An AP-2 element acts synergistically with the cyclic AMP- and phorbol ester-inducible enhancer of the human proenkephalin gene.

An enhancer with two DNA elements, one containing the sequence CGTCA, is required for cyclic AMP- and phorbol ester-inducible transcription of the human proenkephalin gene. We report that an AP-2 element located adjacent to the enhancer acts synergistically with it to confer maximal response to cyclic AMP and phorbol esters.

A common trans-acting factor is involved in transcriptional regulation of neurotransmitter genes by cyclic AMP.

Activation of neurotransmitter receptors can regulate transcription in postsynaptic cells through the actions of second messengers. Trans-synaptic regulation of transcription appears to be an important mechanism controlling the synthesis of molecules involved in neuronal signaling, especially neuropeptides. Proenkephalin, vasoactive intestinal polypeptide, and somatostatin have been shown to be transcriptionally regulated by the second messenger, cyclic AMP (cAMP), as has the catecholamine synthesizing enzyme tryosine hydroxylase. cAMP-inducible elements have been mapped within these genes, and trans-acting factors which bind to several such elements have been identified. With the discovery that individual neurons generally contain multiple transmitters within their synaptic terminals, it has become important to understand in detail the mechanisms by which the synthesis of transmitters can be coregulated. Here we compare the structure and function of the proenkephalin cAMP-inducible enhancer with the mapped cAMP-inducible elements of the vasoactive intestinal polypeptide, somatostatin, and tyrosine hydroxylase genes and a putative cAMP-inducible element in the proto-oncogene c-fos. We have previously shown that the proenkephalin enhancer is composed of two different elements, ENKCRE-1 and ENKCRE-2. We show here that one of these, ENKCRE-2, is structurally similar to elements found within the vasoactive intestinal polypeptide, somatostatin, and tyrosine hydroxylase genes and binds a trans-acting factor that is competed for both in cotransfection experiments (in vivo) and in DNase I footprint assays (in vitro) by these other elements. The c-fos element has similar structural requirements to confer transcriptional induction by cAMP but competes less strongly. Protein purified by affinity chromatography with the ENKCRE-2 sequence binds to each of these elements. A second element within the proenkephalin cAMP-inducible enhancer, ENKCRE-1, binds a factor that is not competed for by these other genes and is therefore distinct. This analysis suggests a potential mechanism of transcriptional coregulation of the neuronally expressed genes investigated in this study and also demonstrates that multiple factors are involved in transcriptional activation by cAMP.

Can computed tomography of the chest stage lung cancer? Yes and no.

To determine the accuracy of computed tomography (CT) of the chest in the staging of lung cancer, we studied 418 patients with primary pulmonary carcinoma between 1979 and 1986. Each had a preoperative scan performed before detailed operative staging. Each CT scan was analyzed for components of the current TNM staging system. Computed tomography sensitivity and specificity for mediastinal lymph node metastasis were 84.4% and 84.1%, with corresponding positive and negative predictive accuracies of 68.7% and 92.9%, respectively. When TNM stages were derived from CT scans, only 190 of 418 (45.4%) completely agreed with operative staging. An additional 53 of 418 (12.7%) predicted the correct stage, although components of the TNM system were incorrect. In 94 of 418 scans (22.5%) CT overestimated the stage, whereas in 81 (19.4%) CT downgraded the stage. Computed tomography suggested metastatic lesions in liver, lung, adrenal gland, bone, or abdominal lymph nodes in 40 of 373 scans (10.7%); only five of 40 (12.5%) had documented metastasis. In summary, CT of the chest cannot accurately stage primary lung carcinoma according to the TNM classification. Because the negative predictive accuracy for mediastinal lymph node metastasis remains high (92.9%), invasive staging can be deferred for definitive thoracotomy when no lymphadenopathy is evident on CT. The high negative predictive accuracy for scans of the chest and upper abdomen makes CT a useful tool for exclusion of metastatic disease.

Computed tomographic evaluation of intrathoracic thyroid malignancy.

As only a few cases of intrathoracic thyroid malignancy with computed tomographic (CT) examination have been described, we reviewed the CT examinations of three patients with primary and five patients with recurrent thyroid malignancy involving the thorax. Irregular border of the thyroid mass, extension of tumor mass into mediastinal fat or chest wall, or lymphadenopathy suggested the malignant nature of the primary tumor. CT examination in recurrent disease demonstrated mediastinal, hilar and retrocrural adenopathy, compression of major vessels with collateral flow, pulmonary and bony metastases. CT was of value both in identifying the extent of disease and documenting response to treatment.

Screens using RNAi and cDNA expression as surrogates for genetics in mammalian tissue culture cells.

We have developed methods for the automation of transfection-grade DNA preparation, high-throughput retroviral preparation, and highly parallel phenotypic screens to establish approaches that will allow investigators to examine in an unbiased manner the roles of proteins in mammalian cells. These methods have been used to raise or lower the levels of individual kinases in individual micro-well cultures either by cDNA or short hairpin RNA expression and will allow investigators to treat mammalian cells in culture in manners that are analogous to genetic screens in yeast. Our proof-of-principle experiments have been performed in human cells using repositories that represent over 75% of the protein, nucleotide, carbohydrate, lipid, and amino acid kinases in the human genome. These initial experiments have demonstrated the feasibility of two general types of screens. We have performed phenotypic screens to identify proteins with specific roles in a chosen function and genetic interaction screens to establish epistatic relations between different proteins. The results suggest that any phenotype that can be scored by a robust assay in tissue culture is amenable to these types of screens and that interactions between mammalian proteins can be established. These results point to the near-term goal of establishing comprehensive, unbiased screens that will allow queries on the roles of all human proteins.

Ultrasonic and computed tomographic features of mucocele of the appendix.

The ultrasonic features in three cases and computed tomographic (CT) findings in one case of mucocele of the appendix are presented. This entity appeared cystic on sonography and may have high-intensity echoes within it. The wall was not thickened and did not contain calcification, unlike previous descriptions. On CT, the mass was of soft tissue density, while previously, it had been described as cystic. Although mucocele of the appendix has a more variable appearance on sonography and CT than previously reported, a correct preoperative diagnosis can be made in most cases. Barium enema examination or calcification in the wall or in the mass itself may be necessary to distinguish this entity from lymphoma if the lesion is of soft tissue density on CT.

Deleterious effects of hepatitis delta virus replication on host cell proliferation.

Hepatitis delta virus (HDV) infection and spread in vivo are dependent upon coinfection by hepatitis B virus (HBV), and dual HDV/HBV infection is frequently more severe than HBV infection alone, raising the possibility that HDV infection may be deleterious to cells. Here we have examined the effects of HDV replication on the long-term growth of cultured cells. Our results show that most cells transfected with HDV cDNA do not give rise to stable cell lines expressing viral antigens or replicative intermediates; in addition, cotransfection of HDV replicons with a plasmid vector expressing a hygromycin resistance marker results in a dose-dependent impairment of hygromycin-resistant colony formation. When cells transfected with replication-competent HDV cDNA are followed prospectively, a progressive decline in viral RNA replication and a steady decrease in the proportion of cells expressing delta antigen are observed. However, in transient transfection assays, no evidence was found to link HDV replication to apoptosis or to cell cycle arrest, nor did HDV replication confer on host cells enhanced sensitivity to inducers of apoptosis. Thus, HDV replication does not appear to be acutely cytotoxic. However, in dividing cells HDV replication is associated with a subtler growth disadvantage, leading to selection in culture for cells displaying diminished HDV expression. This effect would not be expected to cause hepatitis in vivo but might contribute to impaired liver regeneration in the setting of ongoing hepatocellular injury.

Computed tomographic features of esophageal intramural pseudodiverticulosis.

A patient with esophageal intramural pseudodiverticulosis (EIPD) was examined with computed tomography (CT). CT demonstrated marked thickening of the esophageal wall, diffuse irregularity of the esophageal lumen, and intramural gas collections--features typical of this entity. In the proper clinical setting, CT can confirm the diagnosis of EIPD, especially when other studies are equivocal. However, this case also demonstrates some of the limitations of CT in differentiating benign and malignant esophageal disorders.

Computed-tomographic and conventional linear-tomographic evaluation of tracheobronchial lesions for laser photoresection.

Laser therapy is a new modality for treatment of airway lesions. We examined 18 patients prior to laser photoresection of tracheobronchial lesions. Thirteen had cancers involving the distal trachea, carina, and/or proximal bronchi; five had benign lesions of the middle or proximal trachea. Each patient was examined by conventional linear tomography (CLT) and computed tomography (CT). CT was valuable in patients who had lesions of the distal trachea, carina, and/or proximal bronchi. Its particular usefulness, and its advantage relative to CLT, consisted in its ability to delineate vascular structures adjacent to the planned area of photoresection. Neither CLT nor CT was helpful in evaluation of benign lesions of the proximal trachea.

Hemophilus influenzae pneumonia in the adult. Radiographic appearance with clinical correlation.

Hemophilus influenzae septicemia is an important cause of life-threatening pneumonia in an immunocompromised patient. Eleven cases proved by blood culture were analyzed. Multilobar involvement with lobar or segmental consolidation and pleural effusion were common radiographic findings, but there were no signs of lobar expansion, bulging fissures, or cavitation. In general, predisposing factors such as alcoholism and chemotherapy place patients at risk. Radiographic response to therapy is variable but often lags behind clinical improvement.

Computed tomographic findings in penetrating peptic ulcer.

Four cases of peptic ulcer penetrating the head of the pancreas were diagnosed by computed tomography (CT). Findings common to 3 cases included (a) an ulcer crater, (b) a sinus tract, and (c) enlargement of the head of the pancreas. Additional findings, not seen in all patients, included (d) edema involving the base of the ulcer and/or the adjacent bowel wall and (e) loss of fascial planes between the base of the ulcer and the head of the pancreas. Unlike other modalities, the inherent spatial resolution of CT allows a confident diagnosis of this important complication of peptic ulcer disease.

T1N0M0 bronchogenic carcinoma: assessment by CT.

We reviewed medical records and conventional chest radiographs that showed a solitary T1N0M0 nodule in 23 patients who had non-oat-cell bronchogenic carcinoma. No patient had evidence of metastases, either on the chest radiograph or clinically. All patients underwent computed tomography (CT) examination of the thorax, including the adrenal glands. Only one patient (4%) had mediastinal lymph nodes greater than 1 cm in diameter accessible to mediastinotomy; anterior mediastinotomy confirmed metastatic spread in this patient, which precluded curative resection. Three patients each had a mildly enlarged (2 cm or less) adrenal gland; however, follow-up study suggested that metastasis was not the cause of adrenal enlargement in these patients. This study reinforces concern over whether CT is warranted in the preoperative assessment of T1N0M0 bronchogenic carcinoma.

GAL11P: a yeast mutation that potentiates the effect of weak GAL4-derived activators.

A mutant yeast in which a weak GAL4-derived activator functions as a strong activator bears a single mis-sense mutation in GAL11 (a.k.a. SPT13). The first 74 amino acids of GAL4, including the zinc-dependent DNA binding region, attached to an acidic activating sequence, are sufficient to respond both to GAL11 and to our mutant GAL11P (potentiator). PPR1, a yeast activator with a similar zinc finger sequence, also responds to GAL11 and to GAL11P, whereas regulators bearing unrelated DNA binding motifs do not. GAL11 itself works as a strong activator when tethered to DNA by fusion to the bacterial LexA protein, and deletion of GAL11 is known to cause a 5- to 10-fold reduction in GAL4 activity. We suggest that a complex of GAL4 and GAL11 constitutes a particularly strong activator; evidence that the putative GAL4-GAL11 complex ordinarily forms preferentially on DNA suggests a biological rationale for GAL11 action.

CT of renal cell carcinoma in patients on chronic hemodialysis.

The occurrence rate of renal cell carcinoma is significantly higher in the native kidneys of patients undergoing chronic hemodialysis than in kidneys of normal subjects. These carcinomas may be difficult to detect because these kidneys tend to be small and distorted owing to acquired cystic disease. Four cases of renal cell carcinoma detected by CT are presented (three in patients undergoing chronic hemodialysis, and one in a patient with a functioning transplant who had history of dialysis). All kidneys had extensive acquired cystic disease. In two cases, contrast-enhanced scans showed a solid lesion of lower density than the kidney. In another case, in which IV contrast material could not be given, the mass was of higher density than the kidney and retroperitoneal lymphadenopathy was present. In the fourth case, no distinct solid mass could be identified. The only sign of malignancy in this case was extensive lymphadenopathy. Whenever CT examination of the abdomen is performed in a patient undergoing chronic hemodialysis, the kidneys must be evaluated carefully because of the increased frequency of renal cell carcinoma. In a few cases, a distinct solid lesion may not be apparent or may be subtle owing to distortion of renal anatomy by acquired cystic disease.

Small-cell bronchogenic carcinoma: CT evaluation.

CT examinations in 37 patients with proved small-cell bronchogenic carcinoma studied before treatment were reviewed. The distribution of lymphadenopathy in the mediastinal compartments designated by the American Thoracic Society was assessed. The frequency of lymph node enlargement was right hilum (43%), left hilum (49%), one or both hila (84%), right upper paratracheal (32%), right lower paratracheal (54%), right tracheobronchial (65%), left upper paratracheal (14%), left lower paratracheal (38%), left peribronchial (35%), paraesophageal (14%), anterior mediastinum (24%), and subcarinal (65%). We also assessed the frequency of additional findings including pleural effusion (38%), pericardial thickening (38%), displacement or narrowing of either the tracheobronchial tree (68%) or major vessels (68%), and hepatic masses (24%). Mediastinal involvement was present in 92% of cases, as compared with 13% reported in a large series based on conventional radiography. These data show the spectrum of intrathoracic CT findings in proved, untreated cases of small-cell bronchogenic carcinoma.

Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription.

Synthesis of the endogenous opioid precursor, proenkephalin, is regulated by neurotransmitters and membrane depolarization. These events act through second messenger dependent signal transduction pathways via a short inducible DNA enhancer to regulate transcription of the proenkephalin gene. Two DNA elements located within this enhancer are essential for the transcriptional response to cAMP and phorbol ester. Inactivation of either element by mutation or by alteration of their stereospecific alignment eliminates inducible enhancer activity. The promoter distal element, ENKCRE-1, in the absence of a functional adjacent ENKCRE-2 element, has no inherent capacity to activate transcription. However, in the presence of a functional ENKCRE-2 element, this element synergistically augments cAMP and phorbol ester inducible transcription. The promoter proximal element, ENKCRE-2, is essential for both basal and regulated enhancer function. Four different protein factors found in HeLa cell nuclear extracts bind in vitro to the enhancer region. ENKTF-1, a novel enhancer binding protein, binds to the DNA region encompassing ENKCRE-1. The transcription factors AP-1 and AP-4 bind to overlapping sites spanning ENKCRE-2, and a fourth transcription factor, AP-2, binds to a site immediately downstream of ENKCRE-2. The binding of ENKTF-1 to mutant ENKCRE-1 sequences in vitro correlates with the in vivo inducibility of the mutant elements suggesting that ENKTF-1 acts in combination with factors that recognize the ENKCRE-2 domain to regulate cAMP inducible transcription. Together, the two DNA elements, ENKCRE-1 and ENKCRE-2 and the protein factors with which they interact, play a critical role in the transduction and reception of signals transmitted from cell surface receptors to the proenkephalin nuclear transcription complex.

Limitations of CT in evaluation of neoplasms involving chest wall.

We reviewed the CT of 20 patients with peripheral lung malignancies in which CT appearance suggested chest wall invasion on the basis of extension of mass around ribs into fat or muscle of the chest wall, or definite bone destruction. We correlated these findings with other radiographic studies and surgical and autopsy results. All 11 cases in which CT indicated chest wall involvement on the basis of definite bone destruction were confirmed. Tumor extension into the chest wall was disproven in six of the remaining nine cases in which invasion was suggested on the basis of tumor infiltration between ribs or extension of tumor into fat or soft tissue planes. Our findings confirm the lack of reliability of CT findings in determining the extension of malignancy into the chest wall, except when definite bone destruction is present.

Contact with a component of the polymerase II holoenzyme suffices for gene activation.

In yeast strains bearing the point mutation called GAL11P (for potentiator), certain GAL4 derivatives lacking any classical activating region work as strong activators. The P mutation confers upon GAL11, a component of the RNA polymerase II holoenzyme, the ability to interact with a portion of the dimerization region of GAL4. The region of GAL11 affected by the P mutation is evidently functionally inert in ordinary cells, suggesting that this mutation is of no functional significance beyond creating an artificial target for the GAL4 dimerization fragment. From these observations and further analyses of GAL11, we propose that a single activator-holoenzyme contact can trigger gene activation simply by recruiting the latter to DNA.

Chest-wall invasion by carcinoma of the lung: detection by MR imaging.

Nineteen patients with bronchogenic carcinoma were studied by MR imaging to determine the presence of chest-wall invasion. All studies were carried out at 1.5 T, and the results were correlated with chest radiographs or CT scans. All MR studies were interpreted before surgery (13 cases) and without knowledge of the results of other radiologic studies. MR findings indicative of chest-wall invasion included a high-signal focus within the chest wall and/or chest-wall thickening with increased signal on spin-echo (SE) images having a repetition time of 2500 msec and an echo time of 50-100 msec (SE 2500/50-100). In one case, noncontour-deforming high-signal intensity within chest-wall musculature (but no other abnormality) was demonstrated on SE 2500/50-100 images. Coronal or sagittal imaging facilitated identification of tumor contiguity with extrathoracic structures in apical lesions. Contrast differences between normal and invaded chest wall on T2-weighted images were the most helpful in assessing chest-wall involvement. These preliminary observations indicate that MR imaging is useful in the evaluation of chest-wall invasion by carcinoma of the lung.