Normal and Atypical Butyrylcholinesterases in Placental Development, Function, and Malfunction

1. In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous abortion and placental malfunction. The major protein interacting with these compounds is butyrylcholinesterase (BuChE), which attenuates the effects of such xenobiotics by their hydrolysis or sequestration. Therefore, we studied BuChE expression during placental development.2. RT-PCR revealed both BuChEmRNA and acetylcholinesterase (AChE) mRNA throughout gestation. However, cytochemical staining detected primarily BuChE activity in first-trimester placenta but AChE activity in term placenta.3. As the atypical variant of BuChE has a narrower specificity for substrates and inhibitors than the normal enzyme, we investigated its interactions with -solanine and cocaine, and sought a correlation between the occurrence of this variant and placental malfunction.4. Atypical BuChE of serum or recombinant origin presented >10-fold weaker affinities than normal BuChE for cocaine and -solanine. However, BuChE in the serum of a heterozygote and a homozygous normal were similar in their drug affinities. Therefore, heterozygous serum or placenta can protect the fetus from drug or poison exposure, unlike homozygous atypical serum or placenta.5. Genotype analyses revealed that heterozygous carriers of atypical BuChE were threefold less frequent among 49 patients with placental malfunction than among 76 controls or the entire Israeli population. These observations exclude heterozygote carriers of atypical BuChE from being at high risk for placental malfunction under exposure to anticholinesterases.     

Messenger RNA competition in living Xenopus oocytes.

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs this become endogenous mRNAs. These results, together with previous estmates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in the temporarily inactivated form.     

The level of butyrylcholinesterase activity increases and the content of the mRNA remains unaffected in brain of senescence-accelerated mouse SAMP8

Looking at cholinesterases (ChEs) changes in age-related mental impairment, the expression of ChEs in brain of senescence accelerated-resistant (SAMR1) and senescence accelerated-prone (SAMP8) mice was studied. Acetylcholinesterase (AChE) activity was unmodified and BuChE activity increased twofold in SAMP8 brain. SAMR1 brain contained many AChE-T mRNAs, less BuChE and PRiMA mRNAs and scant AChE-R and AChE-H mRNAs. Their content unchanged in SAMP8 brain. Amphiphilic (G(4)(A)) and hydrophilic (G(4)(H)) AChE and BuChE tetramers, besides amphiphilic dimers (G(2)(A)) and monomers (G(1)(A)) were identified in SAMR1 brain and their distribution was little modified in SAMP8 brain. Blood plasma does not seem to provide the excess of BuChE activity in SAMP8 brain; it probably arises from glial cell changes owing to astrocytosis.     

Pollination induces mRNA poly(A) tail-shortening and cell deterioration in flower transmitting tissue

Pollination induces many physiological responses in the flower, including deterioration and death in specific pistil cell types. It is shown here that within the style of tobacco, pollination-induced cell deterioration was restricted to the transmitting tissue while the surrounding cortical tissue was not affected. It was distinct from general senescence since exogenously applying the senescence-inducing hormone ethylene, or its precursor aminocyclopropane-1-carboxylic acid (ACC), to the flower or the pistil induced overall deterioration in the entire flower. Furthermore, both pollen tube growth and ethylene action were needed for the entire spectrum of cellular changes associated with this pollination-induced transmitting tissue deterioration process. It is also shown that pollination-induced mRNA poly(A) tail-shortening for at least three major classes of transmitting tissue-specific mRNAs. As is commonly observed for poly(A) tail-shortened mRNAs, the levels of two of these three mRNA classes decline after pollination. On the other hand, the third class of mRNAs, transmitting tissue-specific (TTs) mRNAs, was maintained at a very high level subsequent to pollination, even after substantial poly(A)-tail shortening. TTS mRNAs encode a pollen tube growth-promoting and -attracting protein needed for optimal in vivo pollen tube growth. The specific preservation of TTS mRNAs in the deteriorating transmitting tissue cells suggests that these cells can distinguish molecules needed in the pollinated styles from those that are dispensable, and protect them from degradation. It is suggested that the pollination-induced mRNA poly(A) tail-shortening and cell death are programmed processes suited to the post-pollination transmitting tissue environment. Results showing that ACC is a candidate signal molecule for the pollination-induced mRNA-shortening which is accentuated by ethylene and mediated via a protein phosphorylation-dependent signal transduction pathway are also presented.     

Relative abundance of thrombospondin 2 and thrombospondin 3 mRNAs in human tissues.

The levels of thrombospondin 2 (TSP2) and thrombospondin 3 (TSP3) mRNAs in a variety of human tissues were determined by analysis of multiple-tissue mRNA dot blots. For TSP2 mRNA, aorta and fetal heart had the greatest relative abundance. High levels were also detected for muscle, fetal, endocrine, immune, and nerve tissues. The pattern of expression of TSP3 mRNA was very different: kidney, pituitary gland, trachea, uterus, and fetal kidney had the greatest abundance. In general, TSP3 mRNA was expressed at high levels in endocrine, muscle, and fetal tissues. In addition to the tissue-specific differences, a more even distribution of TSP3 mRNA among tissues was observed. The high relative abundance of the two mRNAs in a variety of tissues and the tissue-specific differences in expression could be significant for understanding the diverse roles implicated for TSP2 and TSP3.     

Characterization of novel mRNAs encoding enzymes involved in peptide alpha-amidation

The COOH-terminal alpha-amidation of bioactive peptides is a 2-step process catalyzed by two separable enzymatic activities both derived from the peptidylglycine alpha-amidating monooxygenase (PAM) precursor. Two forms of PAM mRNA (rPAM-1 and -2), differing by the presence or absence of optional Exon A, were previously characterized; both encode precursors predicted to have an NH2-terminal signal sequence, an intragranular domain containing both enzymatic activities, and a single transmembrane domain followed by a short, cytoplasmic COOH-terminal domain. In this report, two novel types of PAM mRNA were identified in adult rat atrium. A cDNA of each type was sequenced, and the results indicate that rPAM-3 and -4 could be related to each other and to the previously characterized rat PAM cDNAs by alternative mRNA splicing. Deletion of a 258-nucleotide segment (optional Exon B) encoding the transmembrane domain from rPAM-3 and the presence of a novel 3'-exon in rPAM-4 mean that both rPAM-3 and -4 mRNAs encode precursor proteins that have an NH2-terminal signal peptide but lack a transmembrane domain. The rPAM-4 precursor protein lacks the region of the PAM precursor catalyzing the second step in the alpha-amidation reaction. Low levels of rPAM-3 and -4 type mRNA were detected in atrium. Utilizing the polymerase chain reaction, two major patterns of distribution of forms of PAM mRNA were found. In the heart and central nervous system, PAM mRNAs both containing and lacking optional Exon A were prevalent and almost all of the PAM mRNAs detected contained optional Exon B. In the pituitary and submaxillary glands, PAM mRNAs lacking optimal Exon A were prevalent, as were PAM mRNAs lacking all or part of optional Exon B. Since the distribution of PAM activity between soluble and membrane fractions is tissue-specific and developmentally regulated and since rPAM-4 lacks an enzymatic portion of the PAM precursor, the tissue-specific expression of these forms of rat PAM mRNA is expected to be of functional significance.     

Characterization of cDNAs for stylar transmitting tissue-specific proline-rich proteins in tobacco

The pistil of flowers is a specialized organ which contains the female gametophytes and provides the structures necessary for pollination and fertilization. Pollen deposited on the stigmatic surface of a compatible plant germinates a pollen tube which penetrates the stigmatic papillae and grows intercellularly through the style towards the ovules in the ovary. Pollen tube growth is largely restricted to the transmitting tissue in the style. Therefore the stylar transmitting tissue is extremely important for the migration of the pollen cell towards the ovary. We have isolated two related cDNAs, transmitting tissue-specific (TTS)-1 and TTS-2, derived from two proline-rich protein (PRP)-encoding mRNAs that accumulate specifically in the transmitting tissue of tobacco. The deduced PRP sequences share similarities with proline-rich cell wall glycoproteins found in a variety of plants. TTS-1 and TTS-2 mRNAs are induced in very young floral buds, accumulate most abundantly during the later stages of flower development when style elongation is the most rapid, and remain at relatively high levels at anthesis. These mRNAs become undetectable in maturing green fruits. In situ hybridization shows that TTS-1 and TTS-2 mRNA accumulation is restricted to the transmitting tissue of the style. The possible roles that these transmitting tissue-specific PRPs may play in maintaining the structural integrity of the style or in the function of this organ is discussed.     

Rat aldolase A messenger RNA: the nucleotide sequence and multiple mRNA species with different 5'-terminal regions

The nucleotide sequence of aldolase A mRNA in rat skeletal muscle was determined using recombinant cDNA clones and a cDNA synthesized by primer extension. The sequence is composed of 1343 nucleotides (nt) except for the poly(A) tail. Based on the sequence analysis we have deduced an open reading frame with 363 amino acids (aa) (Mr 39134). The sequence suggests several nt polymorphisms in the mRNA population, one of which causes an aa change. The determined sequence of rat aldolase A mRNA was compared with the published ones of rabbit aldolase A or rat aldolase B mRNAs. The homology between rat and rabbit aldolase A mRNA sequences is greater than that between rat aldolase A and B mRNA sequences. Multiple aldolase A mRNAs having different Mrs were detected in the various tissues, and appeared to be expressed in a tissue-specific manner. Further analysis suggests that differences in mRNA length are due to differences in the 5'-noncoding terminal region.     

Developmental regulation of the mRNAs for elastins a, b and c in foetal-calf nuchal ligament and aorta.

The data presented clearly suggest that relative amounts of mRNAs for elastins a, b and c are developmentally regulated in foetal-calf nuchal ligament and aorta and that this regulation is tissue-specific. In nuchal ligament, at earlier stages of foetal development, the relative amounts of mRNAs for elastins a and b are very low. After the foetal age of about 6 months the relative amount of mRNA for elastin b begins to increase. This is followed by an increase in the relative amount of mRNA for elastin a. In aorta, with increasing foetal age, the relative amounts of mRNAs for elastins b and c increase and decrease alternately. The relative amounts of mRNA for elastin a remain low, with only marginal increases with foetal age. A possible self-aggregation role of elastin a in elastogenesis is proposed.