L-azetidine 2-carboxylic Acid Research Articles

Properties, metabolisms, and applications of l-proline analogues

Due to the unique role of L-proline in the folding and structure of protein, a variety of synthetic proline analogues have been developed. L-Proline analogues have been proven to be valuable reagents for studying cellular metabolism and the regulation of macromolecule synthesis in both prokaryotic and eukaryotic cells. In addition to these fundamental researches, they are useful compounds for industrial use. For instance, microorganisms that overproduce L-proline have been obtained by isolating mutants resistant to L-proline analogues. They are also promising candidates for tuning the biological, pharmaceutical, or physicochemical properties of naturally occurring or de novo designed peptides. Among L-proline analogues, L-azetidine-2-carboxylic acid (L-AZC) is a toxic non-proteinogenic amino acid originally found in lily of the valley plants and trans-4-hydroxy-L-proline (4-L-THOP) is the most abundant component of mammalian collagen. Many hydroxyprolines (HOPs), such as 4-L-THOP and cis-4-hydroxy-L-proline (4-L-CHOP), are useful chiral building blocks for the organic synthesis of pharmaceuticals. In addition, L-AZC and 4-L-CHOP, which are potent inhibitors of cell growth, have been tested for their antitumor activity in tissue culture and in vivo. In this review, we describe the recent discoveries regarding the physiological properties and microbial production and metabolism of L-proline analogues, particularly L-AZC and HOPs. Their applications in fundamental research and industrial use are also discussed.

Calnexin Phosphorylation Attenuates the Release of Partially Misfolded α1-Antitrypsin to the Secretory Pathway

Calnexin is a type I integral membrane phosphoprotein resident of the endoplasmic reticulum. Its intraluminal domain has been deduced to function as a lectin chaperone coordinating the timing of folding of newly synthesized N-linked glycoproteins of the secretory pathway. Its C-terminal cytosolic oriented extension has an ERK1 phosphorylation site at Ser(563) affecting calnexin association with the translocon. Here we find an additional function for calnexin phosphorylation at Ser(563) in endoplasmic reticulum quality control. A low dose of the misfolding agent l-azetidine 2-carboxylic acid slows glycoprotein maturation and diminishes the extent and rate of secretion of newly synthesized secretory alpha1-antitrypsin. Under these conditions the phosphorylation of calnexin is enhanced at Ser(563). Inhibition of this phosphorylation by the MEK1 inhibitor PD98059 enhanced the extent and rate of alpha1-antitrypsin secretion comparable with that achieved by inhibiting alpha-mannosidase activity with kifunensine. This is the first report in which the phosphorylation of calnexin is linked to the efficiency of secretion of a cargo glycoprotein.

Influence of a proton gradient on the transport kinetics of the H +/amino acid cotransporter PAT1 in Caco-2 cells

The recently cloned proton-coupled amino acid transporter 1 (PAT1) not only accepts several amino acids as substrates but also pharmaceutically relevant l-proline or GABA derivatives such as cis-4-hydroxy- l-proline, l-azetidine-2-carboxylic acid (LACA), 3-amino-1-propanesulfonic acid, nipecotic acid, and the antituberculotic agent d-cycloserine. Because human intestine expresses hPAT1 at the brush border membrane, the transporter may serve as a new oral drug delivery route. Using the human intestinal cell line Caco-2, we have investigated the influence of an inwardly directed proton gradient on the kinetic parameters of l-proline uptake. H + altered only the apparent affinity of l-proline transport and not the maximal transport velocity. Similarly, treatment of the cells with diethylpyrocarbonate (DEPC), known to chemically modify histidyl residues and block their function, affected only the K t value of l-proline transport. Both increasing pH and DEPC treatment strongly increased the inhibition constants ( K i) of several drugs at hPAT1. It is concluded that H + stimulates hPAT1 primarily by increasing the substrate affinity with no detectable influence on the maximal transport velocity of the transporter.

Novel function of ascorbic acid as an angiostatic factor.

Endothelial permeability is increased by vascular endothelial cell growth factor and decreased by antioxidants. Whether or not l-ascorbic acid (Asc), which decreases endothelial permeability by stimulating the endothelial barrier function, is anti-angiogenic (angiostatic) remains unknown. We examined the role of Asc on angiogenesis using two assay systems. At first, the potential role of Asc on four steps of angiogenesis was investigated in cultured bovine microvascular endothelial cells. Asc inhibited the formation of vessel-like tubular structures of endothelial cells cultured on Matrigel; however, it did not decrease the activity of plasminogen activator (PA), which creates the space into which vascular vessels extend. Furthermore, even at high concentrations, Asc did not inhibit either the proliferation or migration of endothelial cell cultures. Secondly, whether Asc inhibited in vivo angiogenesis or not was studied on chick chorioallantoic membrane (CAM) during the 4-6 days of embryogenesis when neovascularization is rapid. It also revealed that angiogenesis was dose-dependently inhibited by Asc from 0.5 micro mol/CAM with half-maximal inhibition at 2.5 micro mol/CAM. Because it was previously reported that the endothelial barrier function decreases permeability via the stimulation of collagen synthesis induced by Asc, we treated CAM with the inhibitor of collagen synthesis, l-azetidine 2-carboxylic acid (AzC). This compound partially attenuated the angiostatic function of Asc on CAM. To understand the involvement of an antioxidant activity in the angiostatic function of Asc, we further examined the effect of glutathione (GSH), which is an endogenous antioxidant, on angiogenesis in CAM and endothelial cells. GSH inhibited CAM angiogenesis, as well as the formation of vessel-like tubular structures of endothelial cell cultures on Matrigel. Both Asc and GSH inhibited hydrogen peroxide (H(2)O(2)) induced tubular morphogenesis. These findings suggest that Asc affects angiogenesis through both its antioxidant properties and the stimulation of collagen synthesis. As the angiostatic activity of Asc may be one of the many effects involved in host resistance to the growth or invasiveness of solid cancer, it may be useful as a supplementary therapy in various angiogenic diseases.

The Sarco/Endoplasmic Reticulum Calcium-ATPase 2b Is an Endoplasmic Reticulum Stress-inducible Protein

The sarco/endoplasmic reticulum calcium-ATPase (SERCA) translocates Ca(2+) from the cytosol to the lumen of the endoplasmic reticulum. This Ca(2+) storage is important for cellular processes such as calcium signaling and endoplasmic reticulum (ER)-associated posttranslational protein modifications. We investigated the expression of the SERCA2 and SERCA3 isozymes in PC12 cells exposed to agents interfering with different aspects of the posttranslational protein processing within the ER, thereby activating the ER stress-induced unfolded protein response (UPR). All agents increased the SERCA2b mRNA level 3-4-fold, in parallel with increasing mRNA levels for the ER stress marker proteins BiP/GRP78 and CHOP/GADD153. In contrast, SERCA3 mRNA levels did not change. SERCA2b mRNA stability was not changed, indicating that the mechanism of its up-regulation was transcriptional, in accordance with the presence of ER stress response elements in the promoter region of the SERCA2 gene. SERCA2b was also increased at the protein level upon ER stress treatments. Induction of ER stress by tunicamycin, dithiothreitol, or l-azetidine 2-carboxylic acid did not result in depletion of ER calcium, showing that such depletion was not necessary for up-regulation of SERCA2b expression or UPR activation in general. We conclude that the SERCA2b expression can be controlled by the UPR pathway independently of ER Ca(2+) depletion.

Suppression of thermotolerance development through cycloheximide-induced negative control of stress protein gene expression.

Expression of a luciferase reporter gene by Chinese hamster ovary cells under the control of the human heat shock protein (hsp) 70 gene promoter was suppressed by incubation at 37 degrees C after treatment with cycloheximide (CHX) during 42 degrees C heat shock exposure. The CHX-induced suppression of hsp gene expression induced no development of thermotolerance. However, 42 degrees C heat shock treatment without CHX followed by CHX inhibition of protein synthesis during recovery incubation at 37 degrees C induced thermotolerance expression by inducing enhanced synthesis of hsps including hsp70 in subsequent heat challenge incubation at 43 degrees C. The results demonstrated a direct correlation between suppression (induction) of stress protein gene expression and non-expression (expression) of thermotolerance. Kinetic analysis showed that the CHX suppression of hsp gene induction was greater than the CHX inhibition of protein synthesis, and that it depended on the severity of heat stress: it decreased with increasing heat stress doses. Moreover, prior feeding of the proline analog L-azetidine 2-carboxylic acid abrogated the CHX-induced suppression of hsp gene expression. In addition, CHX treatment during heat cell-killing at 43 degrees C induced protection of cells. These results were well explained by the proposed model of negative or positive control of stress protein gene expression depending on the level of free hsp70, which may be modulated by both the rate of protein synthesis and the severity of heat stress.

Fine structure of extracellular matrix and basal laminae in two types of abnormal collagen production: l-proline analog-treated otocyst cultures and disproportionate micromelia (Dmm/Dmm) mutants

l-Azetidine-2-carboxylic acid (LACA), a naturally occurring vegetable imino acid, can be incorporated into mammalian proteins in place of proline, thereby eliciting an inhibitory effect on collagen secretion. Exposure of explants of the embryonic mouse inner ear to LACA reduces the number of collagen fibrils in the otic capsule, gives rise to a dose-dependent derangement of the basal lamina, and ultimately results in dysmorphogenesis and retarded differentiation of the inner ear. Disproportionate micromelia (Dmm) is an incomplete dominant form of dwarfism characterized by a reduced quantity of type II collagen in the cartilaginous extracellular matrix (ECM). Abnormal morphogenesis in homozygotic Dmm mice resembles the abnormal morphogenesis observed in LACA-exposed otic explants, resulting in malformed inner ears with a bulky cartilaginous capsule and a lack or reduction of defined perilymphatic spaces (Van De Water and Galinovic-Schwartz, 1987). In this study, we examined by ultrastructural analysis LACA-exposed otic explants and inner ears of Dmm/Dmm mouse embryos for abnormalities in the collagenous constituents of the basal laminae and capsular ECM. We demonstrate, in comparison to normal embryonic mouse inner ears, a reduction in collagen fibrils and irregular cytodifferentiation of chondrocytes in the ECM of LACA-exposed and Dmm/Dmm inner ears as well as in the basal laminae of LACA-exposed specimens. In addition, we provide evidence of dysmorphogenesis of the otic capsule and perilymphatic spaces in LACA-exposed explants. Moreover, while previous studies demonstrated the anomalous development of sensory structures in otocyst explants following LACA exposure, in this study we provide evidence of the normal morphogenesis of otic epithelial-derived sensory structures in homozygotic Dmm/Dmm mouse embryos.

Isolation and characterisation of a cDNA encoding rat mitochondrial GrpE, a stress-inducible nucleotide-exchange factor of ubiquitous appearance in mammalian organs

In contrast to the E. coli chaperones DnaK, GroEL and GroES, cDNAs encoding mitochondrial homologues of DnaJ and GrpE from higher eukaryotes have yet to be reported. Based on peptide sequences, we have isolated a cDNA encoding a 217 residue nuclear encoded precursor of rat mitochondrial GrpE (mt-GrpE) including a typical mitochondrial presequence of 27 residues. Western blotting revealed that the 21 kDa GrpE homologue is present exclusively in the mitochondrial fraction where it comprises only ∼0.03% of the total soluble protein, while Northern blotting showed that the mt-GrpE transcript is present in most if not all organs. By contrast to other mitochondrial chaperones, the levels of mt-GrpE and its transcript in cultured cells are only marginally increased in response to the proline analog l-azetidine 2-carboxylic acid but not by heat shock. Furthermore, members of the GrpE family exhibit a much lower degree of sequence identity than do the well studied members of the Hsp70, Hsp60 and Hsp10 families.

Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer

The macromolecular permeability of cultured bovine aortic, bovine venous, and human umbilical vein endothelial cell monolayers was decreased significantly in culture medium containing L-ascorbic acid (Asc Acid; 0.01-0.1 mM) and L-ascorbic acid 2-phosphate (Asc 2-P). Dithiothreitol, which shows reducing activity equivalent to that of Asc Acid, did not affect endothelial permeability. Asc Acid induced a sixfold increase in collagen synthesis by the endothelial cells. The coexistence of L-azetidine 2-carboxylic acid, an inhibitor of collagen synthesis, attenuated the effect of Asc 2-P in a dose-dependent manner. Another collagen synthesis inhibitor, ethyl-3,4-dihydroxybenzoate, also inhibited collagen synthesis and increased endothelial permeability. The decrease in permeability of the endothelial monolayer was dependent on a reduction of the permeability coefficient of the endothelial monolayer. These findings indicate that endothelial barrier function is stimulated by Asc Acid via an increase in collagen synthesis.

Prolyl hydroxylase inhibitor and lysyl hydroxylase inhibitor inhibit spreading of corneal epithelium

The spreading of epithelium is critical in the healing of corneal wounds. Such epithelial spreading requires the continuous production of protein and glycoprotein. To determine whether collagen production is required for the spreading of corneal epithelium, we studied the effects of inhibitors for collagen production on spreading of corneal epithelium in vitro. We examined the effect of two proline analogs, L-azetidine 2-carboxylic acid and cis-hydroxyproline, a prolyl hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, and a lysyl hydroxylase inhibitor, minoxidil, on the spreading of epithelium of organ-cultured rabbit cornea. Both analogs and inhibitors inhibited epithelial spreading in a dose-dependent manner. These observations indicate that collagen production may be involved in the spreading of corneal epithelium.

Induction of the 72-kD Heat Shock Protein in Organ-Cultured Normal Human Skin

To study the induction of heat shock protein (HSP) of normal human skin, the indirect immunofluorescence method, using monoclonal antibody directed against 72-kD HSP, was applied in organ-cultured normal human skin that was treated with heat, UV, or chemicals. The present study provided new evidence that HSP 72 was induced not only by heat and chemical agents, such as L-azetidine 2-carboxylic acid, and sodium arsenite, but also by ultraviolet (UV B and C). The result suggests that normal human skin has an induced protective function against numerous environmental stresses.

Collagen Synthesis in Human Skin Fibroblasts is Stimulated by a Stable Form of Ascorbate, 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid

We evaluated the effect of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) on collagen synthesis in cultured human skin fibroblasts and on proliferation of fibroblasts. At concentrations of 0.1–0.5 mmol/L, AA-2G effectively stimulated collagen synthesis with an effectiveness comparable to that of L-ascorbic acid. On the other hand, 6-O-α-D-glucopyranosyl-L-ascorbic acid showed a weak effect. The stimulation of collagen synthesis by AA-2G was attenuated by the addition of a collagen synthesis inhibitor, L-azetidine 2-carboxylic acid, in a dose-dependent manner. In addition, AA-2G-induced stimulation of collagen synthesis could be completely inhibited by the addition of castanospermine, an inhibitor of neutral α-glucosidase. Relatively high α-glucosidase activity, which would contribute to release of ascorbic acid from AA-2G, could be detected in the lysate of cultured fibroblasts. The stimulatory activity of AA-2G on collagen synthesis was observed after 5 d in culture, whereas L-ascorbic acid tended to lose its stimulatory activity. Continuous supplementation of AA-2G (0.25 mmol/L) to culture medium for 24 d enhanced the cell growth four times that of the control. These results indicate that AA-2G is gradually cleaved by the cellular α-glucosidase to release L-ascorbic acid, which adequately stimulates collagen synthesis and proliferation of human skin fibroblasts.

Effect of l-azetidine 2-carboxilic acid on the activity of the general amino-acid permease from Saccharomyces cerevisiae var. ellipsoideus

Addition of the L-proline analogue L-azetidine 2-carboxylic acid to growing cultures of Saccharomyces cerevisiae var. ellipsoideus promoted fast deactivation of the general aminoacid permease, measured as L-valine uptake, without an immediate decrease in the growth rate. Cells preincubated with the analogue for 3 h were unable to restore either growth ability or general aminoacid permease activity in analogue-free medium. Eadie-Hofstee plots of L-valine uptake in the presence of the analogue are consistent with a strong reduction in the number of active molecules of the general amino-acid permease located in the plasma membrane. Inhibitory effects on protein synthesis were seen after preincubations of the yeast with the analogue for 3 h although a 30 min preincubation had no effect.

L-Ascorbic acid 2-phosphate, a phosphate derivative of l-ascorbic acid, enhances the growth of cultured rabbit keratocytes

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc) on the proliferation of cultured rabbit keratocytes. P-Asc is a phosphate derivative of L-ascorbic acid and has more prolonged vitamin C activity in solution than does L-ascorbic acid. The proliferation of cultured keratocytes was promoted by the presence of P-Asc in culture medium. Transmission electron microscopic observations revealed that cells were more multi-layered after culture in the presence of P-Asc (0.1 mM) for 30 days than were those cultured in the absence of P-Asc. The effect of P-Asc was abrogated by L-azetidine 2-carboxylic acid, which is an analogue of proline that inhibits the production and secretion of collagen. Our observations support a therapeutic role for P-Asc in the repair of corneal stromal damage such as that caused by a corneal chemical burn.

L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts.

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.

Experimental control of the differentiation of Leydig cells in the rat fetal testis.

In the developing fetal testis, in vitro as well as in vivo, two kinds of endocrine cells differentiate successively: Sertoli cells, which produce the Müllerian inhibitor (or anti-Müllerian hormone) and aggregate with germ cells into seminiferous cords; and Leydig cells, which release androgens. Serum added to the synthetic culture medium prevents the morphogenesis of the seminiferous cords but not the cytodifferentiation of the endocrine cells. L-Azetidine 2-carboxylic acid (LACA), a proline competitor, introduced into the medium also prevents differentiation of seminiferous cords. In the present experiments, the effects of LACA on the endocrine cells were studied. It did not suppress production of the Müllerian inhibitor, but it opposed differentiation of Leydig cells. Histochemically detectable 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was virtually absent and the release of testosterone, delta 4-androstenedione, 17-hydroxyprogesterone, or progesterone into the medium became undetectable. Moreover, dibutyryl cAMP added to the medium during the final day in vitro had very little effect on the parameters of steroidogenesis. An excess of proline added to the LACA-containing medium permitted normal morphogenesis of seminiferous cords, normal steroidogenesis, and normal response to cAMP. LACA did not prevent the appearance of 3 beta-HSD activity in the adrenals, nor did it reduce the expression of laminin and fibronectin (data not shown) in the mesonephric structures as much as in the testes. The differentiation of the testis and especially of the Leydig cells appears to have special requirements for proline.

Characterization of the thermotolerant cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression.

Exposure of mammalian cells to a nonlethal heat-shock treatment, followed by a recovery period at 37 degrees C, results in increased cell survival after a subsequent and otherwise lethal heat-shock treatment. Here we characterize this phenomenon, termed acquired thermotolerance, at the level of translation. In a number of different mammalian cell lines given a severe 45 degrees C/30-min shock and then returned to 37 degrees C, protein synthesis was completely inhibited for as long as 5 h. Upon resumption of translational activity, there was a marked induction of heat-shock (or stress) protein synthesis, which continued for several hours. In contrast, cells first made thermotolerant (by a pretreatment consisting of a 43 degrees C/1.5-h shock and further recovery at 37 degrees C) and then presented with the 45 degrees C/30-min shock exhibited considerably less translational inhibition and an overall reduction in the amount of subsequent stress protein synthesis. The acquisition and duration of such "translational tolerance" was correlated with the expression, accumulation, and relative half-lives of the major stress proteins of 72 and 73 kD. Other agents that induce the synthesis of the stress proteins, such as sodium arsenite, similarly resulted in the acquisition of translational tolerance. The probable role of the stress proteins in the acquisition of translational tolerance was further indicated by the inability of the amino acid analogue, L-azetidine 2-carboxylic acid, an inducer of nonfunctional stress proteins, to render cells translationally tolerant. If, however, analogue-treated cells were allowed to recover in normal medium, and hence produce functional stress proteins, full translational tolerance was observed. Finally, we present data indicating that the 72- and 73-kD stress proteins, in contrast to the other major stress proteins (of 110, 90, and 28 kD), are subject to strict regulation in the stressed cell. Quantitation of 72- and 73-kD synthesis after heat-shock treatment under a number of conditions revealed that "titration" of 72/73-kD synthesis in response to stress may represent a mechanism by which the cell monitors its local growth environment.

Collagen type ii in the otic extracellular matrix effect on inner ear development

Immunocytochemistry was used to demonstrate type II collagen distribution during normal development of the mouse inner ear and in two malformed inner ears. Patterns of inner ear abnormalities and type II collagen distribution were compared between the malformed labyrinth of a mouse mutation (disproportionate micromelia, Dmm) and otic expiants exposed to the teratogenic action of an L-proline analog, L-azetidine-2-carboxylic acid (LACA). The results suggest that type II collagen is an important constituent of the developing inner ear's extracellular matrix. Disruptions of the spatial and temporal pattern of collagen type II can adversely affect morphogenesis of the inner ear. A common mechanism of action is postulated for the causation of both the genetic and teratogen-induced inner ear malformations (i.e. disruption of the secretion of collagens to the otic extracellular matrix).

Cellular and biochemical events in mammalian cells during and after recovery from physiological stress.

We have examined and compared a number of cellular and biochemical events associated with the recovery process of rat fibroblasts placed under stress by different agents. Metabolic pulse-labeling studies of cells recovering from either heat-shock treatment, exposure to sodium arsenite, or exposure to an amino acid analogue of proline, L-azetidine 2-carboxylic acid, revealed interesting differences with respect to the individual stress proteins produced, their kinetics of induction, as well as the decay in their synthesis during the recovery period. In the initial periods of recovery, the major stress-induced 72-kD protein accumulates within the altered nucleoli in close association with the pre-ribosomal-containing granular region. During the later times of recovery from stress, the nucleoli begin to regain a normal morphology, show a corresponding loss of the 72-kD protein, and the majority of the protein now begins to accumulate within the cytoplasm in three distinct locales: the perinuclear region, along the perimeter of the cells, and finally in association with large phase-dense structures. These latter structures appear to consist of large aggregates of phase-dense material with no obvious encapsulating membrane. More interestingly we show, using double-label indirect immunofluorescence analysis, that much of the perinuclear and cell perimeter-distributed 72-kD protein coincides with the distribution of the cytoplasmic ribosomes. We discuss the possible implications of the presence of the 72-kD stress proteins within the pre-ribosomal-containing granular region of the nucleolus as well as its subsequent colocalization with cytoplasmic ribosomes in terms of the translational changes which occur in cells both during and after recovery from physiological stress.

Protein Patterns and Synthetic Profiles during Distal Regeneration in Hydra oligactis: (Hydra/regeneration/protein/mesoglea).

Protein patterns and synthetic profiles were examined during distal regeneration in Hydraoligactis. Electrophoretic and radioactive tracer analyses revealed qualitative changes in the general protein profile during regeneration, with a heightened period of protein synthesis between 27-30 hr of regeneration, immediately preceding emergence of the first pair of tentacles. Following this, an increase in collagen-rich mesogleal protein secretion was observed coincident with tentacle initiation. Inhibition of collagen secretion with the proline analog L-azetidine-2-carboxylic acid (LACA) inhibited tentacle formation, and resulted in the development of unique "hypostome buds" at the distal regeneration surface. At the cellular level LACA did not inhibit the nerve cell differentiation that normally precedes tentacle growth, although some predicted decline in cnidocyte production was noted. It is proposed that mesogleal collagen secretion and structural organization may play a major role in the mechanical aspects of Hydra tentacle morphogenesis.