Prenatal and postnatal expression of mRNA coding for rat prothrombin.

The levels of prothrombin mRNA in prenatal and postnatal rat tissues were analyzed in order to determine tissue distribution of prothrombin expression and to determine if increases in liver prothrombin mRNA during development correlated with previously documented developmental increases in plasma prothrombin levels. Maternal tissues were also analyzed in order to determine if prothrombin mRNA levels varied due to gestational or postpartum influences. Northern analysis demonstrated that rat liver prothrombin mRNA levels increased several-fold late in gestation and reached maximal levels by 13 days after birth. Prothrombin mRNA was also expressed in diaphragm, stomach, intestine, kidney, spleen and adrenal tissues during development. In maternal tissues during pregnancy, prothrombin mRNA was expressed in liver, diaphragm, stomach, uterus and placenta. Prothrombin mRNA levels in each of these tissues that were positive by Northern analysis were quantitated by solution hybridization analysis. Between gestational day 18 and postnatal day 13, liver prothrombin mRNA levels increased from approx. 600 to 2100 molecules per cell (a 3.5-fold increase). In maternal liver during pregnancy, between day 18 and day 22, prothrombin mRNA levels increased from approx. 1800 to 2100 molecules per cell. Immediately after delivery, maternal liver prothrombin mRNA levels decreased to approx. 50% of preparturition levels. Prothrombin mRNA levels in placental tissue ranged from approx. 100 to 250 molecules per cell. In other fetal, postnatal and maternal tissues, prothrombin mRNA expression was less than 100 molecules per cell. These results demonstrate that the level and tissue-type expression of prothrombin mRNA varies in response to prenatal and postnatal influences.

Follicle-stimulating hormone (FSH) increases FSH receptor messenger ribonucleic acid while decreasing FSH binding in cultured porcine granulosa cells.

The goal of the present studies was to compare direct effects of porcine FSH (pFSH) on [125I]pFSH-binding sites with effects of pFSH on FSH receptor mRNA in cultured porcine granulosa cells. Cells from immature follicles were cultured on laminin-coated plates in serum-free medium for up to 6 days in the absence or presence of pFSH (1-100 ng/ml) or cholera toxin (0.04-400 ng/ml), which activates adenylyl cyclase independently of the FSH receptor. RRA indicated that [125I] pFSH binding to cells cultured without stimulator increased more than 10-fold with time in culture. Addition of pFSH to cultures resulted in a dose-dependent decrease in binding, assessed after removal of bound pFSH. Equilibrium saturation binding analysis indicated that pFSH (10 ng/ml) caused a 39% decrease in binding sites in cells cultured for 6 days. At the same time, pFSH increased progesterone production 9.5-fold. Cholera toxin (4 ng/ml) increased [125I]pFSH binding 110% and progesterone production 8.9-fold. Northern hybridization analysis of cultured granulosa cell mRNA using a porcine FSH receptor cDNA revealed three transcripts for the FSH receptor [2.2, 3.5, and 4.2 kilobases (kb)], with the major transcript being 4.2 kb in length. Addition of either pFSH (10 ng/ml) or cholera toxin (10 ng/ml) to cultures of granulosa cells increased the intensity of pFSH receptor transcripts compared with control values, with the 4.2-kb message remaining predominant. Hybridization with a porcine LH receptor cDNA revealed different transcripts (2.4, 4.0, 4.7, 7.0, and 11.0 kb), with the major transcript being 4.7 kb in length. Addition of either pFSH or cholera toxin to the cultures increased the intensity of all LH receptor transcripts; however, cholera toxin was more effective than pFSH. pFSH and cholera toxin increased the intensity of each species to different extents, although the 4.7-kb transcript remained predominant. These results indicate that exposure to FSH in culture results in down-regulation of the FSH receptor. Down-regulation is accompanied by increased FSH receptor mRNA levels, suggesting that FSH enhances FSH receptor synthesis.

The effects of vitamin K1 and warfarin on prothrombin expression in human hepatoblastoma (HepG2) cells.

Cultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 micrograms vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 micrograms/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 micrograms/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 micrograms/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240-350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on gamma-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2.

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.

Non-specific effects of aquaMEPHYTON (vitamin K1) on prothrombin expression in human hepatoblastoma (HepG2) cells.

In order to determine the effects of vitamin K1 on prothrombin production, we have treated cultures of human hepatoblastoma cells with an aqueous colloidal suspension of vitamin K1. Dose-response analysis demonstrated increases in secreted prothrombin antigen levels ranging from 3 to 3.7-fold over controls. Time-course analysis demonstrated increases in secreted prothrombin antigen levels over controls up to 6 hours of treatment. Between 6 and 24 hours, secreted prothrombin antigen levels increased at a rate parallel to controls. Vitamin K1 treatment also resulted in a parallel increase in total secreted protein levels. Prothrombin mRNA size (approximately 2.1 kb) and levels (ranging from 390-480 prothrombin mRNA molecules per cell) were determined by Northern and quantitative solution hybridization analysis, respectively, and were unaffected by vitamin K1 treatment. The increases in secreted prothrombin antigen levels most likely result from non-specific effects of vitamin K1 or agents used to emulsify vitamin K1 on protein release from HepG2 cells.

Developmental expression of protein C and protein S in the rat.

In order to better understand the expression of the Protein C/Protein S anticoagulant system, we have isolated and characterized cDNAs coding for rat Protein C and Protein S. These cDNAs were used in Northern analysis to determine tissue-specificity and developmental expression patterns for mRNAs coding for Proteins C and S. In rats, Protein C mRNA is expressed almost exclusively in liver with a small amount of expression in kidney, diaphragm, stomach, intestine, uterus and placenta. Protein C mRNA was not expressed in brain, heart, lung, spleen, small intestine, large intestine, ovary, or urinary bladder. In liver, Protein C mRNA is expressed at very low levels at prenatal day 18 and these levels increased to maximal levels by postnatal day 13. The size of the mRNA coding for rat Protein C is approximately 1.9 kb. Rat Protein S mRNA was expressed in all tissues examined: brain, heart, lung, diaphragm, liver, spleen, stomach, small intestine, large intestine, kidney, adrenal ovary, uterus, placenta, and urinary bladder. Interestingly, there were 4 bands hybridizing with the rat protein S cDNA that were evident in many of the tissues examined, corresponding to mRNA sizes of approximately 3.5, 2.6, 1.8, and 0.3 kb. There was a difference in tissue-specificity of each mRNA. The 1.8 kb band is generally the most prominent autoradiographic band in any tissue. From these results, it is evident that the expression of Protein C mRNA is similar to that of other vitamin K-dependent proteins. The expression of Protein S mRNA, however, is surprisingly complex and may include alternative splicing of mRNA to generate the various sizes evident on Northern analysis.

Three-year follow-up of bibliotherapy for depression.

This study examined the durability of cognitive bibliotherapy for mild to moderately depressed adults by conducting a 3-year follow-up of participants from a previous study (C. Jamison & F. Scogin, 1995). The Hamilton Rating Scale for Depression, Beck Depression Inventory, and questions relating to participants' perceptions of the program were administered. Results indicated that treatment gains were maintained over the 3-year follow-up period and support the usefulness of cognitive bibliotherapy as an adjunct to traditional treatment modalities in a general adult population.

Palmar dermatoglyphics of dyslexia.

Palmar dermatoglyphic prints were taken of 261 dyslexics (173 males and 88 females) and compared against those of 707 controls (372 males and 335 females). Dyslexics of both sexes were found to exhibit greater complexity in terms of ridge count and pattern location than controls, particularly on the left hand. Specifically, both male and female dyslexics exhibited higher left a-b counts, wider atd angles on both palms, and higher frequencies of pattern in left interdigital area IV. Additionally, male dyslexics also had higher right a-b counts and greater frequency of pattern in the left hypothenar area. Dyslexics of both sexes were also found to have more distally located axial triradii, and investigation of bilateral asymmetry found dyslexics to exhibit more directional asymmetry than controls in the variable of a-b count, with the left value for both groups being greater than the right. It was concluded that the study evidenced strong support for the hypothesis that some causative factor relating to the development of dyslexia is operating during the time period in which dermatoglyphic features are formed. The relevance of these findings in terms of the Geschwind hypothesis and the possible importance of prenatal testosterone are discussed.

Mutations in Escherichia coli that effect sensitivity to oxygen.

Fifteen oxygen-sensitive (Oxys) mutants of Escherichia coli were isolated after exposure to UV light. The mutants did not form macroscopic colonies when plated aerobically. They did form macroscopic colonies anaerobically. Oxygen, introduced during log phase, inhibited the growth of liquid cultures. The degree of inhibition was used to separate the mutants into three classes. Class I mutants did not grow after exposure to oxygen. Class II mutants were able to grow, but at a reduced rate and to a reduced final titer, when compared with the wild-type parent. Class III mutants formed filaments in response to oxygen. Genetic experiments indicated that the mutations map to six different chromosomal regions. The results of enzymatic assays indicated that 7 of the 10 class I mutants have low levels of catalase, peroxidase, superoxide dismutase, and respiratory enzymes when compared with the wild-type parent. Mutations in five of the seven class I mutants which have the low enzyme activities mapped within the region 8 to 13.5 min. P1 transduction data indicated that mutations in three of these five mutants, Oxys-6, Oxys-14, and Oxys-17, mapped to 8.4 min. The correlation of low enzyme levels and mapping data suggests that a single gene may regulate several enzymes in response to oxygen. The remaining three class I mutants had wild-type levels of catalase, peroxidase, and superoxide dismutase, but decreased respiratory activity. The class II and III mutants had enzyme activities similar to those of the wild-type parent. Our results demonstrate that mutations in at least six genes can be expressed as oxygen sensitivity. Some of these genes may be involved in respiration or cell division or may regulate the expression of several enzymes.

Effect of prenatal testosterone administration on palmar dermatoglyphic intercore ridge counts of rhesus monkeys (Macaca mulatta).

Dermatoglyphic ridge counts of the prints of 59 rhesus monkeys (Macaca mulatta) whose mothers had been treated with injections of testosterone during their pregnancies were studied to determine the effect of the day the hormone began to be administered, the amount of hormone administered, and the number of days of hormone administration upon the dermatoglyphic variation of the offspring. Of the three hormone variables, only the day of beginning administration (STARTDAY) was significantly associated with dermatoglyphic variation, and its positive significance was demonstrated with the ridge counts of Area I on both the left and right hand, Area II of the left hand, and the total ridge counts of both hands. These results are discussed within the context of the timing of the dermatoglyphic window, and the differences in the findings between the monkey and earlier human studies are addressed.

Dermatoglyphic and anthropometric relationships within the Iñupiat (Eskimo) hand.

On the basis of earlier findings with Easter Islanders suggesting a positive correlation between dermatoglyphic variables and hand anthropometric measurements, the present study was designed to determine if such a relationship could be generalized to another population, namely, Iñupiat (Eskimo). Since some dermatoglyphic and anthropometric variables were available for both sides of the body, the extension of this study to explore the question of asymmetry was also possible. The Iñupiat sample numbered 142 male and 176 female adult inhabitants of five Alaskan North Slope communities. The major findings of this study included, for males, significant negative correlations between left arm length and digital ridge counts and positive relationships between the palmar variable of axial index and hand length on both hands. For females, the hand breadth/length index was negatively related to most of the digital variables. Very little definitive information regarding the relationship of the asymmetry variables between the two types of measures was ascertained.

Characterization of the mouse cDNA and gene coding for a hepatocyte growth factor-like protein: expression during development.

The cDNA and gene coding for mouse hepatocyte growth factor-like protein (HGF-like protein) were isolated and characterized. The size of the gene from the site of initiation of transcription to the polyadenylation site is 4613 bp in length and is composed of 18 exons separated by 17 intervening sequences. The exons range in size from 36 to 227 bp in length, while the intervening sequences range in size from 78 to 613 bp in length. The site of initiation of transcription was identified by primer extension analysis using total RNA isolated from mouse liver. On the basis of these results, the first exon is 146 bp in length and includes 94 bp of 5'-noncoding sequence. The sequence 5'TATGTG3' is present between 34 and 39 bp upstream of the transcription start site and could potentially be the TATA sequence found for many constitutively expressed eukaryotic genes to be the promoter for RNA polymerase II. The sequence 5'GCAAT3' at -96 to -92 may be the CCAAT sequence responsible for stimulation of transcription of some eukaryotic genes. The same sequences in the Genbank and NBRF databases were homologous to similar regions in the genes coding for both human and mouse HGF-like protein (Han et al., 1991). The acyl-peptide hydrolase gene is 410 bp downstream of the mouse HGF-like protein, but is transcribed from the complementary strand. The mouse cDNA for HGF-like protein codes for a putative protein with the same domain structure as its human homologue with four kringle domains followed by a serine protease-like domain. On the basis of the translated sequence of the cDNA, the mouse HGF-like protein would be 716 amino acids in length with a molecular weight of 80K. There are four potential N-linked carbohydrate attachment sites. The DNA and amino acid sequences of mouse HGF-like protein are compared to the human protein. Overall, the two proteins are about 80% identical with each other. In contrast to mRNA for human HGF-like protein, which is 2.4 and 3.0 kilobases in length in human liver, only the smaller species is seen in mouse and rat liver. The expression pattern of mRNA coding for HGF-like protein during development and in maternal rats was determined by Northern analysis. It is apparent that the majority of mRNA coding for HGF-like protein is expressed in liver. Messenger RNA is also expressed at a lower level in lung, adrenal, and placenta.