Morphogenetic movements during axolotl neural tube formation tracked by digital imaging

During neurulation in vertebrate embryos, epithelial cells of the neural plate undergo complex morphogenetic movements that culminate in rolling of the plate into a tube. Resolution of the determinants of this process requires an understanding of the precise movements of cells within the epithelial sheet. A computer algorithm that allows automated tracking of epithelial cells visible in digitized video images is presented. It is used to quantify the displacement field associated with morphogenetic movements in the axolotl (Ambystoma mexicanum) neural plate during normal neural tube formation. Movements from lateral to medial, axial elongations and area changes are calculated from the displacement field data and plotted as functions of time. Regional and temporal differences are identified. The approach presented is suitable for analyzing a wide variety of morphogenetic movements.     

Rank and age related feeding strategy observed through field experiments in the Koshima group of Japanese macaques

The feeding process of Japanese monkeys on soy beans which were scattered over a sandy beach on Koshima islet was studied. Younger monkeys were able to pick up more beans when 8 kg of beans were divided and given two times (“two times feeding”) than when the whole amount (8 kg) of beans was given at one time (“one time feeding”). The effect of saturation of the food intake capacity in younger monkeys at the first feeding in “two times feeding” did not appear at the second feeding one hour later. The minutely intake of soy beans (feeding speed) for each age class was analyzed. The decline of feeding speed in adult females after the peak in “one time feeding” was not related to the decline in density of beans on the ground, and this decline was caused by saturation of their food intake capacity. Adult females were divided into four classes according to their dominance rank order: high, lower-high, higher-low, and low classes. The total amount of intake in “one time feeding” was far larger in the high class than in any of the other classes. The total amount of feeding in the first feeding of “two times feeding” increased in accordance with rise in the dominance rank class, and there was no relation to rank and total feeding amount in the second feeding of “two times feeding.” Differences existed in the process of feeding between the rank classes. The feeding speed of the low class was as high as that of the high class on the curve of minutely intake, while the low class stopped feeding much earlier than the high class. The lower-high class displayed a low feeding speed, and stopped feeding the latest. The order of the duration to stay and to feed in the feeding area was lower-high > high > higher-low > low, and this order did not change under the three different feeding conditions, “one time feeding,” and the first and second feedings of “two times feeding.” Adolescent females tended to stay the longest duration in the feeding area among all age classes. Both the lower-high class females and adolescent females had an unstable social status in the Koshima group, and their social status affected their feeding behaviors. The feeding behaviors were similar in attitude depending on social status, and are considered to be maintained for a fairly long time. The feeding strategy of the lower-high class, in staying a longer duration in the artificial feeding area, and departing later, may be effective under the artificial feeding conditions, but it may be a bad strategy in a natural habitat where the food is not so clumped as in artificial feeding, and where choice of other food patches is possible. The above results agree well with previous reports for the Koshima group, indicating that the rank of the lower-high class females was unstable (Mori et al., 1989), and that their reproductive success was low (Watanabe et al., 1992).     

Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia

We have previously characterized nuclear cDNA clones encoding two RNA binding proteins, CP-RBP30 and CP-RBP-31, which are targeted to chloroplasts in Nicotiana plumbaginifolia. In this report we describe the analysis of the 3-untranslated regions (3-UTRs) in 22 CP-RBP30 and 8 CP-RBP31 clones which reveals that mRNAs encoding both proteins have a very complex polyadenylation pattern. Fourteen distinct poly(A) sites were identified among CP-RBP30 clones and four sites among the CP-RBP31 clones. The authenticity of the sites was confirmed by RNase A/T1 mapping of N. plumbaginifolia RNA. CP-RBP30 provides an extreme example of the heterogeneity known to be a feature of mRNA polyadenylation in higher plants. Using PCR we have demonstrated that CP-RBP genes in N. plumbaginifolia and N. sylvestris, in addition to the previously described introns interrupting the coding region, contain an intron located in the 3 non-coding part of the gene. In the case of the CP-RBP31, we have identified one polyadenylation event ocurring in this intron.     

Mutations in the processing site of the precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit: effects on import,processing, assembly and stability

The small subunit (SSU) of Rubisco is synthesized in the cytosol in a precursor form. Upon import into the chloroplast, it is proteolytically processed at a Cys-Met bond to yield the mature form of the protein. To assess the importance of the Met residue for recognition and processing by the stromal peptidase, we substituted this residue with either Thr, Arg or Asp. The mutant precursor proteins were imported into isolated chloroplasts, and the products of the import reactions were analyzed. Mutants containing Thr or Arg residues at the putative processing site were processed to a single peptide, comigrating with the wild-type protein. N-terminal radio-sequencing revealed that these mutants were processed at the Cys-Thr and the Cys-Arg bond, respectively. After import of the Asp-containing mutant, four processed forms of the protein were observed. Analysis of the most abundant one, co-migrating with the wild-type protein, demonstrated that this species was also a product of correct processing, at the Cys-Asp bond. All the correctly processed peptides were found to be associated with the holoenzyme of Rubisco, and remained stable within the chloroplast, like the wild-type protein. The results of this study, together with previous ones, suggest that proper recognition and processing of the SSU precursor are more affected by residues N-terminal to the processing site than by the residue on the C-terminal side of this site.     

Characterization of a novel plant poly(A) polymerase

We have purified and characterized poly(A) polymerases (PAPs) from Pisum sativum, Brassica juncea, and Zea mays. Through chromatography on DEAE-Sepharose and heparin-Sepharose, these PAPs copurified as a single enzyme along with RNPs that could provide RNA substrates for the enzyme. More extensive purification by chromatography on MonoQ resulted in the resolution of the PAPs into as many as three fractions. One of these (PAP-I) contained a 43-kDa polypeptide immunologically related to the yeast PAP, and two others (PAP-II and PAP-III) contained RNAs that could serve as substrates for polyadenylation. These fractions by themselves possessed little PAP activity, but mixtures containing combinations of these displayed substantial activity. Similar PAP factors (PAP-I and PAP-III) were identified after fractionation of extracts prepared from Brassica juncea and Zea mays. The factors from one plant were completely interchangeable with those from different plants. We conclude that the poly(A) polymerases present in vegetative plant tissues consist of more than one component. In this respect, they are substantially different from other reported plant, mammalian, and yeast PAPs.     

Pro-Thyrotropin-Releasing Hormone Processing by Recombinant PC1

Abstract: Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH₂), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [³H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH_25–152 or preproTRH_25–158) and a 10-kDa C-terminal peptide (preproTRH_154–255 or preproTRH_160–255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (preproTRH_25–76 or preproTRH_25–82) and a 3.8-kDa peptide (preproTRH_83–108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (preproTRH_206–255). The optimal pH for these cleavages was 5.5. ZnCl₂, EDTA, EGTA, and the omission of Ca²⁺ inhibited the formation of pYE₂₇ (preproTRH_25–50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.     

Comparison of the Molecular Forms of the Kex2/Subtilisin-Like Serine Proteases SPC2, SPC3, and Furin in Neuroendocrine Secretory Vesicles Reveals Differences in Carboxyl-Terminus Truncation and Membrane Association

Abstract: The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full-length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full-length 86-kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl-terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane-associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86-kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus-truncated 64-kDa form.     

Processing of Procarboxypeptidase E into Carboxypeptidase E Occurs in Secretory Vesicles

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of bioactive peptides. Like other peptide processing enzymes, CPE is initially produced as a precursor ("proCPE") that undergoes posttranslational processing at a site containing five adjacent Arg residues near the N-terminus and at other sites near the C-terminus of proCPE. The time course of the N-terminal processing step suggests that this conversion occurs in either the Golgi apparatus or the secretory vesicles. To delineate further the site of proCPE processing, pulse/chase analysis was performed under conditions that block transit out of the Golgi apparatus (brefeldin A, carbonyl cyanide m -chlorophenylhydrazone, or 20°C) or that block acidification of vesicles (chloroquine, monensin, or ammonium chloride). The results of these analysis suggest that efficient proCPE processing requires an acidic post-Golgi compartment. To test whether known processing enzymes can perform this cleavage, purified proCPE was incubated with furin, prohormone convertase 1, or a dynorphin converting enzyme, and the products were analyzed on denaturing polyacrylamide gels. Furin cleaves proCPE within the N-terminal region, although the reaction is not very efficient, requiring relatively large amounts of furin or long incubation times. The other two peptide processing enzymes did not cleave proCPE, whereas a relatively small amount of secretory granule extract was able to convert proCPE into CPE. Taken together, these findings suggest that the conversion of proCPE into CPE occurs primarily in secretory vesicles.     

Spinal Cord Dynorphin Precursor Intermediates Decline During Late Gestation

Abstract: This laboratory has previously reported that the maternal opioid analgesia associated with pregnancy and parturition is mediated, at least in part, by a maternal spinal cord dynorphin/κ opioid system. This analgesia is accompanied by an increase in dynorphin peptides (1–17 and 1–8) in the lumbar spinal cord. Levels of trypsin-generated arginine⁶-leucine-enkephalin (Leu-Enk-Arg)-immunoreactive determinants were also determined and used to reflect the content of dynorphin precursor intermediates. In spinal tissue, the amount of dynorphin A (1–17) contained in the form of precursor is, at a minimum, 10-fold higher than the content of mature dynorphin A (1–17) or dynorphin (1–8). During gestational day 22, the content of dynorphin precursor is reduced significantly (∼50%). The decline in the magnitude of dynorphin precursor intermediates in the spinal cord of pregnant rats vastly exceeds the magnitude of increase in the content of dynorphin peptides (1–17 and 1–8). This difference can best be explained by postulating a corresponding increase in the rate of release of spinal cord dynorphin (1–17). It is suggested that enhanced processing of dynorphin precursor intermediates represents the initial biochemical level of adaptation of spinal dynorphin neurons to increased demands of pregnancy.     

Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons: A new tool for dissecting the molecular and cellular basis of LHRH physiology

Summary 1. Two LHRH neuronal cell lines were developed by targeted tumorigenesis of LHRH neuronsin vivo. These cell lines (GN and GT-1 cells) represent a homogeneous population of neurons. GT-1 cells have been further subcloned to produce the GT1-1, GT1-3, and GT1-7 cell lines. While considerable information is accumulating about GT-1 cells, very little is currently known about the characteristics and responses of GN cells.2. By both morphological and biochemical criteria, GT-1 cells are clearly neurons. All GT-1 cells immunostain for LHRH and the levels of prohormone, peptide intermediates, and LHRH in the cells and medium are relatively high.3. GT-1 cells biosynthesize, process, and secrete LHRH. Processing of pro-LHRH appears to be very similar to that reported for LHRH neuronsin vivo. At least four enzymes may be involved in processing the prohormone to LHRH.4. LHRH neurons are unique among the neurons of the central nervous system because they arise from the olfactory placode and grow back into the preoptic-anterior hypothalamic region of the brain. Once these neurons reach this location, they send their axons to the median eminence. With respect to the immortalized neurons, GN cells were arrested during their transit to the brain. In contrast, GT-1 cells were able to migrate to the preoptic-anterior hypothalamic region but were unable correctly to target their axons to the median eminence. These problems in migration and targeting appear to be due to expression of the simian virus T-antigen.5. While GT-1 cells are a homogeneous population of neurons, they are amenable to coculture with other types of cells. Coculture experiments currently under way should help not only to reveal some of the molecular and cellular cues that are important for neuronal migration and axonal targeting, but they should also highlight the nature of the cellular interactions which normally occurin situ.6. GT-1 cells spontaneously secrete LHRH in a pusatile manner. The interpulse interval for LHRH from these cells is almost identical to that reported for release of LH and LHRHin vivo. GT-1 cells are interconnected by both gap junctions and synapses. The coordination and synchronization of secretion from these cells could occur through these interconnections, by feedback from LHRH itself, and/or by several different compounds that are secreted by these cells. One such compound is nitric oxide.7. GT-1 cells have Na⁺, K⁺, Ca²⁺, and Cl^– channels. Polymerase chain reaction experiments coupled with Southern blotting and electrophysiological recordings reveal that GT-1 cells contain at least five types of Ca²⁺ channels. R-type Ca²⁺ channels appear to be the most common type of channel and this channel is activated by phorbol esters in the GT-1 cells.8. LHRH is secreted from GT-1 cells in response to norepinephrine, dopamine, histamine, GABA (GABA-A agonists), glutamate, nitric oxide, neuropeptide Y, endothelin, prostaglandin E₂, and activin A. Phorbol esters are very potent stimulators of LHRH secretion. Inhibition of LHRH release occurs in response to LHRH, GABA (GABA-B agonists), prolactin, and glucocorticoids.9. Compared to secretion studies, far fewer agents have been tested for their effects on gene expression. All of the agents which have been tested so far have been found either to repress LHRH gene expression or to have no effect. The agents which have been reported to repress LHRH steady-state mRNA levels include LHRH, prolactin, glucocorticoids, nitric oxide, and phorbol esters. While forskolin stimulates LHRH secretion, it does not appear to have any effect on LHRH mRNA levels.