Muscle determinants in the ascidian egg are inactivated by UV irradiation and the inactivation is partially rescued by injection of maternal mRNAs

The ascidian egg contains cytoplasmic determinants that specify the fate of larval muscle cells. In a previous study, we developed an experimental system to identify the molecular nature of muscle determinants, in which unfertilized Ciona savignyi eggs were fragmented into four pieces by centrifugation. When inseminated, only nucleated fragments (red fragments) develop into partial embryos that only show differentiation of epidermal cells. One type of enucleated fragment (black fragment) has the remarkable ability to promote muscle differentiation when fused with red fragments. In the present study, using this experimental system, we investigated the molecular nature of muscle determinants. UV irradiation of black fragments suppressed the ability to promote expression of the muscle-specific protein, myosin heavy chain. The wavelength of UV light responsible for the inactivation (250–275 nm) suggested that UV-sensitive targets are nucleic acids. Injection of poly(A)⁺ RNA isolated from an un-irradiated black-fragment-rich fraction into UV-irradiated black fragments partially recovered the ability to promote the expression of myosin heavy chain protein. Poly(A)⁺ RNA from a red-fragment-rich fraction did not rescue the suppression of UV-irradiated black fragments. These results suggest that maternal mRNAs enriched in black fragments are closely associated with muscle determinants in the ascidian egg.     

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

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

Effects of UV-irradiation at Various Wavelengths on Sterilizing Drosophila Embryos

Embryos of Drosophila melanogaster at the early intravitelline nuclear multiplication stage were irradiated with UV light at the posterior pole. The sterility and mortality of these embryos were examined in relation to the dose and wavelength of the UV light.
Sterility, expressed either as the frequency of pole-cell-deficient embryos, or as the frequency of agametic adults, was found to be dependent on the wavelength of UV light. UV-irradiation at 280 nm was found moot effective in causing sterility on Drosophila embryos. The minimum dose of radiation to give a 100% sterility was 200 J/m² at 280 nm, and 400 J/m² at 254 nm. In contrast, mortality showed no dependency on the wavelength.
The possibility that nucleic acids in the posterior region is a target of 280 nm radiation is discussed.     

Mobility of Membrane Lipids and Proteins at the Animal and Vegetal Pole of the Sea Urchin Egg

We have compared the mobility of a fluorescent lipid analogue and of fluorescently labeled membrane proteins at the animal and vegetal poles of the egg of the sea urchin Paracentrotus lividus. Translational diffusion coefficients have been measured by fluorescence microphotolysis ("photo-bleaching") on the egg which was rotated on its poles. Lipid and protein diffusion coefficients averaged 0.8 μm²/sec and 0.04 μm²/sec, respectively at both animal and vegetal pole of the egg. Substances which were known to animalize (Zn⁺⁺) or vegetalize (Li⁺) the sea urchin egg had no significant effect on protein diffusion coefficients.     

Increased efficiency of photoreversal of UV-induced dimers in the DNA of chick embryo fibroblasts with post-UV dark time

The kinetics of photoreversal of UV-induced dimers in the DNA of early passage chick embryo fibroblasts was studied by monitoring disappearance of UV-endonuleae-sensitive sites. Photorepair was found to increase in efficiency when cells were incubated in the dark for several hours at 37°C following the dimer-inducing short-wavelength (254 nm) UV treatment, but prior to the photoreactivating black light (365 nm). Folllowing a UV dose of 10 J/m² it took at least 4 h in the dark to saturate this effect. This UV dose inserts roughly 2.4 dimer/10⁷ daltons of DNA. Dark repair removes about 0.08 dimers /h/10⁷ daltons. After 6 h in the dark, exposure to black light removes an additional 1.4 dimers /10⁷ daltons leaving about 0.5 dimers unaffected by this treatment. After saturation of the dark effect, the amount of photoreactivation depends only on total black light fluence and not on fluence rate for the range of rates studied. This indicates that during 30 min, the maximum time of black light exposure, no appreciable reattachment of the photorepair molecule to additional unrepaired dimer sites occurs. We estimate that the number of effective photorepair molecules per chick chick cell is at least of the order of 2 × 10⁵.     

Adaptations to extreme low light in the fern Trichomanes speciosum

Trichomanes speciosum is a threatened species restricted to sheltered, very humid sites. Uniquely amongst European ferns, differing ecological tolerances of the gametophyte and sporophyte generations are manifested as widely differing distributions. The perennial, vegetatively propagating gametophyte persists in drier, colder, darker habitats than the sporophyte. In sites where the gametophyte grows, light availability was found to be < 1 μmol m² s¹ for approx. 85% of daylight hours, rarely or (in some sites) never rising above 10 μmol m² s¹. Much of the time, light was < 0.01% of full sunlight. Measurements of gas exchange and chlorophyll fluorescence yield show that these plants have optimal photosynthesis at light intensities c . 5–10 μmol m² s¹, the highest light to which they are normally exposed to in their natural environment. The absence of any capacity for reversible nonphotochemical fluorescence quenching means that there is little or no protection of the photosynthetic apparatus from light-induced damage. We conclude that these plants are able to create what are essentially monocultures in their extreme environments only because of a combination of low metabolic rate (at low temperatures) and an ability to make efficient use of what little light is available to them by morphological and physiological means.     

Photophobic stop-response in a dinoflagellate: Modulation by preirradiation

Gyrodinium dorsum Kofoid responds photophobically to flashes of blue light. The photophobic response consists of a cessation of movement (stop-response). Without background light and after a flash fluence above 10 J m⁻², 75–85% of the cells show a stop-response, while only 50% of the cells show this response at 5 J m⁻². With a flash fluence of 5 J m⁻², background light of different wavelengths either increases (614 nm. 5.5–18.2 μmol m⁻² s⁻¹) or decreases (700 nm, 18.4–36.0 μmol m⁻² s⁻¹) the stop-response. Two hypotheses for the mechanism of the modulation by background light of the photophobic response are discussed: an effect of light on the balance of the photosynthetic system (PS I/PS II) or an effect on a phytochrome-like pigment (P_r/P_fr). This study supports the idea that a phytochrome-like pigment works in combination with a blue light-absorbing pigment. It was also found that cells of Gyrodinium dorsum cultured in red light (39.8 μmol m⁻²) had a higher absorption in the red region of the absorption spectra than those cultured in white light (92.7 μmol m⁻²).     

Development of myoplasm-enriched ascidian embryos

The fertilized ascidian egg is thought to be comprised of distinct regions of tissue-specific cytoplasmic determinants. This idea was tested by bisecting fertilized eggs into egg fragments and culturing them until the unoperated controls developed into larvae. Fertilized eggs were bisected using a microsurgical method in which part of the uncleaved zygote was extruded through a hole made in the follicular envelope and the cytoplasmic bridge between the two egg regions was severed. One egg fragment contained all of the egg myoplasm (termed myoplasm-enriched or ME fragment), while the other fragment lacked myoplasm. ME fragments consisting of 40-50% of the total egg volume in many cases cleaved normally and developed into larvae. In a few cases, ME larvae initiated metamorphosis and developed into normal juveniles. Triton-extraction of ME embryos and larvae showed that the myoplasm was redistributed into nonmuscle lineage cells at each stage of development. Despite the redistribution of myoplasm into many of the endoderm cells situated in the head region of ME larvae, the expression of the muscle-specific enzyme acetylcholinesterase (AchE) and a muscle-specific antigen (Mu-2) was restricted to the tail muscle cells. The endoderm cells situated in the head region of ME larvae expressed an endoderm-specific enzyme alkaline phosphatase (AP) as in the controls. Furthermore, cleavage-arrested four- and eight-cell ME embryos expressed AchE activity in the expected number of blastomeres. When a greater quantity of myoplasm was redistributed into cells that normally do not express AchE activity by producing 10-30% ME embryos, in a few cases more than the expected number of blastomeres expressed AchE activity. In conclusion, the main finding of the present investigation, based on the development of ME fragments comprising 40-50% of the total egg volume, is that ascidian embryos are capable of regulative development.     

Short-term thermal acclimation induces adaptive changes in the inner mitochondrial membranes of fish skeletal muscle

In the oxidative muscles (musculi laterales superficiales) of crucian carp Carassius carassius acclimated for 6 weeks to either 5 or 25° C, the volume density and the surface density of fibres per tissue did not differ significantly between the control and experimental groups. The correlation ratio (μ²) for these values was below 50, 39·3 and 43·9 respectively. After acclimation to 5° C, the surface density of outer mitochondrial membrane per fibre increased significantly from 0·93 to 1·23m² cm⁻³ in the summer population but dropped from 0·94 to 0·67 m² cm⁻³ in the winter population. The surface density of outer mitochondrial membrane per mitochondrion increased from 3·24 to 4·52 m² cm⁻³ in summer fish. After acclimation to 25° C, the surface density of inner mitochondrial membranes per muscle fibre decreased from 4·04 to 1·79 m² cm⁻³ in summer fish and from 3·86 to 1·07 m² cm⁻³ in winter fish. The surface density of inner mitochondrial membranes per mitochondrion increased from 14·17 to 15·60 m²cm⁻³ in summer fish but dropped from 13·91 to 10·67 m² cm⁻³ in winter fish. Correlation matrices demonstrate a negative correlation of the surface density of outer mitochondrial membrane per mitochondrion with the volume density of mitochondria per fibre and temperature, suggesting cold-induced proliferation of small mitochondria. It was concluded that short-term cold acclimation increased surface area of the inner mitochondrial membranes in summer fish.     

Effect of light quality (blue, red) and fluence rate on the synthesis of pigments and pigment-proteins in maize and black pine mesophyll chloroplasts

Maize ( Zea mays L. hybrid ZP-704) and black pine ( Pinus nigra Arn.) were grown for five days at low fluence rate (0.4–4.0, μmol m^–2 s⁻¹) in blue or red light. Compared to red light of the same fluence rate, blue light effects in maize were repressive for the accumulation of Chita, b , carotenoids and light-harvesting complex-2 (LHC-2) proteins. The maximal reduction of proteins bound to the light-harvesting complex of photosystem 2 and pigments was attained at different fluence rate levels. In black pine, blue light compared to the red of the same fluence rate level either activated or reduced accumulation of pigments and LHC proteins, the effect being dependent on its fluence rate level. At fluence less than 3.0 μmol m⁻² s⁻¹ blue light was more efficient for the synthesis of Chi a, b and carotenoids, hut for LHC-2 complexes, fluence rates between 0.4 and 1.5 [μmol m⁻² s⁻¹ were more effective. In pine the effects of the two lights on the accumulation of pigments and LHC proteins were demonstrated separately and were dependent on fluence rate level. This suggests irradianoe-controlled activation/deactivation of the photoreceptor at the level of the cell.     

Influence of blue light on the activity of phosphofructokinase in Chlorella kessleri

In crude extracts of Chlorella kessleri Fott and Novákóva cells grown autotrophically in white light the activity of phosphofructokinase (PFK, EC 2.7.1.11) is 62.9 ± 1.5 nmol (mg protein)⁻¹ min⁻¹ under optimized test conditions. It is greatly increased in red [88.3 ± 1.8 nmol (mg protein)⁻¹ min⁻¹], but somewhat decreased [57.0 ± 0.5 nmol (mg protein)⁻¹ min⁻¹] in blue light of equal productivity. Mixtures of blue and red light yield the low activity as long as blue light represents at least 35% of the total quantum fluence rate. The rough wavelength dependence of the counteracting effect of short wavelength light on the increasing effect of red light exhibits a broad peak at 460 nm, reminiscent of action spectra of the blue/UV photoreceptors(s). Upon transfer of red light-grown cells to blue light, the decrease develops slowly within 72 h; it cannot be prevented by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). Since there is less carbohydrate in blue than in red light-exposed cells, correlations between biosynthesis of PFK and level of carbohydrate are discussed, based on the assumption that red light decreases and/or blue light increases the transport of metabolites across the chloroplast envelope.     

Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage-arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three-fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.     

Dosage differential effects of permethrin impregnated into bednets on pyrethroid resistant and susceptible genotypes of the mosquito Anopheles stephensi

Abstract Effects of bednets impregnated with permethrin 200 mg and 500mg/m² on pyrethroid resistant and susceptible strains of Anopheles stephensi and their F hybrid progeny were studied, using free-flying female mosquitoes of these three genotypes, in a room with a human subject under a polyester net, having one of his arms in contact with the treated netting. Unexpectedly an apparently higher feeding rate, but lower knockdown and mortality rates, of mosquitoes were obtained for each of the three genotypes with the higher concentration of 500mg/m² compared with the lower dose of 200mg/m². At the lower dose there was 100% mortality 24 h after exposure of all three genotypes, suggesting that there would not be selection for resistance at this dose. However, at the higher dose there was significantly higher mortality of the susceptible strain than of the F hybrids, suggesting incomplete recessiveness of this resistance and that there would therefore be effective selection for resistance by this dose.
When female mosquitoes were confined in bioassay cones on treated netting, the resistant strain of An.stephensi showed significantly less irritability (scored as the time until first flight take-off) in response to each dose, as compared with the susceptible strain and F, hybrids. The higher dose provoked more irritation of each genotype; this could explain the greater knockdown and mortality rates of mosquitoes exposed to the lower dose which was less irritating and hence more effectively insecticidal. Thus a dose of 200mg/m² is preferable to 500mg/m² for malaria vector control.     

Mechanism of calcium-dependent salt tolerance in cells of Nitellopsis obtusa: role of intracellular adenine nucleotides

Abstract. The tonoplasts of internodal cells of Nitellopsis were removed by perfusing the vacuoles with media containing a Ca² chelator, EGTA. Treatment of tonoplast-free cells with 100 mol m³ NaCl induces a large membrane depolarization, a drastic decrease in the membrane resistance and an increase in Na⁺ influx. These events are identical to those that occur in intact cells subjected to high NaCl. These responses to NaCl are prevented if 10 mol m³ Ca²⁺ is supplied together with 100 mol m³ NaCl. The protective effect of Ca²⁺ is evident only when the intracellular ATP concentration exceeds 0.1 mol m³ and does not occur full when the intracellular ATP is removed. AMP at concentrations greater than 0.5 mol m³ or 0.25 mol m³ AMPPNP can replace ATP. It is concluded that ATP does not act as an energy source nor as a substrate for protein phosphorylation. ATP seems to exert its effects as a coeffector with Ca²⁺ in regulating the Na⁺ permeability of the plasma membrane.     

Characterization of label-retaining cells in the epidermis of a human skin equivalent

Abstract. The human skin equivalent (HSE) is an in vitro reconstructed model that resembles skin morphologically and biochemically. The HSE is formed by overlaying a fibroblast-populated collagen matrix with a suspension of epidermal cells. Basal keratinocytes attach to the dermal equivalent via a newly formed basement membrane and multiply to form a stratified, differentiated epidermis. The aim of the studies described here was to characterize the basal cells of the HSE in terms of their cell cycling potential. The experiments utilized long-term labelling of the cells with tritiated thymidine ([³H]dT), followed by irradiation with ultraviolet light. [³H]dT incorporation was analysed via routine autoradiography. Irradiation with 100 J/m² UV light increased the number of labelled basal cells by 58% over the control, the maximal stimulation observed. Decreased numbers of labelled basal cells were observed at doses of UV light greater than 100 J/m². The maximal number of labelled basal cells was observed on day 14 and decreased over time; the number of labelled suprabasal cells increased concomitantly. Label-retaining cells (12%) persisted in the stratum basale of control HSEs after 32 days in culture. Labelled cells were observed in the apical layers of the stratum granulosum of control HSEs after 22 days in culture. These data suggest that the stratum basale of the HSE contains a population of slow-cycling cells whose characteristics resemble a subpopulation of slowly cycling cells found in normal human skin.     

Direct and indirect effects of light on stomata. I. In Scots pine and Sitka spruce

Abstract. The response of stomatal conductance to broadband blue and red light was measured in whole shoots of Scots pine and Sitka spruce, two species which have low stomatal sensitivity to CO². In Scots pine, blue light was more than three times more effective than red light (on an incident quantum basis) in opening stomata, particularly at low quantum flux densities (<100μmiol m⁻² s⁻¹). However, the apparent quantum yield of net CO² assimilation rate in blue light was only half that in red light. The contrasting effects of red and blue light on conductance and assimilation led to higher intercellular CO² concentrations (C_i) in blue light (up to 100 μmol mol⁻¹ higher) than in red light. Similar results were obtained with Sitka spruce shoots, though differences in the effectiveness of red and blue light were less marked. In both species, both red and blue light increased conductance in normal and CO²-free air, indicating that neither red nor blue light exert effects through changes in C_i or mesophyll assimilation. However, decreases in C_i caused increases in conductance in both red and blue light, suggesting that these direct effects of light are not wholly independent of CO².     

The reproductive success of pike, Esox lucius: aspects of fecundity, egg density and survival

Power-law relationships have been estimated between fecundity and fish length, weight and age for pike from two gravel pit lakes (Main Lake and St Peter's Lake) in Buckinghamshire, England. Relative fecundity, estimated at 17.6 eggs g⁻¹ for Main Lake pike and 19.4 g⁻¹ for St Peter's Lake pike, did not differ significantly between the lakes. Population fecundity for the Main Lake was estimated at 10.6 million eggs and 6.4 million eggs in 1986 and 1987, respectively.
Experimental work involving samples of eggs from 18 artificially fertilized Main Lake female pike showed a significant relationship between egg diameter and female length, and a significant difference between mean egg diameters for 41-cm and 101-cm females. No significant relationships were found between mean egg size and mean fry size, nor between fry length and adult length. Mean size differences between newly hatched, 18-day-old and 41-day-old fry from the 41-cm and 101-cm females were not significant.
Samples of eggs taken at 2-day intervals from 6 April to 5 May in a pike spawning area of the Main Lake revealed clumped distributions of eggs, with average egg densities ranging from 1 to 47 eggs per 0.071 m² (14–671 m ²) on sand and silt substrata. The maximum egg density of 51 per 0.071 m² (729 m⁻²) was found on flooded grass. The stages of development of the eggs were identified over the sampling period. Survival from stage 1 to fry was estimated at 3–6%. Egg losses were estimated at 9–10% day⁻¹.
Egg survival experiments gave overall hatching success rates of 11 % on sand/silt compared with 2.5% on aquatic plants (1986), and 18.9% on undisturbed sand/silt compared with 7.9% on disturbed sand/silt (1987). The effect of siltation on egg hatching is discussed.     

The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal

Rose Bengal was cytotoxic to the following bacteria at the concentrations given in parentheses (highest concentrations of dye in mol/1 at which growth occurred on nutrient medium): Brochothrix thermosphacta and Deinococcus radiodurans (1 times 10^-6 or less); Streptococcus, Micrococcus, Staphylococcus, Bacillus, Arthrobacter and Kurthia spp. (1 times 10^-5–1 x 10^-4), and Pseudomonas spp. and Enterobacteriaceae (5 times 10^-3–1 x 10^-2 or greater). These organisms were killed rapidly when suspended in illuminated (170 μE/m²/s) solutions of Rose Bengal (1 times 10^-4 mol/1) providing oxygen was present. Singlet oxygen was identified as the lethal agent, because the rate of killing was increased by dissolving the dye in deuterium oxide while the organisms were protected against photoinactivation by L-histidine or crocetin. Yeasts from chilled foods were killed in illuminated solutions of Rose Bengal but a light intensity of 315 μE/m²/s was needed for a death rate comparable with that of bacteria. The yeasts present in a range of chilled meat and dairy products failed to form colonies on Rose Bengal (5 times 10^-5 mol/1) media exposed continuously to modest illumination (55–80 μE/m²/s).     

CIRCADIAN GROWTH IN PORPHYRA UMBILICALIS (RHODOPHYTA): SPECTRAL SENSITIVITY OF THE CIRCADIAN SYSTEM

The circadian rhythm in growth of the red macroalga Porphyra umbilicalis (Linnaeus) J. Agardh was investigated under different spectral light conditions in laboratory-grown thalli. A free-running rhythm was observed in constant green or red light at irradiances of 2.5 to 20 μmol photons·m⁻²·s⁻¹, whereas arhythmicity occurred in constant blue light at 6–20 μmol photons·m⁻²·s⁻¹. The circadian oscillator controlling growth rhythmicity in Porphyra uses most of the visible sunlight spectrum and possibly multiple photoreceptors with a high sensitivity for blue light and a lower sensitivity for red light. This was inferred from three experimental results: (1) The free-running period, τ, of the growth rhythm decreased with increasing irradiance, from approximately 25 h at 2.5 μmol photons·m⁻²·s⁻¹ to 22 h at 20 μmol photons·m⁻²·s⁻¹ in red or green light, (2) Dark pulses of 3 h duration, interrupting otherwise continuous green or red light, caused advances during the subjective day and delays during the subjective night; the circadian oscillator in Porphyra can discriminate darkness from green or red light, and (3) Low-irradiance blue light pulses (2.5 μmol photons·m⁻²·s⁻¹) shifted the growth rhythm in red light of higher irradiance (e.g. 10 μmol photons·m⁻²·s⁻¹), and a strong, high amplitude, type 0 phase response curve was obtained that is usually observed with light pulses shifting a circadian rhythm in otherwise continuous darkness.     

Effects of light and temperature on the cell size and some biochemical components in two freshwater cryptophytes

The effects of light and temperature on cell size and cellular composition (chlorophyll, protein, carbohydrate) of two freshwater cryptophytes were studied with batch cultures. Neither of the species had a constant cell size but the size varied with growth conditions. At each temperature the smallest cells were recorded at the lowest experimental photon flux density. The smallest cells of Cryptomonas 979/67 had an average volume of 232 μm³ and the largest ones 1 020 μm³. In Cryptomonas 979/62 the smallest and largest cells measured 4 306 μm³ and 12 450 μm³. Both species increased their cellular chlorophyll content when PFB dropped below 110–120 μmol m^-2 s^-1. The highest and lowest chlorophyll contents of 979/67 were 7.45 fg μm^-3 and 0.55 fg μm^-2 respectively. For 979/62 the corresponding values were 10.23 fg μm^-3 and 0.93 fg μm^-3. In both species the protein content remained stable at PFDs higher than 110–120 μmol m^-2 S^-1. The highest content of protein measured in 979/67 was 638 fg μm^-3 and the lowest 147 fg μm^-3. For 979/62 these values were 1 036 fg μm^-3 and 148 fg μm^-3 respectively. The carbohydrate results were less clear and no pattern either in response to photon flux density or temperature was obvious. The lowest and highest contents recorded for 979/67 were 62 fg μm^-3 and 409 fg μ^-3 and for 979162, 36 fg μm^-3 and 329 fg μm^-3