Chromatin and alternative splicing.

Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

Presence of alpha-globin mRNA and migration of bone marrow cells after sciatic nerve injury suggests their participation in the degeneration/regeneration process.

We have previously reported that in the distal stump of ligated sciatic nerves, there is a change in the distribution of myelin basic protein (MBP) and P0 protein immunoreactivities. These results agreed with the studies of myelin isolated from the distal stump of animals submitted to ligation of the sciatic nerve, showing a gradual increase in a 14 kDa band with an electrophoretic mobility similar to that of an MBP isoform, among other changes. This band, which was resolved into two bands of 14 and 15 kDa using a 16% gel, was found to contain a mixture of MBP fragments and peptides with great homology with alpha- and beta-globins. In agreement with these results, we have demonstrated that the mRNA of alpha-globin is present in the proximal and distal stumps of the ligated nerve. It is also detected at very low levels in Schwann cells isolated from normal nerves. These results could be due to the presence of alpha- and/or beta-globin arising from immature cells of the erythroid series. Also, they could be present in macrophages, which spontaneously migrate to the injured nerve to promote the degradation of myelin proteins. Cells isolated from normal adult rat bone marrow which were injected intraortically were found to migrate to the injured area. These cells could contribute to the remyelination of the damaged area participating in the removal of myelin debris, through their transdifferentiation into Schwann cells or through their fusion with preexisting Schwann cells in the distal stump of the injured sciatic nerve.

Alternative splicing of Xenopus alphafast-tropomyosin pre-mRNA during development: identification of determining sequences.

The Xenopus alphafast-tropomyosin gene contains in its central part a set of mutually exclusive exons, designated 6A and 6B, which are incorporated into mRNA encoding, respectively, nonmuscle and muscle tropomyosins. In this study, we show that usage of both exons is strictly regulated during development, exon 6A being used in the oocyte and nonmuscle tissues of the embryo, while exon 6B is used in muscle tissues. An approach of transient embryo transgenesis was developed to study the mechanisms involved in the splice site choice during development. We demonstrate that a-tropomyosin minigenes driven by tissue-specific promoters that target gene expression in nonmuscle and muscle tissues recapitulate the splicing pattern of the endogenous gene. A mutational analysis showed that regulation occurred at both exons 6A and 6B in muscle and nonmuscle tissues. In this context, we have identified an element located in the intron downstream of 6A that participates in the recognition of the weak 5' splice site of exon 6A and the repression of exon 6B in nonmuscle cells.

Ticarcillin/clavulanate versus imipenem/cilistatin for the treatment of infections associated with gangrenous and perforated appendicitis.

The objective of this study was to compare ticarcillin/clavulanate given at 3.1 g every 6 hours with imipenem/cilistatin given at 500 mg every 6 hours for the treatment of infections associated with gangrenous or perforated appendicitis. One hundred thirty-seven patients were found to have gangrenous or perforated appendicitis and received the study medication for 3 to 5 days in a double-blinded, randomized manner. Clinical success was similar for the two treatment groups, 96.9 and 95.9 per cent in the ticarcillin/clavulanate and imipenem/cilistatin groups, respectively (P=0.99; 95% confidence interval for the difference was -5.6% to 7.6%). Bacteriologic success at the end of therapy was similar in the two groups, 100 and 98.4 per cent in the ticarcillin/clavulanate and imipenem/ cilistatin groups, respectively (P=0.99; 95% confidence interval for the difference was -1.8% to 4.7%). The occurrence of adverse events related to treatment was similar for the two groups (P=0.31) and led to study withdrawal for four patients (one with ticarcillin/clavulanate and three with imipenem/ cilistatin). Ticarcillin/clavulanate given at 3.1 g every 6 hours is as effective and as safe as imipenem/ cilistatin given at 500 mg every 6 hours for treatment of gangrenous or perforated appendicitis.

Hürthle cell neoplasms of the thyroid: are there factors predictive of malignancy?

OBJECTIVE: To determine if any preoperative or intraoperative factors can reliably predict malignancy in patients with Hürthle cell neoplasms. SUMMARY BACKGROUND DATA: Most experienced surgeons recommend total thyroidectomy for Hürthle cell carcinomas and reserve thyroid lobectomy for Hürthle cell adenomas. However, delineation between Hürthle cell adenoma versus carcinoma often cannot reliably be made either before or during surgery. METHODS: Medical records from 57 consecutive patients who underwent thyroid resections for Hürthle cell neoplasms between October 1984 and April 1995 at The Johns Hopkins Hospital were analyzed to determine if any factors were predictive of malignancy. RESULTS: Of the 57 patients with Hürthle cell neoplasms, 37 had adenomas and 20 had carcinomas, resulting in a 35% prevalence of malignancy. Patients with adenomas did not differ from those with carcinoma with respect to age, sex, or history of head and neck irradiation. However, patients with Hürthle cell carcinomas had significantly larger tumors (4.0 +/- 0.4 cm vs. 2.4 +/- 0.2 cm, p < 0.005). Furthermore, although the incidence of malignancy was only 17% for tumors 1 cm or less and 23% for tumors 1 to 4 cm, tumors 4 cm or greater were malignant 65% of the time (p < 0.05). Both fine-needle aspiration and intraoperative frozen section analysis had low sensitivities in the detection of cancer (16% and 23%, respectively). With up to 9 years of follow-up, there has been no tumor-related mortality. CONCLUSIONS: These data demonstrate that the size of a Hürthle cell neoplasm is predictive of malignancy. Therefore, at the time of initial exploration for large Hürthle cell neoplasms (>4 cm), definitive resection involving both thyroid lobes should be considered because of the higher probability of malignancy.

Alpha-tropomyosin gene expression in Xenopus laevis: differential promoter usage during development and controlled expression by myogenic factors.

Tropomyosins (TMs) constitute a group of contractile proteins encoded by a multigene family showing distinct cell-type-specific and developmental expression patterns. In mammals and birds, the alpha-TM gene is the most complex and can produce several muscle and non-muscle isoforms. We report here the characterization of the 5' region of the Xenopus laevis alpha-TM gene and its developmental expression. The 5' region of the gene is structurally related to the avian and mammalian cognates and presents two promoters flanking a pair of alternatively spliced exons, 2a/2b, where exon 2a is a smooth-muscle-specific exon. The internal promoter is used to generate a non-muscle low molecular weight TM whilst muscle TM isoforms originate from the distal promoter. RNase protection analysis shows that the two promoters have distinct temporal programs of activation. The internal promoter is activated early in oogenesis and non-muscle transcripts are found throughout oogenesis, embryogenesis and in adult tissues. Only low molecular weight non-muscle TM-encoding mRNAs are expressed in oogenesis. The distal promoter is silent during oogenesis, and the skeletal muscle alpha-TM transcripts accumulate from stage 15 in the embryo and are expressed in adult striated muscle tissues. In situ hybridization indicates that these transcripts are expressed in both the somites and heart of the embryo. Ectopic expression of myogenic factors, but not the MEF2 myocyte-specific enhancer factor 2 factors SL1 and SL2, can induce the expression of the alpha-TM gene suggesting that the gene is a direct target for myogenic but not for MEF2 factors. The amphibian alpha-TM gene constitutes a gene marker for studying the developmental control expression of muscle genes in the different myogenic lineages.

[Visceral leishmaniasis in patients with HIV infection. Our experience]. / Leishmaniosi viscerale in paziente con infezione da HIV. Nostra esperienza.

The authors describe a case of visceral leishmaniasis in an intravenous drug abuser, HIV seropositive patient. Visceral leishmaniasis was the first opportunist infection and diagnosis was based on smear from bone marrow. A treatment with N-methylglucamine and allopurinol was successful. The authors stress to take into account visceral leishmaniasis in HIV-infected patient in act resident or in past lived in endemic for Leishmania areas even in presence of atypical clinical features and propose to include visceral leishmaniasis as an opportunist infection in the IVC-2 group of clinical classification of HIV infection.

The MLC1f/3f gene is an early marker of somitic muscle differentiation in Xenopus laevis embryo.

cDNAs clones encoding the MLC1f and MLC3f proteins of Xenopus laevis have been isolated from a stage 42 cDNA library. Sequence analysis reveals that the amphibian MLC1f and MLC3f isoforms are similar to the mammalian and avian cognates. The two isoforms share a common 141-amino-acid carboxy-terminal regions. These are 49 and 9 residues long for the MLC1f and MLC3f isoforms, respectively. This suggests a genomic organization similar to the mammalian and avian genes, with two promoters and alternative splicing. The developmental expression of the MLC1f/3f mRNAs was studied by Northern blot and RNase protection and their spatial expression analyzed by in situ hybridization. Both the MLC1f and MLC3f mRNAs can be detected in the developing embryo from the end of gastrulation and accumulate rapidly in the somitic mesoderm. Expression of the MLC1f/3f gene can also be detected in animal cap explants which have been induced to form mesodermal derivatives by exposure to activin A or bFGF. However, unlike other muscle-specific markers, neither transcript from the MLC1f/3f gene can be detected in embryonic or adult cardiac muscle, their expression being restricted to somitic muscle. Together, these data demonstrate that expression of the MLC1f/3f gene provides a sensitive and specific marker for skeletal muscle differentiation. Ectopic expression of myogenic factors in animal caps induces the expression of the MLC1f/3f gene, suggesting that the amphibian gene, like its mammalian and avian counterparts, is a regulatory target for members of the MyoD family of transcription factors.

[Micro-morphology of the ovipositor in Hymenoptera and evolution from phytophagous Symphyta to parasitoid Apocrita]. / Micromorphologie de l'ovipositeur des hyménoptères et évolution des symphytes phytophages aux apocrites parasitoïdes.

The ovipositor of 1 Symphyta and 12 primitive parasitoid Apocrita belonging of the family of Ichneumonidae has been studied with the scanning electron microscope (SEM) and for 2 species, semi-thin sections were used. The study shows the presence of closed trachea in the 3 pairs of valvulae and a secretory system in the 2 pairs of valvulae interlocked into a piercing stylus. We discuss the role of trachea and a secretory system leading to excretory pores on the lancets of valvulae which occupy very precisely the site of sensory chemoreceptors known in more advanced species lacking this secretory system. The micromorphological data support the phylogeny within Hymenoptera, from Symphyta to primitive parasitoid Apocrita.

The Xenopus laevis TM-4 gene encodes non-muscle and cardiac tropomyosin isoforms through alternative splicing.

A full-length cDNA clone coding for a 248-amino-acid tropomyosin (TM) was isolated from a Xenopus laevis (Xl) oocyte cDNA library. This TM is very similar to the members of the non-muscle (nm) TM family that includes rat TM-4, human TM30pl and chicken cardiac fibroblast FT-C. An RNase protection assay showed that the corresponding transcript is expressed ubiquitously and revealed the presence of an alternative transcript in adult heart RNA. A full-length cDNA clone was isolated from an adult heart cDNA library using a nm TM-encoding cDNA probe. It encodes a muscle TM homologous to chicken FT-C and the corresponding mRNA is expressed in adult RNA and to a very low level in adult skeletal muscle. The two cDNAs correspond to two alternatively spliced isoforms of TM generated from the Xl TM-4 gene. These data, together with published observations, demonstrated that, in this amphibian, the cardiac muscle TM are synthesized from the TM alpha and TM-4 genes.

Occult hypoadrenalism in critically ill patients.

No clear criteria exist to rule out hypoadrenal shock by cosyntropin (alpha 1-24-corticotropin, a synthetic subunit of adrenocorticotropic hormone) testing in persons who have critical nonadrenal illness. Four patients in the surgical intensive care unit with critical multisystem disease and refractory high cardiac output, low vascular resistance shock had significantly diminished or terminated vasopressor requirements after institution of hydrocortisone sodium succinate infusion in doses simulating physiologic stress response (100 to 300 mg of hydrocortisone per day). In each case, cosyntropin testing revealed serum cortisol levels higher than those usually associated with hypoadrenal shock. Positive response was defined as maintenance of blood pressure with a decrease to less than 25% of baseline pressor requirements within 48 hours of treatment. We hypothesize a syndrome of functional hypoadrenalism in patients with multisystem critical illness and refractory shock responsive to glucocorticoid administration in doses simulating physiologic stress response despite cosyntropin stimulation test results that would rule out hypoadrenalism in a normal person.

Preventing postoperative acute bleeding of the upper part of the gastrointestinal tract.

Two hundred and ninety-eight critically ill patients at risk for the development of postoperative stress ulcers and bleeding were randomized into three groups. The first group comprised 85 patients who received meciadanol, a new bioflavonoid, 500 milligrams every six hours through a nasograstric tube; the second group comprised 100 patients who received sucralfate (crushed tablets), 1,000 milligrams every six hours through a nasogastric tube, and the third group comprised 113 patients who received an antacid (Maalox [magnesium aluminum hydroxide gel]) through a nasogastric tube at an initial dose of 15 milliliters every hour. The gastric pH was measured hourly and titrated to a pH greater than or equal to 4.0 in patients in the group receiving the antacid. The gastric pH was measured every two hours in the other two groups. Bleeding in the upper part of the gastrointestinal tract was determined visually (frank blood in gastric contents) or by guaiac testing. Bleeding occurred in seven patients receiving meciadanol, nine receiving sucralfate and six receiving the antacid. The difference in rates of bleeding was not statistically significant. Correlation between the severity of illness index and the development of bleeding was poor, at least in the low and intermediate index range. In contrast, there was a strong correlation between the age of the patient and the development of bleeding. Only one patient younger than 50 years had bleeding develop. Apparently, meciadanol exerts its action by a mechanism other than pH control. It may, therefore, fill an important gap in the ability to prevent postoperative stress ulcers and bleeding.

Not all "occult" papillary carcinomas are "minimal".

"Occult" papillary carcinomas are characterized as small papillary tumors of less than 1.5 cm in maximum diameter, with or without bulky metastatic deposits in cervical nodes. The primary lesion is usually not palpable, and although the clinical behavior usually follows a benign course, tumors with unfavorable histologic features (invasiveness, multifocality) or extrathyroidal disease or a combination of both may not do so. In this report six cases are presented to illustrate this entity. No patient had a history of irradiation to the head or neck. All had primary lesions smaller than 1.5 cm. None had a palpable nodule or abnormal thyroid scan results, and the diagnosis of thyroid cancer was based on cervical lymph node or lung biopsy specimens, which revealed papillary thyroid cancer. All of the patients underwent total or near-total thyroidectomies and were found to have small, invasive papillary lesions with additional metastases to cervical nodes noted at the time of thyroidectomy. Adjunctive treatment consisted of a 5 mCi iodine-131 scan, ablative iodine-131 therapy, and suppression with L-thyroxine. Although distant metastasis to lung or other organs is uncommon and the mortality rate is low (as in larger papillary cancers), these invasive lesions--despite their small size--have a high propensity for recurrence and should be considered to behave more like encapsulated papillary tumors with extrathyroidal extension than like their small, unencapsulated intrathyroidal counterparts.

Single photon emission computed tomography of the thyroid.

Single photon emission computed tomography (SPECT) entails imaging at multiple positions along an arc and computed-assisted reconstruction of transaxial, coronal, and sagittal images. To determine if SPECT imaging of the thyroid augments information provided by planar imaging, both SPECT and pinhole planar scans were performed in 52 patients with thyroid disease. After 123I (n = 45), Tc99m (n = 5), or thallium-201 (n = 2) administration, images were acquired at 30 intervals along a 180 degree arc. Nodules that were hypofunctioning on planar scans (n = 4) were equally well shown on SPECT images. SPECT analysis of 123I uptake by 5 palpable nodules with completely normal 123I planar scans demonstrated the nodular tracer uptake was increased (n = 1), equivalent (n = 3), or decreased (n = 1) in comparison with that of extranodular tissue. In 6 of 12 multinodular goiters, transaxial SPECT images showed tracheal compression, which was confirmed by neck x-ray, flow volume loops, or surgery. A neck phantom study was performed to assess the accuracy of SPECT-determined functional thyroid volume. SPECT-estimated volumes (30-173 mL; n = 11) correlated well with true volumes (r = 0.996; P less than 0.001). Our observations suggest that SPECT is a useful adjunct to planar imaging in 1) estimating functional volume for 131I dosimetry; 2) evaluating the size, extension, and tracheal compression of multinodular goiter; and 3) determining the functional properties of palpable nodules with normal 123I planar pinhole images.

Neutrophil intracellular kill following thermal injury. Different bactericidal capability for patients' organisms and laboratory organisms.

Sixteen patients with septic complications of severe thermal injury were studied with respect to neutrophil intracellular-killing power against clinical isolates from the patients themselves and against other laboratory organisms. Simultaneous measurements of neutrophil chemotaxis, helper/suppressor lymphocyte ratios, and serum IgG concentrations were also carried out. Neutrophils from patients who survived had diminished intracellular-killing capacity for their own organisms, but normal capacity for killing laboratory organisms either matched or unmatched with the patients' own isolate's species. In these patients, the chemotactic index, the lymphocyte helper/suppressor ratio, and the serum IgG concentration remained within normal limits. Neutrophils from patients who died failed to kill their own, as well as laboratory, organisms. In these patients, the chemotactic index, lymphocyte helper/suppressor ratio, and IgG concentration were significantly diminished. The biological implications of these findings are noted.

AIDS and the surgeon.

Several issues related to the AIDS virus concern the surgeon. This article discusses the common presentations of AIDS in each body system with special emphasis on conditions that require surgical intervention, alternatives to surgical procedures for diagnosis, and precautions for the handling of tissue and body secretions of individuals suspected of harboring the AIDS virus.

Iodine-123 SPECT of the thyroid in multinodular goiter.

Iodine-123 single photon emission computed tomography (SPECT) imaging of the thyroid was performed in two patients with multinodular goiter and swallowing difficulty to provide the functional and anatomic orientation of the goiter in relation to the airway. Transaxial slices showed the retrolaryngeal extension of the enlarged thyroid and the tracheal compression by the goiter in both patients. Sagittal and coronal sections confirmed the posterior extension of the goiter. Tracheal displacement was confirmed by roentgenography of the neck in both patients. Vocal cord paralysis demonstrated by fiberoptic laryngoscope and esophageal compression shown by esophagography were found in a patient with toxic multinodular goiter with coexisting papillary carcinoma of the thyroid. In this patient, both the tracheal compression noted in SPECT imaging and the tracheomalacia suggested by the flow volume loop pattern in pulmonary function test were confirmed at the time of thyroidectomy. Our observation suggests that SPECT imaging of large multinodular goiter may be useful in preoperative delineation of the functional anatomy and the extension of goiter in relation to the airway.

Primary cutaneous aspergillosis associated with Hickman intravenous catheters.

We describe nine patients with underlying hematologic cancer in whom primary cutaneous aspergillosis developed at the sites of Hickman intravenous catheters. Our patients, 17 to 74 years of age, were all immunocompromised either from their primary disease or from chemotherapy, and the Hickman catheters had been placed to provide venous access for chemotherapy or hyperalimentation or both. Clinical signs of infection included erythema, induration, and cutaneous or subcutaneous necrosis at the point of entry into the subclavian vein, in the subcutaneous tunnel, or at the exit site from the skin. Diagnosis was confirmed by positive wound culture for Aspergillus flavus in all but one patient. Treatment consisted of intravenous amphotericin B, oral flucytosine, and local wound care. Three patients recovered completely without operative débridement; three others recovered after operative débridement and delayed grafting. Two patients died of disseminated aspergillosis, and one died of unrelated causes while recovering from primary cutaneous aspergillosis. Successful treatment required resolution of aplasia or leukopenia, catheter removal, systemic treatment with amphotericin B, and local wound care. We conclude that primary cutaneous aspergillosis, a rare infection, may occur at the sites of Hickman catheters in immunocompromised patients, and that it is a serious complication requiring prompt diagnosis and treatment.