Role of calmodulin as an endogenous initiatory factor in compensatory lung growth after pneumonectomy

Compensatory lung growth after pneumonectomy is a well established phenomenon in young humans and experimental animals. To date, the cellular initiating and/or regulatory factor(s) responsible for this growth response have yet to be established. We have studied changes in lung content and activity of calmodulin, a calcium-dependent regulatory protein in relation with lung mass and DNA content during postpneumonectomy compensatory lung growth in 4-week-old rats. We observed that after left pneumonectomy, right lung calmodulin content (measured by radioimmunoassay) and calmodulin activity (measured by cyclic nucleotide phosphodiesterase activation) were increased at days 1 and 2 after surgery in the pneumonectomized rats, preceding the increase of lung mass and DNA content which started at day 3. Treatment of the pneurnonectomized rats with a highly specific calmodulin antagonist, trifluoperazine, immediately after the surgery, resulted in diminished lung calmodulin activity, sparing the calmodulin content, and in concomitant reduction of lung mass and DNA content to values intermediate between those of controls and the pneumonectomized animals. Based on these findings, we conclude that calmodulin may be an important intracellular (possibly, autocrine) initiatory or facilitatory factor in compensatory hyperplastic lung growth after pneumonectomy. © 1993 Wiley-Liss, Inc.     

Rapid stimulatory effect of insulin on binding of glycolytic enzymes to cytoskeleton of C-6 glial cells, and the antagonistic action of calmodulin inhibitors

Insulin was shown in our previous experiments to induce an increase in binding of glycolytic enzymes to muscle cytoskeleton. We show here the same stimulatory effect of insulin in C-6 glial cells in culture. In these cells, like in muscle, a short time of incubation with insulin (1-10 min) induced an increase in cytoskeleton bound phosphofructokinase and aldolase. This stimulatory effect of insulin could be prevented by treatment with calmodulin antagonists trifluoperazine, thioridazine or CGS 9343 B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that calmodulin is involved in this action of insulin. Our previous experiments have shown that growth factors and Ca(2+) also induce a rapid, calmodulin-mediated stimulation of binding of glycolytic enzymes to cytoskeleton. The present and previous results suggest that the rapid binding of glycolytic enzymes to cytoskeleton, may be a general mechanism, in different cells, in signal transduction of insulin, growth factors and other Ca(2+) -mobilizing hormones. The accelerated cytoskeletal glycolysis will supply local ATP, which is required for the rapid cytoskeletal-membrane rearrangements following the binding of hormone to its receptor.     

Calmodulins with deletions in the central helix functionally replace the native protein in yeast cells.

Deletion of Glu-84, Glu-83 and Glu-84, or Ser-Glu-Glu-Glu (residues 81-84) from the central helix of mammalian calmodulin is known to result in a 5-7 times decrease in its apparent in vitro affinities for three calmodulin-dependent enzymes. However, based on in vitro experiments, it is difficult to estimate how these deletions might affect in vivo cellular function. The yeast Saccharomyces cerevisiae, which requires calmodulin for growth, provides an excellent system to evaluate these deletion proteins in vivo. Based on its ability to restore normal growth characteristics to yeast cells, mammalian calmodulin is functionally identical to the yeast protein; herein we evaluate the effect of deleting residues 84, 83 and 84, or 81-84 from the central helix. Sequences encoding the deletion proteins and an unaltered control sequence were introduced by means of a yeast shuttle vector and were expressed under control of the yeast calmodulin promoter. The deletion and control calmodulins are produced at levels similar to that observed for the yeast protein, and they completely restore normal growth characteristics. This result suggests that the regions deleted from the central helix are not critical for activation of any yeast calmodulin target normally required for cell growth or division. It is likely that there are twisting and shortening motions associated with the deletions from the central helix that alter significantly the spatial relationship between the two lobes of calmodulin. The abilities of the deletion calmodulins to restore completely normal growth characteristics to yeast cells suggest that the lobes of all the deletion proteins can still be appropriately positioned in calmodulin-target complexes. This is consistent with the hypothesis that the central helix of calmodulin is analogous to a flexible tether rather than to a rigid connector between the two lobes of the molecule.     

Phenotypic characterization of malignant progenitor cells in patients with idiopathic myelofibrosis

Objective/BackgroundIdiopathic myelofibrosis (IM) is a clonal hematological malignancy originating from pluripotent hematopoietic stem cells (HSC). HSC are very rare potent cells that reside in the bone marrow (BM) and at a lower level in peripheral blood (PB). Previous studies showed that IM PB CD34⁺ cells contain not only BM repopulating cells belonging to the malignant clone but also residual normal HSC.MethodsIn the current study, we separated the subpopulations of IM PB CD34⁺ cells using IL-3Rα/CD123 labeling and further characterized them by genetic and functional analyses.ResultsWe differentiated IM PB CD34⁺ cells into three subpopulations (IL-3Rα^high, IL-3Rα^low, and IL-3Rα^negative). IL-3Rα^high CD34⁺ cell subgroup represents a small population in IM PB CD34⁺ cells which was not seen in normal G-CSF mobilized CD34⁺ cells. IM IL-3Rα^high CD34⁺ cells contained significant higher percentage of cells bearing marker chromosome detected by fluorescence in situ hybridization (FISH) analysis. In the absence of growth factors, IM IL-3Rα^high CD34⁺ cells exhibited abnormal colony forming ability and carried greater percentage of JAK2V617F mutant allele compared with IL-3Rα^low and IL-3Rα^negative CD34⁺ cells.ConclusionThese data indicate that IL-3Rα^high CD34⁺ cells from IM enriched for the malignant progenitor cells and IL-3Rα/CD123 may be a potential biomarker and therapeutic target for IM. Our findings will be further validated in future studies with a larger sample size and serial transplant in murine models.     

Gastric cancer risk according to the distribution of intestinal metaplasia and neutrophil infiltration

Background and Aim: Gastritis and intestinal metaplasia (IM) have long been known to be risk factors for and precursors of gastric cancer. We aimed to elucidate the association between gastric cancer risk and the distribution of precancerous lesions in the stomach by histological analyses. Methods: We analyzed patients from whom two biopsy specimens (one from the antrum and one from the corpus) were obtained by upper gastrointestinal endoscopy. Specimens were assessed for Helicobacter pylori, IM, and neutrophil infiltration (NI). Patients were classified into three groups based on the presence of IM. Patients were also classified into four groups based on the presence of NI. The prevalence of gastric cancer was compared between groups. Results: A total of 1395 patients were analyzed. Of these, 54 had gastric cancer (34 intestinal and 20 diffuse type). A multivariate analysis showed that male sex and the distribution of IM were independent risk factors for intestinal‐type cancer. Compared with patients without IM (n = 1005), the odds ratio (OR) for patients with IM in the antrum only (n = 240) was 2.34 (95% confidence interval: 1.08–4.96), and that for patients with IM in the corpus (n = 150) was 5.84 (2.92–11.8). However, NI was related to diffuse‐type cancer. Compared with patients without NI (n = 899), the OR for patients with NI in the corpus only (n = 122) was 3.66 (1.02–12.2). Conclusions: The histological pattern and distribution of gastric mucosal change assessed by two biopsy specimens were related to gastric cancer.     

Absence of bcl-2 expression by activated CD45RO+ T lymphocytes in acute infectious mononucleosis supporting their susceptibility to programmed cell death

bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death (apoptosis). There is now increasing evidence that regulation of bcl-2 expression is a determinant of life or death in normal lymphocytes. We have recently described that activated (CD45RO+) CD4+ and CD8+ T cells in acute infectious mononucleosis (IM) undergo apoptotic cell death on culturing, indicating an activation-driven cell death of mature T cells. In this work, we examine bcl-2 expression by activated T cells in acute IM using a flow-cytometric analysis with an anti-bcl-2 monoclonal antibody (MoAb). It was consistently observed that most T cells from acute IM patients displayed only much less bcl-2, while normal T cells expressed bcl-2 relatively strongly. Multicolor analysis showed that bcl-2- lacking T cells in acute IM were restricted to the CD45RO+ (activated) populations of CD4+, as well as CD8+ T cells. In contrast, the relatively intense levels of bcl-2 were expressed in both CD45RO+ and CD45RO- T-cell populations from normal subjects. This marked difference in bcl-2 expression of CD45RO+ T cells between acute IM and normal controls was also confirmed by Western blot analysis. Activated (CD45RO+) T cells with low bcl-2 expression, but not bcl-2-expressing CD45RO- T cells, in acute IM patients were found to die easily when cultured without added growth factors. However, in normal individuals, both CD45RO+ and CD45RO- T cells were relatively stable on culturing. These findings suggest that lack of bcl-2 expression by activated (CD45RO+) T cells in acute IM might be associated with their susceptibility to programmed cell death.     

Bile reflux gastritis and intestinal metaplasia at the cardia

BACKGROUND AND AIMS: Intestinal metaplasia (IM) at the cardia is likely to be a precursor of cardia cancer. Previous work has shown that it is associated with chronic inflammation attributable to either gastro-oesophageal reflux disease (GORD) or Helicobacter pylori infection. An alternative aetiological factor is bile reflux. Duodenogastric reflux brings about histological changes in the gastric mucosa that can be graded and used to calculate a bile reflux index (BRI). We used the BRI to assess whether reflux of bile plays a part in the development of cardia IM. METHODS: Histological changes in simultaneous gastric antrum and cardia biopsies from 267 dyspeptic patients were independently graded by two pathologists. The association between cardia IM and age, sex, clinical group, H pylori status, increased BRI (>14), and inflammation at the cardia were evaluated using logistic regression. RESULTS: A total of 226 patients had adequate cardia and antral biopsies; 149 had GORD and 77 had non-ulcer dyspepsia. Cardia IM was present in 66 (29%) patients, of whom 28 (42%) had complete IM. Increasing age, male sex, chronic inflammation, and a high BRI emerged as significant independent associations with cardia IM. Clinical group and H pylori status were not independent risk factors. CONCLUSIONS: Histological evidence of bile reflux into the stomach is associated with cardia IM. This could have an important bearing on carcinogenesis at this site.     

Durable responses in chronic myeloid leukemia patients maintained with lower doses of imatinib mesylate after achieving molecular remission

In the present report, we address the question if the reduction of standard dosage of imatinib mesylate (IM) in imatinib-intolerant chronic myeloid leukemia (CML) patients with undetectable residual disease may impair their outcome. Four patients are described. The median follow up from the beginning of IM therapy was 35 months (33–59). The median duration of real-time quantitative polymerase chain reaction (RQ-PCR) negativity on IM 200 mg daily was 17months (4–37). We hypothesize that in IM intolerant CML patients with complete molecular remission, the compound dosage might be safely reduced to a lower than standard dose without to lose the response. A tight molecular monitoring of such patients should be required.     

Multiple Epstein-Barr virus strains in patients with infectious mononucleosis: comparison of ex vivo samples with in vitro isolates by use of heteroduplex tracking assays

Recent work using a heteroduplex tracking assay (HTA) to identify resident viral sequences has suggested that patients with infectious mononucleosis (IM) who are undergoing primary Epstein-Barr virus (EBV) infection frequently harbor different EBV strains. Here, we examine samples from patients with IM by use of a new Epstein-Barr nuclear antigen 2 HTA alongside the established latent membrane protein 1 HTA. Coresident allelic sequences were detected in ex vivo blood and throat wash samples from 13 of 14 patients with IM; most patients carried 2 or more type 1 strains, 1 patient carried 2 type 2 strains, and 1 patient carried both virus types. In contrast, coresident strains were detected in only 2 of 14 patients by in vitro B cell transformation, despite screening >20 isolates/patient. We infer that coacquisition of multiple strains is common in patients with IM, although only 1 strain tends to be rescued in vitro; whether nonrescued strains are present in low abundance or are transformation defective remains to be determined.     

Factors predicting progression of gastric intestinal metaplasia: results of a randomised trial on Helicobacter pylori eradication

BACKGROUND AND AIMS: Gastric intestinal metaplasia (IM) is generally considered to be a precancerous lesion in the gastric carcinogenesis cascade. This study identified the risk factors associated with progression of IM in a randomised control study. SUBJECTS AND METHODS: A total of 587 Helicobacter pylori infected subjects were randomised to receive a one week course of anti-Helicobacter therapy (omeprazole, amoxicillin, and clarithromycin (OAC)) or placebo. Subjects underwent endoscopy with biopsy at baseline and at five years. Severity of IM was graded according to the updated Sydney classification and progression was defined as worsening of IM scores at five years in either the antrum or corpus, or development of neoplasia. Backward stepwise multiple logistic regression was used to identify independent risk factors associated with IM progression. RESULTS: Of 435 subjects (220 in the OAC and 215 in the placebo group) available for analysis, 10 developed gastric cancer and three had dysplasia. Overall progression of IM was noted in 52.9% of subjects. Univariate analysis showed that persistent H pylori infection, age >45 years, male subjects, alcohol use, and drinking water from a well were significantly associated with IM progression. Duodenal ulcer and OAC treatment were associated with a reduced risk of histological progression. Progression of IM was more frequent in those with more extensive and more severe IM at baseline. With multiple logistic regression, duodenal ulcer (odds ratio (OR) 0.23 (95% confidence interval (CI) 0.09-0.58)) was found to be an independent protective factor against IM progression. Conversely, persistent H pylori infection (OR 2.13 (95% CI 1.41-3.24)), age >45 years (OR 1.92 (95% CI 1.18-3.11)), alcohol use (OR 1.67 (95% CI 1.07-2.62)), and drinking water from a well (OR 1.74 (95% CI 1.13-2.67)) were independent risk factors associated with IM progression. CONCLUSION: Eradication of H pylori is protective against progression of premalignant gastric lesions.     

H. pylori genotypes and cytokine gene polymorphisms influence the development of gastric intestinal metaplasia in a Chinese population

BACKGROUND: Cytokine gene polymorphisms and Helicobacter pylori (HP) genotypes have been linked to gastric cancer development in Western countries. We determined the role of host cytokine polymorphisms and bacterial virulent factors in the development of gastric intestinal metaplasia (IM) in a Chinese population with a high background gastric cancer incidence. METHODS: Three hundred two HP-infected noncancer individuals living in Shandong province of China with available DNA were studied. Polymorphisms in different loci of inflammatory cytokines Interleukin IL-1B, IL-1RN, Interleukin IL-8, IL-10, IL-18, tumor necrosis factor-A (TNF-A), and Transforming growth factor (TGF-B), were determined by allelic discriminating TaqMan polymerase chain reaction (PCR) or a variable number of tandem repeats. Presence of HP virulence factors in cagA, vacA, and babA2 were determined by PCR. Baseline gastric biopsies were assessed for the presence of IM. RESULTS: Among HP-infected subjects, carriers of the IL-1B-511 T allele were associated with a modestly greater prevalence of IM (adjusted OR 2.0, 95% CI 1.0-3.7). There was no association between the presence of IM and polymorphisms in other inflammatory cytokines. Although most subjects from this region harbored the virulent HP strains, carriage of the vacA m1 strain was associated with a significantly higher prevalence of IM (adjusted OR 1.8, 1.1-3.0). The presence of both host (IL-1B-511 T) and HP (vacA m1) genotypes further increased the risk of IM (OR 5.7, 2.0-16) when compared with individuals with the low-risk genotype. CONCLUSION: The carriage of proinflammatory IL-1B-511 and HP vacA m1 genotypes was associated with the development of gastric IM in the Chinese.     

胃黏膜肠化生的逆转性问题

胃黏膜肠化生作为胃癌前病变,与胃癌的发病存在密切联系.关于肠化生是否具有逆转性,目前尚有争议.但来自流行病学的证据显示,经过长期随访研究,肠化生可以逆转,但改变程度较小.除H pylori感染外,维生素C缺乏、胃酸减少和/或胆汁返流等亦是其发病因素.肠化生的发病机制尚处于探索阶段,H pylori毒力因子、肠道特异性转录因子、微卫星不稳定性等均参与其发病环节,但尚不能肯定肠化生是由干细胞突变引起的胃上皮细胞表型的改变.肠化生在诊断上存在诸多困难,严格的内镜评估以及正确的取检部位尤为重要.单独根除H pylori似乎不足以逆转肠化生,联合应用其他化学阻断剂以及中医药,可能是一条新的治疗途径.     

A role for calmodulin in the growth of human hematopoietic progenitor cells.

A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.     

Diagnosis and Management of High Risk Group for Gastric Cancer

Gastric cancer is associated with high morbidity and mortality worldwide. To reduce the socioeconomic burden related to gastric cancer, it is very important to identify and manage high risk group for gastric cancer. In this review, we describe the general risk factors for gastric cancer and define high risk group for gastric cancer. We discuss strategies for the effective management of patients for the prevention and early detection of gastric cancer. Atrophic gastritis (AG) and intestinal metaplasia (IM) are the most significant risk factors for gastric cancer. Therefore, the accurate selection of individuals with AG and IM may be a key strategy for the prevention and/or early detection of gastric cancer. Although endoscopic evaluation using enhanced technologies such as narrow band imaging-magnification, the serum pepsinogen test, Helicobacter pylori serology, and trefoil factor 3 have been evaluated, a gold standard method to accurately select individuals with AG and IM has not emerged. In terms of managing patients at high risk of gastric cancer, it remains uncertain whether H. pylori eradication reverses and/or prevents the progression of AG and IM. Although endoscopic surveillance in high risk patients is expected to be beneficial, further prospective studies in large populations are needed to determine the optimal surveillance interval.     

Functional ABCG2 is overexpressed on primary CML CD34+ cells and is inhibited by imatinib mesylate

Imatinib mesylate (IM) therapy for chronic myeloid leukemia (CML) has transformed the treatment of this disease. However, the vast majority of patients, despite major responses, still harbor Philadelphia chromosome-positive (Ph(+)) cells. We have described a population of primitive Ph(+) cells that are insensitive to IM and may be a source of IM resistance. Cell line studies have suggested that the drug transporter ABCG2 may be a mediator of IM resistance, however there is considerable debate about whether IM is an ABCG2 substrate or inhibitor. We demonstrate here that primitive CML CD34(+) cells aberrantly overexpress functional ABCG2 but that cotreatment with IM and an ABCG2 inhibitor does not potentiate the effect of IM. We definitively show that IM is an inhibitor of, but not a substrate for, ABCG2 and that, therefore, ABCG2 does not modulate intracellular concentrations of IM in this clinically relevant cell population.     

Reduced-intensity allogeneic hematopoietic stem cell transplantation combined with imatinib has comparable event-free survival and overall survival to long-term imatinib treatment in young patients with chronic myeloid leukemia

The relative merits of reduced intensity hematopoietic stem cell transplantation (RIST) for chronic myeloid leukemia (CML) in the first chronic phase (CP) in imatinib era have not been evaluated. The study was designed to compare the outcomes of combination therapy of RIST plus imatinib (RIST + IM) vs. imatinib (IM) alone for young patients with early CP (ECP) and late CP (LCP). Of the patients, 130 were non-randomly assigned to treatment with IM alone (n = 88) or RIST + IM (n = 42). The 10-year overall survival (OS) and event-free survival (EFS) were comparable between RIST + IM and IM groups. LCP, high Sokal score, and no complete cytogenetic response at 3 months were adverse prognostic factors for survival, but only the time from diagnosis to IM was an independent predictor after multivariate analysis. For ECP, IM was similar to RIST + IM, with 10-year EFS rates of 77.2 vs. 81.6% (p = 0.876) and OS rates of 93.8 vs. 87.9% (p = 0.102), respectively. For LCP, both treatments resulted in similar survival, but more patients in the imatinib group experienced events (10-year EFS 40.8 vs. 66.7%, p = 0.047). The patients with higher EBMT risk scores had an inferior survival than those with lower scores (69.2 vs. 92.9%, p = 0.04). We concluded that RIST + IM was comparable to IM in terms of OS and EFS. However, RIST + IM was more affordable than IM alone in a 10-year scale. Thus, RIST + IM could be considered as an alternative treatment option, especially when the patients have low EBMT risk scores and demand a definite cure for CML.     

Overexpressions of RHOA,CSNK1A1, DVL2, FZD8, and LRP5 genes enhance gastric cancer development in the presence of Helicobacter pylori

Background and study aimsIntestinal metaplasia (IM), and Helicobacter pylori (HP) infection can be shown as risk factors in the development of gastric cancer (GC). WNT signaling pathway plays a critical role in carcinogenesis. However, the literature studies are limited on the significance of this pathway for the transition from IM to GC.Patients and methodsWe aimed to investigate the importance of the genes of WNT signaling pathways diagnostic and prognostic markers in the presence and absence of HP in conversion from IM to GC. 104 patients, (GC group n = 35, IM group n = 45, control group n = 25) were included in this case-control study. Expression of genes in WNT signalling were searched in study groups with qRT-PCR array and qRT-PCR method. Data were analysed using PCR array data analysis software.ResultsStatistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was detected in the GC and IM groups compared to the control group (p < 0.05). Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was observed in patients with metastatic GC compared to patients with GC without metastasis (p < 0.05). It was found that the RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes were statistically significantly over-expressed in diffuse GC patients compared to non-diffuse GC patients (p < 0.05). Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was detected in HP positive IM patients compared to HP negative IM patients (p < 0.05).ConclusionOverexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes in IM may suggest that these genes are important markers in the development of IM and inflammation with HP. In addition, these genes are linked to tumor burden in the GC group. Consequently, we can conclude that these genes are poor prognosis biomarkers for GC and have the potential to be used as markers for future treatment monitoring.     

Extracellular calmodulin and its association with epidermal growth factor in normal human body fluids

In this study we describe the occurrence of a calmodulin-like protein in normal human biological fluids. Extraction of the calmodulin-like protein from breast milk, saliva, serum and urine provided an extract with enhanced calmodulin immunoreactivity which, in the case of milk and saliva, showed a protein band comigrating with authentic calmodulin (Mr 17,000) on sodium dodecylsulphate-polyacrylamide gel electrophoresis. However, in milk, saliva and serum a major protein band of Mr 14,000-15,000 was always observed, which we speculate may be related to calmodulin, possibly as a partially degraded form. Estimates of biologically active calmodulin in most normal extracellular fluids were of the order which we have found will stimulate cell division when added to the extracellular medium of cells in culture. Levels ranged from 0.03 nmol/l in urine to 18.6 nmol/l in breast milk, and exhibited a quantitative relationship (r = 0.79, P less than 0.01) to epidermal growth factor (EGF) levels in fluids. Where EGF concentrations varied from normal (increased in saliva 24 h after oral surgery and reduced in the urine of patients with renal failure) calmodulin concentrations were similarly affected. The presence of calmodulin in serum may in part be attributable to its release from platelets which are particularly rich in calmodulin. Release of calmodulin from the platelet was associated with that of EGF and other platelet products.     

Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants.

Transgenic plants were constructed that express two foreign calmodulins (VU-1 and VU-3 calmodulins) derived from a cloned synthetic calmodulin gene. VU-1 calmodulin, similar to endogenous plant calmodulin, possesses a lysine residue at position 115 and undergoes posttranslational methylation. VU-3 calmodulin is a site-directed mutant of VU-1 calmodulin that is identical in sequence except for the substitution of an arginine at position 115 and thus is incapable of methylation. Both calmodulin genes, under the control of the cauliflower mosaic virus 35S promoter, were expressed in transgenic tobacco. Foreign calmodulin protein accumulated in plant tissues to levels equivalent to that of the endogenous calmodulin. All transformed lines of VU-1 plants were indistinguishable from untransformed controls with respect to growth and development. However, all transformed lines of VU-3 plants were characterized by decreased stem internode growth, reduced seed production, and reduced seed and pollen viability. The data suggest that these phenotypes are the result of the expression of the calmodulin mutant rather than the position of transferred DNA insertion or the overall alteration of calmodulin levels. Analyses of the activity of the purified transgenic calmodulins suggest that calmodulin-dependent NAD kinase is among the potential targets that may have altered regulation in VU-3 transgenic plants.