclerotiorum under in vitro systems. Applications of KHCO₃ greater than 8 mM significantly inhibited (P < 0.001) the growth of both fungi. Concentrations of KHCO₃ lower than 25 mM did not affect the antagonistic effect of Trichoderma on the growth of S. sclerotiorum; however, this fungal interaction was not observed when exposed to 50 mM KHCO₃ because of its strong inhibition of fungal growth. In addition, KHCO₃ concentrations higher than 8 mM caused significant (P < 0.001) reduction of the sclerotium formation of S. sclerotiorum. Sclerotium germination and de novo sclerotium formation were significantly (P < 0.001) inhibited as the concentrations of KHCO₃ increased. Results show the potential benefits of potassium bicarbonate for controlling both growth and development of S. sclerotiorum, although it also exerts negative effects on the Trichoderma strain that is a natural antagonist to S. sclerotiorum.     

Effect of garlic and allium-derived products on the growth and metabolism of Spironucleus vortens

Spironucleus is a genus of small, flagellated parasites, many of which can infect a wide range of vertebrates and are a significant problem in aquaculture. Following the ban on the use of metronidazole in food fish due to toxicity problems, no satisfactory chemotherapies for the treatment of spironucleosis are currently available. Using membrane inlet mass spectrometry and automated optical density monitoring of growth, we investigated in vitro the effect of Allium sativum (garlic), a herbal remedy known for its antimicrobial properties, on the growth and metabolism of Spironucleus vortens, a parasite of tropical fish and putative agent of hole-in-the-head disease. The allium-derived thiosulfinate compounds allicin and ajoene, as well as an ajoene-free mixture of thiosulfinates and vinyl-dithiins were also tested. Whole, freeze-dried garlic and allium-derived compounds had an inhibitory effect on gas metabolism, exponential growth rate and final growth yield of S. vortens in Keister’s modified, TY-I-S33 culture medium. Of all the allium-derived compounds tested, the ajoene-free mixture of dithiins and thiosulfinates was the most effective with a minimum inhibitory concentration (MIC) of 107 μg ml⁻¹ and an inhibitory concentration at 50% (IC_50%) of 58 μg ml⁻¹. It was followed by ajoene (MIC = 83 μg ml⁻¹, IC_50% = 56 μg ml⁻¹) and raw garlic (MIC >20 mg ml⁻¹, IC_50% = 7.9 mg ml⁻¹); allicin being significantly less potent with an MIC and IC_50% above 160 μg ml⁻¹. All these concentrations are much higher than those reported to be required for the inhibition of most bacteria, protozoa and fungi previously investigated, indicating an unusual level of tolerance for allium-derived products in S. vortens. However, chemically synthesized derivatives of garlic constituents might prove a useful avenue for future research.     

Differential susceptibility ofphialophora gregata ff.sp.adzukicola andsojae to antimicrobial chemicals

The susceptibility ofPhialophora gregata ff.sp.adzukicola andsojae to antimicrobial chemicals was investigated. The minimum inhibitory concentrations (MICs) of benomyl, chloramphenicol, CuSO₄, cycloheximide and perchlorate for mycelial growth were the same for the two formae speciales. The MIC of hygromycin against f.sp.adzukicola was slightly lower than that against f.sp.sojae, and the latter was more resistant to iprodion than the former. Susceptibility to nystatin was markedly different: ff.sp.adzukicola andsojae had relative growth values of 3–20% and 59–93% at 100 µg/ml, respectively, and this difference could be used to differentiate the two formae speciales.     

Effects of E2 levuglandins on the contractile activity of the rat uterus

The effects of E₂ levuglandins on the contractile activity of rat uterine horns were studied. LGE₂, AnLGE₂, Δ⁹-LGE₂ and the synthetic epimer, 8-epi-Δ⁹-LGE₂ all induced contractions in a dose-response fashion. AnLGE₂ gave decreased response with increased bath concentrations. Paired comparision showed potent and selective inhibitory effects of AnLGE₂ on the uterotonic activity of prostaglandins. An LGE₂ inhibited the uterotonic activity of PGe₂ at a 0.1:1 ratio, of PGD₂ at 1:1 ratio, but did not inhibit the activity of PGF_2α. Exposure of sponteneously contracting uteri to high concentrations of AnLGE₂, or prolonged exposure to lower concentrations, suppressed contractions.     

Synthesis and antituberculosis activity of derivatives of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> glycoside steviolbioside and diterpenoid isosteviol containing hydrazone,hydrazide, and pyridinoyl moieties

Conjugates of the antituberculosis drug isoniazid (isonicotinyl hydrazine) and isomeric hydrazides of nicotinic and α-picolinic acid with glycoside steviolbioside from the Stevia rebaudiana plant and the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. In addition, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties joined by a dihydrazide linker were obtained. The parental compounds and their synthetic derivatives were found to inhibit the in vitro growth of Mycobacterium tuberculosis (H₃₇R_V). The measured minimal concentrations of stevio-side and steviolbioside, at which the growth of M. tuberculosis was inhibited by 100% (MIC), were 7.5 and 3.8 μg/ml, respectively. MIC values for steviolbioside and isosteviol conjugates with hydrazides of pyridine carbonic acid were within the ranges of 5–10 and 10–20 μg/ml, respectively. The maximal inhibitory effect against M. tuberculosis was shown by the isosteviol conjugates with adipic acid dihydrazide (MIC 1.7 and 3.1 μg/ml). Antituberculosis activities of the tested compounds were higher than the activity of antituberculosis drug Pyrizanamide (MIC 20 μg/ml) but lower than that of antituberculosis drug isoniazid (MIC 0.02–0.04 μg/ml).     

Intrinsic rate of natural increase in Neotropical forest mammals: relationship to phylogeny and diet

Summary The relationship of diet and phylogeny to the intrinsic rate of population increase (r _max) was examined in a sample of 39 mammalian species that live in Neotropical forests. Diets of species did not predict their r _max, contrary to published predictions based on associations between basal metabolic rate and diet and between basal metabolic rate and r _max. Phylogeny did however, apparently because life history characteristics and susceptibility to predation vary predictably with phylogeny and with one another.     

Toxic effect of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether on growth of soil isolate <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> T1/1

The toxic and growth inhibiting effects of methyl tert-butyl ether (MTBE) on the hydrocarbon-degrading Pseudomonas veronii T1/1 strain (isolated from gasoline contaminated soil) were studied. In our experiments, the MIC of MTBE was found to be 60 mM and the EC₅₀ was 51.7 mM. In the concentration range 0–30 mM, MTBE did not significantly influence the growth parameters of this bacterium, but at concentrations over 30 mM MTBE exerted a significant growth inhibiting effect. In the presence of 70 mM MTBE, the specific growth rate dropped from 0.4731 to 0.1201 h⁻¹, while the length of the lag period increased from 5.41 to 17.01 h and the yield coefficient declined from 0.2652 to 0.0718 g g⁻¹. MTBE at 100 mM inhibited the growth of this strain completely. These findings may have important environmental implications, as high concentrations of MTBE could influence the efficiency of soil and groundwater bioremediation processes significantly.     

The antimicrobial action of chitosan,low molar mass chitosan,and chitooligosaccharides on human colonic bacteria

Antibacterial effect of chitooligosaccharides (COS) and low molar mass chitosans (LMWC) is considered as one of the most important characteristics of chitosan (CS) hydrolysates. Here, we show the in vitro effect of different COS, LMWC, and CS on representative anaerobic bacteria isolated from human colon as a possibility of targeting modification of colonic microflora composition by supplementation of dietary CS products by humans. Specific growth rate of seven selected nonpathogenic anaerobic bacterial strains (Clostridium paraputrificum, Clostridium beijerinckii, Roseburia intestinalis, Bacteroides vulgatus, Bacteriodes thetaiotaomicron, Faecalibacterium prausnitzii and Blautia coccoides) was determined in the presence of 0.25 and 0.5% COS (2, 3, and 6 kDa), 0.025 and 0.05% of LMWC (10 and 16 kDa), and 0.025 and 0.1% of CS in vitro. The growth rate decreased in all strains in the presence of COS and LMWC in higher concentrations in comparison to control incubations. A relatively higher resistance to CS hydrolyzates was detected in R. intestinalis and F. prausnitzii, and more susceptible were bacteria belonging to Bacteoides sp. and Clostridium sp. The antimicrobial activity, minimum inhibitory concentrations (MIC), and minimal bactericidal concentrations (MBC) were determined. The antimicrobial activity increased with the degree of polymerization (DP). MIC ranged from 0.25 to 4.5% in dependence on bacterial strain and DP of CS/LMWC. MBC also decreased with DP. The most effective antimicrobial action was detected in LMWC with 16 kDa and CS. Weak antimicrobial activity was found in COS with small molecules (2 and 3 kDa).     

Regulation of μ-Opioid Receptor in Neural Cells by Extracellular Sodium

Abstract: SH-SY5Y neural cells expressing μ- and δ-opioid receptors were maintained viable in isotonic, sodium-free buffer in vitro. Intracellular sodium levels were manipulated by various methods, and ligand binding to intact cells was studied. In physiological buffer containing 118 mM sodium, [³H]Tyr-d -Ala-Gly-(Me)Phe-Gly-ol ([³H]-DAMGO) and [³H]naltrexone bound to μ receptor with K_D values of 3.1 and 0.32 nM and B_max values of 94 and 264 fmol/mg of protein, respectively. Replacement of sodium by choline decreased the affinity of the antagonist and increased B_max for [³H]DAMGO, without significantly affecting the other corresponding binding parameters. Depolarizing concentrations of KCl (34 mM) in physiological buffer decreased the intracellular sodium levels by 67%, but this did not decrease the [³H]DAMGO binding to the cells. Incubation of cells with monensin and ouabain increased the intracellular sodium levels dramatically (from 78 to 250 and 300 nmol/mg, respectively), with no changes in agonist binding parameters. Ethylisopropylamiloride inhibited [³H]DAMGO and [³H]naloxone binding to intact cells with EC₅₀ values of 24 and 3,600 nM, respectively. Adenylyl cyclase activities measured in intact cells, at different concentrations of sodium, showed the physiological significance of this ion in signal transduction. Potency of DAMGO in inhibiting the forskolin-stimulated adenylyl cyclase activity was significantly higher at lower concentrations of sodium. However, inhibition reached the maximal level only at 50 mM sodium, and typical sigmoidal dose-response curves were obtained only in the presence of 118 mM sodium. Furthermore, even at low or high intracellular sodium levels, DAMGO inhibition of cyclic AMP levels was normal. These results support a role for extracellular sodium in regulating not only the ligand interactions with the receptor, but also the signal transduction through the μ receptor.     

Shade-induced response and recovery of the seagrass Posidonia sinuosa

The effect of shading on the seagrass Posidonia sinuosa Cambridge et Kuo was investigated to identify mechanisms that prolong its survival during periods of low light and permit its subsequent recovery. We also tested whether the responses were consistent in plants growing at different depths. Shade treatments were low (LS; 70 – 100% of ambient Photosynthetic Photon Flux Density), medium (MS; 12 – 39%) and heavy (HS; 5 – 4%) at the shallow (3 – 4 m) site, whilst the deep (7 – 8 m) site had no HS treatment. HS at the shallow and MS at the deep site were below minimum light requirements (MLR) for the long-term survival of P. sinuosa. Physiological, morphological and growth attributes were repeatedly measured during 198 d of shade treatments and a subsequent 384 d recovery period at ambient PPFD. Shoot density declined by 82% within 105 d under HS treatment, though 6% of shoots remained after 198 d. We estimate that complete shoot loss in HS would have taken 2 years. Rhizome sugar concentrations declined to 32 – 52% of the controls at the end of the most severe shading treatments but after shoot loss, sugar concentrations declined more slowly or increased, suggesting a return to positive carbon balance. In the treatments below MLR, shading induced changes in physiological, morphological and growth characteristics, including reduced leaf length and width, reduced δ¹³C and photosynthetic adaptation to low light (increased α, reduced E_k and ETR_max), though not consistently. After removal of shading, photosynthetic characteristics became more typical of high light adaptation, possibly induced by greater light penetration through the thinned canopy, including reversal of the changes in α, E_k and ETR_max and induction of non-photochemical quenching. Carbohydrate concentrations increased to ambient concentrations within 115 d at ambient PPFD. Recovery of shoot density was slow, remaining significantly reduced in the MS and HS treatments after 384 d recovery. Shoot density at the end of shading is an important determinant of the rate seagrass meadows will recover and we estimated that the moderately and heavily shaded meadows would require 3.5 to 5 years to recover.