Templating irreversible covalent macrocyclization by using anions.

Inorganic anions were used as templates in the reaction between a diamine and an activated diacid to form macrocyclic amides. The reaction conditions were found to perform the macrocyclization sufficiently slow to observe a template effect. A number of analytical methods were used to clarify the reaction mechanisms and to show that the structure of the intermediate plays a decisive role in determining the product distribution. For the macrocyclization under kinetic control, it was shown that the amount of a template, the conformational rigidity of building blocks, and the anion affinities of reaction components and intermediates are important parameters that one should take into consideration to achieve high yields.

Subcellular resolution three-dimensional light-field imaging with genetically encoded voltage indicators.

Significance: Light-field microscopy (LFM) enables high signal-to-noise ratio (SNR) and light efficient volume imaging at fast frame rates. Voltage imaging with genetically encoded voltage indicators (GEVIs) stands to particularly benefit from LFM's volumetric imaging capability due to high required sampling rates and limited probe brightness and functional sensitivity. Aim: We demonstrate subcellular resolution GEVI light-field imaging in acute mouse brain slices resolving dendritic voltage signals in three spatial dimensions. Approach: We imaged action potential-induced fluorescence transients in mouse brain slices sparsely expressing the GEVI VSFP-Butterfly 1.2 in wide-field microscopy (WFM) and LFM modes. We compared functional signal SNR and localization between different LFM reconstruction approaches and between LFM and WFM. Results: LFM enabled three-dimensional (3-D) localization of action potential-induced fluorescence transients in neuronal somata and dendrites. Nonregularized deconvolution decreased SNR with increased iteration number compared to synthetic refocusing but increased axial and lateral signal localization. SNR was unaffected for LFM compared to WFM. Conclusions: LFM enables 3-D localization of fluorescence transients, therefore eliminating the need for structures to lie in a single focal plane. These results demonstrate LFM's potential for studying dendritic integration and action potential propagation in three spatial dimensions.

Transforming DNA sequences present in human prolactin-secreting pituitary tumors.

Five human PRL-secreting pituitary tumors were tested for the presence of DNA-transforming sequences. After calcium phosphate transfection to NIH-3T3 mouse fibroblast cells, DNA samples derived from two prolactinomas induced foci of morphologically transformed cells which subsequently grew in soft agar. After retransfection of transformant DNA, resulting secondary transformants elicited rapidly growing solid tumors in nude mice. Southern analysis of transformant DNA revealed the integration of Alu-positive human DNA sequences into the mouse fibroblast NIH-3T3 cells, as judged by hybridization to a Blur-8 probe. The Alu signal became increasingly more difficult to detect with the multiple passaging (greater than 20) of transformant cells in culture. Alu polymerase chain reaction (PCR) was, therefore, used to selectively amplify human DNA sequences from the NIH-3T3 rodent background. PCR using a human Alu-specific primer resulted in amplification of an Alu-containing DNA region within these transformants. The transformant DNA did not hybridize to human genomic probes for genes known to evoke focus formation in this assay, including H-ras, K-ras, N-ras, trk, ret, ros, or met. Further identification of the Alu-containing region revealed that it contained sequences from the human hst gene, a member of the fibroblast growth factor family. The presence of human hst was demonstrated by strong hybridization to a 40-mer oligonucleotide probe to the second exon of hst, by amplification of this region with human hst-nested amplimers within the first and second introns, and finally by direct sequencing.(ABSTRACT TRUNCATED AT 250 WORDS)

Purine-binding factor (nm23) gene expression in pituitary tumors: marker of adenoma invasiveness.

To determine the molecular distinction between invasive and non-invasive pituitary adenomas, we evaluated expression of the metastasizing suppressor gene, nm23, in tumors of varying stages. The nm23 gene was recently identified on the basis of reduced expression in highly metastic cancer compared with its expression in low metastatic potential tumors. Twenty-two pituitary tumors (10 nonfunctioning, 9 acromegaly, 2 prolactinomas, and 1 Cushing) were studied. H1 and H2 isoform expression of nm23 was investigated using a ribonuclease protection assay. nm23 H2 messenger ribonucleic acid expression was significantly reduced in invasive tumors and correlated highly (P = 0.0016) with cavernous sinus invasion. In these invasive tumors, sequencing of the nm23 gene did not reveal a mutation. Invasive tumors also demonstrated markedly reduced immunostaining for nm23 H2. These results show the relevance of nm23 gene expression to behavior of these benign tumors. High expression of nm23 H2 is associated with noninvasive pituitary adenomas and may restrain tumor aggression. This molecular defect distinguishing invasive from noninvasive tumors is shown to be a sensitive marker of adenoma invasiveness and may be a predictor for postoperative management plans.

Molecular screening of pituitary adenomas for gene mutations and rearrangements.

Although pituitary tumors arise as benign monoclonal neoplasms, genetic alterations have not readily been identified in these adenomas. We studied restriction fragment abnormalities involving the GH gene locus, and mutations in the p53 and H-, K- and N-ras genes in 22 human GH cell adenomas. Twenty two nonsecretory adenomas were also examined for p53 and ras gene mutations. Seven prolactinoma DNA samples were tested for deletions in the multiple endocrine neoplasia-1 (MEN-1) locus, as well as for rearrangements in the hst gene, a member of the fibroblast growth factor family. Pituitary adenoma tissue and lymphocytes were obtained from patients at the time of transsphenoidal surgery. In DNA from GH-cell adenomas, identical GH restriction patterns were detected in both pituitary and lymphocyte DNA in all patients and in one patient with a mixed GH-TSH cell adenoma. Using polymerase chain reaction (PCR)-single stranded conformation polymorphism analysis, no mutations were detected in exons 5, 6, 7, and 8 of the p53 gene in GH cell adenomas nor in 22 nonsecretory adenomas. Codons 12/13 and 61 of H-ras, K-ras, and N-ras genes were also intact in GH cell adenomas and in nonsecretory adenomas. Site-specific probes for chromosome 11q13 including PYGM, D11S146, and INT2 were used in 7 sporadic PRL-secreting adenomas to detect deletions of the MEN-1 locus on chromosome 11. One patient was identified with a loss of 11p, and the remaining 6 patients did not demonstrate loss of heterozygosity in the pituitary 11q13 locus, compared to lymphocyte DNA. None of these patients, demonstrated hst gene rearrangements which also maps to this locus. These results show that p53 and ras gene mutations are not common events in the pathogenesis of acromegaly and nonsecretory tumors. Although hst gene rearrangements and deletions of 11q13 are not associated with sporadic PRL-cell adenoma formation, a single patient was detected with a partial loss of chromosome 11, including the putative MEN-1 site.

Clonal origin of pituitary adenomas.

As the pathogenesis of pituitary adenomas remains unclear, the tumor clonal composition of these common neoplasms was studied. Clonality was determined in female patients by analysis of restriction fragment length polymorphisms of the X-chromosome genes hypoxanthine phosphoribosyl transferase and phosphoglycerate kinase in conjunction with their respective methylation patterns. Peripheral lymphocyte DNA was screened from 62 female patients undergoing transsphenoidal surgery for pituitary adenoma. Eleven patients were heterozygous for the BglI site on PGK, 4 for the BamHI site on HPRT, and 1 patient for both sites. Of these 16 patients, 3 had acromegaly, 4 had Cushing's disease, 7 had hyperprolactinemia, and 2 were clinically nonfunctional. After surgery, morphological study, including immunohistochemistry and electron microscopy of the pathological specimens, allowed a direct comparison between clonality and tumor cell type. Control fresh normal pituitary tissue was found to be polyclonal. The following tumors were monoclonal: all 3 somatotroph adenomas, 4 of 4 lactotroph tumors, 3 of 4 corticotroph cell adenomas, a gonadotroph adenoma, and a nonsecretory adenoma. A mixed plurihormonal adenoma was polyclonal, as were 2 tumors consisting of adenomatous lactotrophs interspersed with nontumorous adenohypophyseal pituitary tissue and one corticotroph adenoma mixed with normal pituitary tissue. Functional pituitary adenomas derived from somatotrophs, corticotrophs, or lactotrophs and nonsecretory tumors are monoclonal in nature, suggesting that somatic cell mutations precede clonal expansion of these cells and play a major role in pituitary tumorigenesis.

Serum prolactin after chest wall surgery: elevated levels after mastectomy.

Galactorrhea is a recognized sequel of chest injury, but serum PRL levels in these patients have not been systematically documented. Therefore, we examined the PRL responses over 5 days in patients undergoing either mastectomy (10 patients) or thoracotomy (10) and in seven patients undergoing elective laparotomy (controls). Basal serum PRL levels were normal in every subject. There were no consistent or significant alterations in PRL levels after laparotomy or thoracotomy. After mastectomy, PRL levels rose from a mean preoperative level of 7.1 +/- 1.3 to 16.0 +/- 3.3 ng/ml (P < 0.01) on the first postoperative day. Mean levels continued to rise to 35.6 +/- 6.6 ng/ml (P < 0.005) on day 5; levels were supranormal in eight subjects. Hyperprolactinemia persisted in the four subjects evaluated 4 weeks postoperatively and in one of five patients evaluated at 6 months. In a retrospective study, serum PRL levels were measured months to years after thoracotomy (31 patients) and mastectomy (53 patients) and compared to levels in 41 normal female controls. Mean serum PRL levels were 8.4 +/- 1.3 ng/ml in the control group, 13.1 +/- 0.9 ng/ml in the thoracotomy group (P < 0.005), and 20.6 +/- 3.1 ng/ml in the mastectomy group (P < 0.001). One thoracotomy patient and 18 mastectomy patients (34%) had supranormal PRL levels. It is concluded that mastectomy acutely stimulates PRL secretion in most subjects, and levels may remain elevated for months, perhaps for years, in a proportion of patients. Both the acute and chronic hyperprolactinemic states are probably the result of neurogenic PRL release mediated via the suckling reflex.

Deletion of 20p 11.23----pter with normal growth hormone-releasing hormone genes.

Using a molecular analysis of the DNA from a patient with a deletion of chromosome 20 [46,XX,del(20)(p 11.23)], we have excluded the growth hormone-releasing hormone (GHRH) gene from the region 20p11.23----pter. The patient had minor facial anomalies. Rieger eye anomaly, a congenital heart defect, severe failure to thrive, and a neurosecretory problem in growth hormone (GH) secretion. Since the GHRH gene was previously mapped to chromosome 20, we used molecular genetic methods to determine whether the growth abnormalities were due to the deletion of this gene. DNAs of the patient and 2 normal control subjects were analyzed by quantitative Southern blotting using a DNA probe for the GHRH gene and 2 reference DNA probes mapping to chromosome 21. The GHRH gene was found to be present in 2 copies in the patient. This indicates that the gene for GHRH maps to the region outside the patient's deletion, in 20p11.23----qter. Furthermore, our results suggest that genes other than GHRH on 20p are important for developmental steps leading to normal neurosecretory function of GH and may also be involved in generating Rieger eye anomaly. Finally, GH deficiency and Rieger eye anomaly should be sought in other patients with deletions of 20p.

Clinical and biochemical responses to nadolol and clonidine in hyperthyroidism.

Some features of hyperthyroidism mimic sympathetic nervous system overactivity. We have compared the clinical (scored on the Wayne Therapeutic Index), hemodynamic (blood pressure and heart rate) and biochemical (plasma epinephrine, norepinephrine, glucose, free fatty acids, insulin, growth hormone and free thyroxine index) effects of clonidine (alpha 2-agonist, which reduces plasma catecholamine levels) with those of nadolol (non-selective beta adrenergic receptor antagonist) in ten female hyperthyroid patients. Each patient received nadolol for 1 week followed by clonidine for 1 week in a single-blind manner. All measurements were made before treatment and then repeated at the end of the nadolol and clonidine treatment periods. Thyroid function remained unaltered during the study. Both agents caused significant clinical improvement--the mean Wayne Index score was 18 pretreatment, 2 on nadolol and 6 on clonidine (P less than .003 for each). Heart rate was reduced by both drugs, but blood pressure was unchanged. Side effects occurred in eight out of ten patients while on clonidine. Nadolol increased plasma concentrations of epinephrine from 47 +/- 18 pg/mL to 87 +/- 24 pg/mL, and norepinephrine from 241 +/- 154 pg/mL to 338 +/- 224 pg/mL (P less than .001 for each). In contrast, clonidine depressed norepinephrine levels from 241 +/- 154 pg/mL to 110 +/- 49 pg/mL (P less than .001) without lowering plasma epinephrine significantly. Plasma free fatty acids tended to fall on both agents compared to pretreatment levels. The blood glucose, insulin and growth hormone concentrations were unaffected by either drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary hydatid disease: a four-year experience in an urban black hospital.

Intrathoracic hydatid disease is rarely seen in black patients hospitalized in the Johannesburg area. Eleven patients with pulmonary hydatid disease were treated during 4 years at a major referral center for black patients. The clinical features and management of these patients are described.

Inaccuracy of nitrogen balance determinations in thermal injury with calculated total urinary nitrogen.

Many burn centers use nitrogen balance studies to estimate the adequacy of nutritional support. Nitrogen loss includes the sum of urinary urea nitrogen, nonurea urinary nitrogen, and losses from skin, wound, and stool. Urinary urea nitrogen is often used to calculate total urinary nitrogen by multiplying the urinary urea nitrogen by a factor of 1.25 to account for nonurea urinary nitrogen. This formula is appropriate when applied to a nonstressed individual who has fasted overnight but is not appropriate for patients who have undergone surgery or experienced trauma. We have undertaken this study to assess the predictability of this formula in patients with thermal injuries. Twenty-seven patients with major thermal injuries had random 24-hour urine collections, which were analyzed for both urinary urea nitrogen and total urinary nitrogen. In these patients with burns we found that urinary urea nitrogen represented approximately 65% of the directly measured total urinary nitrogen rather than 80% as assumed by the formula. This increase in the nonurea nitrogen loss is greater than that found after surgery or trauma. Individual measurements may underestimate losses by 20% to 60%. Directly measured total urinary nitrogen should replace calculated total urinary nitrogen as the index of urine nitrogen losses for nitrogen balance studies in patients with burns.

[Short-term complications of transtympanic aerators]. / Complications à court terme des aérateurs transtympaniques.

Three hundred and thirteen transtympanic aerators (TTA), including 309 T-tubes, were inserted between november 1985 and April 1987 at the Bretonneau hospital, Paris. Twenty-three (7%) were expelled spontaneously, 16 (5%) became obstructive and in 6 cases a change of TTA was required. The commonest complication was otorrhea. Younger children tended to develop early otorrhea. There were 7 cases (2%) of early otorrhea and 50 (16%) of secondary otorrhea. By contrast the onset of secondary otorrhea was independent of age. A relationship with penetration of water into the middle ear through the TTA could not be confirmed. At the present time the authors permit bathing without any special precautions. In parallel, between november 1985 and august 1987, 84 bacteriological samples were obtained in cases of otorrhea related with a TTA, whether or not the latter had been inserted at Bretonneau. Predominant organisms were Hemophilus influenzae (30%), Pseudomonas aeruginosa (30%) and Staphylococcus (18%). On the basis of results of antibiotic sensitivity studies using the organisms discovered in these samples, the authors suggest local therapy based upon polymyxin or rifamycin as treatment of first choice.

[Treatment of blood vessel injuries in the extremities]. / Osetrovanie poranení koncatinových ciev.

The authors give an account of a group of 53 patients with injuries of the acral blood vessels, treated at the hospital and policlinic in Banská Bystrica between Jan. 1, 1979 and Dec. 31, 1988. They discuss the frequency, incidence, site of injury, method of treatment of acral blood vessels and the results, as well as the number of diseases treated by surgeons with different specializations.

Regional techniques as an adjunct to general anesthesia for pediatric extremity and spine surgery.

The purpose of this review is to evaluate the safety of regional anesthesia techniques performed for postoperative analgesia in anesthetized children. Pediatric regional anesthesia techniques, such as nerve blocks and neuraxial injections of either local anesthetics or narcotics, can potentially reduce postoperative pain for all children undergoing surgery. However, children may react differently to anesthesia than adults, and they usually cannot tolerate the administration of regional anesthesia unless they are under general anesthesia. During a 5-year period (1999-2004) at the Shriners Hospitals for Children Northern California, 2236 regional anesthetic procedures were performed in 1809 patients. All of the regional procedures were performed with patients under general anesthesia. Ninety-one percent (1641) of patients were for orthopaedic extremity or spine surgeries. Patients ranged from 2 months to 20 years old, with 65% (1169) between the ages of 6 months and 12 years. One thousand eleven procedures were lower extremity blocks, 646 were upper extremity blocks, and 579 were neuraxial injections. Four hundred fifty-four peripheral nerve blocks were performed in patients aged 3 years or younger. Two self-limiting complications possibly related to peripheral nerve blocks were noted. No complications were noted in patients who received neuraxial injections. This retrospective review indicates that regional anesthesia techniques performed 'under general anesthesia have a low rate of complications in children. A prospective trial is recommended to establish the efficacy and safety of this practice.

The cardiomyopathy of primary hypoparathyroidism. A case report.

A case of hypocalcaemic cardiomyopathy associated with primary hypoparathyroidism is described. The cardiac failure, which initially proved resistant to conventional therapy, resolved with correction of the serum calcium and magnesium levels.

Determination of quinolone antibiotics in growth media by reversed-phase high-performance liquid chromatography.

A simple, accurate, precise, and versatile high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of three quinolone antibodies in Mueller-Hinton broth. The fluoroquinolone agents studied were ciprofloxacin, ofloxacin, and sparfloxacin; other quinolone agents have been identified using this method but not validated in this matrix (levofloxacin, clinafloxacin, temafloxacin, and trovafloxacin). In addition, several other biological growth mediums have been investigated (human serum, human urine, Todd-Hewitt growth media, Ensure enteral feeding solution, and Haemophilus growth media). This method uses UV detection (280 nm), a simple, one-step protein precipitation extraction, and separation using a C18 column with an isocratic, ion-pairing mobile phase. An appropriate internal standard was obtained by using another quinolone antibiotic of differing retention time. The calibration curves were linear (r2> or =0.999) over a concentration range of 0.0625-20.0 microg/ml with a lower limit of quantification of 0.1 microg/ml. The intra-day and inter-day coefficients of variation were less than 15%.

Large-vessel disease in a black diabetic: a case report.

A case of a middle-aged Black woman with maturity-on-set diabetes is described. The patient had had a cerebrovascular accident and subsequently developed thrombosis of the descending thoracic aorta, with extension into the left subclavian artery and emboli in the right femoral and left brachial arteries. Occlusive large-vessel disease rarely occurs in Black diabetics. Vascular disorders and haemostatic factors in Black and White diabetics are discussed.