Investigation of the regulation of transcriptional changes in Ancylostoma caninum larvae following serum activation, with a focus on the insulin-like signalling pathway.

The exit from dauer in the free-living nematode Caenorhabditis elegans is under the control of a single amphidial neuron (ASJ) of the insulin-like signalling pathway. Mutations of this pathway have the ability to suppress entry into the dauer stage. It has been postulated that insulin-like signalling plays a significant role in the response to serum stimulation in vitro of the third-stage larvae (L3s) of the canine hookworm Ancylostoma caninum. To test for the possible involvement of the insulin-like signalling cascade in the response to serum stimulation, the effects of two signalling stimulants (8-bromo cGMP and arecoline) and four inhibitors, namely 4,7-phenanthroline, phosphoinositide-3 kinase (PI3K), Akt inhibitor IV and rapamycin on feeding and on levels of selected activation-associated mRNAs in serum-stimulated L3s were explored. L3s of A. caninum were pre-incubated with or without the appropriate inhibitor/agonist. Following serum-stimulation, the feeding activity was assessed. The transcription levels of a number of activation-associated mRNAs linked to particular expressed sequence tags (ESTs) were investigated by reverse transcription, real-time PCR (rtPCR). The treatment of worms with 4,7-phenanthroline completely suppressed feeding and significantly reduced the differential levels of most activation-associated mRNAs, whereas the treatment with cGMP resulted in the resumption of feeding in almost 85% of the L3s and yielded a specific transcriptional profile consistent with that following serum stimulation. The treatment of L3s with arecoline resulted in the resumption of feeding in approximately 85% of L3s, but did not result in a transcriptomic profile consistent with activation. A complete reduction in feeding was recorded in the presence of the PI3K inhibitor LY294002 (1mM) and resulted in a pronounced dampening of differential transcription in response to serum stimulation for the molecules examined. Akt inhibitor IV resulted in a approximately 70% reduction in feeding but had almost no effect on the level of any of the activation-associated mRNAs studied. Rapamycin was shown to have a weak effect on feeding, and several of the mRNAs studied exhibited greater than expected transcription following treatment. The complexities of activation-associated transcription could not be addressed using the current approach. A larger number of mRNAs needs to be investigated in order to predict or identify regulatory mechanisms proposed to function in the insulin-like signalling pathway in A. caninum.

E2F binding is required but not sufficient for repression of B-myb transcription in quiescent fibroblasts.

We have previously shown in mouse NIH3T3 fibroblasts that transcription of the B-myb gene, which encodes a transcription factor required for S phase entry, is repressed through a promoter E2F site in G0/early G1. Transcription repression at this stage of the cell cycle was correlated with binding of a specific p107/E2F complex to this site. We report here, however, that transfection of cells with the known components of this complex, p107, E2F-4 and DP-1, did not repress the B-myb promoter in cycling NIH3T3 cells, although p107 inhibited transcription transactivation by E2F-4/DP-1. To establish definitively the contribution of E2F to repression, the effects of further mutations within and surrounding the E2F site were examined. It was evident that E2F binding and repression were closely correlated, lending greater weight to the contention that E2F itself is implicated in this activity. These studies also identified a closely linked site, designated the downstream repression site (DRS), which was not required for E2F binding or transactivation but which was necessary for repression. These findings indicated that E2F-dependent repression and activation are independently regulated phenomena and suggest that repression involves additional interactions determined by the promoter context.

Cell-cycle regulation of human B-myb transcription.

We demonstrate here that activity of the human B-myb promoter is regulated during the cell cycle by the E2 transcription factor (E2F). Comparison of the human B-myb promoter sequence with that of its murine counterpart revealed an evolutionally conserved sequence that contains an E2F-binding site. In transiently transfected murine NIH3T3 and human HaCaT cells, luciferase (Luc) reporter activity directed by the human B-myb promoter was found to increase significantly in late G1/S phase of the cell cycle. Mutation of the promoter E2F site resulted in significantly greater Luc activity in NIH3T3 and HaCaT cells made quiescent by serum deprivation, indicating that E2F repressed transcription of this gene during G0. Analysis of E2F DNA-binding activity in G0 HaCaT cells revealed a distinct complex that apparently contained neither the retinoblastoma gene protein, pRb, nor the related p107 protein. De-repression of transcription in S phase was accompanied by the disappearance of this G0 E2F complex and the appearance of a distinct complex containing p107. In addition, complexes containing pRb were detected at both stages of the cell cycle.

Disruption of the ATM gene in breast cancer.

Mutations in the ATM gene, which maps to 11q22-23, cause the multisystem recessive syndrome ataxia-telangiectasia (AT). Breast cancer has been reported in AT patients and carriers. Sporadic breast cancer is associated with loss of heterozygosity at or in the region of ATM and chromosomal abnormalities involving 11q23. We have investigated the chromosomes, nuclei and released chromatin fibers from nine primary breast carcinoma and eight cell lines by fluorescence in situ hybridization with four fluorochrome-labeled cosmids spanning the ATM gene. The ATM gene was disrupted in one primary breast carcinoma and in the cell lines MDA-MB-231 and MCF-7. The role of these aberrations in breast carcinomas, which may lead to gene dosage or dominant negative effects on gene function, requires further investigation.

Measurement of dose distribution around Fletcher-Suit-Delcos colpostats using a Therados radiation field analyzer (RFA-3).

Using a small silicon diode and the Therados RFA-3 automated dosimetry system, the relative dose distribution around Fletcher-Suit-Delcos colpostats was measured. Thermoluminescent dosimeters (LiF) were used for absorbed dose measurements. These data provide the basis for computerized dose calculations which will account for the attenuation effects of the rectal and bladder shieldings. The stainless-steel housing attenuated the radiation of a Cs-137 source by 6%. The tungsten shields caused a further dose reduction of 15%-25%.

An absorptiometry method for the determination of arterial blood concentration of injected iodinated contrast agent.

A single photon absorptiometry method to measure the arterial concentration of injected iodinated contrast agent was developed. A prototype absorptiometry unit was built which consists of either a square or circular cross section acrylic (polymethylmethacrylate) cuvette connected to an arterial catheter at one end and a paristaltic pump at the other via PE60 surgical tubing. At opposing ends of the length of the cuvette were a 0.4 GBq 125I source and a scintillation crystal/photomultiplier tube assembly. This assembly was connected to a single-channel analyser (SCA)/scaler unit to count the transmitted photons through the cuvette. The scaler was interfaced to an IBM PC and counts accumulated in preset time intervals were transmitted to the computer via a serial interface. Experiments were performed to calibrate the unit for measurement of blood concentration of contrast agent (Isovue 300) and to determine the dispersion characteristics of the unit. Deconvolution was used to correct the measured concentration waveform for the dispersion introduced by passage through the lead-in tubing and the cuvette. The precision of concentration measurements was determined to be between 5 and 10% using computer simulations and theoretical calculations. The method was used successfully in a number of patient and animal studies to measure the contrast concentration in blood following intravenous injection of contrast agent.

Fibrous histiocytoma of the larynx.

Fibrous histiocytoma has a variable, unpredictable behaviour and potential for malignancy. The treatment of choice is wide local excision, which, in the larynx, entails total laryngectomy. A case of fibrous histiocytoma of the larynx, treated successfully by radiotherapy, is presented. A recurrence was treated with cryosurgery and the patient is well at 10-year follow-up. The pathological characteristics of fibrous histiocytoma and the difficulties of classification are reviewed.

Symptomatic hypocalcemia and hypoparathyroidism in two infants of mothers with hyperparathyroidism and familial benign hypercalcemia.

Neonatal hypocalcemia secondary to maternal hypercalcemia is relatively rare. We describe two infants with hypocalcemia and generalized seizures at 2 weeks and at 5 days of age, respectively. In the first case the infant was delivered of a mother who was subsequently diagnosed as having hyperparathyroidism. In the second case the infant was a member of a kindred with familial benign hypercalcemia. The evaluation of late-onset neonatal hypocalcemia should include an evaluation of maternal hyperparathyroidism and, in some cases, an evaluation for familial calcium disorders.

Multinomial logit analysis of injury severity in U.S. underground bituminous coal mines, 1975-1982.

Relationships among mine and injured miner characteristics and degrees of injury are examined for 91 404 injuries in underground bituminous coal mines in the United States from 1975 through 1982. Injury severity varies by the mining system, geographical region, circumstances surrounding the injury, the injured miner's age, whether the miner was using powered equipment, the year in which the injury occurred and the location in the mine where the injury happened. Injury severity is not related to the injured miner's total mining experience, job experience and experience in the mine in which the injury occurred.

Cytopathic effect of lentogenic strains of Newcastle disease virus in cultured chicken muscle cells.

The ability of lentogenic strains of Newcastle disease virus (NDV) to inhibit the development of embryonic chicken muscle cells in vitro is described. In contrast to the uninfected control, which displayed a thick network of branched multinucleated muscle fibers, cultures infected with NDV exhibited a severely reduced potential to differentiate. The few differentiated muscle fibers that formed in the presence of NDV were poorly developed, displayed an attenuated morphology, and detached from the substrate 2 to 3 days before uninfected cultures showed signs of cell aging. When NDV-infected cultures were stained before muscle fiber detachment with a fluorescent-labeled NDV-specific antibody, the infected muscle fibers reacted strongly relative to neighboring myoblasts and fibroblasts. All six strains of NDV induced a similar inhibitory effect, and no strain-specific differences were detectable. These results indicate that differentiating muscle fibers are highly susceptible to infection by NDV in vitro and that this tissue exhibits an early NDV-induced cytopathic effect.

Virus characterization and sequence of the fusion protein gene cleavage site of recent Newcastle disease virus field isolates from the southeastern United States and Puerto Rico.

Nine Newcastle disease virus (NDV) isolates obtained from Puerto Rico, Georgia, Alabama, Mississippi, and Texas were analyzed for in vivo pathogenicity, biological properties (hemagglutination of mammalian erythrocytes), and for sequence variation at the amino acid and sense RNA level of the fusion protein cleavage site. Intracerebral pathogenicity index values ranged from 0 to 0.3 and the intravenous pathogenicity index obtained for all isolates was 0. Four isolates hemagglutinated bovine erythrocytes, whereas no hemagglutination was observed using equine erythrocytes. By direct sequencing of reverse transcription polymerase chain reaction products, all the isolates had a predicted fusion cleavage sequence comparable to lentogenic NDV strains. Based on nucleotide sequence, the viruses could be grouped phylogenetically with the B1 vaccine-type virus. However, nucleotide sequences were not 100% similar to the B1 or La Sota NDV strains, indicating that minor genetic heterogeneity occurs among lentogenic field isolates of NDV.

A novel oligonucleotide probe for the detection of Newcastle disease virus.

An approach that possesses high specificity and broad applicability was used to obtain a DNA probe with potential diagnostic value. By utilizing synthesized oligonucleotide DNA, termed NDV probe, all 14 strains of NDV tested under high-stringency conditions were recognized in a slot-blot hybridization assay. The sequence of the NDV probe was generated from a highly conserved region of the NDV genome. No hybridization was observed with RNA isolated from other avian viruses, including avian influenza, infectious bursal disease, and infectious bronchitis. The specificity inherent in using an oligonucleotide probe offers advantages over probes obtained from cloned DNA fragments.

The Burn Wheel: a practical guide to fluid resuscitation.

During preparation for mass casualties of burn victims in the Gulf War (1990-91), a 'Battlefield Burns Table' was devised to allow rapid calculation of fluid replacement. By standardizing weights and using only Hartmann's Solution BP, a regimen could be prescribed by the non-specialist without resort to mathematical calculations. By means of a revolving disc, this has now been refined for use in a civilian setting where expert burn advice may not be immediately available.

Hemodynamic assessment of local anesthetic administration by laser Doppler flowmetry.

OBJECTIVE: The hemodynamic effects of local anesthetic administration with and without a vasoconstrictor were compared by using laser Doppler flowmetry. STUDY DESIGN: Seventeen people participated in a single study session in which they were given 2 intraoral injections. The injections, which were administered in random order, consisted of 1.8 mL lidocaine (2%) with epinephrine (1:100,000) and mepivacaine (3%). Hemodynamic parameters consisting of blood pressure, heart rate, and laser Doppler flowmetry were reordered at regular intervals. RESULTS: The laser Doppler flowmeter detected changes in the peripheral perfusion of the finger that were not detected by changes in blood pressure and heart rate. The greatest change was associated with anxiety and occurred just before the injection. The inclusion of epinephrine in the local anesthetic resulted in a persistence of these changes. CONCLUSION: This investigation has confirmed the sensitivity of laser Doppler flowmetry as an investigational tool for assessing hemodynamic changes associated with anxiety and the administration of local anesthesia.

Emergency drug therapy. Drugs and routes of administration.

Management of medical emergencies requires a thorough knowledge of specific medications and their routes of administration. This article briefly discusses the medications that should be included in a basic emergency cart and routes in which to administer them.

Polymorphonuclear leukocytes labeled with technetium-99m HMPAO. A potential bone marrow imaging agent.

A case of multifocal avascular necrosis of the bone complicating systemic lupus erythematosis is presented in which the extent of involvement was incidentally identified by Tc-99m HMPAO leukocyte imaging. Leukocytes labeled with Tc-99m HMPAO may be an excellent bone marrow imaging agent.

High tracheostomy and other errors--revisited.

Timely cricothyrotomy may be life-saving, but it is not without its complications. Together with tracheostomy performed too high, there are high incidences of stenosis and voice changes afterwards-often neglected because the patient has so many other problems. Jackson warned of these problems over 70 years ago-his message is still relevant.

Primary amyloidosis of the larynx.

The history, examination and operative findings of primary amyloidosis of the larynx are very suggestive of carcinoma, indicating the need for careful histological examination. Staining with Congo red shows a characteristic birefringence. Systemic amyloidosis may be present.

Emergency cricothyrotomy.

The indications for the ruling for the Gulf Forces that emergency cricothyrotomy is to be performed where the airway is thought to be compromised are reviewed. The advantages of this procedure are outlined and some of the likely consequences regarding the incidence of complications and their management predicted.

Draffin and his rods.

Draffin's rods or bipods are now a well-known, internationally accepted ENT instrument. Before their invention in 1951, the attendant anaesthetist or nurse was obliged to support the mouthgag during tonsillectomy. Research into the life of their originator, David Alexander Draffin, reveals that in addition to his evident design skills, he was a surgeon of courage, wit and great charm.