Determination of epidermal G2-chalone in epidermal-type tissues

By means of monospecific immune serum, using the indirect method by Coons, the epidermal G2-chalone was revealed in the corneal epithelium, in the transitory epithelium of the urinary bladder, renal pelvis, as well as in stromal epithelial cells of the cortical substance and in thymic bodies, the facts that suggest epithelial nature of these tissues. In tracheal epithelium the method mentioned failed to reveal G2-chalone. Analysing localization of the epidermal G2-chalone in various tissues of the epidermal origin, it has been stated that in the non-cornified multistratified flat epithelium it is present in cellular cytoplasm of all layers, while in the cornified epithelium - it is predominantly detected in basal and scupular cells. A suggestion is made that distribution of the intratissue epidermal G2-chalone depends on the process of cornification. A possibility to use G2-chalone as a marker for tissues of the epidermal type is discussed.     

Cell cycle specificity and reversibility of the inhibitory effect of epidermal G1-chalone on DNA synthesis in partially synchronized RTE-2 keratinocytes in vitro

The specific action of a pig skin fraction enriched in epidermal G1-chalone, a tissue-specific inhibitor of epidermal DNA synthesis, was investigated by means of flow cytofluorometry. The results indicate that G1-chalone inhibits progression of partially synchronized rat tongue epithelial cells (line RTE-2) through the cell cycle at a point 2 h prior to the beginning of the S-phase. Approximately 8 h after chalone addition, the cells can overcome the inhibition and begin to enter the S-phase. The duration of this delay is concentration-independent, but the fraction of cells affected is proportional to the chalone concentration. The progression of cells which already have entered S-phase is not affected. In contrast to the G1-chalone preparation, aphidicolin, a potent inhibitor of DNA polymerase alpha, clearly shows S-phase-specific inhibition. These results indicate that the epidermal G1-chalone inhibits epidermal cell proliferation in a fully reversible manner by a highly specific effect on cell cycle traverse.     

Changes in the level of epidermal G2-chalone and mitotic activity in the rat vaginal epithelium]

The content of tissue-specific inhibitor of mitosis in epidermal epithelium (G2-chalone) was estimated by a single radial immunodiffusion test in the rat vagina during various stages of the estrous cycle. The level of chalone was found to correlate with the mitotic index (MI) of vaginal epithelium. The lowest level of G2-chalone is detected in proestrus and the highest one in estrus. The level of G2-chalone in vaginal epithelium was shown to be significantly decreased in aging rats (14--16 month-old) with regular cycles as compared to that in young normal cycle rats (3--4 month-old). The single injection of estradiol benzoate (1 microgram/100 g) into ovariectomized rats led to an increase in the MI following 18 hours. The increased MI is preceeded by a substantial drop of the G2-chalone level 12 hours after estrogen injection.     

Immunomorphologic analysis of G2-chalone localization in epidermal-type tissues

Localization of the epidermal G2-chalone in tissues has been established by means of indirect method of Coons using a monospecific immune serum. It has been found in dermal epithelia of the back, external ear, tongue, esophagus, forestomach, vagina, hairy follicles, but it has not been found in the sebaceous glands and derma. These findings are fully in agreement with the results obtained by the method of double diffusion after Ouchterlony. Tissue specificity of G2-chalone is proved by the fact that at places where epithelia differing histogenetically join with each other, it is found only in the epithelia of the epidermal type. Within the epithelial layer G2-chalone is mainly localized in the spinous and partly in the basal cells. Possible mechanisms on regulation of the mitotic activity are discussed in connection with the data obtained.     

Immunomorphological analysis of G2-chalone localization during the process of rat epidermis histogenesis

The appearance of G2-chalone in the cytoplasm of the intermediate cell layer and partly in the periderm of 17-day-old rat embryo epidermis has been demonstrated by the indirect method of Coons using a monospecific antiserum. G2-chalone was absent from the basal cell layer of 17--21-day-old embryos and of the newborn rats. It was found in all the epidermal layers in 2--5-day-old postnatal rats, while in 6--9-day-old animals it was primarily detected in the cytoplasm of spinous and basal cells. Thus the localization of epidermal G2-chalone typical for defined tissue becomes stabilized at the end of epidermis histogenesis.     

Inhibition by epidermal extracts of the 12-O-tetradecanoylphorbol-13-acetate induced peak of ornithine decarboxylase activity in the mouse epidermis

The time course of induction of epidermal ornithine decarboxylase (E.C. 4.1.117) (ODC) activity following a single topical application of 17 nmoles of 12-O-tetradecanoylphorbol-13-acetate (TPA) on hairless mouse skin was established. Prior intraperitoneal (i.p.) administration of a crude epidermal extract prepared from hairless mouse epidermis led to a time-dependent, 50% inhibition of the peak level of TAP-induced ODC activity. Maximum inhibition was observed when the extract was injected 1.5 h before TPA treatment. The crude epidermal extract did not affect ODC activity in vitro. Following the administration of epidermal extracts, the inhibition of the TPA-induced ODC-response correlated positively with the presence of epidermal G2-chalone activity (determined by a stathmokinetic method) whereas myocardial, skeletal muscle, or heat-inactivated epidermal extracts with no epidermal G2-chalone activity, had no effect on TPA-induced ODC activity. These results indicate a possible relationship between ODC-activity and the control of mitotic rate by G2-chalone.     

Purification of epidermal G2-chalone using immunoaffinity chromatography

Epidermal G2-chalone was purified from rat skin extract by immunoaffinity chromatography with the use of mono-specific antibodies coupled to CNBr-activated sepharose 4B. During SDS PAAG electrophoresis, it produced a single band with a molecular weight of 13000 dalton. G2-chalone purified in this way had a 60% antimitotic inhibitory effect on external ear epidermocytes in mice in a dose of 2 micrograms/g bw but had no effect on the mitosis in the sebaceous glands.     

A system for the study of epidermal G1-chalone in cell culture. The rat tongue epithelial cell line RTE2

A pig-skin preparation enriched in epidermal G1-chalone when administered to cells of the rat tongue epithelial line RTE2 at concentrations of 3-300 micrograms/ml (dry mass) caused a 60% reduction in cell number. Three other cell lines showed essentially no growth inhibition during chalone treatment. The kinetics of chalone inhibition were similar to those observed in mouse epidermis in vivo. Five hours after the addition of chalone preparation in fresh medium a decrease in the rate of DNA synthesis was observed. Maximum inhibition at 12 h was followed by a subsequent increase in DNA synthesis, reaching control values again after 30 h. The inhibitory effect was dose-dependent up to 3 micrograms/ml. At higher concentrations the degree of inhibition remained constant at about 50% of the control up to 300 micrograms/ml. Removal of added chalone by changing the medium at the time of maximum inhibition gave rise to a complete recovery within 9 h. These results indicate a cell-line specific, non-toxic and reversible inhibitory effect of the chalone preparation which resembles that observed in the living animal. The RTE2 cell line may thus be considered to provide a highly sensitive experimental system suitable for more detailed studies on the mechanism of action of epidermal G1-chalone.     

Cell cycle specificity and reversibility of the inhibitory effect of epidermal G1-chalone on DNA synthesis in partially synchronized RTE-2 keratinocytes in vitro

The specific action of a pig skin fraction enriched in epidermal G₁-chalone, a tissuespecific inhibitor of epidermal DNA synthesis, was investigated by means of flow cytofluorometry. The results indicate that G₁-chalone inhibits progression of partially synchronized rat tongue epithelial cells (line RTE-2) through the cell cycle at a point 2 h prior to the beginning of the S-phase. Approximately 8 h after chalone addition, the cells can overcome the inhibition and begin to enter the S-phase. The duration of this delay is concentrationindependent, but the fraction of cells affected is proportional to the chalone concentration. The progression of cells which already have entered S-phase is not affected. In contrast to the G₁-chalone preparation, aphidicolin, a potent inhibitor of DNA polymerase α, clearly shows S-phase-specific inhibition. These results indicate that the epidermal G₁-chalone inhibits epidermal cell proliferation in a fully reversible manner by a highly specific effect on cell cycle traverse.     

Change in the activity of epidermal chalones during skin reaction to the effect of a carcinogen and epilation]

The biological activity of an epidermal G2-chalone extracted from the rat back skin was found to decrease 1 to 2 days after the treatment with methylnitrosourea (a carcinogen), and one day after the epilation. Results of quantitative recording and morphological analysis of the skin serving as the source of chalones, pointed to different mechanisms which underlay this effect.     

Regulation of mitotic activity in rat vaginal epithelium: relationships between the level of its inhibitor (G2-chalone) and estrogens.

The level of a tissue-specific inhibitor of mitotic activity (G2-chalone) and mitotic activity in the vaginal mucosa of cycling rats of varying age and castrated rats were studied. A direct correlation between the level of the inhibitor and mitotic index is found in cycling animals. Both parameters are maximal during estrus and minimal in proestrus, when estrogen level in blood circulation is the highest. The undulating variations in G2 inhibitor level during estrous cycle are less pronounced and the concentrations of the inhibitor in relevant phases are significantly lower in aged females than in adult rats. Administration of estradiol benzoate (1 microgram/100 g) to castrated female rats was followed by a significant decrease in mitotic inhibitor level in vaginal mucosa within 12 hrs. This, in turn, was followed by a rise in mitotic activity 18 hr after estrogen administration. Therefore, the estrogen exerts its effect on mitotic activity in target tissue after it has induced a decrease in the level of the antimitotic factor (G2-chalone).     

Effect of rat liver chalones on liver cell proliferation at different times after partial hepatectomy]

The action of rat's liver ethanol extract (72--81 per cent saturation) on cell proliferation of this organ at various periods after a partial hepatectomy has been studied. The most sensitive periods of the action of G1- and G2-chalone were, resp., the time of cell transformation, and the middle of the premitotic period of cell cycle. The action of G2-chalone used is organ-specific, since the drug decreased the mitotic activity of both hepatocytes and stromal cells. At the same time, the proliferation of ear, tongue and small bowel epithelial cells remained unchanged.     

Integration of tissue-specific inhibitors and thyroid hormones in the regulation of proliferation of gastric gland epithelial cells]

G1- and G2-chalone effects of pig's stomach mucosa extract were registered in mice stomach gland epithelium. The inhibitory activity of chalones on cell's proliferation reduced following increased level of thyroid hormones. Local (chalones) and organism (thyroid hormones) factors cooperate in control of gland epithelial cell proliferation.     

Interactions between glycosaminoglycans and proteins, with particular reference to a new technique for isolating serum glycoproteins

1. Chondroitin sulphate–serum protein complexes (A, B and C), successively precipitated by adding chondroitin sulphate to serum at three arbitrary descending pH values (5·2, 4·3 and 3·1), were dissociated at pH 6·7 and chromatographed on DEAE-Sephadex, when the liberated serum proteins were simultaneously freed of chondroitin sulphate and separated into five fractions. Evidence that serum proteins were precipitated as a result of electrostatic interactions with dissociated carboxylate groups on the glycosaminoglycan is presented. 2. Serum proteins (fraction G), unable to form complexes with chondroitin sulphate, contained 4·4% of sialic acid and accounted for 4 and 26% of the total protein and protein-bound sialic acid in serum respectively. This fraction interacted electrostatically with chondroitin sulphate only when rendered more basic by removal of sialic acid residues with neuraminidase. The heat stability, solubility properties and high carbohydrate content of fraction G classified it as a seromucoid fraction. 3. Fraction G contained several glycoprotein and hexuronic acid-containing fractions, including a hitherto undetected brown-pigmented high-molecular-weight serum component, which migrated in starch gel between the origin and Sα₂-globulin and contained 3·1 and 4·1% of sialic acid and hexuronic acid respectively. 4. Glycosaminoglycan–protein interactions are discussed in relation to protein fractionation. By prior removal of less acidic proteins by these interactions, a new technique is available for isolating serum seromucoids in higher yields and under milder conditions than existing methods.     

Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins

Normal and pathological turnover of proteoglycans in articular cartilage involves its cleavage close to the N-terminal G1 domain responsible for aggregation. A fragment containing G1 and G2 N-terminal domains of pig cartilage proteoglycans was therefore used as a substrate to investigate its degradation by the metalloproteinase stromelysin and related recombinant stromelysin enzymes. The stromelysins produced an apparent single cleavage yielding a G1 fragment of 56 kDa and a G2 fragment of 110 kDa. Rabbit bone stromelysin was much more active against the G1-G2 fragment and against proteoglycan aggregates than recombinant human stromelysin-1 and stromelysin-2. All metalloproteinase preparations were active against proteoglycan and the G1-G2 fragment at acid (pH 5.5) and neutral pH (7.4). N-terminal sequencing of the G2 fragment derived from the action of recombinant human stromelysin-1 revealed that cleavage between G1 and G2 occurred at the N-terminal end of the interglobular domain, close to the last cysteine in G1. The specific cleavage site was between an asparagine and a pair of phenylalanine residues, where the asparagine corresponds to residue 341 in human and rat mature core protein sequence.     

On the existence of 'arrested G2 cells' in mouse epidermis

It has been postulated that mouse epidermis contains two populations of resting cells, one of which is blocked at the G1-S boundary and the other between G2 and mitosis. The 'arrested G2 cells' were estimated, by the labelled mitosis method, to comprise 5-10% of the epidermal population and presumed to function as a 'reserve pool' which could be activated by wounding. A comprehensive search has now been carried out for arrested G2 cells in mouse epidermis using the direct methods of single cell and flow through cytophotometry. No evidence was obtained which supports the existence of such a cell compartment. Suitable control experiments were carried out to ensure that G2 cells were not lost during the isolation of epidermal nuclei.     

The comparative leaf anatomy of Goniothamus andersonii, G. macrophyllus, G. malayanus and G. velutinus

The leaf anatomy of Goniothalamus andersonii, G. macrophyllus, G. malayanus and G. velutinus from the peat swamps of Sarawak is compared. Sufficient anatomical differences exist to differentiate the four species. G. velutinus has many points of difference from the other three species and G. macrophyllus is readily identified by the presence of fibre-sclereids in the lamina mesophyll. G. andersonii and G. malayanus are similar to each other, but G. andersonii can be distinguished, in particular by the more pronounced multiple projections of the outer periclinal walls of the epidermal cells and the presence of thick and cutinised outer ends of the anticlinal walls of the epidermal cells.     

Ribosomal leaky scanning through a translated uORF requires eIF4G2

eIF4G2 (DAP5 or Nat1) is a homologue of the canonical translation initiation factor eIF4G1 in higher eukaryotes but its function remains poorly understood. Unlike eIF4G1, eIF4G2 does not interact with the cap-binding protein eIF4E and is believed to drive translation under stress when eIF4E activity is impaired. Here, we show that eIF4G2 operates under normal conditions as well and promotes scanning downstream of the eIF4G1-mediated 40S recruitment and cap-proximal scanning. Specifically, eIF4G2 facilitates leaky scanning for a subset of mRNAs. Apparently, eIF4G2 replaces eIF4G1 during scanning of 5′ UTR and the necessity for eIF4G2 only arises when eIF4G1 dissociates from the scanning complex. In particular, this event can occur when the leaky scanning complexes interfere with initiating or elongating 80S ribosomes within a translated uORF. This mechanism is therefore crucial for higher eukaryotes which are known to have long 5′ UTRs with highly frequent uORFs. We suggest that uORFs are not the only obstacle on the way of scanning complexes towards the main start codon, because certain eIF4G2 mRNA targets lack uORF(s). Thus, higher eukaryotes possess two distinct scanning complexes: the principal one that binds mRNA and initiates scanning, and the accessory one that rescues scanning when the former fails.     

G proteins as regulators in ethylene-mediated hypoxia signaling

Waterlogging or flooding are frequently or constitutively encountered by many plant species. The resulting reduction in endogenous O₂ concentration poses a severe threat. Numerous adaptations at the anatomical, morphological and metabolic level help plants to either escape low oxygen conditions or to endure them. Formation of aerenchyma or rapid shoot elongation are escape responses, as is the formation of adventitious roots. The metabolic shift from aerobic respiration to anaerobic fermentation contributes to a basal energy supply at low oxygen conditions. Ethylene plays a central role in hypoxic stress signaling, and G proteins have been recognized as crucial signal transducers in various hypoxic signaling pathways. The programmed death of parenchyma cells that results in hypoxia-induced aerenchyma formation is an ethylene response. In maize, aerenchyma are induced in the absence of ethylene when G proteins are constitutively activated. Similarly, ethylene induced death of epidermal cells that cover adventitious roots at the stem node of rice is strictly dependent on heterotrimeric G protein activity. Knock down of the unique Gα gene RGA1 in rice prevents epidermal cell death. Finally, in Arabidopsis, induction of alcohol dehydrogenase with resulting increased plant survival relies on the balanced activities of a small Rop G protein and its deactivating protein RopGAP4. Identifying the general mechanisms of G protein signaling in hypoxia adaptation of plants is one of the tasks ahead.Key words: submergence, hypoxia, ethylene, G protein, reactive oxygen species, H₂O₂