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PubMed1978

Neonatal islet cell transplantation in the diabetic rat: effect on the renal complications.

PVG/C rats were made diabetic with streptozotocin and after 1 mth received a single intraperitoneal transplant of isogeneic collagenase digested pancreatic tissue. Renal changes have been studied in transplanted and control diabetic rats using light and electron microscopy and immunological techniques. Following transplantation, renal lesions did not increase in severity and progressive basement membrane thickening was prevented. Ultrastructurally many glomeruli showed a significant reduction in the mesangial matrix and the tubular and mesangial cell changes reverted to normal. Immunofluorescent studies demonstrated a similar reduction in the glomerular deposits of IgG. Possibilities for the treatment of diabetes mellitus in human patients are discussed.

Animals↗
PubMed2001

Biological Control of Pseudomonas syringae pv. glycinea by Epiphytic Bacteria under Field Conditions.

The efficacy of a bacterial strain as a biocontrol agent in the field may be related to the ecological similarity between the biocontrol agent and the target pathogen. Therefore, a number of different Pseudomonas syringae strains were evaluated for their antagonistic activities in vitro (agar-diffusion assay) and in planta (greenhouse assay) against the target pathogen, Pseudomonas syringae pv. glycinea. Six strains of five different pathovars were found to be antagonistic in vitro as well as in planta. The epiphytic fitness of the antagonistic Pseudomonas syringae strain 22d/93 and its two antibiotic-resistant mutants were examined on soybean plants in the fields. After adaptation the parental strain and its mutants had the ability to establish and maintain large epiphytic populations (about 106 cfu/g FW) over the whole growing season after a single spray inoculation. The epiphytic behaviors of the mutants and the parent were not significantly different. The introduced bacteria did not influence the total bacterial population size. When the antagonist was coinoculated with the pathogen, the development of the pathogen was significantly reduced during the whole growing season. When the antagonistic strain was inoculated 4 weeks in advance of the pathogen, this antagonistic effect could be markedly enhanced. The final population size of the pathogen reached just 104 cfu/g FW and was significantly reduced to 0.12% compared to the pathogen alone. This study demonstrates that biological control of foliar pathogens through colonization of the host plants with near isogenic or ecologically similar antagonistical strains seems to be a realistic goal.

Journal Article↗
PubMed2006

Varied growth, biomass and cellulose content in tobacco expressing yeast-derived invertases.

The effects of the expression of yeast-derived apoplastic (AI) and cytosolic (CI) invertases (EC 3.2.1.26) on biomass and structural carbohydrate accumulation in tobacco (Nicotiana tabacum L. cv. Xanthi) were evaluated. Transgenic tobacco plants expressing AI or CI under the control of either a tandem repeat of the Cauliflower Mosaic Virus 35S promoter (2X35S), or a promoter that drives xylem-localized expression (Petroselinum crispum 4-coumarate:CoA ligase promoter; 4CL) were generated. Yeast-derived invertase transcript levels, invertase protein, enzyme activity, growth parameters as well as both structural and soluble carbohydrates of stem tissue of all transformed lines were quantified. Transgenic tobacco lines expressing invertase under the control of 4CL displayed severe growth retardation with both yeast-derived isogenes. Similarly, several transformed lines expressing either AI or CI regulated by the 2X35S promoter were also shorter than wild-type (WT) plants. Despite the decreases in height, some transformed lines had significant increases in biomass. One line (2X35S::AI-1) had a biomass/height increase of 88% and an increase in stem diameter of over 40%, while a second line (2X35S::CI-5) had a biomass/height increase of 21%. A separate line (2X35S::AI-2) had a 36% increase in cellulose content, while two others (4CL::AI-2 and 4CL::AI-3) displayed significant decreases in cellulose content. The observed phenotypes can be in part explained by the levels of foreign invertase present, subcellular localization and the carbohydrate status of the tissues.

Biomass↗
PubMed2000

A low CD34+ cell dose results in higher mortality and poorer survival after blood or marrow stem cell transplantation from HLA-identical siblings: should 2 x 10(6) CD34+ cells/kg be considered the minimum threshold?

We studied the effect of the CD34+ cell dose on transplant-related mortality (TRM) and survival in 39 patients randomized to receive lenograstim-mobilized PBSCT (n = 20) or BMT (n = 19) from HLA-identical siblings. Both marrow and blood were harvested, and one infused in a double-blind fashion. The median nucleated (7.0 vs 3.2 x 10(8)/kg; P < 0.0001), CD34+ (3.7 vs 1.5 x 10(6)/kg; P = 0.002), CFU-GM (42 vs 19 x 10(4)/kg; P = 0.002), and CD3+ (1.9 vs 0.3 x 10(8)/kg; P < 0.0001) cell doses with PBSCT were higher. Thirteen patients (6 BMT and 7 PBSCT) experienced TRM at 15-733 days (median 57); 10 of 20 receiving <2 x 10(6) CD34+ cells/kg compared with three of 19 receiving > or =2. Eight of 20 patients receiving <2 x 10(6) CD34+ cells/kg are alive compared with 14 of 19 receiving > or =2. In Cox analysis, CD34+ cell dose > or =2 x 10(6)/kg was associated with lower TRM (RR 0.2, P = 0.01), and higher overall (RR 3.7, P = 0.01) and event-free (RR 3.2, P = 0.02) survival. Other cell populations and the source of stem cells did not affect TRM or survival. We conclude that 2 x 10(6) CD34+ cells/kg may be the ideal minimum cell dose for allogeneic transplantation although lower doses do not preclude successful therapy. Since the likelihood of obtaining this threshold CD34+ cell number is significantly greater from blood than marrow, PBSCT may be preferable to marrow for allografts from HLA-identical siblings.

Adolescent↗
PubMed1998

Acute and chronic effects of nicotine on anastomotic patency following ischemia/reperfusion.

The effect of nicotine on anastomotic patency was studied using an isogenic rat-knee transplantation model. Animals were divided into experimental and control groups, with experimental animals exposed to either 40 days of twice-daily nicotine injections (Group 1-chronic exposure) or acute nicotine exposure by graft perfusion (Group 2-acute exposure). Four and 6 hr warm ischemia times were studied. Syngeneic rat knees were transplanted from donor to recipient by microvascular anastomosis of the femoral vessels. There was a statistically significant difference (p < 0.05) in anastomotic patency after 4 hr of ischemia in Group 1 animals exposed to nicotine (33 percent patency) vs. controls (89 percent patency). In addition, there was a statistically significant difference in anastomotic patency after 4 hr of ischemia in Group 2 animals exposed to nicotine (11 percent patency) vs. controls (67 percent patency). No significant differences between controls and Group 1 animals were noted in anastomotic patency after 6 hr of ischemia. Both acute and chronic nicotine exposure reduces anastomotic patency after short intervals of warm ischemia in vascularized composite tissue grafts. This detrimental effect is lost when the ischemic interval is prolonged to periods with poor anastomotic patency rates related to the ischemia/reperfusion itself.

Anastomosis, Surgical↗
PubMed1995

The role of DNA repair genes in recombination between repeated sequences in yeast.

The presence of repeated sequences in the genome represents a potential source of karyotypic instability. Genetic control of recombination is thus important to preserve the integrity of the genome. To investigate the genetic control of recombination between repeated sequences, we have created a series of isogenic strains in which we could assess the role of genes involved in DNA repair in two types of recombination: direct repeat recombination and ectopic gene conversion. Naturally occurring (Ty elements) and artificially constructed repeats could be compared in the same cell population. We have found that direct repeat recombination and gene conversion have different genetic requirements. The role of the RAD51, RAD52, RAD54, RAD55, and RAD57 genes, which are involved in recombinational repair, was investigated. Based on the phenotypes of single and double mutants, these genes can be divided into three functional subgroups: one composed of RAD52, a second one composed of RAD51 and RAD54, and a third one that includes the RAD55 and RAD57 genes. Among seven genes involved in excision repair tested, only RAD1 and RAD10 played a role in the types of recombination studied. We did not detect a differential effect of any rad mutation on Ty elements as compared to artificially constructed repeats.

Adenosine Triphosphatases↗
PubMed2005

Polyglutamine represses cAMP-responsive-element-mediated transcription without aggregate formation.

Transcriptional dysregulation, particularly cAMP-responsive-element-mediated transcriptional repression, has been implicated in expanded polyglutamine diseases. However, it has not been clarified whether this transcriptional repression is a cause or result of neurodegeneration. Furthermore, the association between aggregates of expanded polyglutamine stretches and transcriptional repression is not clear. We established isogenic cell lines with polyglutamine stretches, which also expressed d2EGFP under the control of cAMP-responsive elements. In this system, the polyglutamine stretch repressed cAMP-responsive-element-mediated transcription without the formation of macroscopic expanded polyglutamine aggregates. Furthermore, aggregate formation did not have an adverse effect on the repression of transcriptional activity. The results demonstrated that the repression of cAMP-responsive-element-mediated transcription is an early event caused by a soluble form of polyglutamine stretch.

Cell Line↗
PubMedDate not supplied

Influence of microbiota in experimental cutaneous leishmaniasis in Swiss mice.

Infection of Swiss/NIH mice with Leishmania major was compared with infection in isogenic resistant C57BL/6 and susceptible BALB/c mice. Swiss/NIH mice showed self-controlled lesions in the injected foot pad. The production of high levels of interferon-gamma (IFN-gamma) and low levels of interleukin-4 (IL-4) by cells from these animals suggests that they mount a Th1-type immune response. The importance of the indigenous microbiota on the development of murine leishmaniasis was investigated by infecting germfree Swiss/NIH in the hind footpad with L. major and conventionalizing after 3 weeks of infection. Lesions from conventionalized Swiss/NIH mice were significantly larger than conventional mice. Histopathological analysis of lesions from conventionalized animals showed abscesses of variable shapes and sizes and high numbers of parasitized macrophages. In the lesions from conventional mice, besides the absence of abscess formation, parasites were rarely observed. On the other hand, cells from conventional and conventionalized mice produced similar Th1-type response characterized by high levels of IFN-gamma and low levels of IL-4. In this study, we demonstrated that Swiss/NIH mice are resistant to L. major infection and that the absence of the normal microbiota at the beginning of infection significantly influenced the lesion size and the inflammatory response at the site of infection.

Animals↗
PubMed2002

The role of thymidylate synthase induction in modulating p53-regulated gene expression in response to 5-fluorouracil and antifolates.

Thymidylate synthase (TS) is a critical target for chemotherapeutic agents such as 5-fluorouracil (5-FU) and antifolates such as tomudex (TDX),multitargeted antifolate, and ZD9331. Using the MCF-7 breast cancer line, we have developed p53 wild-type (M7TS90) and null (M7TS90-E6) isogenic lines with inducible TS expression (approximately 6-fold induction compared with control after 48 h). In the M7TS90 line, inducible TS expression resulted in a moderate approximately 3-fold increase in 5-FU IC-50(72 h) dose and a dramatic >20-fold increase in the IC-50(72 h) doses of TDX, multitargeted antifolate, and ZD9331. S-phase cell cycle arrest and apoptosis induced by the antifolates were abrogated by TS induction. In contrast, cell cycle arrest and apoptosis induced by 5-FU was unaffected by TS expression levels. Inactivation of p53 significantly increased resistance to 5-FU and the antifolates with IC-50(72 h) doses for 5-FU and TDX of >100 and >10 microM, respectively, in the M7TS90-E6 cell line. Furthermore, p53 inactivation completely abrogated the cell cycle arrest and apoptosis induced by 5-FU. The antifolates induced S-phase arrest in the p53 null cell line; however, the induction of apoptosis by these agents was significantly reduced compared with p53 wild-type cells. Both inducible TS expression and the addition of exogenous thymidine (10 microM) blocked p53 and p21 induction by the antifolates but not by 5-FU in the M7TS90 cell line. Similarly, inducible TS expression and exogenous thymidine abrogated antifolate but not 5-FU-mediated up-regulation of Fas/CD95 in M7TS90 cells. Our results indicate that in M7TS90 cells, inducible TS expression modulates p53 and p53 target gene expression in response to TS-targeted antifolate therapies but not to 5-FU.

Breast Neoplasms↗
PubMed2006

Influence of parathyroid mass on the regulation of PTH secretion.

In advanced uremia, parathyroid hormone (PTH) levels should be controlled at a moderately elevated level in order to promote normal bone turnover. As such, a certain degree of parathyroid gland (PG) hyperplasia has to be accepted. No convincing evidence of apoptosis or of involution of PG hyperplasia exists. However, even considerable parathyroid hyperplasia can be controlled when the functional demand for increased PTH levels is abolished. When 20 isogenic PG were implanted into one parathyroidectomized (PTX) rat normalization of Ca(2+) and PTH levels and normal suppressibility of PTH secretion by high Ca(2+) was obtained. Similarly, normal levels of Ca(2+) and PTH and suppressibility of PTH secretion were obtained when Eight isogenic PG from uremic rats were implanted into normal rats or when long-term uremia and severe secondary hyperparathyroidism (sec. HPT) was reversed by an isogenic kidney transplantation. Normalization of PTH levels after experimental kidney transplantation took place despite a persistent decrease of vitamin D receptor (VDR) mRNA and calcium sensing receptor (CaR) mRNA in PG. Thus, in experimental models PTH levels are determined by the functional demand and not by parathyroid mass, per se. When non-suppressible sec. HPT is present in patients referred to PTX, nodular hyperplasia with differences in gene expression between different nodules has been observed in most cases. An altered expression of some autocrine/paracrine factors has been demonstrated in the nodules. Enhanced expression of PTH-related peptide (PTHrP) has been demonstrated in PG from patients with severe secondary HPT. PTHrP has been shown to stimulate PTH secretion in vivo and in vitro. PTH/PTHrP receptor was demonstrated in the parathyroids. The low Ca(2+) stimulated PTH secretion was enhanced by 300% by PTHrP 1-40. The altered quality of the parathyroid mass and not only the increased parathyroid mass, per se, might be responsible for non-controllable hyperparathyroidism in uremia and after kidney transplantation.

Animals↗
PubMed2003

Global effects of virulence gene regulators in a Bacillus anthracis strain with both virulence plasmids.

Control of anthrax toxin and capsule synthesis, the two major virulence factors of Bacillus anthracis, has been associated with two regulatory genes, atxA and acpA, located on virulence plasmids pXO1 and pXO2, respectively. We used transcriptional profiling to determine whether atxA and/or acpA control genes other than those already described and to investigate functional similarities of the regulators. Transcription was assessed in a pXO1(+) pXO2(+) parent strain and in isogenic mutants in which one or both regulatory genes were deleted. We determined that in addition to the toxin and capsule genes, atxA controls expression of numerous other genes on both plasmids and the chromosome. Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed. Certain atxA-regulated genes were affected synergistically in an atxA acpA mutant. Yet overall, acpA appears to be a minor regulator with fewer targets than atxA. In contrast to previous reports of acpA function in attenuated strains, acpA had a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain. Surprisingly, acpA expression was positively affected by atxA, although atxA-activated capsule gene transcription is not acpA dependent. The newly discovered atxA-regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling. Regulation of chromosomal genes by atxA is particularly intriguing, given that many of the target genes have homologues in other Bacillus species that lack atxA homologues. Given the global effect of atxA on gene expression in B. anthracis, previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identified atxA-regulated genes should be investigated.

Bacillus anthracis↗
PubMedDate not supplied

Effect of cyclosporine on the uptake of monoclonal antibody to cardiac myosin.

Monoclonal antibody to cardiac myosin labeled with indium-111 diethylenetriamine pentaacetic acid holds promise as a noninvasive marker of cardiac graft rejection. Uptake of antibody has correlated with histologic evidence of rejection in nonimmunosuppressed animals. Whether this correlation will apply with immunosuppression has important clinical implications. Fifty-two heterotopic heart transplantations were performed between isogeneic and nonisogeneic strains of rats. Cyclosporine-treated (15 mg/kg day subcutaneously for 9 days) and untreated control animals were killed on day 9, 48 hours after injection of radiolabeled antibody. Donor and recipient hearts were submitted for scintillation scanning and histologic analysis. In untreated animals, antibody uptake was significantly greater in nonisogeneic than in isogeneic donor hearts, correlating with a significantly higher rejection score and increased myocyte necrosis in the former. Between isogeneic groups, cyclosporine-treated donor hearts had significantly higher antibody uptake and donor/native antibody uptake ratios than did untreated isogeneic hearts. There was, however, no significant difference in the histologic degree of rejection or myocyte necrosis between isogeneic groups. Between cyclosporine-treated and untreated nonisogeneic animals, donor heart antibody uptake and donor-native heart antibody uptake ratios were not significantly different. Nonetheless, the histologic grade of rejection and presence of myocyte necrosis was significantly greater in untreated than in treated nonisogeneic hearts. There were no abnormalities in the native hearts. In this model, cyclosporine treatment correlates with an increased uptake of antimyosin antibody in both isogeneic and nonisogeneic donor hearts, out of proportion to histologic evidence of rejection or myocyte necrosis. This effect may lead to false-positive results in clinical tests utilizing antimyosin antibody uptake as a marker of rejection in the presence of cyclosporine therapy.

Animals↗
PubMed1978

[Research on the artificial regulation of sex in animals in the USSR].

The problem of artificial sex control was cardinally solved only for the silkworm among the domestic animals. The artificial sex control is realized by means of several methods. The artificial ameiotic parthenogenesis allows to obtain isogenic parthenogenetic progeny of only female sex. The meiotic parthenogenesis, on the contrary, gives only male progeny characterized by homozygosis by all genes. As a result of androgenesis with the participation of ooplasm and male nuclear material, only male progeny develops as well. Sex marked strains in which the female and male eggs are coloured in different may were obtained by means of translocation of the dominant genes controlling the egg colour on W chromosome determining the female sex. This allows readily to pick out the eggs of desirable sex only with the help of photoelectric equipment. The males of a specially raised strain balanced by two non-allelic sex-linked lethals, when being crossed with the females of any other strain, give male progeny only. The developed methods of sex control have found wide application in theoretical investigations of various directions and practical sericulture.

Animals↗
PubMed2006

The AlgT-dependent transcriptional regulator AmrZ (AlgZ) inhibits flagellum biosynthesis in mucoid, nonmotile Pseudomonas aeruginosa cystic fibrosis isolates.

Pseudomonas aeruginosa is a microorganism associated with the disease cystic fibrosis. While environmental P. aeruginosa strains are generally nonmucoid and motile, isolates recovered from the cystic fibrosis lung frequently display a mucoid, nonmotile phenotype. This phenotypic conversion is mediated by the alternative sigma factor AlgT. Previous work has shown that repression of fleQ by AlgT accounts for the loss of flagellum biosynthesis in these strains. Here, we elucidate the mechanism involved in the AlgT-mediated control of fleQ. Electrophoretic mobility shift assays using purified AlgT and extracts derived from isogenic AlgT(+) and AlgT(-) strains revealed that AlgT inhibits fleQ indirectly. We observed that the AlgT-dependent transcriptional regulator AmrZ interacts directly with the fleQ promoter. To determine whether AmrZ functions as a repressor of fleQ, we mutated amrZ in the mucoid, nonmotile P. aeruginosa strain FRD1. Unlike the parental strain, the amrZ mutant was nonmucoid and motile. Complementation of the mutant with amrZ restored the mucoid, nonmotile phenotype. Thus, our data show that AlgT inhibits flagellum biosynthesis in mucoid, nonmotile P. aeruginosa cystic fibrosis isolates by promoting expression of AmrZ, which subsequently represses fleQ. Since fleQ directly or indirectly controls the expression of almost all flagellar genes, its repression ultimately leads to the loss of flagellum biosynthesis.

Bacterial Proteins↗
PubMed1993

Genetically engineered microorganisms to rescue plants from frost injury.

Ice nucleation active bacteria belonging to genera Pseudomonas, Xanthomonas and Erwinia contribute to frost damage to plants by initiating the formation of ice in plants that would otherwise supercool and avoid the damaging ice formation. The biological control of frost injury can be achieved by the application of non-ice nucleation active bacteria to the plant surfaces before they become colonized by Ice+ species. ice genes have been cloned from Pseudomonas and isogenic Ice- derivatives constructed via genetic manipulations. These genetically engineered microorganisms (GEMs) have been released into the environment to control the frost damage. The incidence of frost injury to the plants has, thereby, been reduced by 50-85% during natural frosts. These GEMs do not survive in soil and show no aerial dispersal in the environment.

Amino Acid Sequence↗
PubMed2000

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases.

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.

Animals↗
PubMed2000

Characterization of the sigma(B) regulon in Staphylococcus aureus.

The sigma(B)-dependent stress regulon in gram-positive bacteria might fulfill a physiological role in stress response and virulence similar to that of the sigma(S) regulon in Escherichia coli and other gram-negative bacteria. In order to obtain evidence for the function of the sigma(B) regulon of Staphylococcus aureus, especially in virulence control, sigma(B)-dependent stress genes were identified. The two-dimensional protein pattern of wild-type cells of S. aureus COL was compared with that of an isogenic sigB mutant. By this approach, we found that the synthesis of about 27 cytoplasmic proteins seemed to be under the positive control of sigma(B). N-terminal sequencing of 18 proteins allowed the identification of their genes on the almost finished genome sequence of S. aureus COL and the analysis of the promoter structure. Transcriptional analyses of 11 of these genes confirmed their sigma(B) dependency, and moreover, about 7 additional sigma(B)-dependent genes were found which are cotranscribed with the newly detected genes, forming operons. Altogether, we identified 23 sigma(B)-dependent genes and their corresponding proteins. Among them are proteins probably involved in the generation of NADH or in membrane transport mechanisms. Furthermore, at least one clpC-homologous gene was localized on the S. aureus sequence solely transcribed by sigma(B). In contrast, a second clpC-homologous gene in S. aureus forming an operon with ctsR, yacH, and yacI was sigma(B) independently expressed.

Bacterial Proteins↗
PubMed1998

Cyclic nucleotide phosphodiesterase 4 subtypes are differentially expressed by primary keratinocytes and human epidermoid cell lines.

Cyclic AMP phosphodiesterase (PDE) type 4 has been the subject of considerable interest as a potential molecular target for treating cutaneous inflammatory and allergic disorders; however, little is known regarding the expression of PDE 4 isogenes in human keratinocytes, the predominant cell type present in the epidermis. In this study, we investigated the expression of PDE 4 subtypes in epidermal cells (primary keratinocytes derived from breast skin, epidermoid cell lines A431, KB, and HaCaT) using reverse transcriptase polymerase chain reaction. Analysis of the amplified cDNA showed that there were differences in the expression of PDE 4 isogenes in the epidermal cells. Constitutive expression of the PDE 4 isozymes was detected in untreated epidermal cells at different levels. Transcripts for PDE 4B and 4D were abundantly expressed in breast skin-derived keratinocytes, whereas transcripts for PDE 4A, 4C, and 4D were present in HaCaT cells and transcripts for all the PDE 4 isotypes were present in A431 and KB. PDE 4A and 4C were detectable in HaCaT cells in equal amounts, but PDE 4C was only marginally expressed in the other cells. Of the four isogenes, PDE 4D was abundantly expressed in all the cells. Elevation of intracellular levels of cAMP by forskolin increased the expression of all the hPDE 4 isogenes 2-3-fold as revealed by semiquantitative reverse transcriptase polymerase chain reaction. Western blot analysis of extracts from control and forskolin or dibutyryl cAMP-treated A431 and KB cells demonstrated the presence of proteins with a molecular mass identical to that corresponding to recombinant human PDE 4B. Together, these findings show that PDE 4 isogenes are expressed in keratinocytes to a different degree and that their expression can be modulated by intracellular levels of cAMP.

Blotting, Western↗