Low Steady-state Levels Research Articles

The murine Spam1 gene: RNA expression pattern and lower steady-state levels associated with the Rb(6.16) translocation.

Recently we mapped the murine Spam1 gene to the proximal region of chromosome 6 (MMU 6). Based on the map location and physiological characteristics of its encoded sperm antigen, the gene is an attractive candidate for the sperm dysfunction seen in Rb(6.16) translocation heterozygotes and the reduced fertility of homozygotes. We have analyzed the expression of Spam1 mRNA in normal and Rb(6.16) mice. The expression of Spam1 mRNA was found to be: 1) tissue specific; it is expressed exclusively in testis; and 2) developmentally regulated, with a haploid expression. Notably, the steady-state mRNA level of Spam1 in Rb(6.16) homozygotes was 25-30% of that in chromosomally normal consomic mice and those homozygous for Rb(2.8) (7.18). In Rb(6.16) and Rb(6.15) heterozygotes the levels were 61% and 66% of the normal. Studies are currently under way to determine the protein levels and gene structure of Spam1, to detect the underlying cause of the mRNA reduction associated with these translocations.

Molecular Mechanism of a Mild Phenotype in Coagulation Factor XIII (FXIII) Deficiency: A Splicing Mutation Permitting Partial Correct Splicing of FXIII A-Subunit mRNA

AbstractCongenital factor XIII (FXIII) deficiency is potentially a severe bleeding disorder, but in some cases, the symptoms may be fairly mild. In this study, we have characterized the molecular mechanism of a mild phenotype of FXIII A-subunit deficiency in a Finnish family with two affected sisters, one of whom has even had two successful pregnancies without regular substitution therapy. In the screening tests for FXIII deficiency, no A-subunit could be detected, but by using more sensitive assays, a minute amount of functional A-subunit was seen. 3H-putrescine incorporation assay showed distinct FXIII activity at the level of 0.35% of controls, and also the fibrin cross-linking pattern in the patients clotted plasma showed partial γ-γ dimerization. In Western blot analysis, a faint band of full-length FXIII A-subunit was detected in the patients' platelets. The patients have previously been identified as heterozygotes for the Arg661 → Stop mutation. Here we report a T → C transition at position +6 of intron C in their other allele. The transition affected splicing of FXIII mRNA resulting in low steady state levels of several variant mRNA transcripts. One transcript contained sequences of intron C, whereas two transcripts resulted from skipping of one or two exons. Additionally, correctly spliced mRNA lacking the Arg661 → Stop mutation of the maternal allele could be detected. These results demonstrate that a mutation in splice donor site of intron C can result in several variant mRNA transcripts and even permit partial correct splicing of FXIII mRNA. Further, even the minute amount of correctly processed mRNA is sufficient for producing protein capable of γ-γ dimerization of fibrin. This is a rare example of an inherited functional human disorder in which a mutation affecting splicing still permits some correct splicing to occur and this has a beneficial effect to the phenotype of the patients.

A respiration-deficient Chinese hamster cell line with a defect in mitochondrial protein synthesis: rapid turnover of some mitochondrial transcripts.

Translational initiation and elongation in mammalian mitochondria is still poorly understood, and genetic approaches are expected to be helpful in the elucidation of the mechanism. This study describes a further characterization of a Chinese hamster mutant cell line which is severely defective in mitochondrial protein synthesis. Additional proof is provided that the mutation is nuclear. It is shown that there is no dramatic depletion of mitochondrial DNA in these cells, and the mtDNA appears to have neither significant deletions nor rearrangements. Transcription is not affected, and the polycistronic transcripts are processed normally. However, mt mRNAs are differentially quite unstable, and some are at very low steady state levels. Several nuclear transcripts encoding mitochondrial proteins were also investigated and found to be present at normal levels, and in particular, it was demonstrated that complex II (and succinate dehydrogenase activity), encoded entirely by nuclear genes, was only moderately affected by the absence of all the other complexes of the electron transport chain.

Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast.

To investigate the function of the nucleolar protein Nop2p in Saccharomyces cerevisiae, we constructed a strain in which NOP2 is under the control of a repressible promoter. Repression of NOP2 expression lengthens the doubling time of this strain about fivefold and reduces steady-state levels of 60S ribosomal subunits, 80S ribosomes, and polysomes. Levels of 40S subunits increase as the free pool of 60S subunits is reduced. Nop2p depletion impairs processing of the 35S pre-rRNA and inhibits processing of 27S pre-rRNA, which results in lower steady-state levels of 25S rRNA and 5.8S rRNA. Processing of 20S pre-rRNA to 18S rRNA is not significantly affected. Processing at sites A2, A3, B1L, and B1S and the generation of 5' termini of different pre-rRNA intermediates appear to be normal after Nop2p depletion. Sequence comparisons suggest that Nop2p may function as a methyltransferase. 2'-O-ribose methylation of the conserved site UmGm psi UC2922 is known to take place during processing of 27S pre-rRNA. Although Nop2p depletion lengthens the half-life of 27S pre-RNA, methylation of UmGm psi UC2922 in 27S pre-rRNA is low during Nop2p depletion. However, methylation of UmGm psi UC2922 in mature 25S rRNA appears normal. These findings provide evidence for a close interconnection between methylation at this conserved site and the processing step that yields the 25S rRNA.

A constitutively active mutant of the alpha 1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins G9 alpha and G11 alpha than the wild-type receptor.

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.

Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica.

The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic theory of eukaryote origins.

Technology, convergence, and growth in the European Union

This paper contains an empirical analysis of growth and convergence in the European Union using a cross-country data set covering the period 1950–92. It seeks an answer to the question why some countries in Europe manage to catch up, while others, most notably the poorest ones, apparently do not. The empirical evidence provided in the paper points to several responsible factors. The distance of the economy to the technological leader differed across economies, which contributed to differences in convergence and growth behavior. In addition, the finding of conditional convergence implies that economies converge to different steady state levels of income per capita. Poor economies, like Portugal, Greece, Spain, and Ireland, presumably converge to a lower steady state level of income per capita, which leads to persistent differences in income per capita.

Fluorescence induction curves registered from individual microalgae cenobiums in the process of population growth

Registration of chlorophyll fluorescence induction curves (IC) from individual microalgae cenobiums was performed during Scenedesmus quadricauda culture growth. Emphasis was placed on the analysis of patterns of the slow phase of IC, since these slow fluorescence transitions reflect complex interactions between primary and secondary photosynthetic processes. A classification was performed of the ICs obtained according to the patterns of their slow phase. Four different types of such patterns were distinguished. The microalgae population structure with respect to IC patterns was investigated at different stages of culture growth. The distribution of microalgae cenobiums over the patterns of IC was found to change in accordance with the stage of population development. At the stage of the population growth enhancement, nonmonotonous IC dominated with a high steady-state level of fluorescence. The stage of linear growth was characterized by IC with monotonous decay kinetics and low steady-state level of fluorescence. At the third stage including the phases of growth inhibition, stationary state and the beginning of cell death the population structure was the most heterogeneous, with all IC patterns observed.

Identification of Sequences in c-myc mRNA That Regulate Its Steady-State Levels

The level of cellular myc proto-oncogene expression is rapidly regulated in response to environmental signals and influences cell proliferation and differentiation. Regulation is dependent on the fast turnover of c-myc mRNA, which enables cells to rapidly alter c-myc mRNA levels. Efforts to identify elements in myc mRNA responsible for its instability have used a variety of approaches, all of which require manipulations that perturb normal cell metabolism. These various approaches have implicated different regions of the mRNA and have led to a lack of consensus over which regions actually dictate rapid turnover and low steady-state levels of c-myc mRNA. To identify these regions by an approach that does not perturb cell metabolism acutely and that directly assesses the effect of a c-myc mRNA region on the steady-state levels of c-myc mRNA, we developed an assay using reverse transcription and PCR to compare the steady-state levels of human myc mRNAs transcribed from two similarly constructed myc genes transiently cotransfected into proliferating C2C12 myoblasts. Deletion mutations were introduced into myc genes, and the levels of their mRNAs were compared with that of a near-normal, reference myc mRNA. Deletion of most of the myc 3' untranslated region (UTR) raised myc mRNA levels, while deletion of sequences in the myc 5' UTR (most of exon 1), exon 2, or the protein-coding region of exon 3 did not, thus demonstrating that the 3' UTR is responsible for keeping myc mRNA levels low. Using a similar reverse transcription-PCR assay for comparing the steady-state levels of two beta-globin-myc fusion mRNAs, we showed that fusion of the myc 3' UTR lowers globin mRNA levels by destabilizing beta-globin mRNA. Surprisingly, fusion of the protein-coding region of myc exon 3 also lowered globin mRNA steady-state levels. Investigating the possibility that exon 3 coding sequences may play some other role in regulating c-myc mRNA turnover, we demonstrated that these sequences, but not myc 3' UTR sequences, are necessary for the normal posttranscriptional downregulation of c-myc mRNA during myoblast differentiation. We conclude that, while two elements within c-myc mRNA can act as instability determinants in a heterologous context, only the instability element in the 3' UTR regulates its steady-state levels in proliferating C2C12 cells.

Molecular Basis of the Altered Antigenic Expression of RhD in Weak D (Du) and RhC/e in RN Phenotypes

The RH blood group locus is composed of two sequence-related genes, RHD and RHCE, encoding the D, Cc, and Ee antigens in common Rh-positive phenotypes. In this report, we have analyzed the molecular basis of Rh antigens expression in weak D (Du) and RN donors, in whom there is a severe reduction of the D and C/e antigens, respectively. Genomic and transcript analysis of three unrelated low-grade weak D (Du) variants indicated that the very low expression of the D antigen is not the result of rearrangement or mutation in the coding sequence of the RHO gone. Accordingly, weak D (Du) erythrocytes should carry a normal RhD polypeptide, which is in agreement with the observation that these variants never produce anti-D antibodies. Comparative polymerase chain reaction analysis showed a lower steady-state level of RhD transcripts in weak D (Du) reticulocytes, as compared with normal RhD-positive controls, thus providing direct evidence that the difference between the D antigen of D-positive and weak D (Du) red blood cells is quantitative only. Conversely, analysis of the molecular genetic basis of the RN phenotype Indicated that the severely decreased expression of the RhC and Rhe antigens in three variants is associated with a qualitative alteration identified as a segmental DNA exchange between the RHCE and RHD genes. These genomic rearrangements, which resulted in hybrid RhCe-D-Ce proteins expressing the low frequency Rh32 but not the high incidence Rh46 antigens, involved either axon 4 alone or both exons 3 and 4. These findings show that an identical phenotypical alteration of Rh antigens (reduced expression) may result either from a quantitative or a qualitative alteration of the RH genes expression.

The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis

We have previously described the fact that the individual expression of influenza virus PA protein induced a generalized proteolysis (J.J. Sanz-Ezquerro, S. de la Luna, Ortin, and A. Nieto, J. Virol. 69:2420-2426, 1995). In this study, we have further characterized this effect by mapping the regions of PA protein required and have found by deletion analysis that the first 247 amino acids are sufficient to bring about this activity. PA mutants that were able to decrease the accumulation levels of coexpressed proteins also presented lower steady-state levels due to a reduction in their half-lives. Furthermore, the PA wild type produced a decrease in the stationary levels of different PA versions, indicating that is itself a target for its induced proteolytic process. All of the PA proteins that induced proteolysis presented nuclear localization, being the sequences responsible for nuclear transport located inside the first 247 amino acids of the molecule. To distinguish between the regions involved in nuclear localization and those involved in induction of proteolysis, we fused the nuclear localization signal of the simian virus 40 T antigen to the carboxy terminus of the cytosolic versions of PA. None of the cytosolic PA versions affected in the first 247-amino-acid part of PA, which were now located in the nucleus, were able to induce proteolysis, suggesting that conservation of a particular conformation in this region of the molecule is required for the effect observed. The fact that all of the PA proteins able to induce proteolysis presented nuclear localization, together with the observation that this activity is shared by influenza virus PA proteins from two different type A viruses, suggests a physiological role for this PA protein activity in viral infection.

HERV-K: the biologically most active human endogenous retrovirus family.

The human genome contains a wide variety of endogenous retrovirus-like sequences. The human endogenous retrovirus type K (HERV-K) family comprises 30-50 members per haploid genome in humans and is highly conserved in Old World monkeys and apes. Some proviruses are displaying open reading frames (ORF) with coding capacity for viral particles. HERV-K sequences most likely code for the previously described human teratocarcinoma-derived virus (HTDV) and correlated expression of HERV-K Gag has been demonstrated by immunoelectron microscopy studies. Protease, but not yet reverse transcriptase (RT), enzymatic activity was demonstrated for recombinant HERV-K proteins. However, an ultrasensitive RT assay revealed specific polymerase activity associated with the HTDV particles. HERV-K transcription is specifically regulated by viral long terminal repeats and RNA is expressed at low steady-state levels in a variety of human tissues and tumours. In teratocarcinoma cell lines, HERV-K is highly expressed in a complex pattern showing full-length as well as subgenomic envelope (env) and two alternatively spliced small transcripts. The doubly spliced 1.8-kb mRNA codes for cORF protein which resembles Rev of HIV-1 and is located in the nucleolus. In addition, the cORF sequence acts as a leader and is essential for effective expression of glycosylated HERV-K Env protein. Although HERV-K sequences code for all necessary retroviral proteins, infectious particles could not yet be demonstrated. The putative implication of HERV sequences in pathophysiological processes, for example, testicular malignancies, remains to be elucidated.

Bone mineral metabolism in T lymphocyte–deficient and –replete strains of rat

The immune and skeletal systems are known to interact. We have repeatedly shown that in contrast to in vitro data, the administration of T lymphocyte immunosuppressants, such as cyclosporin A, leads to an increase in bone resorption and a high turnover osteopenia. The purpose of this study was to characterize the bone metabolism of the T lymphocyte deficient Rowett athymic homozygous (rnu/rnu) nude rat. We wished to determine whether these rats share the bone abnormalities of cyclosporin A-treated rats. Eleven 10-week-old Sprague-Dawley rats and 12 similarly aged nude rats were studied over a 4-week period. Metaphyseal cancellous bone histomorphometry was similar in the two groups of rats and only differed with regard to percentage eroded perimeter (lower in nude rats, p = 0.0008) and longitudinal growth rate (49% lower in nude rats, p < 0.001). The nude rats had less body mass (p < 0.001) but nevertheless gained the same percentage of their body weight over the study period. The athymic rats had lower levels of serum, 1,25-dihydroxyvitamin D (p < 0.014) and serum osteocalcin(p < 0.009), and at the age of 14 weeks the nude rats had lower concentrations of serum creatinine (p = 0.001) and blood ionized calcium (p = 0.0002), yet serum PTH was similar throughout. RNA isolated from the contralateral tibias revealed that the nude group had lower steady-state levels of osteocalcin mRNA despite similar rates of bone formation. In its entirety, the data suggest that T cell deficiency per se is not necessarily associated with high turnover osteopenia.

Changes in steady-state levels of tryptophan hydroxylase protein in adult rat brain after neonatal 6-hydroxydopamine lesion.

A recently developed technique of immunoautoradiography on nitrocellulose transfers of serial frozen sections was used to determine tryptophan hydroxylase concentration in selected areas of the adult rat brain following neonatal 6-hydroxydopamine destruction of nigrostriatal dopamine neurons. Particular attention was paid to the neostriatum, known to be serotonin-hyperinnervated under these conditions, and to the nucleus raphe dorsalis, containing the cell bodies of origin for these nerve terminals. The hippocampus was also investigated as a territory of structurally intact serotonin innervation arising primarily from the nucleus raphe medianus. Tryptophan hydroxylase protein was measured at successive transverse levels across the entire caudorostral extent of all these regions. Similar measurements of tyrosine hydroxylase protein across the substantia nigra and the neostriatum verified the disappearance of the nigrostriatal dopamine neurons. The average tryptophan hydroxylase tissue concentration in the dorsal third of the serotonin-hyperinnervated neostriatum was up by 36% above control, i.e. significantly less than the number of its serotonin axon terminals or varicosities. This was therefore indicative of a lowering of the tryptophan hydroxylase protein content per serotonin ending. Interestingly, a tight correlation between the respective level-by-level concentrations of tryptophan hydroxylase and tyrosine hydroxylase protein in the control neostriatum allowed the prediction the tryptophan hydroxylase concentration after dopamine denervation with a serotonin hyperinnervation. Tryptophan hydroxylase concentration was also significantly reduced in both the nucleus raphe dorsalis and nucleus raphe medianus, notably at those raphe dorsalis levels known to give rise to the serotonin hyperinnervation of neostriatum. It is hypothesized that the lower steady-state level of tryptophan hydroxylase inside the terminals and cell bodies of hyperinnervating serotonin neurons was the result of a feedback inhibition of the synthesis of the enzyme by its end-product, presumably because of the increased amount of serotonin in these terminals.

Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent Ki values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO2 fixation follows. When leaves are maintained in constant darkness at 15 °C until CO2 output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO2 fixation even though the phosphorylation state of PEPCase does not change.

Calcitonin acutely increases tyrosyl-phosphorylation of proteins in human osteosarcoma (SaOS-2) cells.

In order to test the hypothesis that salmon calcitonin has direct effects to modulate tyrosyl-protein phosphorylation in human osteosarcoma cells, SaOS-2 cells (with very high steady-state levels of skeletal alkaline phosphatase) were exposed to calcitonin, in duplicate serum-free cultures, at concentrations ranging from 10(-13) to 10(-9) mol/liter, for 0-60 minutes at 37 degrees C. Phospho-tyrosyl proteins were identified by autoradiography of Western blots after incubation with 125I-labeled antiphosphotyrosine antibodies (or with unlabeled antibodies and 125I-labeled protein A) and quantitated by laser densitometry. The results of these studies revealed (1) time-dependent effects of salmon calcitonin (sCt) (at 3 x 10(-12) mol/liter) to increase the level of tyrosylphosphorylation of at least six proteins, with apparent molecular weights of 20, 25, 27, 41, 48, and 135 kD (P < 0.05 for each); and (2) dose-dependent effects of sCt (during 15 minutes of exposure) to increase the level of tyrosyl-phosphorylation of at least 10 proteins with apparent molecular weights of 19, 20, 27, 35, 41, 102, 135, 195, 220, and 244 kD (P < 0.05 for each). A supplementary study of calcitonin effects on tyrosyl-protein phosphorylation in a subpopulation of SaOS-2 cells with very low steady-state levels of skeletal alkaline activity revealed similar responses--time and dose-dependent increases in the tyrosyl-phosphorylation of at least seven proteins with apparent molecular weights of 44, 48, 57, 62, 101, 244, and 280 kD (P < 0.05 for each).(ABSTRACT TRUNCATED AT 250 WORDS)

Bactericidal properties of hydrogen peroxide and copper or iron-containing complex ions in relation to leukocyte function

Various combinations of hydrogen peroxide, reductant (ascorbic acid and superoxide ion), and copper or iron salts and their coordination complexes were examined to determine their cytotoxicity toward several bacteria with diverse metabolic capabilities and cell envelope structures. Four sets of bactericidal conditions were identified, comprising: (1) high concentration levels (5–100 mM) of H 2O 2 in the absence of exogenous metal ions and reductant; (2) ferrous or ferric coordination complexes plus enzymatically generated O 2 ·− and H 2O 2 at relatively low steady-state concentration levels; (3) cupric ion plus low concentration levels of H 20 2 (1 μM-1 μM) and ascorbate (10μM-4 mM); (4) cuprous ion (or cupric ion plus ascorbate) in the absence of 0 2 and H 20 2. Rates of losses in viabilities increased proportionately with increases in the concentration of H 2O 2 in metal-free environments and with each of the components in the Cu 2+/ascorbate/H 2O 2 bactericidal assay system. Oxidant levels required for equivalent killing increased with increasing cell densities of the bacterial suspensions over the range investigated (2 × 10 7-2 × 10 9 cfu/ml). Other experimental conditions or other combinations of reagents, most notably Fe 3+/ascorbate/H 2O 2 systems, did not generate bactericidal environments. The patterns of response of the three organisms tested, Streptococcus lactis, Escherichia coli, and Pseudomonas aeruginosa, were similar, suggesting common bactericidal mechanisms. However, preliminary evidence suggests that the lethal lesions caused by the various bactericidal conditions are distinct. As discussed, each of the four bactericidal conditions could conceivably be attained within the phagosomes of leukocytes, although none has as yet been identified.

Members of the Nuclear Factor κB Family Transactivate the Murine c-myb Gene

Expression of the c-myb proto-oncogene is primarily detected in normal tissue and tumor cell lines of immature hematopoietic origin, and the down-regulation of c-myb expression is associated with hematopoietic maturation. Cell lines that represent mature, differentiated hematopoietic cell types contain 10-100-fold less c-myb mRNA than immature hematopoietic cell types. Differences in steady-state c-myb mRNA levels appear to be primarily maintained by a conditional block to transcription elongation that occurs in the first intron of the gene. The block to transcription elongation has been mapped, using nuclear run-on analysis, to a region of DNA sequence that is highly conserved between mouse and man. Two sets of DNA-protein interactions, flanking the site of the block to transcription elongation, were detected that exhibited DNA-binding activities that strongly correlated with low steady-state c-myb mRNA levels. Several criteria demonstrated that members of the nuclear factor kappa B (NF-kappa B) family of transcription factors were involved in the DNA-protein interactions identified in these two sets. Surprisingly, cotransfection experiments demonstrated that coexpression of members of the NF-kappa B family, specifically p50 with p65 and p65 with c-Rel, transactivated a c-myb/chloramphenicol acetyltransferase reporter construct that contained 5'-flanking sequences, exon I, intron I, and exon II of the c-myb gene. Transactivation by these heterodimer combinations was dependent on regions of the c-myb first intron containing the NF-kappa B-binding sites. These findings suggest that NF-kappa B family members may be involved in either modifying the efficiency of transcription attenuation or acting as an enhancer-like activity to increase transcription initiation. Thus, the regulation of c-myb transcription may be quite complex, and members of the NF-kappa B family likely play an important role in this regulation.

Transforming growth factor-beta reverses a posttranscriptional defect in elastin synthesis in a cutis laxa skin fibroblast strain.

Skin fibroblasts from two cases of autosomal recessive cutis laxa (CL), having insignificant elastin production and mRNA levels, were challenged with transforming growth factor beta-1 (TGF-beta 1). Elastin production was brought from undetectable values to amounts typical of normal human skin fibroblasts in a dose-dependent fashion. Basic fibroblast growth factor (100 ng/ml) alone or in combination with TGF-beta 1 reduced elastin production and mRNA expression in CL skin fibroblasts more extensively than in normal cells. In situ hybridization showed that these effects were at the transcript level. One of the CL strains was examined in detail. Transcription rates for elastin were similar in normal and CL and unchanged by TGF-beta 1 or TGF-beta 2 (10 ng/ml), while in CL elastin mRNA half-life was increased > 10-fold by TGF-beta 2 and reduced 6-fold after TGF-beta 2 withdrawal, as compared with a control strain. Cycloheximide partially reversed elastin mRNA instability. These data are consistent with a defect in elastin mRNA stability that requires synthesis of labile factors or intact translational machinery, resulting in an extremely low steady state level of mRNA present in this strain of CL. Furthermore, TGF-beta can relieve elastin mRNA instability in at least one CL strain and elastin production defects in both CL strains.

Naturally Occurring Antisense Transcripts Are Present in Chick Embryo Chondrocytes Simultaneously with the Down-regulation of the α1(I) Collagen Gene

It has previously been shown that very low steady state levels of alpha 1 (I) collagen mRNA are present in chick embryo sternal chondrocytes (Askew, G. R., Wang, S., and Lukens, L. N. (1991) J. Biol. Chem. 266, 16834-16841), yet nuclear run-on experiments with double-stranded cDNA probes indicated a high transcription rate at this locus. These findings were investigated in this study using single-stranded probes, where nuclear run-on experiments showed that antisense transcription of the alpha 1 (I) collagen gene was occurring in chondrocytes, while sense strand transcription was down-regulated. Treatment of these chondrocytes with 5-bromo-2'-deoxyuridine (BrdU), which causes the cells to resemble their mesenchymal precursors, resulted in an antiparallel situation, where antisense transcription was lost, and instead, sense strand transcription was acquired, suggesting that the reverse switch from sense to antisense transcription occurs during chondrogenesis. Very large (> 10 kilobases) and heterogeneous antisense transcripts of moderate stability were shown to span both ends of the gene in chondrocytes, while their absence was shown in BrdU-treated chondrocytes, chick embryo fibroblasts, and a variety of other tissues. The function of these antisense transcripts is so far unknown, but their unusual chondrocyte-specific appearance, concurrent with little or no sense strand transcription, suggests a possible functional role in the down-regulation of the alpha 1 (I) collagen gene.