Real time observations of polylysine,dextran and polyethylene glycol induced mutual adhesion of erythrocytes held in suspension in an ultrasonic standing wave field

A technique which enables cells to be observed in suspension for times of the order of minutes (employing acoustic radiation foreces in a 1 MHz ultrasonic standing wave field) is described. Video recordings of the mutual adhesion of human erythrocytes in suspension have been analysed. Concave-ended cell doublets and linear rouleaux developed in 0.5–1.5% w/v Dextran T500 by a gradual (2.5–17 s) increase in the area of cell contact over the cell cross-section. The concave-ended rouleaux form was not seen in polylysine or in polyethylene glycol. In 5–7% dextran and in 20g/ml polylysine mutual adhesion was a two stage process. Cells first form a strong local contact which persists (without apparently growing in area) for a number of seconds following which the cell surfaces move suddenly to form a spherical doublet. The average initial contact time and engulfment time for cells in 7% Dextran T500 are 18 and 2.7 s, respectively. The corresponding values for cells in 20 g/ml, 14 kDa, polylysine are 2.7 and 0.3 s. There was no initial contact delay during spherical doublet formation in mg/ml polylysine. Electron microscopy showed that the intercellular seam for spherical doublets formed with all three agglutinating molecules was bent in a wavy (4 m) profile. The thickness of the intercellular space varied in a spatially periodic way (0.8 m) for cells in polylysine. Examples of periodic intercellular spaces were seen by light microscopy in polyethylene glycol induced clumps. The role of interfacial instability in the adhesion processes is discussed.     

Multiple shoot-bud formation and plantlet regeneration on Castanea sativa Mill. seeds in culture

Primordial initiation and development of shoot-buds has been accomplished by using shoots derived from chestnut (Castanea sativa Mill) seedlings cultured with added 6-benzylaminopurine (BAP). Germination of chestnut seeds in the presence of BAP (4 – 40 M) stimulated varying numbers of shoot-buds in those areas of the main axis that were favorably altered. When excised single shoots from these treated seeds were subcultured on a fresh medium containing BAP (4 – 40 M) continual shoot production was observed. Bud growth and shoot elongation were stimulated by transferring cultures to a reduced concentration of BAP (2 M) plus indole-3-butyric acid (IBA 0.4 M). Plant regeneration occurred in the presence of IBA (0.8 M) after a preconditioning treatment in which naphthaleneacetic acid (NAA 50 M) and kinetin (k 2 M) were applied to the tissue culture shoots for 7 days in light.     

Fabrication of Stable Packed Capillary Reversed-Phase Columns for Protein Structural Analysis

Capillary column (320-m ID) liquid chromatography is an essential tool for the separation and concentration of low-picomole amounts of proteins and peptides for mass-spectrometric based structural analysis. We describe a detailed procedure for the fabrication of stable and efficient 50- to 180-m ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reversed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-silica particles were employed for column end-frit construction. The ability of these columns to withstand high back pressures (300–400 bar) enabled their use for rapid chromatography (>3400 cm/hr; i.e., 40 l/min for 200-m ID columns) and the loading of large sample volumes (up to 500 l). The accurate low flow rates (0.4–4.0 l/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr. 599, 119–130]. Column performance was evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

A decrease in intracellular zinc level precedes the detection of early indicators of apoptosis in HL-60 cells

Low extracellular zinc concentrations have been associated with the induction of apoptosis. To assess the relationship between intracellular zinc concentration and the rate of apoptosis, cells were grown in media containing 0.5, 25, or 50 M zinc and analyzed by flow cytometry or fluorescence microscopy. Cells grown in 0.5 M zinc medium over 48 h showed a successive decrease in intracellular zinc concentration measured by the zinc-specific fluorophore, zinquin. After 18 h in 0.5 M zinc medium, rhodamine 123 retention decreased. However, the addition of 10 M zinc to the 0.5 M medium before 16 h in culture restored rhodamine retention in the cells. After 30 h there was an increase in the number of cells cultured in 0.5 M zinc medium that bound annexin V-FITC. These data indicated that decreased intracellular zinc concentration preceded early markers of apoptosis, with alterations in mitochondrial transmembrane potential preceding the loss of polarity in the cell membrane.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Trois nouveaux Nadejdolepis Spasskii & Sasskaya, 1954 (Cestoda: Hymenolepididae) parasites de Charadrii (Aves) de Tasmanie

Three species of Nadejdolepis from Tasmania, Australia, are described and illustrated. N. burgessi n. sp., a parasite of Charadrius ruficapillus, is 4-6 mm long, with rostellar nitiduloid hooks 63-66 m long, a short evaginated cirrus 13-16 m long with a short collar of thin spines 1 m long, a narrow and tubular sclerotinoid vagina 40-50 long and 3-4 m in diameter with a little ampulla 3-5 m in diameter at the proximal end, and a membranous atrial segment with smooth, short (1 m) and compact spines which are sometimes difficult to observe. N. smithi n. sp., a parasite of 40-50 long and 3-4 m in diameter with a little ampulla 3-5 m in diameter at the proximal end, and a membranous atrial segment with smooth, short (1 m) and compact spines which are     

Different effects of inorganic and triethyl lead on growth and ultrastructure of lily pollen tubes

Summary Pollen tubes ofLilium longiflorum growingin vitro were treated for 1 h with inorganic lead (Pb) and with triethyl lead (TriEL) and studied by light and electron microscopy. Pb was considerably more toxic in relation to inhibition of pollen tube growth (EC₅₀=6 M Pb) than was TriEL (EC₅₀=60 M TriEL). On the other hand, at almost the entire concentration range tested (25-500 M) TriEL caused aberrant tubes and tube swellings. Pb did not cause tube swellings, even at highly growth-impairing concentrations. Pb (60 M) predominantly affected the ultrastructure of the growing cell walls without impairing the distribution of the cell organelles in the tube tips. In contrast, 50 and 100 M TriEL did not visibly influence cell wall ultrastructure but it severely damaged dictyosomes; 100 M TriEL also disturbed the original order of cell organelles in the tube tips. Cortical microtubules were selectively and completely destructed by TriEL at concentrations (50 M) where no effect on polar organization of the tube tips occurred but they remained unimpaired by 60 M Pb, indicating selective and effective interaction of TriEL with these cell organelles.Abbreviations EC⁵⁰ effective lead concentration causing 50% inhibition of pollen tube growth - MTs microtubules - Pb inorganic lead - TriAL trialkyl lead - TriEL triethyl lead     

Zinc protects against arsenic-induced apoptosis in a neuronal cell line,measured by DEVD-caspase activity

Acute and chronic arsenic exposure results in toxicity in humans and causes many neurological and other manifestations. For the first time the present study reports that zinc decreases arsenic-induced apoptosis and also confirms a single report of apoptosis induced by arsenic in a neuronal cell line. Apoptosis measured by DEVD-caspase activity peaked between 10 M and 20 M of arsenic trioxide. Higher concentrations of arsenic up to 40 M caused increasing cell death with diminishing DEVD-caspase activity. The beneficial effect of zinc was proportional to its concentration with a significant decrease in arsenic-induced DEVD-caspase activity at 50 M and 75 M zinc (P<0.05). This finding may be of therapeutic benefit in people suffering from chronic exposure to arsenic from natural sources, a global problem especially relevant to millions of people on the Indian subcontinent.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: Some methodological aspects

The time course of endogenous phosphorylation in vitro of total or separted synaptic plasma membrane proteins (SPM) has been correlated with that of hydrolysis of the phosphate donor (ATP) in the incubation medium. The ATP/SPM ratio in the medium was varied. In a low-ratio medium (7.5 M ATP; 2.2 g SPM/l) a complete hydrolysis of ATP occurred almost instantaneously as was measured by the release of free phosphate in and the disappearance of ATP from the medium. As a consequence, only a very short peak of phosphorylation, followed by dephosphorylation was observed. However, when higher ATP/SPM ratios were used (200 M ATP; 0.4 g SPM/l and 500 M ATP; 0.4 g SPM/l), the incorporation of phosphate into SPM proteins was linear for 20 sec, and the maximum level of phosphate incorporation was increased. Similar results were obtained after separation of³²P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained fromone SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.     

Uptake and Retention of Selenite and Selenomethionine in cultured K-562 cells

The selenium uptake and retention have been studied in K-562 cells exposed to selenite or selenomethionine. In the uptake experiments the cells were exposed to two doses of selenite (5 or 50 M) or selenomethionine (10 or 50 M). In the retention study the cells were treated for 2 h with the above mentioned doses of the selenocompounds before being observed at different times. The selenium uptake in cells exposed to selenite 5 M began to saturate at 8 h, but increased again between 48 and 96 h. In cells exposed to selenite 50 M the selenium uptake never reached a maximum, however, at 48 and 96 h the cell viability decreased strongly. The two doses of selenite showed different retention patterns, with a relatively small cellular decrease of selenium after treatment with selenite 5 M compared to treatment with 50 M of selenite. The selenium uptake in cells exposed to selenomethionine 10 M or selenomethionine 50 M began to saturate at 24 h and 48 h, respectively. The retention patterns were similar for both selenomethionine doses with a continuous decrease of the selenium concentration during the whole observation period. The results indicated a more controlled uptake and retention pattern of selenomethionine compared to selenite.     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Coccidia of Brazilian edentates: Eimeria cyclopei n.sp. from the silky anteater,Cyclopes didactylus (Linn.) and Eimeria choloepi n.sp. from the two-toed sloth,Choloepus didactylus (Linn.)

Summary Eimeria cyclopei n.sp. is described from the silky anteater, Cyclopes didactylus, from Pará State, north Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in seven days at 26 to 28°C. Oocysts are ellipsoidal to sub-spherical, with a mean size of 28.1 × 23.6 m: the wall is 1.5 to 2.0 m thick, apparently with an outer thin, colourless membrane and two inner, thicker, striated and yellowish layers. There is no micropyle, oocyst residuum or polar body. The mean measurements of sporocysts are 19.0 × 9.0 m, and they are slightly asymmetrical, elongate pear-shape, with a plug-shaped Steida body projecting beyond the end of the sporocyst. Sporozoites are as long as or longer than the sporocysts: The sporocyst residuum is scattered between sporozoites in younger specimens and becomes condensed into rounded mass in older ones. The endogenous stages occur in the epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Uninucleate meront, microgamont and macrogamont precursors are recognizable morphologically. Mature meronts are 20.0 × 15.7 m some produce 12 to 20 merozoites which are 8.7 × 2.0 m, and others 10 to 26 merozoites which are 11.4 × 2.0 to 15.0 × 3.0 m. Mature microgamonts which are 27.5 × 24.1 m, produce from 150 to 170 microgametes of 7.1 × 1.0 m: microgametes have two flagella of unequal length. Mature macrogamonts are 28.4 × 24.5 m Eimeria choloepi n.sp. is recorded from the two-toed sloth, Choloepus didactylus, from the same area of Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in 23 days at 26 to 28°C. Oocysts with a mean size of 23.0 × 20.3 m, have a wall 2.0 to 2.5 m thick which is composed of two thick, yellowish and striated outer layers and a delicate, colourless inner one. There is no micropyle, oocyst residuum or polar granule. Mature sporocysts with a mean size of 11.3 × 7.1 m, are ellipsoidal to egg-shaped and have a poorly developed Steida body. The sporocyst residuum is composed of a small number of large globules: The sporozoites are longer than the sporocyst and strongly recurved. The endogenous stages occur in epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Dimorphic meronts produce 8 to 18 merozoites which are either 13.0 × 2.0 m or 13.0 × 3.0 m. Microgamonts produce 50 to 80 microgametes of 8.0 × 1.0 m. Mature macrogamonts are 18.3 × 17.9 m. ac]19820212     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Effects of GABA antagonists,SR 95531 and bicuculline,on GABAA receptor-regulated chloride flux in rat cortical synaptoneurosomes

Synaptoneurosomes isolated from cerebral cortices of male Sprague-Dawley rats were used for studying GABA_A receptor-regulated chloride influx. The in vitro effects of GABA antagonists, SR 95531 (a pyridazinyl GABA derivative) and bicuculline, on pentobarbital-stimulated, muscimol-stimulated or flunitrazepam-enhanced, muscimol-stimulated chloride uptake were studied. The chloride uptake was determined at 30°C, for 5 sec. Pentobarbital and muscimol produced a maximal stimulation of chloride uptake in cortical synaptoneurosomes at 500 M and 50M, respectively. SR 95531 as well as bicuculline had no effect on the basal uptake of chloride. Whereas, SR 95531 (0.3–30 M) and bicuculline (0.1–100 M), when added 5 min before muscimol (50 M), produced a significant concentration-dependent inhibition of muscimol (50 M)-stimulated chloride uptake (IC₅₀ s of 0.89±0.11 M and 13.45±2.10M, respectively). In studies of the inhibitory effects of SR 95531 and bicuculline on pentobarbital (500 M)-stimulated chloride uptake, the IC₅₀ s were 0.81±0.12 M and 3.86±1.14 M, respectively. SR 95531 exhibited a more potent inhibitory effect than bicuculline on flunitrazepam-enhanced, muscimol-stimulated chloride uptake. The results revealed that SR 95531 has a more potent antagonistic effect than bicuculline on GABA_A-regulated chloride flux.     

Induction of apoptosis by gemcitabine in BG-1 human ovarian cancer cells compared with induction by staurosporine, paclitaxel and cisplatin

A variety of chemotherapeutic agents induce cell death via apoptosis. We had shown previously that gemcitabine (2,2-difluorodeoxycytidine) induced an atypical apoptosis in BG-1 human ovarian cancer cells; therefore, further studies were conducted to characterize more precisely gemcitabine-induced apoptosis in BG-1 cells compared to a general inducer of apoptosis, staurosporine. BG-1 cells exposed to 0.5, 1.0 and 10 M gemcitabine for 8 h, or staurosporine (1.0 M) for 6 h, exhibited high molecular weight DNA fragmentation (50 kbp); however, only staurosporine treatment produced internucleosomal DNA fragments (200 bp) in a laddered pattern on the agarose gel. Staurosporine (1.0 M) rapidly induced phosphatidylserine plasma membrane translocation that increased linearly with time as measured by annexin V-FITC binding, and similar kinetics were observed for caspase activation by staurosporine in BG-1 cells. In contrast, 10 M gemcitabine increased phosphatidylserine expression in a small fraction of cells (5–10%) vs. untreated controls over the course of 48 h and significant caspase activity was detected within 12 h of drug exposure. Time-lapse video microscopy of BG-1 cells exposed to 1.0 M staurosporine or 10 M gemcitabine for up to 72 h showed that the morphologic changes and kinetics of cell death induced by these agents differed significantly. We also evaluated the apoptosis induced by paclitaxel (a mitotic poison) and cisplatin (an agent not dependent on cell cycle functions) in BG-1 cells by these methods because these drugs are used clinically to treat ovarian cancer. Our findings demonstrate that the kinetics of apoptotic cell death induced by gemcitabine and other chemotherapeutic agents should be taken into account when designing treatment strategies for ovarian cancer.     

Aluminum-induced secretion of both citrate and malate in rye

Aluminum (Al)-resistant mechanisms responsible for Al-induced secretion of organic acids are poorly understood. In this study, we characterized the Al-induced secretion of both citrate and malate from rye (Secale cereale L. cv. King). Secretion of organic acids increased with increasing concentration (10, 30 and 50 M) and duration of Al treatments. Neither phosphorous (P) deficiency up to 15 days nor addition of 50M lanthanum, 50 M lead, 10 M cadmium, or 200 M manganese caused secretion of organic acids, suggesting that this secretion was a specific response to Al stress. Aluminum activated citrate synthase, the main enzyme for the synthesis of citrate, but its activation occurred only in the root tip. The elongation of roots of an Al-sensitive cultivar of wheat (Tritium aestivum L. cv. Scout 66) was not inhibited by 50 M Al in the presence of externally applied 50 M citrate or 400 M malate. The secretion of citrate and malate from intact rye roots exposed to 50 M Al corresponded to 31.3 ± 1.7 M and 11.5 ± 2.5 M, respectively, in the rhizosphere based on an assumption of a 2 mm thick unstirred layer around root tips. This result indicated that Al-resistance in rye was achieved by the Al-induced synthesis of citrate in root apices followed by Al-induced specific secretion of citrate from root tips.     

Fine structure of early previtellogenic oocytes in Mugil (Liza) auratus Risso, 1810 (Teleostei,Mugilidae)

Summary Sequential cytological events at the onset of previtellogenesis were studied in oocytes from 12 m to 70 m in diameter of golden grey mullet. The main cytological changes observed (increase in size of cell, nucleus, nucleolus and increase in number of nucleoli, RNP particles, nuage and mitochondria) provide evidence for important synthetic processes in an early preparatory phase of oocyte development.Somatic cells (pre-follicle, follicle and thecal) are also described.