Volume 59, Issue 1-2

Stem bark extracts of Boerhavia erecta L. (erect spiderling) and Amaranthus spinosus L. (spiny amaranth), two wild growing weed plants used in traditional African medicine, were characterized with respect to their phenolic profile including the betalains. While the main betalains in A. spinosus were identified as amaranthine and isoamaranthine, the major betacyanins in B. erecta were betanin, isobetanin together with neobetanin. The latter showed higher betalain concentrations amounting to 186 mg/100 g, while the former contained 24 mg betacyanins in 100 g of the ground plant material. Extracts of A. spinosus were found to contain hydroxycinnamates, quercetin and kaempferol glycosides, whereas catechins, procyanidins and quercetin, kaempferol and isorhamnetin glycosides were detected in B. erecta. The amounts of these compounds ranged from 305 mg/100 g for A. spinosus to 329 mg/100 g for B. erecta.

Two new diterpenes namely verbenacine (1) and salvinine (2) have been isolated from the aerial parts of Salvia verbenaca. Their structures have been elucidated on the basis of chemical and spectral data as 3α-hydroxy-19-carboxykaur-15-ene and 19-hydroxy-12,14-dioxo labda-15,17-diene.

Acetone extract of aerial parts of Euphorbia marschalliana collected from Iran has been subjected to different chromatography techniques for fractionation and purification. The stereo- structures of the myrsinol esters 15-O-acetyl-3-O-propionyl-5-O-butanoyl-7-O-nicotinoylmyrsinol (1) and 15-O-acetyl-3,5-O-dibutanoyl-7-O-nicotinoylmyrsinol (2) have been probed using ROESY spectroscopy and modified for the stereochemistry at C-6, C-12 and C-13. β-Sitosterol (3), 29-norcycloart-5-ene (4), 5,8-lanostadiene-3β-ol (5), 3β,24(S),25-trihydroxycycloartane (6), 3β,24(R),25-trihydroxycycloartane (7) and 24-methylenecycloartan-3β-ol (8) were identified for the first time in this plant.

Two new secondary metabolites, the phenylpropanoid 3-(2′,4′,5′-trimethoxyphenyl)propanoic acid (1) and the sesquiterpene (+)-1β,4β,6α-trihydroxyeudesmane (2) were isolated from the heartwood of Cordia trichotoma Vell., along with the known sesquiterpenes (D)-1β,4β,7α- trihydroxyeudesmane (3) and (+)-1β,4β,11-trihydroxyoppositane (4). Their structures were elucidated by means of spectroscopic data interpretation, mainly 1D and 2D NMR and mass spectrometry.

A new phenylbutanone glucoside, dorycnioside, was isolated from the methanol extract of the aerial parts of Dorycnium pentaphyllum subsp. herbaceum Vill. (Rouy) and identified as 4-(4′-O-β-d-glucopyranosyl-3′,5′-dimethoxyphenyl)-2-butanone (1). In addition, two known phenylbutanone glucosides, five flavonoids, one cyanogenic glucoside, one cyclitol and one hydroquinone glucoside were also isolated and identified. The major constituent of the methanol extract was found to be myricitrin. The structure of 1 was elucidated on the basis of its spectroscopic data. It is the first time that derivatives of phenylbutanone are isolated from the Leguminosae family.

Micellar electrokinetic chromatography (MEKC) was used for the separation and determination of two iridoid glycosides, aucubin and catalpol, in several Plantago species growing in Croatia: P. altissima L., P. argentea Chaix, P. coronopus L., P. holosteum Scop. (subsp. depauperata, subsp. holosteum and subsp. scopulorum), P. lagopus L., P. lanceolata L., and P. maritima L. Hot water extraction (HWE) was applied for the isolation of iridoid substances. Significant differences appeared between the iridoid contents in the examined species. The yield of aucubin and catalpol was up to 0.27% and 1.81% of the dry mass of the leaves, respectively. Besides aucubin and catalpol, two related compounds were determined in the plant samples.

The major aromatic components of the essential leaf oil of the New Zealand lemonwood tree Pittosporum eugenioides are octyl acetate (33%), terpinen-4-ol (13%), decanol (6%) and (Z)-hex-3-enol (5%). These products are responsible for the characteristic Citrus-like aroma which is detected when the leaves are crushed, a phenomenon which provided the species with its common name. The major component of the oil, octyl acetate is also an abundant component of the essential oils of Heracleum and Boswellia species.

The Raukaua genus of the family Araliaceae consists of 3 species, R. anomalus, R. edgerleyi and R. simplex, all endemic to New Zealand. The essential oil of R. edgerleyi and R. simplex consisted largely of monoterpenes. Limonene was the most abundant (23%) in R. edgerleyi and myrcene the most abundant (34%) in R. simplex. The oil of R. simplex lacked any significant sesquiterpenoids whereas bicyclogermacrene constituted 12% of the oil of R. edgerleyi. The oil of R. anomalus contained monoterpenes in low abundance and the sesquiterpenoids germacrene-B (15%) and γ-muurolene (16%) dominated the composition of this oil. The oil of R. edgerleyi had a pleasant fresh green aroma but the low yields of all three oils excluded them from potential commercial use.

Essential oils from three of the eleven endemic New Zealand species of Pseudopanax, P. arboreus, P. discolor and P. lessonii, were found to have a fairly uniform composition which was different from that of the oils of Raukaua species that were formerly classified in the Pseudopanax genus. Oils of the three Pseudopanax species all contained significant proportions of viridiflorol and a closely related unidentified hydroazulene alcohol in common. In addition, the oil of P. arboreus contained bicyclogermacrene, linalool and long chain hydrocarbons. The oil of P. discolor contained nerolidol in abundance (36.3%) together with linalool and epi-α-muurolol. The oil of P. lessonii contained a complex mixture of sesquiterpene alcohols including epi-α-muurolol and a mixture of long chain hydrocarbons. Nerolidol and linalool provided the oil of P. discolor with a pleasant floral aroma, but the yield of oil was very low (0.01%).

From the needles of Taxus baccata the following flavonoids were isolated: 3-O-rutinosides quercetin, myricetin and kaempferol, 7-O-glucosides kaempferol and quercetin, kaempferol, quercetin, myricetin. The composition of flavonols and biflavones in some of the species of the genus Taxus, namely T. celebica, T. cuspidata, T. media and cultivar varieties T. baccata ‘Aurea’, T. baccata ‘Aurea decora’, T. baccata ‘Elegantissima’, T. baccata ‘Fastigiata’, T. baccata ‘Pyramidalis’, T. media ‘Hatfieldii’ were compared by HPLC separation.

A rapid in vitro propagation system leading to the formation of shoots, calli, roots, cell suspensions and plantlets was developed for the Andean medicinal plant Fabiana imbricata (Solanaceae). Massive propagation of shoots and roots was achieved by the temporary immersion system (TIS), morphogenesis and maintenance of cell suspensions by standard in vitro culture techniques. Oleanolic acid (OA), rutin, chlorogenic acid (CA) and scopoletin content in aerial parts of wild growing Fabiana imbricata plants as well as in plantlets regenerated in vitro, callus cultures, cell suspensions and biomass, obtained by the TIS system was assessed by HPLC. On a dry weight basis, the OA content in the aerial parts of the plant ranged between 2.26 and 3.47% while in vitro plantlets, callus and root cultures presented values ranging from not detected up to 0.14%. The rutin content of the samples presented a similar trend with maxima between 0.99 and 3.35% for the aerial parts of the plants to 0.02 to 0.20% for plantlets, 0.12% for cell suspensions and 0.28% for callus. Rutin was not detected in the roots grown by the TIS principle. The CA and scopoletin content in the aerial parts of F. imbricata ranged between 0.22-1.15 and < 0.01-0.55%, respectively. In the plantlets, the concentration of CA was 0.29 to 1.48% with scopoletin in the range 0.09 to 0.64% while in the callus sample, the CA and scopoletin content were 0.46 and 0.66%, respectively. A very different result was found in roots grown by TIS, where both OA and rutin were not detected and its main secondary metabolite, scopoletin was found between a range of 0.99 and 1.41% with CA between of 0.11 and 0.42%.

The influence of culture temperature and the concentration of sodium nitrate (NaNO 3 ) on the gas-chromatographic profile of the fatty acids of the filamentous cyanobacterium Spirulina platensis was evaluated. We found that temperature was the most important factor and that the greatest amount of gamma-linolenic acid (GLA) was obtained at 30 °C, the fatty acid profile of the Spirulina cultivated showing that (in order of abundance) palmitic, linolenic and linoleic acids were most prevalent.

The effect of temperatures (15 and 5 °C) on adsorption parameters of phytohormones at monolayers prepared from a mixture of phospholipids extracted from non-embryogenic (NE) and embryogenic (E) winter wheat calli initiated from inflorescences (inf) and embryos (emb) was studied. The surface parameter values, i.e. limiting area and collapse pressure, were determined using the Langmuir method. Phytohormones 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), kinetin, zeatin and zearalenone were investigated. The phytohormones, at a concentration of 0.2 μg/ml dissolved in water, were injected into the subphase. Phospholipids, at the concentration of 2 mg/ml, were spread at the water surface and the monolayer was compressed. The anomalous temperature effect was observed, especially, in non-embryogenic systems. In monolayers obtained from E phospholipids, the temperature effect was dependent on the kind of tissue from which the callus was initiated. Among all the examined phytohormones, the greatest changes (monolayer expansion) were found for IAA and zearalenone. However, this activity depended strongly on the kind of tissue from which the phospholipid mixture was extracted.

A GC/MS analysis of alkaloids from leaves, bulbs and roots of Pancratium maritimum was performed. From the identified 16 alkaloids, 5 alkaloids were reported for the first time for this plant. Several compounds with pharmacological activity were found. Haemanthamine was main alkaloid in the leaves and bulbs whereas galanthane was found to be main alkaloid in roots.

A new strain Renibacterium salmoninarum 27BN was isolated for its capacity to utilize nhexadecane as sole substrate. Growth on n-hexadecane was accompanied with the production of glycolipid surface active substances detected by surface pressure lowering and emulsifying activity. Glycolipid detection by thin layer chromatography and infrared spectra analyses showed for the first time that Renibacterium salmoninarum 27BN secretes the two rhamnolipids RLL and RRLL typical for Pseudomonas aeruginosa. Growth of Renibacterium salmoninarum 27BN on n-hexadecane depended on the bioavailability of the substrate and the secreted rhamnolipids appeared to be efficient in increasing hexadecane availability for the cells.

The genus Thymus (Lamiaceae) is represented by 38 species (64 taxa) in Turkey, and 24 of which are endemic to Turkey. Aerial parts of Thymus longicaulis subsp. chaubardii var. chaubardii, T. zygioides var. lycaonicus, T. longicaulis subsp. longicaulis var. subisophyllus and T. pulvinatus collected from three different localities in Balikesir province were subjected to hydrodistillation to yield essential oils which were subsequently analysed by GC and GC/ MS. The main constituents of the oils were identified, and antimicrobial bioassay was applied. Thymol (56.6%, 42.8%, 36.9%) was the main component in the oils of T. longicaulis subsp. chaubardii var. chaubardii (chemotype I), T. longicaulis subsp. chaubardii var. chaubardii (chemotype II) and T. zygioides var. lycaonicus respectively. The oil of T. longicaulis subsp. longicaulis var. subisophyllus contained carvacrol (60.0%) and the oil of T. pulvinatus borneol (27.9%) as main constituents.

Two new steroidal saponins were isolated from the rhizomes of Costus spiralis Rosc. Their structures were established as (3β,25R)-26-(β-ᴅ-glucopyranosyloxy)-22-hydroxyfurost-5- en-3-ylO-d-apio-β-ᴅ-furanosyl-(1→2)-O-[α-ʟ-rhamnopyranosyl-(1→4)]-β-ᴅ-glucopyranoside (1) and (3β,25R)-26-(β-ᴅ-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl O-ᴅ-apio-β-ᴅ-furanosyl-( 154)-O-[α-ʟ-rhamnopyranosyl-(1→2)]-β-ᴅ-glucopyranoside (2). Their structural identifications were performed using detailed analyses of 1 H and 13 C NMR spectra including 2D NMR spectroscopic techniques (DEPT, COSY, HETCOR and COLOC) and chemical conversions. The steroidal saponins were evaluated for anti-inflammatory activity.

Compounds isolated from the hexane extract of the leaves of Syzygium samarangense (Blume) Merr. & L. M. Perry were tested for inhibitory activity against the following serine proteases: trypsin, thrombin and prolyl endopeptidase. The compounds were identified as an intractable mixture of α-carotene and β-carotene (1), lupeol (2), betulin (3), epi-betulinic acid (4), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (5), 2′-hydroxy-4′,6′-dimethoxy-3′- methylchalcone (6), 2′,4′-dihydroxy-6′-methoxy-3′,5v-dimethylchalcone (7), 2′,4′-dihydroxy- 6′-methoxy-3′-methyldihydrochalcone (8) and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (9). Hydrogenation of compounds 5, 6 and 7 yielded compound 8, 2′-hydroxy-4′,6′-dimethoxy- 3′-methyldihydrochalcone (10) and 2′,4′-dihydroxy-6′-methoxy-3′,5-dimethyldihydrochalcone (11), respectively. The hydrogenated products of compounds 6 and 7 were also tested for enzyme inhibitory activity. In addition, β-sitosterol (12) and β-ᴅ-sitosterylglucoside (13) were also isolated. This is the first report of the isolation of compounds 1-6, 8 and 13 from this plant. Compounds 3-8 and 10 exhibited significant and selective inhibition against prolyl endopeptidase among three serine proteases. This is the first report of this kind of activity for all these compounds.

Porphyrins are known to be efficient photosensitizer molecules and the combined action of light and porphyrins in Propionibacterium acnes have a lethal action on the cells. Identification and quantification of in situ porphyrins in P. acnes have been done using an integrating sphere connected to an ordinary absorption spectrophotometer, and the amounts of porphyrins in the cells were quantified by measuring scattering free absorption spectra of the cell suspensions. The concentration of porphyrins in P. acnes cells were increased in either of two ways; by the addition of δ-aminolevulinic acid (ALA), which lead to the formation of coproporphyrin III under the incubation conditions used in these experiments, or by the addition of protoporphyrin IX (PPIX) to the cell suspension. In the latter case, PPIX molecules are taken up by the cells in a membrane-mediated uptake mechanism, and accumulate in the cells either on a monomeric or a particular aggregate form. The fraction of porphyrins on aggregate form increased with increasing PPIX additions. In the case of ALA induced porphyrin production, only monomeric porphyrins were stored in the cells. In both cases, the cells have a limited binding capacity of monomeric porphyrins, which is estimated to be 3 × 10 5 molecules/cell, or one porphyrin molecule to every 100st lipid molecule in the cell membrane.

A mutant Rhodotorula rubra with enhanced carotenoid-synthesizing activity for synthesizing total carotenoids and β-carotene was obtained by N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. When co-cultivated with yogurt starter bacteria (Lactobacillus bulgaricus + Streptococcus thermophilus) in whey ultrafiltrate it produced 15.7 mg total carotenoids l -1 culture fluid or 946 μg total carotenoids g -1 dry cells of which 71% was β-carotene. Grown as a monoculture in glucose substrate, the mutant shown 1.4 times lower carotenoid-synthesizing activity, and the relative share of β-carotene in the total carotenoids was lower (63%). The individual pigments torulene and torularhodin were identified, whose mass fractions were (29% and 7%) and (24% and 4%), respectively, for the mutant grown as a monoculture and as a mixed culture with the yogurt bacteria.

As a result of chemical investigation on the ethanolic extract of fresh fruit coatings of Azadirachta indica A. Juss. (neem), twenty-seven compounds were identified in non-polar to less polar fractions which showed pesticidal activity determined by WHO method against Anopheles stephensi Liston. These identifications were basically made through GC-EIMS and were further supported by other spectroscopic techniques, including 13 C NMR, UV and FTIR as well as retention indices. Thus sixteen n-alkanes, 1-16; three aromatics 2,6-bis-(1,1- dimethylethyl)-4-methyl phenol (17), 2-(phenylmethylene)-octanal (20), 1,2,4-trimethoxy-5- (1Z-propenyl)-benzene (27); three benzopyranoids 3,4-dihydro-4,4,5,8-tetramethylcoumarin (18), 3,4-dihydro-4,4,7,8-tetramethylcoumarin-6-ol (19), 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl- cyclopenta[g]-2-benzopyran (22); one sesquiterpene methyl-3,7,11-trimethyl-2E,- 6E,10-dodecatrienoate (21); three esters of fatty acids methyl 14-methyl-pentadecanoate (23), ethyl hexadecanoate (24), ethyl 9Z-octadecenoate (25) and one monoterpene 3,7-dimethyl- 1-octen-7-ol (26) were identified. Except 6, 8, 24 and 25 all these compounds were identified for the first time from the pericarp and fifteen of these, 1-3, 7, 9, 10, 17-23, 26, 27, are hitherto unreported previously from any part of the tree. Although this tree is a rich source of various natural products, it is the first report of identification of mono- and sesquiterpenes 26 and 21 and a potent antioxidant, 17.

The Mediterranean sponges Aplysina aerophoba and A. cavernicola accumulate brominated isoxazoline alkaloids including aplysinamisin-1 (1), aerophobin-2 (2), isofistularin-3 (3) or aerothionin (4) at concentrations up to 10% of their respective dry weights. In laboratory feeding experiments employing the polyphagous Mediterranean fish Blennius sphinx crude extracts of both Aplysina sponges were incorporated into artificial fish food at their physiological concentrations (based on volume) and offered to B. sphinx in choice feeding experiments against untreated control food. In addition to the Aplysina sponges, extracts from nine other frequently occurring Mediterranean sponges were likewise included into the experiments. Both Aplysina species elicited strong feeding deterrence compared to the other sponges tested. Bioassay-guided fractionation of A. cavernicola yielded the isoxazoline alkaloids aerothionin (4) and aplysinamisin-1 (1) as well as the 3,4-dihydroxyquinoline-2-carboxylic acid (8) as major deterrent constituents when tested at their physiological concentrations as present in sponges. Aeroplysinin-1 (5) and dienone (6), however, which are formed in A. aerophoba and A. cavernicola from isoxazoline precursors through bioconversion reactions upon tissue injury showed no or only little deterrent activity. Fractionation of a crude extract of A. aerophoba yielded aerophobin-2 (2) and isofistularin-3 (3) as major deterrent constituents against B. sphinx. We propose that the isoxazoline alkaloids 1-4 of Mediterranean Aplysina sponges as well as the 3,4-dihydroxyquinoline-2-carboxylic acid (8) (in the case of A. cavernicola) act as defensive metabolites against B. sphinx and possibly also against other predators while the antibiotically active bioconversion products aeroplysinin-1 (5) and dienone (6) may protect sponges from invasion of bacterial pathogens.

Thyroid hormones exert a major role in growth and differentiation of almost all types of tissues in animals, particularly in amphibian metamorphosis, through its specific nuclear receptor activation followed by gene expression. However, its function in mature tropical amphibians is less studied. The present study revealed the existence of a single class of specific nuclear receptor(s) in the liver nuclei of mature tropical toad, Bufo melanostictus, with a dissociation constant of (3.7 ± 0.9) × 10 -10 molar and maximum binding capacity of 0.074 d 0.013 pmol/mg DNA. The percentage of relative binding affinities for the specific nuclear l-T3 binding site in the liver nuclei of toad were l-triiodothyronine (l-T3) > triiodothyroacetic acid (TRIAC) > l-thyroxine (l-T4) = tetraiodothyroacetic acid (TETRAC) > 3,3′,5′- triiodothyronine (r-T3) > Diiodothyrtonine (l-T2) (100 > 75 > 19.4 = 19.4 > 3.7 > 0.39) and the relative ED50 values (in nanomolar) were 0.33 < 0.44 < 1.7 = 1.7 < 9 < 83.

Cyclopentenone prostaglandins (PGs) exhibit antiviral activity against RNA and DNA viruses in mammalian cell lines, and this effect has been associated with the induction of a heat shock protein (hsp70). We investigated the effect of prostaglandin A 1 (PGA 1 ) on the replication of vesicular stomatitis virus (VSV) in Aedes albopictus (mosquito) cells. PGA 1 was found to inhibit VSV replication dose dependently. Virus yield was reduced to 50% (3 μg PGA 1 /ml) and to 95% with 8 μg PGA 1 /ml. Even with the dramatic reduction of virus production observed in cells treated with PGA 1 , VSV-specific protein synthesis was unaltered. Treatment of cells with PGA 1 (5 μg/ml) stimulated the synthesis of a polypeptide identified as a heat-shock protein (hsp) by immunoblot analysis. PGA 1 induced hsp70 synthesis in uninfected cells. However, in VSV-infected cells the induction of hsp70 by PGA 1 was reduced. This is the first report of antiviral effects of PGs affecting the replication of VSV in a mosquito cell line.

The protein and lipid composition of a vitellin isolated from eggs of Sparus aurata were characterized by SDS PAGE, N-terminal sequence analysis and lipid analysis by thin layer chromatography and gas chromatography. The lipoprotein complex contains proteins with apparent molecular weights of 69, 59, 23, 21 and 12 kDa and were characterized as vitellinogenin fragments by N-terminal sequencing. Lipid extraction and analysis indicate an association of cholesterol and phospholipids with the protein subunits. The phospholipids contain fatty acids with 14, 16 and 18 carbon atoms as determined by GC/MS.

Termites have become an important pest of Eucalyptus and Pinus reforestations, sugarcane and other cultures. An alternative for the control of this pest would be the use of attractive traps that take in account the social behavior of these insects. Diverse factors are important for the insects in the localization of the habitat and the choice of the food and specific odors can facilitate this. Studies referring to Heterotermes tenuis (Isoptera: Rhinotermitidae) are scarce. The objective of this work was to analyze the tergal cuticular extract of H. tenuis and determine the selectivity and sensitivity of its antennae to the components of this extract by electroantennography (EAG). The composition of the cuticular extract was determined by GC-MS analysis. The hydrocarbons found were restricted to linear alkanes, being most abundant C 24 to C 27 that comprises ca. 65% of the total. Olefins were not detected. EAG and behavioral test responses to the cuticular hydrocarbons were greater and significantly different from the control and the high selectivity of the antennae to the extract indicates its potential as chemical messenger. Cuticular hydrocarbons mixture is species-specific and can be used to identify a given taxon without the diagnostic castes, soldiers or imagoes. Difference in the composition appears to relate with the type of habitat of specie.

Seventeen pure aerobic microbial isolates were obtained from soil samples of three regions of Antarctica: Casey Station, Dewart Island and Terra Nova Bay. Most of them were gram positive coryneform bacteria. Isolates were tested for their ability to grow on mineral salt agar plates supplemented with one of the following model n-alkanes or aromatic hydrocarbons: hexane, heptane, paraffin, benzene, toluene, naphthalene and kerosene. Cell hydrophobicity, the ability to produce anionic glycolipids and extracellular emulsifying activity were also determined and assessed on the basis of growth of soil isolates on hydrocarbons. This study revealed degraders with broader abilities to grow on both types of hydrocarbons, good production of glycolipids and emulsifying activity. On this basis, a mixed culture of strains is proposed, which may find application for bioremediation at temperate temperature of soil environments polluted with different hydrocarbons.

Diphenyloxide (DPO) is one of many, rather toxic pollutants produced by combustion of fossil fuels, which are emitted to the atmosphere with flue gases and brought to ground water by rain and snow. Its decomposition is investigated by ozonolysis at room temperature and the major products like phenol, resorcinol, hydroquinone, dihydroxy-benzoic acid as well as the total yield of aldehydes and carboxylic acids were determined as a function of applied ozone concentration. In addition, the DPO-degradation was studied by a combined action of γ-ray under continuous bubbling of a known ozone concentration. In this case the formation of the same products is observed, but their yields differ from the above ones. Based on the synergistic action of ozone and γ-ray the DPO-radiolysis is rather efficient, leading to an initial-G-value of 11.3. Some probable reaction mechanisms are presented for explanation of the degradation process.

Here I systematically examine the information complexity of all primary sequences of natural proteins deposited in the Swiss-Prot database. The sequence complexity is assessed by determining the frequency of occurrence of each amino acid type on sequence windows of fixed length, calculating the Shannon entropy of the window and then averaging over all windows covering the sequence. The minimum value in information content obtained from the present-day record imposes a lower limit in the number of letters that a primeval amino acid alphabet must have had.