S. gallolyticus Aortic Valve Endocarditis with Mitral Valve Leaflet Aneurysm.

S. gallolyticus is one of the pathogenic agents of endocarditis, and mitral valve aneurysm is a rare but potentially devastating complication. We present a case of S. gallolyticus aortic valve endocarditis with concomitant anterior mitral valve leaflet aneurysm. Patient underwent surgery before aneurysm perforation, and postoperative course was uneventful. Time of surgery is crucial to avoid severe complications due to aneurysm rupture.

Imbibition of Femtoliter-Scale DNA-Rich Aqueous Droplets into Porous Nylon Substrates by Molecular Printing.

This work presents the first reported imbibition mechanism of femtoliter (fL)-scale droplets produced by microchannel cantilever spotting (µCS) of DNA molecular inks into porous substrates (hydrophilic nylon). Differently from macroscopic or picoliter droplets, the downscaling to the fL-size leads to an imbibition process controlled by the subtle interplay of evaporation, spreading, viscosity, and capillarity, with gravitational forces being quasi-negligible. In particular, the minimization of droplet evaporation, surface tension, and viscosity allows for a reproducible droplet imbibition process. The dwell time on the nylon surface permits further tuning of the droplet lateral size, in accord with liquid ink diffusion mechanisms. The functionality of the printed DNA molecules is demonstrated at different imbibed oligonucleotide concentrations by hybridization with a fluorolabeled complementary sequence, resulting in a homogeneous coverage of DNA within the imbibed droplet. This study represents a first step toward the µCS-enabled fabrication of DNA-based biosensors and microarrays into porous substrates.

The worrisome liaison between left ventricular systolic dysfunction and mitral annulus calcification in type 2 diabetes without coronary artery disease: data from the SHORTWAVE study.

BACKGROUND AND AIM: Mitral annulus calcification (MAC) is a marker for coronary artery disease (CAD) and predicts poor outcome in the general population. No data are available on MAC in patients with type 2 diabetes. In these patients we assessed prevalence of MAC and the relation between MAC and left ventricular (LV) systolic function. METHODS AND RESULTS: As many as 386 patients with type 2 diabetes without CAD were studied with Doppler echocardiography. LV systolic dysfunction was defined by analyzing 120 healthy subjects. Stress-corrected midwall shortening (sc-MS) and mitral annular peak systolic velocity (peak S') were considered as indexes of LV circumferential and longitudinal shortening and classified low if <89% and <8.5 cm/s, respectively (10th percentiles of controls). Patients who had MAC (107 = 28%) were older with longer duration of DM and were receiving more anti-hypertension medications than those who had not. At echocardiographic evaluation patients with MAC showed higher LV mass, larger left atrial volume (LAV), reduced sc-MS (88.4 ± 14.9 vs 92.6 ± 14.3%; p = 0.01) and peak S' (8.9 ± 2.2 vs 10.0 ± 2.0 cm/s; p < 0.001) than patients without MAC. Multiple logistic regression demonstrated older age (OR 1.03 [IC 1.01-1.06], p = 0.009), larger LAV (OR 1.19 [IC 1.11-1.28], p < 0.001) and combined reduction in sc-MS and peak S' (OR 3.00 [IC 1.57-5.72], p = 0.001) as independent factors associated with MAC. CONCLUSIONS: MAC is detectable in one fourth of patients with type 2 diabetes without CAD and is mostly related to LV systolic dysfunction expressed as combined impairment of LV circumferential and longitudinal fibers, independent of age and LAV.

Analysis of the thymidylate synthase gene structure in colorectal cancer patients and its possible relation with the 5-Fluorouracil drug response.

Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and it is the target for the 5-Fluorouracil (5-FU) activity. Barbour et al. showed that variant structural forms of TS in tumour cell lines confer resistance to fluoropyrimidines. We planned to perform the whole TS gene structure by means of sequencing techniques in human colorectal cancer (CRC) samples to try to identify the presence of any possible TS variant form that could be responsible of fluoropyrimidines drug resistance and of the worse prognosis. We performed the TS-DNA gene sequence in 68 CRC from patients of A, B, and C Dukes' stages and different histological grade, but we did not find any mutation in the TS-DNA structure. In the future we intend to widen the TS structure analysis to the metastatic CRCs, because due to their higher genomic instability, they could present a TS variant form responsible of the fluoropyrimidines drug resistance and the worse prognosis.

Evidence for a role of inducible nitric oxide synthase in gastric relaxation of mdx mice.

Alterations of gastric mechanical activity have been reported in mdx mouse, animal model for Duchenne muscular dystrophy. This study examined if alterations in the vasoactive intestinal polypeptide (VIP) system are present in mdx stomach. Gastric mechanical activity was recorded in vitro as changes of endoluminal pressure and neurally or pharmacologically evoked relaxations were analysed in mdxvs normal stomach. Reverse-transcription polymerase chain reaction was used to detect inducible nitric oxide synthase (iNOS) expression. Relaxations to sodium nitroprusside in mdx stomach showed no difference in comparison with normal preparations. In normal stomach, VIP produced relaxation, which was reduced by VIP6-28, antagonist of VIP receptors, but was not modified by Nomega-nitro-L-arginine methyl ester (L-NAME), 1-H-oxodiazol-[1,2,4]-[4,3-a]quinoxaline-1-one (ODQ) or by N-(3-(aminomethyl)-benzyl)acetamidine (1400W) and aminoguanidine, inhibitors of iNOS. In contrast, in mdx stomach VIP responses were antagonized not only by VIP6-28, but also by L-NAME, ODQ, 1400W or aminoguanidine. In normal stomach, the slow relaxation evoked by stimulation at high frequency was reduced by VIP6-28, but it was unaffected by 1400W or aminoguanidine. In mdx stomach, it was reduced by VIP6-28 or 1400W, which did not show additive effects. iNOS mRNA was expressed only in mdx stomach. The results suggest that in mdx gastric preparations, iNOS is functionally expressed, being involved in the slow relaxation induced by VIP.

The 'real' woman with heart failure. Impact of sex on current in-hospital management of heart failure by cardiologists and internists.

AIM: To identify differences between sexes in the clinical profile, use of resources, management and outcome in a large population of 'real world' patients with heart failure (HF). METHODS: A prospective cross-sectional survey was conducted on 2127 consecutive patients (47% women) admitted with HF to 167 cardiology and 250 internal medicine departments between February 14 and 25, 2000. RESULTS: Women were older, had a higher prevalence of atrial fibrillation, and more frequently a hypertensive or valvular aetiology. Females were admitted more frequently in Medical than in Cardiology Departments. The rate of invasive and non-invasive procedures was lower in women than in men, slightly higher if managed by cardiologists. Women were less frequently prescribed ACE-inhibitors, amiodarone, and spironolactone, and more frequently prescribed digoxin. In-hospital mortality was similar, without difference between health-care providers. A 6-month follow-up was performed in 56.4% of the cases in both setting, but less frequently in women. Event rates were similar with nearly half of patients re-hospitalised at least once. CONCLUSION: The 'real' HF woman has generally a more severe disease; she is an old lady who is more frequently hospitalised in a medical unit, receives few diagnostic, and cardiovascular procedures and pharmacological therapy, has a relatively low probability of dying in hospital, but a high likelihood of requiring readmission.

Tale of two churches: differential impact of a church-based diabetes control programme among Pacific Islands people in New Zealand.

AIMS: To compare the impact on weight and exercise of a 2-year church-based diabetes risk reduction programme in four churches in South Auckland, New Zealand. METHODS: A prospective non-randomized controlled study of a modular lifestyle and diabetes awareness intervention programme applying community development principles. The study involved four complete church congregations, two Samoan and two Tongan, with 516 participants at commencement. Risk of Type 2 diabetes is high among both ethnic groups. RESULTS: Overall, 285 subjects were available for their second assessment. In one intervention church, weight gain was controlled (vs. control 0 +/- 4.8 vs. +3.1 +/- 9.8 kg, respectively; P=0.05), diabetes knowledge (+46 +/- 26% vs. +4 +/- 17%; P<0.001) and regular exercise (at least 3 days per week: +22% vs. -8%; P=0.032) increased and readiness to change weight (P=0.007) shifted towards maintenance (e.g. maintenance +41% vs. +8%, respectively). The other intervention church increased diabetes knowledge (+19 +/- 24 vs. +8 +/- 25; P<0.024), but no other significant personal changes occurred. Attendance and perceived utility of the programme were greater in the first intervention church. CONCLUSIONS: A moderate intensity, community-based, structured diabetes awareness and lifestyle programme can reduce diabetes risk, but increasing diabetes knowledge alone is not necessarily associated with healthier lifestyle choices. Continuous and detailed monitoring of penetration of interventions may be essential to help guide the timing of interventions and identify the need for additional strategies to increase participation and motivation.

Thymidylate synthase gene promoter polymorphisms are associated with TSmRNA expressions but not with microsatellite instability in colorectal cancer.

BACKGROUND: Microsatellite instability (MSI) is a biological characteristic of most tumours, being involved in 85% of hereditary non-polyposis colorectal cancer (HNPCC). It also occurs in 10-15% of sporadic colorectal cancers (CRC). HNPCC appears to be caused by germline mutations in mismatch repair (MMR) genes, which are responsible for repairing single base-pair mismatches. MSI is also associated with a better response of CRC to adjuvant chemotherapy with fluoropyrimidines. We investigated any relationship between the MSI status and the TSmRNA expression, the polymorphisms of 5-Fluorouracil (5-FU cellular target, the enzyme thymidylate synthase (TS) and TS expression evaluated by means of immunohistochemistry. MATERIALS AND METHODS: A series of 80 colorectal cancers was evaluated for MSI and polymorphisms in the 3'UTR and the 5'UTR of the TS gene by a PCR assay. TSmRNA was quantified by real-time PCR and the TS expression by immunohistochemical assay. RESULTS: There was no significant association between the polymorphisms in the TS gene and the MSI or between the TSmRNA expression and the MSI status. CRC with a 3R/3R or 2R/3R genotype showed a significantly higher TSmRNA expression than those with the 2R/2R genotype (p = 0.001 and p = 0.028, respectively). Another significant association was found between the TSmRNA expression and the TS immunohistochemical determination (p = < 0.05). No association was found between the polymorphism of the 3'UTR and the TSmRNA expression. CONCLUSION: Our data show that there is no association between MSI status and the polymorphisms in the 3' and 5' UTRs and the TS expression. Tumour samples displaying the 3R/3R or 2R/3R genotype of TS have higher TSmRNA levels than the 2R/2R genotype. Polymorphic variant of the 3'UTR does not influence the TSmRNA level. We found a relationship between the TSmRNA expression, evaluated by real-time PCR, and with the TS level determined by immunohistochemical assay. Thus, genotyping of the 5'UTR and quantification of the TSmRNA expression in human CRC could be considered as predictors for response to SFU-based chemotherapy. The evaluation of the TS expression by means of immunohistochemistry assay remains a safe and reliable assay in CRC.

[Risk of sudden death in heart failure patients]. / Rischio aritmico nei pazienti con scompenso cardiaco cronico.

Sudden death is one of the more important cause of mortality in patients with chronic heart failure. The highest risk occurs among patients with less severe functional impairment, whereas patients in NYHA class IV usually die of progression of heart failure. Predictors of sudden death have been evaluated. Nevertheless, current methods of risk stratification for sudden death are still inadequate, especially in patients with advanced heart failure. Low left ventricular ejection fraction is widely used for the risk stratification, but it lacks of sensitivity and specificity in distinguishing patients with an increased arrhythmic mortality from those with an increased mortality due to pump failure. Unsustained ventricular tachycardia and inducibility at electrophysiological study may help identifying high-risk patients, requiring more aggressive therapy, as the ICD implantation. Heart rate variability and baroreflex sensitivity analysis have been utilized to obtain information on autonomic modulation, but with uncertain conclusion on the identification of high-risk patients. Increased QT dispersion, the presence of T-wave alternans and abnormal signal-averaged electrocardiography have also been proposed, but, up-to-now, any of these parameters showed a strong predictor power. In conclusion, our capability to identifying heart failure patients at risk for arrhythmic death is still far from being satisfactory.

Functional and psychological recovery during intensive hospital rehabilitation following cardiac surgery in the elderly.

UNLABELLED: The recovery process in the elderly after cardiac surgery is influenced not only by clinical cardiac conditions, but also by comorbidity, cognitive decline and disability. We evaluated the relationship between clinical objective and self-perceived factors and their influence on functional recovery in 204 consecutive, over-70s pts who were admitted into an intensive hospital rehabilitation program following cardiac surgery. The variables taken into consideration were: comorbidity (Charlson index), length of hospital stay and complications in cardiac surgery and rehabilitation, disability (nursing needs score index), functional status (6-min walking test), left ventricular EF, number of training sessions, self-perceived health status (EuroQol questionnaire), emotional impairment (anxiety/depression, CBA-2.0/interview). RESULTS: Functional capacity: the distance walked was 198 +/- 103 m at admission and 287 +/- 121 m at discharge (p < 0.0001). Only the nursing needs score index resulted as a weak, independent predictor of the distance walked at admission (r2 = 0.14, p < 0.001, beta = 0-.21), which (beta = 0.49), together with complications during rehabilitation (beta = -0.15), self-perceived health status at discharge (beta = 0.15) and number of training sessions (beta = 0.20), was independently correlated with the distance walked at time of discharge (r2 = 050, p < 0.0001). Patients mood: anxiety correlated with depression. Emotional scores did not correlate with functional measures. Patients self-perceived health status: only the nursing needs score index was a weak, independent predictor of well-being at entry (r2 = 0.15, p < 0.0001, beta = -0.29), which, in turn, was the only predictor of perception at discharge (r2 = 0.33, p < 0.0001, beta = 0-.42). CONCLUSIONS: In an intensive hospital rehabilitation program following cardiac surgery in patients over 70 a) there was no correlation between clinical and psychological variables; b) anxiety and depression were associated, but neither influenced the recovery process nor correlated to health status perception; c) functional impairment was strongly influenced by nursing needs which also affected the self-perceived health status; d) both functional and perception recovery were influenced by disability at time of admission and reacted positively after rehabilitation.

[Educating and communicating: non-pharmacologic treatment for patients with chronic heart failure?]. / Educare e comunicare: terapie non farmacologiche per il paziente con scompenso cardiaco cronico?

Chronic heart failure shows an increasing prevalence and extremely high rate of both hospital admission and readmission. Thus, in recent years there has been a growing interest in the development of more effective strategies for disease management. The existing literature shows programs involving multidisciplinary teams, specialized clinics, that employ a systematic approach and provide continuity of care, frequently involving a specialized nurse dedicated to comprehensive management. These experiences reported a favourable effects on clinically relevant outcome, including readmission, quality of care and cost of care. A recent randomized trial conducted by Krumholtz and colleagues emphasises the importance of patient's education and support intervention, without medical management components. This model was effective in reducing readmissions and in-hospital costs. On the basis of these data, patients education and support should be considered a "non-pharmacological therapy" for heart failure patients.

Anti-inflammatory effects of chemically modified tetracyclines by the inhibition of nitric oxide and interleukin-12 synthesis in J774 cell line.

We investigated the effects of chemically modified tetracyclines (CMTs) on the production of nitric oxide (NO) and on the synthesis of some cytokines: tumour necrosis factor alpha (TNF-alpha), interleukin(IL)-10 and IL-12 in lipopolysaccharide (LPS)-treated J774 cell line. Furthermore, we studied the ability of these drugs to modify the viability in LPS-stimulated J774 macrophages. CMTs decreased, in a dose-dependent manner, inducible NO synthase (iNOS) activity and, consequently, nitrite formation in J774 cultures. The CMT-induced decrease in NO production is due to the inhibition of enzyme activity rather than to a direct effect on enzyme expression. The absence of the inhibition in mRNA accumulation indicates that the inhibiting activity is mainly post-transcriptional. CMTs were unable to modulate TNF-alpha and IL-10 synthesis and they were not effective in modifying the transcription of relative mRNA in J774 macrophages. On the contrary, IL-12 mRNA expression was significantly increased by CMT-1 and CMT-8 with LPS activation. Since IL-12 protein secretion was inhibited by CMTs, these compounds interfere in the blocking of post-transcriptional events. The studies on cell viability showed that various CMTs induced a dose-dependent decrease in J774 macrophage viability. The cytotoxic activity was present even though NO production was inhibited by CMTs. These compounds appear to be able to activate apoptosis in aNO-independent way. Altogether, these results indicate that CMTs can exert anti-inflammatory effects by inhibiting NO synthesis, and they are able to modify cell viability by exerting a strong apoptotic activity.

Conserved structure and promoter sequence similarity in the mouse and human genes encoding the zinc finger factor BERF-1/BFCOL1/ZBP-89.

We have characterized the genomic structure of the mouse Zfp148 gene encoding Beta-Enolase Repressor Factor-1 (BERF-1), a Kruppel-like zinc finger protein involved in the transcriptional regulation of several genes, which is also termed ZBP-89, BFCOL1. The cloned Zfp148 gene spans 110 kb of genomic DNA encompassing the 5'-end region, 9 exons, 8 introns, and the 3'-untranslated region. The promoter region displays the typical features of a housekeeping gene: a high G+C content and the absence of canonical TATA and CAAT boxes consistent with the multiple transcription initiation sites determined by primary extension analysis. Computer-assisted search in the human genome database allowed us to determine that the same genomic structure with identical intron-exon organization is conserved in the human homologue ZNF 148. Functional analysis of the 5'-flanking sequence of the mouse gene indicated that the region from nucleotide -205 to +144, relative to the major transcription start site, contains cis-regulatory elements that promote basal expression. Such sequences and the overall promoter architecture are highly conserved in the human gene. Furthermore, we show that the complex transcription pattern of the Zfp148 gene might be due to a combination of alternative splicing and differential polyadenylation sites utilization.

Your cardiac patient wants to become a mother. Risk considerations and advice Part I--your cardiac patient asks advice on a possible pregnancy.

In this part, the risks and complications of pregnancy in women with active or corrected, congenital or acquired heart disease are reviewed, in order to allow individual counseling on a possible pregnancy or treatment recommendations on contraception.

ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1).

The Myc promoter-binding protein-1 (MBP-1) is a 37-38 kDa protein that binds to the c-myc P2 promoter and negatively regulates transcription of the protooncogene. MBP-1 cDNA shares 97% similarity with the cDNA encoding the glycolytic enzyme alpha-enolase and both genes have been mapped to the same region of human chromosome 1, suggesting the hypothesis that the two proteins might be encoded by the same gene. We show here data indicating that a 37 kDa protein is alternatively translated from the full-length alpha-enolase mRNA. This shorter form of alpha-enolase is able to bind the MBP-1 consensus sequence and to downregulate expression of a luciferase reporter gene under the control of the c-myc P2 promoter. Furthermore, using alpha-enolase/green fluorescent protein chimeras in transfection experiments we show that, while the 48 kDa alpha-enolase mainly has a cytoplasmic localization, the 37 kDa alpha-enolase is preferentially localized in the cell nuclei. The finding that a transcriptional repressor of the c-myc oncogene is an alternatively translated product of the ENO1 gene, which maps to a region of human chromosome 1 frequently deleted in human cancers, makes ENO1 a potential candidate for tumor suppressor.

C4, BF, C3 allele distribution and complement activity in healthy aged people and centenarians.

The aim of this study was to examine the complement system and the distribution of some human leukocyte antigen (HLA) class III alleles (C4, BF) in healthy aged people (77 centenarians and 89 elderly subjects). We have also studied the alleles of C3, a complement component genetically unrelated to HLA, the immunochemical levels of C4 and C3 and serum functional hemolytic activity for classical (CH50) and alternative (AP50) complement pathway. The levels of C3 and C4 and the CH50 and AP50 were found to be within the normal range. The frequencies of C3, BF, and C4A alleles were similar in the cohorts that have been studied. For C4B null allele (C4BQ0) a trend toward an increase in the older cohort was observed, although the differences were not significant after statistical correction. Our data suggest that the complement system is well preserved in centenarians and elderly subjects and class III HLA antigens are equally distributed in aged cohorts and in young healthy individuals.

Chimeric amplicons containing the c-myc gene in HL60 cells.

The major amplicon present in HL60 cells is chimeric in nature being composed of 70 kb of DNA sequence derived from the MYC locus linked to 80 kb of novel DNA sequence derived from a non contiguous region located telomeric to the c-myc gene at 8q24 (Feo et al., 1996). Here we show by fluorescence in situ hybridization (FISH) that these coamplified sequences, MCR (Myc Coamplified Region), are derived from a locus located 3-4 Mb telomeric to the c-myc gene in the q24.2-24.3 region of chromosome 8. Genomic cloning and Southern blot analysis indicate the arrangement of chimeric amplicons are in tandem arrays. Analysis of the DNA sequences at the juncture of the MYC locus and the MCR suggest that these non syntenic regions were joined by nonhomologous recombination events. Visualization of the organization of the amplified DNA by fiber-FISH analysis illustrates we have cloned the complete amplicon. This is the first complete mammalian amplicon to be cloned and have its structure visualized. In addition to the major class of tandemly repeated amplicons, a second class of amplicons was detected by fiber-FISH in which the extent of the MCR component is about twice the size of the MCR component in the major amplicon. These longer amplicons most likely contain inverted repeats of MCR and MYC region sequences. Whether the amplicons contain mixtures of these two types of structures or separate amplicons only contain one type of structure has not yet been resolved. Properties of the MCR sequences responsible for retention in the chimeric HL60 amplicons upon long term passage are discussed.

Tetracycline inhibits the nitric oxide synthase activity induced by endotoxin in cultured murine macrophages.

Here we investigate the effects of tetracycline base and of a semi-synthetic tetracycline derivative, doxycycline, on the induction of inducible nitric oxide synthase and, hence, on the production of nitric oxide (NO) by lipopolysaccharide in J774 macrophage cultured in vitro. The treatment of J774 line with tetracycline base (6.25-250 microM) or doxycycline (5-50 microM) dose-dependently decreased the lipopolysaccharide-stimulated (1 microg/ml) inducible NO synthase activity and, consequently, nitrite formation. For instance, the inhibition was 70% for tetracycline base at 250 microM and 68% for doxycycline at 50 microM. The inhibitory effect of tetracyclines was due neither to a reduction in the viability of the cells, studied as colorimetric 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, nor to an indiscriminate inhibition of total protein synthesis, but to a specific decrease in inducible NO synthase protein content in the cells, as attested by the significant reduction of the expression of inducible NO synthase, assayed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. However, no effect of tetracyclines on inducible NO synthase mRNA accumulation could be demonstrated in lipopolysaccharide-stimulated macrophage line, suggesting that the inhibitory effect of tetracyclines on NO synthesis involves post-transcriptional events. The reduction in lipopolysaccharide-stimulated nitrite accumulation produced by tetracyclines was significantly less when they were applied 6 h after lipopolysaccharide and absent 12 h after lipopolysaccharide, indicating that tetracyclines modify an early event in inducible NO synthase activation operating after mRNA transcription. The findings presented in this study indicate that the modulation of NO synthesis is another possible pathway by which tetracyclines may function as anti-inflammatory compounds.

A pilot urban church-based programme to reduce risk factors for diabetes among Western Samoans in New Zealand.

We have assessed the impact of a 2-year pilot church-base diabetes risk reduction programme on major lifestyle predictors of future Type 2 diabetes mellitus: exercise and weight control in a prospective non-randomized controlled study of a modular lifestyle and diabetes awareness intervention programme using a community development model. The study involved two complete church congregations from an ethnic group at high risk of diabetes (Western Samoans) (intervention church n = 78; control church n = 144). Weight remained stable (0+/-4.8 kg) in the intervention church but increased by 3.1+/-9.8 kg in the control church (p = 0.05). In the intervention church, there was an associated reduction in waist circumference (-4+/-10 cm vs +2+/-7 cm in control, p < 0.001), an increase in diabetes knowledge (46+/-26% vs 4+/-17% in control, p < 0.001) and an increase in the proportion exercising regularly (+22% vs -8% in control, p < 0.05). Consumption of key fatty foods was also reduced in the intervention church. We conclude that diabetes risk reduction programmes based upon lifestyle change, diabetes awareness, and empowerment of high risk communities can significantly reduce risk factors for future Type 2 diabetes.

Negative regulation of beta enolase gene transcription in embryonic muscle is dependent upon a zinc finger factor that binds to the G-rich box within the muscle-specific enhancer.

We have previously identified a muscle-specific enhancer within the first intron of the human beta enolase gene. Present in this enhancer are an A/T-rich box that binds MEF-2 protein(s) and a G-rich box (AGTGGGGGAGGGGGCTGCG) that interacts with ubiquitously expressed factors. Both elements are required for tissue-specific expression of the gene in skeletal muscle cells. Here, we report the identification and characterization of a Kruppel-like zinc finger protein, termed beta enolase repressor factor 1, that binds in a sequence-specific manner to the G-rich box and functions as a repressor of the beta enolase gene transcription in transient transfection assays. Using fusion polypeptides of beta enolase repressor factor 1 and the yeast GAL4 DNA-binding domain, we have identified an amino-terminal region responsible for the transcriptional repression activity, whereas a carboxyl-terminal region was shown to contain a potential transcriptional activation domain. The expression of this protein decreases in developing skeletal muscles, correlating with lack of binding activity in nuclear extract from adult skeletal tissue, in which novel binding activities have been detected. These results suggest that in addition to the identified factor, which functionally acts as a negative regulator and is enriched in embryonic muscle, the G-rich box binds other factors, presumably exerting a positive control on transcription. The interplay between factors that repress or activate transcription may constitute a developmentally regulated mechanism that modulates beta enolase gene expression in skeletal muscle.