A clinical profile of otitis media.

An otitis media with effusion algorithm developed by Paradise et al and tested by Cantekin et al has become the basis for many studies of otitis media. However, a two-point scale algorithm (otitis media with effusion-no otitis media with effusion) may be too ambitious (ie, low specificity) and too optimistic (ie, absence of fluid does not necessarily mean normal ear). We propose a four-point profile that characterizes the condition of the middle ear, and we report the validation of the profile against findings at myringotomy. Statistically, a four-point scale profile would substantially increase powers of statistical tests, compared with a two-point scale algorithm (in studies of the same size), when used to study changes of the middle-ear condition over time or to compare treatment effects.


Bacteremia during tonsillectomy.

To determine the risk of bacteremia during tonsillectomy, we cultured blood specimens that were taken from 32 children during surgery and tonsillar swabs that were obtained just before excision, and compared the results with quantitative cultures of the excised tonsillar tissue. Twenty-five children had Haemophilus influenzae within the tonsillar tissue (density range, 10(3) to 10(8) colony-forming units per gram), and seven had Streptococcus pyogenes (density, 10(3) colony-forming units per gram in one case, 10(5) colony-forming units per gram in one case, and 10(6) colony-forming units per gram in five cases). Twelve perioperative blood cultures were positive; H influenzae was found nine times, and Micrococcus species was found one time, and alpha-hemolytic streptococci were found two times. Haemophilus influenzae was always present in the corresponding tonsillar specimens, although there was no apparent relationship between the density of colonization of the tonsillar tissue and a positive blood culture.


Nasal mucociliary transport of chronic sinusitis in children.

Nasal mucociliary function is one of the most important and indispensable mechanisms of the respiratory tract, providing protection against the atmospheric environment. We previously found mucociliary dysfunction in the noses of adult patients suffering from chronic sinusitis. In this study, using the saccharin method, we determined nasal mucociliary function in normal children and in children with chronic sinusitis. The mean (+/- SD) value of saccharin transit time in the nose was 28.2 +/- 19.9 minutes in patients with chronic sinusitis, this being significantly slower than that in the control group of children. The incidence of abnormally slow nasal mucociliary transport time (> 30 minutes) in patients was significantly higher than in controls of the same age. Mucociliary dysfunction may initiate a vicious cycle of self-mediated inflammation and may be important in recovery from chronic respiratory inflammation.


Oronasal reconstruction with local mucoperiosteal and free latissimus dorsi flaps.

We describe the use of maxillary sinus mucoperiosteum as a pedicled flap to reconstruct the floor of the nose following resection of cancer involving the palate, buccal mucosa, and mandible. Complete inferior turbinectomy allows access to the roof of the maxillary sinus to harvest the mucoperiosteum as a vascularized flap pedicled under the remaining middle turbinate. A free latissimus dorsi myocutaneous flap suspended from the floor of the orbit and nasal septum is then used to obliterate the maxillary sinus, reconstruct the palate, and support the floor of the nose. A reconstruction employing this combination of flaps is presented, including postoperative endoscopic intranasal photography and magnetic resonance imaging. Despite the elevated level of the floor of the nose and the absence of the inferior turbinates, rhinomanometry performed 3 months postoperatively demonstrates adequate nasal airflow with normal responses to nasal decongestants.


Sebaceous carcinoma of the head and neck. Case report and literature review.

Sebaceous carcinoma is uncommon, with fewer than 400 cases reported. Usually, lesions arise in the meibomian glands of the eyelid; however, extraocular lesions within the head and neck have been reported. Regardless of the location, sebaceous malignancies must be considered aggressive neoplasms with a potential for regional and distant metastasis. Diagnosis may be difficult, given the low incidence and inconsistencies in histopathologic classification. Recently, needle aspiration cytologic characteristics have been delineated, with this procedure becoming increasingly useful in establishing the diagnosis. Treatment requires wide surgical excision with removal of involved regional lymph nodes. Opinions are divided regarding the use of postoperative irradiation or chemotherapy. Records of all patients with sebaceous carcinoma of the head and neck treated at UCLA Medical Center, Los Angeles, over the last 35 years were reviewed. The clinical and pathologic features are discussed, and the literature is summarized.


The utility of magnetic resonance imaging in the diagnosis of intranasal meningoencephaloceles.

We present three patients in whom the diagnosis of intranasal meningoencephalocele was made by magnetic resonance imaging. The initial clinical evaluation and computed tomographic examinations of these patients failed to distinguish between chronic inflammation and intranasal meningoencephalocele. Although both computed tomography and magnetic resonance imaging are used to distinguish between normal, inflammatory, and neoplastic tissue in the nasal cavity and paranasal sinuses, limitations do exist and these are the focus of our communication. A clear understanding of the efficacy of these radiographic modalities will enhance surgical planning and can preclude severe complications.


Histopathology of the temporal bone following multichannel cochlear implantation.

The temporal bone histopathologic findings of a patient having received a Nucleus 22-channel cochlear implant (Cochlear Corp, Englewood, Colo) following bilateral transverse otic capsule fractures is presented. The case was complicated by perilymph fistulas, labyrinthitis, meningitis, and death due to drowning. The left cochlea revealed complete labyrinthitis ossificans; the implanted right cochlea demonstrated new bone formation in the basal turn of the scala tympani, degeneration of the organ of Corti, but normal ganglion cell counts. Results of psychophysical and speech perception 4 months after implantation are presented.


Cellular senescence.

The ageing of cells, cellular senescence, is an event that is encountered in all normal cells. Cells grown in vitro have a limited life span and do not grow well after a certain number of divisions. They cease to divide and eventually die. In accordance with this, the life expectancy of an established cell culture depends on the age of the donor. Cells that have undergone immortalization via a crisis period of transformation by chemicals or viruses, as well as malignant cell lines in general, have an ability to divide indefinitely. A distinct form of cell death, apoptosis or programmed cell death, is encountered in many physiological situations like in keratinocyte differentiation.


Progress in the treatment of childhood leukaemias.

The Nordic Society for Pediatric Hematology and Oncology (NOPHO) has run a population-based registry on all cases of acute leukaemias in the Nordic countries since 1981. Data on close to 2000 children with these diagnoses are presented and used as a background for a general discussion of progress in the therapy of these challenging conditions. Our material is unique in that it is population based. The results obtained are comparable to those obtained by all other major cooperative groups. Since January 1992, the treatment protocols for all types of acute leukaemias in childhood have been harmonized in the Nordic countries.


Leukocyte cell adhesion proteins: from molecular dissection to clinical applications.

Leukocyte adhesion is needed for a number of leukocyte functions like immunoglobulin synthesis, T and NK-cell-mediated cytotoxicity, phagocytosis by granulocytes, and cellular accumulation in inflamed tissue. Several cell surface molecules involved in leukocyte-leukocyte and leukocyte-target cell interactions have recently been identified and characterized. Both the polypeptide and carbohydrate portions are important in leukocyte interactions. It is becoming increasingly apparent that it is possible to interfere with the normal functions of the leukocyte adhesion glycoproteins, and such applications may become important in medicine.


Toxoplasma pericarditis mimicking systemic lupus erythematosus. Diagnostic and treatment difficulties in one patient.

A life-threatening T. gondii pericarditis developed in a patient with symptoms corresponding to systemic lupus erythematosus (SLE) with high concentrations of antinuclear antibodies and lymphadenopathy. The diagnosis would have been SLE-associated serositis, had not pericardial fluid been inoculated into mice, because pericarditis is frequently seen in SLE and false positive toxoplasma seroreactions may occur in ANA positive patients. High IgG T. gondii antibodies without increased IgM antibodies indicated reactivation rather than primary infection. Prolonged high-dose treatment with pyrimethamine-sulphadiazine was needed. Interestingly, the patient's SLE symptoms, including high ANA antibodies, declined to an unexpected remission after treatment for toxoplasmosis. This may not be mere coincidence, but may point to a causative role of toxoplasmosis in some cases of SLE.


Inhibition of colonic motility by cholecystokinin.

Distal colonic motor activity was measured in 12 control subjects and seven constipation-predominant irritable bowel syndrome patients to examine the effects of intravenous administration of cholecystokinin. In the basal state, no significant motility differences were noted between these two groups. Following the intravenous administration of the hormone cholecystokinin, a statistically significant reduction in colonic motility in control subjects and a non-significant decrease in motility in irritable bowel syndrome patients was seen. Our results do not suggest an exaggeration of the colonic motor response to cholecystokinin occurs in irritable bowel syndrome.


Genetic variants of apolipoprotein B: relation to serum lipid levels and coronary artery disease among the Finns.

The genetic variation of apolipoprotein B has been studied extensively in Finns with the aid of protein and DNA polymorphisms. Population association studies confirmed significant associations between the XbaI polymorphism of the apolipoprotein B gene and elevated serum cholesterol levels, and dietary studies suggested that the XbaI and ins/del polymorphisms might modify the serum lipid response to dietary fat intake. Studies comparing patients and controls, however, did not confirm previous studies suggesting that the multi-allelic variation at the 3'VNTR region of the apolipoprotein B gene was associated with coronary artery disease. The results suggest that the apolipoprotein B gene is involved in the regulation of serum lipid levels. A yet unknown mutation in linkage disequilibrium with the XbaI site may contribute to this regulation.


Molecular genetics of familial hypercholesterolaemia: common and rare mutations of the low density lipoprotein receptor gene.

Mutations of the low density lipoprotein (LDL) receptor gene give rise to familial hypercholesterolaemia (FH), one of the most common single-gene diseases in the world. Approximately 150 different LDL receptor gene mutations have been reported until now and the list seems to be continuously growing. Although hampering molecular genetic diagnosis of FH, this wide variability at the DNA level provides a useful tool to population genetics and may ultimately lead to better understanding of the variation in disease manifestations from family to family. The Finns are among the few populations in which one or two mutant LDL receptor genes explain the majority of FH cases. Either of the two 'Finnish-type' LDL receptor gene deletions, FH-Helsinki or FH-North Karelia, is present in more than 60% of the Finnish FH patients; there are no reports on their existence in other ethnic groups. Assays for these mutations were shown to markedly complement clinical diagnosis of FH in Finland.


Molecular basis for apo A-IV polymorphisms.

Apolipoprotein (apo) A-IV is involved in the metabolism of triglyceride-rich lipoproteins and high-density lipoproteins and may thus play an important role in lipid metabolism, although the precise physiological function of apo A-IV is unknown. Structural and functional alterations of apolipoproteins, caused by a variety of mutations, have been shown to influence lipoprotein metabolism in several ways. Genetic studies are consistent with a single locus for apo A-IV with two main codominant alleles A-IV1 and A-IV2. The basis behind some of these electrophoretic variants has been determined on a molecular level by the aid of the polymerase chain reaction and sequencing of the amplified DNA. For instance, apo A-IV1 and apo A-IV2 alleles are explained by glutamine for histidine change of amino acid 360. Furthermore, several polymorphisms not leading to charge differences have been characterized. The genetic variability of apo A-IV may be of help in elucidating the functional role of apo A-IV in lipoprotein metabolism. In our study, the frequencies of the alleles due to Gln360-->His and Thr347-->Ser substitutions in a Finnish population sample were similar to those reported from other populations. In addition we have identified a novel genetic polymorphism of apo A-IV, Asn127-->Ser. In the Finnish population the asparagine for serine change of amino acid 127 was the most common apo A-IV polymorphism. There are no reports from other populations concerning this polymorphism.(ABSTRACT TRUNCATED AT 250 WORDS)


Twin studies in metabolic diseases.

The traditional role of twin studies has been to assess the relative role of genetic factors as a first step in defining the genetic architecture of complex traits. This has been based on the realization that monozygotic pairs (MZ) share all their genes, while dizygotic pairs (DZ) share 50% of their genes on average. Thus, greater similarity of MZ pairs compared to DZ pairs has been taken as prima facie evidence of the role of genetic factors. This is true provided the environmental similarity of MZ pairs is not greater than for DZ pairs for effects relevant to the trait in question. This first step in genetic studies was carried out long ago in many research areas, but not in others. More detailed knowledge of the genetic architecture of traits is then obtained by other means. In this paper, we give a brief overview of some results for metabolic diseases (ischaemic heart disease, hypertension, subarachnoid haemorrhage, NIDDM and IDDM) using the classical twin approach in a large, unselected population-based twin cohort. We also outline approaches to using twins that we believe will continue to be useful, particularly for the study of environmental effects.


Multipoint analysis for radiation hybrid mapping.

Radiation hybrid mapping is a somatic cell genetic technique that permits construction of fine-structure maps of human chromosomes. Radiation hybrid mapping uses X-ray breakage of chromosomes to order genetic loci and to estimate distances between them. Cox and colleagues described radiation hybrid mapping and derived methods for locus ordering based on two-point statistical analysis of radiation hybrid mapping data. In this paper, I describe alternative non-parametric and likelihood methods for radiation hybrid mapping that make use of information on many loci simultaneously, and efficiently include information on partially typed radiation hybrids. Locus orders are selected by minimum number of obligate chromosome breaks or maximum likelihood. I demonstrate the utility of these methods with an application to data on 14 markers and 99 hybrids for human chromosome 21.


Empirical Bayes methods for testing associations with large numbers of candidate genes in the presence of environmental risk factors, with applications to HLA associations in IDDM.

Standard regression models for disease incidence data can be used to test for associations between a disease and measured genetic and environmental factors and their interactions. Complications arise when the gene is not observed, requiring segregation and linkage analysis approaches, or when the candidate gene(s) are found to be highly polymorphic, as in the HLA region. We propose a Bayesian approach to the latter problem, in which the log relative risks for all alleles at a given locus are taken to be independent and exchangeable, assuming there is no preferential zygotic assortment and negligible recombination. Multi-locus problems can be addressed either by adding exchangeable interaction terms or by adopting a multivariate prior for haplotype effects. Some simulations based on our current work on family studies of IDDM are discussed.


Genetic epidemiology of insulin-dependent diabetes mellitus: international comparisons using molecular genetics.

International comparative molecular studies of insulin-dependent diabetes mellitus can be viewed from an immunogenetical and an epidemiological perspective. Each approach independently contributes to our knowledge concerning the etiology and prevention of the disease. However, the combination of these perspectives, representing an interface between molecular biology and epidemiology, will achieve more in terms of scientific advancement and public health than either perspective alone. An excellent example of the development of molecular epidemiology has been the evolution of international comparative research through the WHO Multinational Project for Childhood Diabetes. Case-control molecular studies are currently being conducted to test the hypothesis that population variation in the frequency of HLA class II susceptibility alleles is the primary determinant of the global patterns of insulin-dependent diabetes mellitus incidence. Preliminary data suggest that this hypothesis is correct. Future international molecular epidemiological studies, encompassing host-environment interactions, will lead to a better understanding of the relationships among specific risk factors within populations and across the world. Molecular epidemiology represents a new paradigm for studying the etiology and epidemiology of infectious and non-communicable diseases, and no doubt represents a major challenge for the 1990s.


The future of multilocus linkage analysis.

Current limitations and needs of linkage analysis are discussed. Possible future developments in analysis methods and computer implementation are outlined.


Transgenic animals in lipoprotein research.

Lipid metabolism is a complex system involving the interaction of several lipid-transporting and -transferring serum proteins, enzymes regulating lipoprotein synthesis and modification, and lipoprotein receptors. Several epidemiological studies have demonstrated that many abnormalities in lipid metabolism are associated with coronary heart disease risk. There are at least 17 different genes that code for proteins directly involved in lipoprotein metabolism. Naturally occurring mutations in many of these genes have been described that affect either the quantity or the quality of these proteins. Such genetic alterations aid in the precise evaluation of the function of these gene products. Transgenic animal technology has made it possible to create genetic alterations and to study the consequences of overexpression of a gene and expression of a mutated protein, and also to study species-specific differences between proteins.


Approaches to gene therapy of complex multigenic diseases: cancer as a model and implications for cardiovascular disease and diabetes.

The general concept of gene therapy is now well established and accepted by the medical, scientific and public policy communities, and is rapidly being implemented in human experimental studies. In addition to the initial models of single gene defects, target diseases have now come to include multigenic and multifactorial diseases such as human cancer, neurodegenerative diseases such as Parkinson's disease and firms of cardiovascular disease. While many conceptual and technical obstacles must still be overcome before therapy for disorders such as coronary artery disease and diabetes mellitus will easily be approached at the genetic level, the early results with several multigenic disease models gives some cause for optimism that gene therapies for even those complicated disorders will eventually become available.


Stability of the intelligence quotient-language quotient relation: is discrepancy modeling based on a myth?

Diagnostic and service eligibility decisions are often made on the basis of the relation between assessments of cognitive ability and assessments of language ability. A discrepancy between cognitive and language performance, with cognition being higher, is thought to be a positive predictor of ability to benefit from intervention. This triage model assumes reasonable stability of cognitive and language measures over time. We evaluated the stability, over 1- and 2-year periods, of the relation between a cognitive measure and two language measures. We examined whether the cognitive-language relation for these measures changed significantly over time and whether children remained in the same service eligibility category, based on the discrepancy model, over time. Results indicated substantial changes in the cognitive-language relation over time. Children's eligibility for service, based on the discrepancy model, also fluctuated greatly over time. We conclude that a discrepancy model of triage based on broad cognitive and language measures is not an effective means of determining who should receive service. Alternative triage methods were presented.


Development and training of referential communication in children with mental retardation.

In referential communication, the speaker must communicate to the listener about how a referent is different from other stimuli (i.e., employ the "difference rule"--Whitehurst & Sonnenschein, 1985). In Experiment 1 we compared use of the difference rule by children with and without mental retardation matched for Quick Test MA. Participants with mental retardation were less likely to employ the difference rule. Experiment 2 demonstrated that perceptual feedback training, which teaches use of the difference rule, enhances referential communication of children with mental retardation.


Degree of associative relatedness and sentence processing by adolescents with and without mental retardation.

Previous research has suggested that individuals with mental retardation are less likely than individuals without mental retardation to access and incorporate information about the relations between words of sentences in the representations of those sentences in memory (e.g., Merrill & Bilsky, 1990; Merrill & Jackson, in press). A cued recall study and a semantic verification study were conducted to determine whether the magnitude of this group difference could be made smaller by increasing the degree to which the words in the sentences were semantically related. In both experiments, individuals with mental retardation exhibited an ability to utilize contextual information to a greater extent when the words were related. In the highly related conditions, the differences between groups was virtually eliminated.


Profile of the prelinguistic intentional communicative behaviors of children with profound mental retardation.

The prelinguistic intentional communicative behaviors of 10 noninstitutionalized children with profound mental retardation were analyzed according to five profile parameters: communicative rate, communicative function, discourse structure, communicative means, and syllable shape. Subjects used a predominance of regulatory communicative functions, initiated discourse structure, gestural communicative means, and nontranscribable vocalizations/vocalizations without consonants. Findings were compared to previously reported data from children without mental retardation and institutionalized children with severe and profound mental retardation.


Linguistic imitation in children with Down syndrome.

Rate of linguistic imitation by 48 children with Down syndrome was compared to that of 57 children without mental retardation. Both groups were taken from different corpora within the Child Language Data Exchange System (CHILDES, MacWhinney, 1991). All speech data were collected from spontaneous speech samples of mothers interacting with their children. Three types of imitative utterances (exact, expanded, and reduced) were coded using an automatic data analysis program called CHIP (Sokolov & MacWhinney, 1990). Multiple regression was utilized to test for group differences while controlling for variability in MLU. The results indicated that children with Down syndrome imitated slightly less, but the exact nature of this difference was related to language level and the source of the imitation. As MLU increased, the rate of imitation decreased at a much steeper rate for children without mental retardation than for children with Down syndrome. In addition, children with Down syndrome showed a different pattern of results for imitations of their mothers than for self-repeated imitations. The results suggest that children with Down syndrome develop differently with respect to linguistic imitation.


A rapid in situ respiration test for measuring aerobic biodegradation rates of hydrocarbons in soil.

An in situ test method to measure the aerobic biodegradation rates of hydrocarbons in contaminated soil is presented. The test method provides an initial assessment of bioventing as a remediation technology for hydrocarbon-contaminated soil. The in situ respiration test consists of ventilating the contaminated soil of the unsaturated zone with air and periodically monitoring the depletion of oxygen (O2) and production of carbon dioxide (CO2) over time after the air is turned off. The test is simple to implement and generally takes about four to five days to complete. The test was applied at eight hydrocarbon-contaminated sites of different geological and climatic conditions. These sites were contaminated with petroleum products or petroleum fuels, except for two sites where the contaminants were primarily polycyclic aromatic hydrocarbons. Oxygen utilization rates for the eight sites ranged from 0.02 to 0.99 percent O2/hour. Estimated biodegradation rates ranged from 0.4 to 19 mg/kg of soil/day. These rates were similar to the biodegradation rates obtained from field and pilot studies using mass balance methods. Estimated biodegradation rates based on O2 utilization were generally more reliable (especially for alkaline soils) than rates based on CO2 production. CO2 produced from microbial respiration was probably converted to carbonate under alkaline conditions.


The correlation of middle ear aeration with mastoid pneumatization. The mastoid as a pressure buffer.

Atelectatic ears, which by definition are poorly aerated, are also usually associated with poor mastoid pneumatization. On the other hand, otosclerotic patients, whose middle ears are usually exceptionally well aerated, also have excellent mastoid pneumatization. Three unusual cases are presented, in which partial atelectasis developed in stapedectomized patients. In each case the mastoid was later found to be nonpneumatized, and further analysis revealed that their stapes fixation had in effect most probably been of non-otosclerotic origin. Thus, although these three cases had at first appeared to represent exceptions to the general rule of otosclerotics having a well-aerated middle ear, in fact they support the association between atelectasis and poor pneumatization. The linkage of good middle ear aeration with large mastoid pneumatization and vice versa may suggest that the mastoid plays a role of a pressure buffer in the middle ear, which is a system of a gas pocket with fluctuating pressures. Also, otosclerosis may be considered to be an unlikely cause of conductive deafness in cases of poor pneumatization.


Late effects of radiotherapy on hearing.

The effect of radiotherapy on hearing was studied in 30 patients who were treated by surgery and radiotherapy for a parotid neoplasm. Functions of the irradiated ear were compared with those of the non-irradiated ear in the same patient. Tympanometry showed a small but significant reduction of static compliance on the irradiated side when compared with the non-irradiated side. Audiometry showed a significant increase in hearing loss in the 1-2, 4-8 and 10-20 kHz ranges that increased with frequency. This hearing loss was mainly sensorineural in contrast to hearing loss at 250-500 Hz, where it was more of a conductive type. There appeared to be a significant dose-effect relation in sound perception at 4-8 kHz. Doses to the cochlea of less than 55 Gy seldom caused a hearing loss, in contrast to doses exceeding 65 Gy. Overall, radiotherapy was found to cause significant, mainly sensorineural hearing losses, which were partly dose-dependent.


Mode of action of photodynamic therapy with sulfonated aluminum phthalocyanine in induced squamous cell carcinomas in animal models.

In order to investigate the mechanism of action of photodynamic therapy (PDT) with sulfonated aluminum phthalocyanine (AlSPc) in squamous cell carcinoma, animal experiments were performed in induced carcinomas of the mucosa of the hamster's cheek pouch and skin of the laboratory mouse. Histological examinations revealed signs of massive interstitial bleeding, indicating a vascular response to PDT with AlSPc. It was also possible to induce similar change adjacent to newly formed vessels at the margin of an inflammatory reaction in the cheek pouch of five hamsters in the absence of tumor cells. Implanted human squamous cell carcinoma cells in athymic nude mice showed that carcinoma cells removed immediately following PDT remained viable, while tumors left in situ became necrotic. These results suggest that the primary effect of PDT with AlSPc in vivo is not the malignant cell itself, but the vascular stroma of the tumor or in the immediate vicinity of the latter.


Kinin generation following methacholine nasal airway challenge of non-allergic subjects.

Following nasal challenges with the muscarinic neurotransmitter methacholine in 14 patients with chronic non-allergic rhinitis, kinin generation was induced. This occurrence was inhibited by pretreatment with anticholinergic agents (ipratropium bromide) and by an overdose of neurotransmitter, indicating that a muscarinic receptor is stimulating the liberation of kinin. These observations suggest that patients with chronic non-allergic rhinitis exhibit cholinergic neurotransmitter-induced kinin generation.


Changes in voice characteristics after uvulopalatopharyngoplasty.

Previous reports have warned that tonsillectomy or uvulopalatopharyngoplasty (UPPP) may alter patients' speech by increasing the amount of nasal resonance as well as by changing voice timbre due to enlargement of the vocal tract. However, very few objective investigations, excluding nasality problems, have been carried out. We studied eight patients who underwent surgery for UPPP and recorded pre- and postoperative speech. The speech samples were then rated by seven experienced listeners, and acoustic spectra of two long vowels,/a/ and /e/, were analyzed using a computer program (MacSpeech Lab II). The listeners were unable to make a distinction between pre- and postoperative voice samples in the recordings studied. Acoustic analysis showed that the fundamental frequency as well as the first and second formants remained essentially unchanged. Present findings show that UPPP should not have a significant effect on voice characteristics as long as excessive nasality is not produced.


Maturation of binaural interaction components in auditory brainstem responses of young guinea pigs with monaural or binaural conductive hearing loss.

Reversible conductive hearing loss created during the first 4 weeks post partum caused marked alterations in the maturation of binaural interaction components in the auditory brainstem responses of guinea pigs. In untreated control animals all three components investigated demonstrated postnatal development in terms of latency shortening that was completed during the first 3 weeks of life. Plugging of both external ear canals caused a significant delay in the maturation of the late component DN2, where latency values of the controls were reached only 2 weeks after the end of the treatment, i.e. after 6 weeks of life. Monaural deprivation likewise led to a retarded development of peak latencies during the phase of imbalanced sensory input. After the end of the one-sided conductive hearing loss the maturation process was markedly enhanced, even resulting in latency values for DN2 and DP1 that were significantly shorter than those of the controls. This phenomenon persisted until the end of the study period and was the case for both plugged and untreated ears in this group of animals. The time course of latencies in two other groups of experimental animals which were deprived in the same way as adults suggests that the effects observed are due to a sensitive period in the maturation process of the auditory pathway.


Long-term hearing results of one-stage tympanoplasty for chronic otitis media.

A study of 277 ears with chronic otitis media undergoing one-stage tympanoplasty has been carried out. The mean follow-up period was 6.4 years. Ears with intact ossicular chains were excluded. Ossiculoplasty was performed using autologous ossicles or cortical bone. Mean hearing gain was 10.8 dB. Closure of the postoperative air-bone gap to within 20 dB was achieved in 51% of cases. The best results were obtained in ears with intact stapes, while cholesteatomatous ears showed poorer results than other chronic ears. The present findings show that autologous ossicle and cortical bone are still suitable for ossicular reconstruction in chronic ears, especially when one-stage surgery is preferred.


The anterior inferior cerebellar arterial network supplying the rat cochlea and its role in autoregulation of cochlear blood flow.

The sensitivity of the cochlea is dependent upon maintenance of a delicate homeostatic environment. One mechanism which participates in providing this environment is the autoregulation of cochlear blood flow. This autoregulation is ensured through the interaction of sympathetic, peptidergic and hemodynamic mechanisms. The current study demonstrates an adaptation that also participates in cochlear blood flow autoregulation. Specifically, an anterior inferior cerebellar arterial network is described and the relative contributions of each of its vessels to total cochlear blood flow is measured using laser Doppler flowmetry. The results show that each collateral vessel contributes to the blood supply of the cochlea and that reperfusion is accompanied by hyperemia. These findings suggest an adaptation that provides stable blood flow through redundancy and compensatory potential. Additionally, these observations have implications for experimental models of ischemia.


Surgical lengthening of the short columella: division of the depressor septi muscles.

A new surgical procedure is described to correct the difficult problem of the short columella without resorting to cartilage grafts. The ready recourse to these grafts, so much in vogue at the present time in primary rhinoplasties, should be carefully and completely re-examined, since the final result very frequently yields no real benefits and may permanently deface the area from which the cartilage has been taken. The present technique combines trimming of the upper lateral nasal cartilages with approximation of the alar cartilages and excisions of the depressor septi muscles and/or contiguous orbicularis oris muscle.


A histofluorescent study of sympathetic innervation of human palatine tonsils.

The glyoxylic catecholaminergic histofluorescence method was employed on human palatine tonsil specimens in order to study the sympathetic innervation present. One percent neutral red was used as a counterstain. Abundant sympathetic fibers were demonstrable around the blood vessels of the medulla and capsule. However, few sympathetic fibers were found around the vessels of the subepithelial connective tissue and interfollicular septa. In the areas of the follicle and extrafollicle where B and T lymphocytes were located, sympathetic fibers were not found. These findings indicate that if sympathetic innervation can affect T and B cells, it will do so indirectly. Results also show that there is a higher norepinephrine content in focally infected tonsils that is not due to hyperactivity of the sympathetic nerve, but may be due to other mechanisms. Finally surgical dissection at the capsule during tonsillectomy will reduce bleeding, perhaps because vessels there have an abundant sympathetic innervation that leads to good vessel contraction.


Induction of transformation of human respiratory epithelium in vitro. Preliminary investigation.

Malignancy is the result of multistep transformational changes of normal somatic cells. In the case of respiratory epithelial malignancies this process lasts for several years. Many methods have been explored to mimic this process in an extracorporal model. In the present investigation we combined several of these methods. Organ cultures were prepared from tracheal specimens and were then consecutively treated with human papilloma virus, benzo(a)pyrene, methylnitronitrosoguanine and tetradecanoyl phorbol acetate. Identical numbers of organ cultures from the same specimen were maintained without exposure to carcinogens. After 6 weeks these cultures were further cultivated either in mixed cultures (MC) with autologous isotopic fibroblasts or under the kidney capsule of the nude mouse (SRC). These two methods were combined after a few months: MC cells were transplanted under the SRC or SRC transplants were explanted in cell culture. This long-term selection procedure revealed striking differences between control and treated organ cultures. Three-dimensional structures containing epithelial cells were isolated from both organ cultures but survived more than 3 months only from treated cultures. Only MC from treated organ cultures produced nodules under SRC. The incidence and morphology of the nodules in the SRC were directly related to carcinogen treatment, with more nodules with pronounced epithelial cell atypia obtained from treated organ cultures. MC and SRC showed the importance of a time factor for selecting cells with changed growth behavior--increased time increased the incidence of such cells.


Clinical experience with the low-resistance Groningen button.

Since its introduction in 1980, the standard Groningen button prosthesis has been of proven value for post-laryngectomy voice rehabilitation. Its relatively high airflow resistance has, however, been the cause of failure in achieving good post-laryngectomy tracheoesophageal shunt speech in some patients. The low-resistance Groningen button (LRGB) was therefore designed, thus reducing airflow resistance by 50% when compared to the standard device. The clinical performance of the LRGB, and the effect of prophylactic amphotericin, was assessed in 32 patients. The device lifetime, intratracheal phonatory pressures and patients' subjective acceptances of the prosthesis were recorded. Present findings showed that the majority of the patients preferred the LRGB as it required less effort to operate than the standard device. The intra-tracheal phonatory pressure was found to increase with time, but this change was prevented with the prophylactic use of amphotericin to inhibit concurrent colonization by Candida spp, which also helped to prolong the prosthesis lifetime.


Hepatic regulation of renal function.

Despite apparently conflicting reports in the past, the bulk of evidence presently available points to a significant role for the liver in the regulation of renal function. Hepatic regulation of renal function may involve both a hepatorenal reflex and a liver-borne diuretic factor (LBDF and/or 'glomerulopressin'). The hepatorenal reflex is elicited by an increase in intrahepatic pressure, and/or certain amino acids in portal venous blood. It is transmitted by serotonin in the liver and presumably by noradrenaline in the kidney. It leads to a marked decrease in renal blood flow, glomerular filtration and urinary flow rate. The evidence for the LBDF is still circumstantial. The LBDF may be stimulated by glucagon and adenosine. It leads to a marked increase of renal blood flow, glomerular filtration rate and urinary output. Amongst the conditions presumed to be associated with altered hepatic regulation of renal function are postprandial hyperfiltration, and the deterioration of renal function which occurs in liver disease, cardiac insufficiency and cardiovascular shock.


Position sense at the proximal interphalangeal joint is distorted in patients with rheumatoid arthritis of finger joints.

The results of this investigation demonstrate, using a position matching paradigm, that the ability of subjects to detect changes in the position of the proximal interphalangeal joint is impaired in patients with rheumatoid arthritis affecting this joint. In this group there is a systematic flexion bias in position judgements compared to an age- and sex-matched control group. This bias becomes progressively more pronounced at more extended positions. These results suggest that chronic inflammatory joint disease significantly alters proprioceptive sensations at finger joints and this may be due to the loss or distortion of afferent feedback from mechanoreceptors innervating the affected joint.


A study of the expression of laminin in the spinal cord of the frog during development and regeneration.

In the present experiments, we have used an affinity-purified polyclonal antibody to laminin to determine the time course of expression of laminin in the central nervous system (CNS) of Rana temporaria tadpoles during normal development and during restoration of the dorsal columns of the spinal cord. Immunoblotting analysis indicated that in the peripheral nervous system (PNS) of adult frogs the antibody recognized proteins of molecular weights 350-400 and 205-220 kDa, corresponding to the A and B chains respectively of mammalian laminin. Immunohistochemistry with our antibody suggested that laminin was absent from the tadpole spinal cord, and did not appear even in the basal lamina of the blood vessels within the spinal cord until after metamorphosis. Furthermore, there was no evidence of laminin expression in the dorsal columns after hemisection of the spinal cord. However, throughout development laminin was present in basal lamina outside the CNS, in particular in the pial membranes, and in the basal lamina of blood vessels and sheath cells in the dorsal and ventral roots. Electron microscopy showed that the blood vessels of CNS capillaries had basal laminae throughout development that was morphologically indistinguishable from that seen in peripheral vessels.


The role of cholecystokinin in inhibition of gastric emptying by peptone in the rat.

It is clear that the intestinal hormone cholecystokinin (CCK) inhibits gastric emptying, but doubts remain about the physiological significance of this action. Evaluation of the apparently conflicting data is complicated by the fact that little is known of the duration of action of CCK-releasing meals in delaying emptying. We have studied this issue by following the emptying of the second of two successive liquid test meals instilled into the stomach in conscious gastric fistula rats. Prior administration of peptone, but not saline, delayed the emptying of subsequently administered saline and delayed still further the emptying of subsequently administered peptone. The action of isotonic peptone lasted about 10 min from the initial instillation into the stomach. Radioimmunoassay of plasma CCK indicated a significant increase 5 min after intragastric peptone, and a still further rise occurred 5 min after administration of the second of two consecutive peptone meals; 21 min after the first meal, plasma CCK had returned to basal levels. Intravenous infusion of CCK in a dose that matched the inhibition of gastric emptying caused by peptone gave plasma concentrations about 35% higher than those seen 5 min after the second of two consecutive peptone meals. It is concluded that a liquid test meal of peptone delays gastric emptying in part through release of CCK and that the response lasts 10 min or less. The relatively short duration of action of endogenous CCK released by a single protein-rich meal in the rat should be kept in mind in interpreting the significance of studies on the physiology of CCK.


Interdigestive gastric blood flow: the relation to motor and secretory activities in conscious dogs.

Blood flow in the left gastric artery was measured with an electromagnetic flowmeter in nine conscious beagle dogs. In the fasting state gastric motility and secretion exhibited periodical changes with an average cycle interval of 115.4 +/- 9.7 min. During a quiescent period, when gastric motility and secretion were minimal, the mean blood flow was stable at 33.9 +/- 3.8 ml/min. During the contracting phase each peristaltic contraction was coupled with a rapid fall and rise in blood flow (from 10.5 +/- 1.9 ml/min below to 21.2 +/- 3.8 ml/min above the precontraction levels) in 20-30 s. In addition there was a sustained elevation in blood flow (58.6 +/- 6.4 ml/min at the peak) lasting for 29.1 +/- 2.8 min. The onset of sustained blood flow elevation was preceded by that of motility in 63% of the cycles. In 23% of the cycles blood flow started to rise before the contracting phase began. Pepsin peaks coincided with blood flow peaks in two dogs and preceded the latter in the others. Feeding abolished periodic increases in motility and blood flow. It is concluded that left gastric arterial blood flow is not steady but exhibits dynamic changes in phase with periodic motor and secretory activities of the stomach in fasting conscious dogs.


Effect of cortisol on blood pressure and vascular reactivity in the ovine fetus.

The aim of this study was to investigate the cardiovascular effects of exogenous cortisol in fetal sheep, (a) between 100 and 120 days of gestation when cortisol production is minimal and (b) after 130 days when endogenous plasma cortisol starts to rise. Chronically cannulated ovine fetuses (103-120 days, n = 9; 130-137 days, n = 7), received sequentially a 24 h infusion of vehicle (0.9% sodium chloride) and a 24 h infusion of cortisol at 100 micrograms/h. Blood pressure and heart rate changes to bolus injections each of angiotensin II and noradrenaline (0.2, 0.5, 1.0, 2.0 micrograms) were measured before and after the saline and cortisol infusions. Fetuses in each age group, served as additional controls receiving 48 h saline infusions. In both immature and mature age groups, the cortisol infusion increased basal fetal blood cortisol concentrations by 33.7 and 35.4 nmol/l respectively. In the immature group, cortisol, but not saline, caused significant 14.3 and 15.3% increases in basal systolic and diastolic pressures respectively. Basal blood pressure was higher in the mature group, but did not increase further despite the increase in cortisol levels. Furthermore, vascular responsiveness to angiotensin II but not to noradrenaline was significantly enhanced following the cortisol infusion, at both ages. Fetal heart rate did not change following the cortisol infusion. Exogenous cortisol contributes to the regulation of fetal blood pressure in the immature fetus, when other mechanisms have not developed. Cortisol might achieve this, in part, by enhancing vascular sensitivity to angiotensin II.


Myoplasmic Mg2+ concentration in Xenopus muscle fibres at rest, during fatigue and during metabolic blockade.

Intracellular free Mg2+ concentration ([Mg2+]i) was measured in isolated single fibres of Xenopus muscle using the fluorescent Mg2+ indicator furaptra. In resting muscle the [Mg2+]i was 1.7 mM in a Mg(2+)-free Ringer solution. There was no significant change in [Mg2+]i over 2 h in Mg(2+)-free Ringer solution. Elevating extracellular [Mg2+] to 40 mM for 5 min caused a small rise (0.13 mM) in [Mg2+]i. There was no detectable rise in [Mg2+]i after 5 min in Na(+)-free Ringer solution. These results suggest that the membrane is relatively impermeable to Mg2+ and that there was no detectable Na(+)-Mg2+ exchange over 5 min. When muscle fibres were fatigued by repeated tetani continued until force declined to about 40% of control, [Mg2+]i showed characteristic changes. During the early period of fatigue when force first showed a small decline and then became almost stable, [Mg2+]i was unchanged; during the final period of fatigue when force declined more rapidly, [Mg2+]i increased by 0.8 mM. Recovery of [Mg2+]i took about 30 min. Recovery of force was complex: tetanic force first declined (post-contractile depression) and then slowly recovered to control. Since the minimum force occurred at about the time when [Mg2+]i had recovered, it seems unlikely that post-contractile depression is caused by elevated [Mg2+]i. Rigor, produced by inhibiting oxidative phosphorylation and glycolysis, was associated with a larger increase (1.6 mM) in [Mg2+]i than fatigue. The rise in [Mg2+]i during fatigue and metabolic blockade could be explained as release of Mg2+ normally bound to ATP. A model of the metabolic changes and the resulting increase in [Mg2+]i explains our results reasonably well.


Electrical resistances of cultured bovine arterial endothelium in solutions of various resistivities.

Relationships between the electrical resistance (Rm) of cultured endothelium and the resistivity (rho) of the bathing solution have been investigated, using eight monolayers of bovine arterial endothelium. Bathing solutions of differing rho were prepared by mixing culture medium and isosmolar (290 mM) sucrose in various proportions. Relationships between 1/Rm and 1/rho were linear, as predicted, with a mean 1/Rm intercept of zero. The slopes of the relationships give a mean fractional area per unit path length (Ap/delta chi) of 12.7 cm-1 for the paracellular pathways of the cultured endothelium. This resembles previous estimates of Ap/delta chi for mammalian endothelium in arteries. The 1/Rm intercept of zero indicates that the major electrical conducting pathways of cultured arterial endothelium are paracellular.


The protective effect of damaging eccentric exercise against repeated bouts of exercise in the mouse tibialis anterior muscle.

Using a damaging eccentric exercise regime of the mouse tibialis anterior (TA) muscle we have investigated the extent and time course of protection afforded by one bout of exercise against damage resulting from a second bout of activity. Maximal force and fibre morphology were preserved if the exercise was repeated within 21 days, but by 84 days muscles once again became susceptible to damage. Low-frequency force loss had a shorter time course of protection against repeated exercise, lasting less than 21 days. The results provide evidence for different mechanisms contributing to the development of muscle damage following eccentric exercise and provide a basis for characterizing the adaptive response of muscle to damaging exercise.


Intracerebroventricular angiotensin II-induced thirst and sodium appetite in rat are blocked by the AT1 receptor antagonist, Losartan (DuP 753), but not by the AT2 antagonist, CGP 42112B.

In the rat, intakes of water and 1.8% NaCl induced by I.C.V. angiotensin II were inhibited by prior I.C.V. injection of the angiotensin subtype 1 receptor antagonist, Losartan, but not by the subtype 2 receptor antagonist, CGP 42112B. Drinking induced by I.C.V. carbachol was unaffected by either antagonist.


An investigation into the extent to which flare in human skin crosses the mid-line.

We investigated whether flare crossed the mid-line in various positions in humans using laser Doppler flowmetry to confirm visual observations. On the back and forehead, flare crossed the mid-line. However, on the posterior part of the neck, flare did not cross the mid-line, an observation that may be related to the elongated shape of flare in this region.


Whole mount in situ detection of low abundance transcripts of the myogenic factor qmf1 and myosin heavy chain protein in quail embryos.

We describe a whole mount in situ hybridization procedure to detect and localize low abundance transcripts (qmf1) and myosin heavy chain proteins in quail embryos. The critical factor in transcript detection was the extent of proteinase K digestion. Optimal digestion increased probe accessibility and reduced background. The use of digoxigenin-labeled probes and immunological detection with anti-digoxigenin-alkaline phosphatase conjugated antibody allowed signal development in 6-10 h, unlike the 7 days required using 35S-labeled probes. Myosin heavy chain proteins were detected using immunofluorescence and confocal laser scanning microscopy to allow precise localization of signal within developing embryos.


Single-cell cDNA-PCR: removal of contaminating genomic DNA from total RNA using immobilized DNase I.

A procedure utilizing immobilized DNase I that allows the efficient amplification of cDNA by PCR from a single cell in the absence of contaminating genomic DNA is described. DNase I treated, total RNA derived from single cells was reverse transcribed into cDNA followed by PCR using beta-actin and c-fos specific primers that recognize different exons of the respective genes. Amplification products corresponding to cDNA, but not to genomic sequences, were detected after treatment with immobilized DNase I in samples previously shown to be contaminated with genomic DNA. This method allows the efficient removal of DNA contaminating total RNA derived from a single cell.


A simple and rapid method for screening transformant yeast colonies using PCR.

A simple and rapid procedure for screening transformant yeast colonies is described. In this method, a trace amount of plasmid DNA is isolated from a small amount of yeast cell mass; then, the presence of the exogenous DNA in each yeast colony is detected by PCR amplification.


DNA elution and amplification by polymerase chain reaction from dried blood spots.

A quick, sensitive and easily automatizable method for PCR amplification of genomic DNA eluted from dried blood spots is described. DNA is eluted from a 3-mm spot routinely used for neonatal screening of inherited diseases either by boiling or by sonication. A preliminary and brief spot-autoclaving step is mandatory to ensure optimal and reproducible PCR amplifications. Only 1% of the eluted DNA is required for PCR analysis allowing the execution of multiple genetic tests on the same blood spot. The method has been successfully applied to the detection of a known phenylketonuria-causing mutation and will facilitate the analysis of the genetic repository provided by Guthrie's cards stored in neonatal screening laboratories.


Periodic acid/Schiff staining of glycoproteins immobilized on a blotting matrix.

A simple and sensitive method for the detection of glycoproteins and glycopeptides in solution and in polyacrylamide gels is described. This method combines the well-known periodic acid/Schiff stain with protein blotting. Compared with direct staining of a polyacrylamide gel, the sensitivity is considerably increased. Using a set of glycoproteins, we have found the sensitivity to be about 4 ng carbohydrate.


A visual method for rapid screening of xylose-fermenting ethanolic strains of Klebsiella pneumoniae.

A simple and rapid screening method for selecting hyper-ethanolic strains of Klebsiella pneumoniae is described. The method involves a novel biological screening marker, namely, the yeast Candida ethanothermophilum. The screening marker was seeded on an agar plate to the surface of which agar blocks, each containing a colony of K. pneumoniae, were subsequently fixed. This seeded plate lacked sources of carbon and energy. Ethanol formed in the agar blocks by the K. pneumoniae colonies diffused into the seeded medium and served as a carbon and energy source for the ethanotrophic yeasts. Colonies of yeasts appeared around the agar blocks in regions of ethanolic diffusion. Hyper-ethanolic strains of K. pneumoniae were thus selected on the basis of the number of colonies of the screening marker that appeared around the blocks containing the ethanolic colonies of K. pneumoniae.


Production of recombinant porcine tumor necrosis factor alpha in a novel E. coli expression system.

DNA sequences encoding porcine tumor necrosis factor alpha (TNF alpha) were reconstructed from a genomic-derived PCR product for expression in Escherichia coli. A synthetic DNA primer containing most of exon III was fused to exon IV sequences by means of PCR. The fused product was then inserted into the novel FLAG vector by restriction and ligation. This initial recombinant construct was propagated in single-strand form through use of a helper phage and subjected to oligonucleotide-directed mutagenesis for the purpose of introducing additional coding sequences from exons II and III. The final construct encoded a fusion protein consisting of the Omp-A signal peptide, a seven-amino acid FLAG peptide and the soluble form of porcine TNF alpha. Bacteria containing this construct produced a protein which was recognized by anti-FLAG monoclonal antibody in Western blots and which was purified by anti-FLAG immunoaffinity chromatography. The purified material was cleaved with enterokinase to remove the FLAG peptide. Both the enterokinase-cleaved form and the uncleaved form were shown to have TNF activity in a WEHI cell cytotoxicity assay.


A PCR-based method for manipulation of the vaccinia virus genome that eliminates the need for cloning.

A general method is described for altering specific genes of vaccinia virus (VV). We demonstrate and evaluate the procedure by gene inactivation, using a dominant selectable marker in conjunction with recombinant polymerase chain reaction (PCR). Primers based on the sequence of the target gene enable amplification of flanking arms and their subsequent attachment to the gpt cassette that confers resistance to mycophenolic acid. Linear PCR constructs are transfected into cells infected with wild-type vaccinia virus. Mutant viruses with gpt inserted into the target gene by homologous recombination are then selected by growth in the presence of MPA. This technique was applied to the vaccinia virus thymidine kinase gene and compared to the traditional method of constructing gpt-containing plasmids by cloning. The PCR scheme was found to be highly efficient and could theoretically be used to insert any foreign DNA element into any nonessential target gene for which partial or complete sequence information is available. The procedure can potentially be used for a wide variety of genetic modifications, including the insertion of foreign genes, with poxviruses and other DNA viruses. Genomes of microorganisms, such as bacteria and yeast that can be transformed with linear DNA, are also candidates for manipulation by this methodology.


A rapid and reliable method to create tandem arrays of short DNA sequences.

Tandemly polymerized regulatory elements, antisense RNA segments or ribozymes are potentially useful in selective gene silencing. However, existing methods of tandemly polymerizing short DNA segments are laborious. We present a procedure that can create cloned arrays of 40-70 monomer units in two steps. We have created long arrays of regulatory elements and potential ribozyme sequences. Silencing of human immunodeficiency virus (HIV-1) activation by tandem arrays of a regulatory element in human immune system cells and in other human and monkey cells is discussed.


Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.


Cyclin B in fish oocytes: its cDNA and amino acid sequences, appearance during maturation, and induction of p34cdc2 activation.

Under the influence of maturation-inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation-promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. Using monoclonal antibodies against Escherichia coli-produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), a natural MIH in fish. Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown. Cyclin B that appeared during oocyte maturation was labelled with [35S]methionine, indicating its de novo synthesis. Introduction of E. coli-produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation. Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17 alpha, 20 beta-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF.


Unexpected position-dependent expression of H-2 and beta 2-microglobulin/lacZ transgenes.

In order to study sequences involved in the developmentally regulated and tissue-specific expression of the class I Major Histocompatibility Complex (MHC) genes, we have constructed several H-2/lacZ transgenic lines in which the 5' regulatory sequences of the H-2Kb gene are linked to the Escherichia coli beta-galactosidase (lacZ) gene. In five H-2/lacZ lines, the pattern of lacZ expression, detected histochemically varied greatly from line to line. None of the H-2/lacZ transgenes were transcribed in cells normally expressing a high level of endogenous H-2 molecules, although these H-2 regulatory sequences have been shown to be sufficient to drive tissue-specific expression of other reporter genes. Interestingly, when constructs containing 5' beta 2-microglobulin (beta 2m) regulatory sequences linked to lacZ were used to derive transgenic lines, similar results were obtained. A survey of lacZ labeling in H-2/lacZ and beta 2m/lacZ transgenic mice strongly suggests that these transgenes are very sensitive to position effect, lacZ expression being controlled by endogenous chromosomal regulatory elements specific for each insertion site. Here we describe the complex pattern of lacZ expression in the different transgenic lines during development; we discuss the unusual properties of these transgenes and underline their potential use for developmental studies and characterization of genomic sequences involved in spatiotemporal gene expression.


Secretion of bovine alpha-lactalbumin into the milk of transgenic rats.

Bovine alpha-lactalbumin (b alpha LA) gene prepared by polymerase chain reaction was introduced into rat genomes by microinjection. Out of 17 transgenic rat lines, 11 secreted b alpha LA into their milk at concentrations higher than 0.2 micrograms/ml. Of these, three lines secreted b alpha LA at concentrations higher than those in bovine milk. The highest concentration of b alpha LA secreted into rat milk was 2,400 micrograms/ml.


Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.


F-actin in guinea pig spermatozoa: its role in calmodulin translocation during acrosome reaction.

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS-PAGE and Western blots immunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility.


Porcine oocyte zona pellucida M(r) 55,000 glycoproteins: identification of O-glycosylated domains.

The distribution of O-linked oligosaccharides on the M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of the porcine oocyte zona pellucida was examined. Purified preparations of endo-beta-galactosidase digested ZP3 alpha and ZP3 beta were reduced and carboxamidomethylated and digested with trypsin. When the trypsin digests were mapped by HPLC, each glycoprotein yielded only one N-acetylgalactosamine containing glycopeptide. Purification of the O-glycopeptides was achieved by a two-step protocol. Tryptic digests were applied to jacalin-agarose and specifically-bound O-glycopeptides (alpha OGP and beta OGP) were eluted with buffer containing 50 mM alpha-methylgalactoside as the haptenic sugar. Further purification of each O-glycopeptide was accomplished by reverse phase HPLC. Purified O-glycopeptides were characterized with respect to amino acid and carbohydrate compositions and sequenced by automated Edman degradation; alpha OGP was a 41-residue glycopeptide with three O-linked sugar chains. Sequence comparisons revealed a 75% identity between alpha OGP and a corresponding segment of rabbit rec55 zona protein; beta OGP was a 25-residue glycopeptide characterized by the presence of one N-linked and five O-linked sugar chains and a trypsin-resistant internal arginine residue. Sequence alignments revealed an 80% or greater identity between beta OGP and internal peptides of mouse, hamster and human ZP3 zona proteins. These studies demonstrate that in the case of ZP3 alpha and ZP3 beta, the pig homologues of rabbit rec55 and mouse ZP3, respectively, O-linked oligosaccharides are confined within delimited domains rather than widely dispersed on the polypeptide backbone. Such clustering of O-linked oligosaccharides may represent an essential determinant of the structure and biological activity of zona proteins.


Detection of chromosome 17- and X-bearing human spermatozoa using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with DNA probes specific to chromosomes 17 and the X has been applied to human ejaculated sperm. After sperm nuclei were decondensed with EDTA and DTT, biotinylated alpha satellite DNA probes TR17 and TRX were separately used on preparations from thirteen healthy donors. After hybridization 96% of sperm were labelled with the TR17 probe and 48% of sperm were labelled with the TRX probe. Frequencies of 0.33% disomic 17 and 0.29% disomic X sperm were found. The frequencies of diploid sperm were assessed as 0.37% using the TR17 probe and 0.20% using the TRX probe which labelled only one half of the sperm; after correcting the result from the X-probe to 0.40% the two frequencies are very similar.


Structural changes in sperm from the fiddler crab, Uca tangeri (Crustacea, Brachyura), during the acrosome reaction.

The acrosome reaction (AR) was induced in sperm from the brachyuran crustacean Uca tangeri either by mixing male and female gametes in filtered seawater or by treating the spermatozoa with the divalent cation ionophore A23187. This latter method provided a sufficient number of reacted spermatozoa to allow a detailed ultrastructural study of the AR. The process consists of two separate phases: a) initial release of the acrosomal vesicle contents, and b) further elongation of the acrosomal filament, which causes reversal of the rigid capsule limiting the acrosomal vesicle contents. The elongate acrosomal filament consists of an apical perforatorium and a basal columnar structure called here the proximal piece. The former derives from the perforatorium of the uninduced sperm stage with only small ultrastructural changes. The proximal piece forms from myelin-like membrane layers which are initially distributed all around the subacrosomal region and then accumulate in a column at the perforatorial base, thus promoting a sudden forward projection of the perforatorium. The AR in brachyurans is thought to be a passive mechanism that utilizes the negative pressure exerted on the nucleus--caused by emptying of the acrosomal vesicle--for an organized accumulation of membrane-rich material immediately behind the perforatorium, with the final result of the raising of a 3 microns long acrosomal filament.


A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.

Superovulated hamster oocytes were cryopreserved and thawed according to our carefully designed procedures. More than 90% (92 +/- 4%) of oocytes survived freezing and thawing. They were proven to be well conserved, showing excellent performance comparable to freshly ovulated oocytes in the human sperm penetration test (proportion of penetrated ova: 94.7% vs. 93.6%) and human sperm chromosome analysis (proportion of metaphasic ova: 81.8% vs. 83.6%). There was no statistically significant difference in the incidences of sperm chromosome aberrations between assays using fresh and frozen-thawed oocytes. In addition, there was no statistically significant increase of aberrations in female pronuclear (hamster) chromosomes. This freezing-thawing method was found to be reliable, yielding viable hamster oocytes of high quality.


Vitrification of mouse oocytes using short cryoprotectant exposure: effects of varying exposure times on survival.

The effects on oocyte viability of varying the duration of exposure to cryoprotectants before rapid cooling to -196 degrees C were examined, using the vitrification protocol of Nakagata. A very short exposure (15 sec) was found to be optimal, resulting in an overall rate of development from vitrified oocytes to hatching blastocysts of 31.8%. Very high rates of survival (77-89%) of oocytes exposed to the cryoprotectant media, but without the vitrification, together with extreme variability in results between straws in the vitrified groups, suggest that losses in viability during vitrification may result from ice damage during devitrification of the medium.


Behavior of sperm nuclei incorporated into parthenogenetic mouse eggs prior to the first cleavage division.

When artificially activated mouse eggs are inseminated in the middle of the first cell cycle, sperm nuclei remain condensed until the first mitosis. During mitosis of the first cleavage division sperm nuclei decondense, subsequently recondense and are passively displaced to the daughter blastomeres. In the 2-cell embryos sperm nuclei form interphase nuclei which are able to replicate DNA and to condense into discrete chromosomes during the following mitotic division. These observations suggest that the mitotic cytoplasm of 1-cell embryos creates similar conditions for the transformation of sperm nuclei into male pronuclei as the cytoplasm of metaphase II oocytes.


A study by in situ hybridization of the stage of appearance and disappearance of the transition protein 2 and the mitochondrial capsule seleno-protein mRNAs during spermatogenesis in the mouse.

Transition protein 2 is a basic chromosomal protein which functions as an intermediate in the replacement of histones by protamines, and the mitochondrial capsule seleno-protein is a constituent of the outer membrane of mitochondria which functions in constructing the mitochondrial sheath surrounding the flagellum. To determine precisely the stages in spermatogenesis when these mRNAs are present, paraffin sections of sexually mature testes were hybridized to 35S- and 3H-labeled antisense RNAs and exposed to autoradiographic emulsion. The cell types hybridizing to probes in situ were determined by staining with hematoxylin and periodic acid Schiff. The in situ hybridizations reveal that the transition protein 2 mRNA is first detectable in step 7 round spermatids, persists at high levels through step 13, and is degraded before step 14. By contrast, the mitochondrial capsule seleno-protein mRNA is first detected in step 3 round spermatids and persists at high levels until step 16, the end of spermiogenesis. The mitochondrial capsule seleno-protein mRNA appears to be expressed only in haploid cells since low levels could not be detected in Northern blots of RNA from pachytene primary spermatocytes from 18 day prepubertal mice. These results demonstrate that the transition protein 2 and mitochondrial capsule seleno-protein mRNAs are transcribed and degraded at different times during the haploid phase of spermatogenesis.


Protein dynamics in sperm membranes: implications for sperm function during gamete interaction.

A number of mammalian sperm plasma membrane antigens have been implicated as playing a functional role in sperm-egg interaction, by virtue of the fact that antibodies against these antigens interfere with fertilization. Two such mouse sperm plasma membrane antigens are M42, a 200/220 kD glycoprotein doublet, and M5, a 150-160 kD glycoprotein. We show that both of these antigens are concentrated on the posterior region of caudal epididymal and capacitated mouse sperm heads and are relatively diffusible, as determined by fluorescence recovery after photobleaching measurements (D = 3-8 x 10(-9) cm2/s with approximately 23% diffusing). Crosslinking of these antigens with bivalent antibodies causes them to redistribute into the anterior region (acrosomal crescent) of the sperm head. In contrast, we describe a third antigen, P220, which is also localized to the posterior region of the sperm head on caudal epididymal sperm but which exhibits very little diffusion and does not redistribute upon crosslinking. Bivalent anti-M42 blocks the ZP3-induced acrosome reaction. We have found that monovalent Fab fragments of anti-M42 do not block the ZP3-induced acrosome reaction, but that inhibition is restored by addition of a second antibody which crosslinks the Fabs. Thus, crosslinking is required for both inhibition of the acrosome reaction and redistribution. This suggests that redistribution of antigen away from the posterior region of the head may be part of the mechanism of inhibition of the ZP3-induced acrosome reaction.


castor encodes a novel zinc finger protein required for the development of a subset of CNS neurons in Drosophila.

Using an enhancer detection screen, we have identified castor, a new gene required for embryonic CNS development in Drosophila. Embryos that lack castor expression have a diminished CNS axonal network and express engrailed aberrantly late in CNS development. castor is unique among the previously described genes involved in Drosophila neurogenesis in that its expression is restricted to a subset of delaminated CNS neuroblasts and to ventral midline glial precursor cells. The putative castor gene product contains a novel zinc-binding domain and multiple transcriptional activation domains, suggesting that it acts as a transcription factor necessary for the development of a subset of CNS neuronal precursors.


Intracellular movements of fluorescently labeled synaptic vesicles in frog motor nerve terminals during nerve stimulation.

We stained synaptic vesicles in frog motor nerve terminals with FM1-43 and studied changes in the shape and position of vesicle clusters during nerve stimulation. Each stained vesicle cluster appeared as a fluorescent spot. During repetitive nerve stimulation the spots gradually dimmed, most without changing shape or position. Occasionally, however, a spot moved, appearing in some cases to stream toward and coalesce with a neighboring spot. This suggests the existence of translocation mechanisms that can actively move vesicles in a coordinated fashion between vesicle clusters. Within single clusters, we saw no signs of such directed vesicle movements. Fluorescent spots in terminals viewed from the side with a confocal microscope did not shrink toward the presynaptic membrane during nerve stimulation, but dimmed uniformly. This suggests that vesicles continuously mix within a cluster during destaining and provides no evidence of active vesicle translocators within single vesicle clusters for moving vesicles to the presynaptic membrane.


Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa.

We inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodopsin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration.


Fasciclin IV: sequence, expression, and function during growth cone guidance in the grasshopper embryo.

Monoclonal antibody 6F8 was used to characterize and clone fasciclin IV, a new axonal glycoprotein in the grasshopper, and to study its function during growth cone guidance. Fasciclin IV is dynamically expressed on a subset of axon pathways in the developing CNS and on circumferential bands of epithelial cells in developing limb buds. One of these bands corresponds to the location where the growth cones of the Ti1 pioneer neurons make a characteristic turn while extending toward the CNS. Embryos cultured in the 6F8 antibody or Fab exhibit aberrant formation of this axon pathway. cDNA sequence analysis suggests that fasciclin IV has a signal sequence; long extracellular, transmembrane, and short cytoplasmic domains; and shows no homology with any protein in the available data bases. Thus, fasciclin IV appears to be a novel integral membrane protein that functions in growth cone guidance.


Single amino acid substitutions in EGF-like elements of Notch and Delta modify Drosophila development and affect cell adhesion in vitro.

Notch locus EGF-like element mutations spl, altering eye development, and AxE2, affecting wing and sensilla development, are modified by mutations at Delta. It is shown that two allele-specific suppressors of spl involve single amino acid substitutions in the 4th (Dlsup5) and 9th (Dlsup4) EGF-like elements of the Delta protein. Cultured cells producing spl or AxE2 aggregate with cells producing wild-type Delta or Dlsup5 protein, and Dlsup5-producing cells adhere to cells producing wild-type Notch protein. However, spl,AxE2, and Dlsup5 are each defective in promoting these cell affinities, as none of the mutant proteins can compete with the corresponding wild-type proteins for formation of cell aggregates. Thus, widely separated EGF-like elements of Notch and Delta appear to participate in functional molecular interactions between the proteins. Dlsup5 does not improve adhesiveness of spl in vitro, so suppression in vivo may involve altered developmental signaling by spl-Dlsup5 complexes, rather than modified cell adhesion.


Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs.

The subunit stoichiometry of the mammalian K+ channel KV1.1 (RCK1) was examined by linking together the coding sequences of 2-5 K+ channel subunits in a single open reading frame and tagging the expression of individual subunits with a mutation (Y379K or Y379R) that altered the sensitivity of the channel to block by external tetraethylammonium ion. Two lines of evidence argue that these constructs lead to K+ channel expression only through the formation of functional tetramers. First, currents expressed by tetrameric constructs containing a single mutant subunit have a sensitivity to tetraethylammonium that is well fitted by a single site binding isotherm. Second, a mutant subunit (Y379K) that expresses only as part of a heteromultimer contributes to the expression of functional channels when coexpressed with a trimeric construct but not a tetrameric construct.


The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.

In CNS synapses, the synaptic junctional complex with associated postsynaptic density is presumed to contain proteins responsible for adhesion between pre- and postsynaptic membranes and for postsynaptic signal transduction. We have found that a prominent, brain-specific protein (PSD-95) enriched in the postsynaptic density fraction from rat brain is highly similar to the Drosophila lethal(1)discs-large-1 (dlg) tumor suppressor protein. The dlg protein is associated with septate junctions in developing flies and contains a guanylate kinase domain that is required for normal control of cell division. The sequence similarity between dlg and PSD-95 suggests that molecular mechanisms critical for growth control in developing organisms may also regulate synapse formation, stabilization, or function in the adult brain.


Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice.

SV40 T antigen (Tag) expression directed to cerebellar Purkinje cells resulted in the generation of three transgenic mouse lines that displayed ataxia, a neurological phenotype characteristic of cerebellar dysfunction. Onset of symptoms and cerebellar pathology, characterized by specific Purkinje cell degeneration, appeared to be directly dependent upon transgene copy number. The SV5 line (containing > 30 transgene copies), exhibited embryonic transgene expression that caused selective death of immature Purkinje cells and a subsequent block in cerebellar development and ataxia at 2 weeks. The developmental effect of the disruption of Purkinje cells in SV5 mice suggests that a normal complement of these cells is required for early development of the cerebellar cortex, especially granule cell proliferation and migration from external to internal layers. Transgene expression in a second line, SV4 (10 copies), was detectable during the second postnatal week. Death of mature Purkinje cells in the SV4 line resulted in onset of ataxia at 9 weeks. Ataxia in a third line, SV6 (2 copies), was detected after 15 weeks. The distinct cerebellar phenotypes of the SV4-6 lines correlate with specific Tag-induced Purkinje cell ablation as opposed to tumorigenesis.


Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus.

The mechanisms responsible for long-lasting, activity-dependent decreases in synaptic efficacy are not well understood. We have examined the initial steps required for the induction of long-term depression (LTD) in CA1 pyramidal cells by repetitive low frequency (1 Hz) synaptic stimulation. This form of LTD was synapse specific, was saturable, and required activation of post-synaptic NMDA receptors. Loading CA1 cells with the Ca2+ chelator BAPTA prevented LTD, whereas lowering extracellular Ca2+ resulted in the induction of LTD by stimulation that previously elicited long-term potentiation. Following LTD, synaptic strength could be increased to its original maximal level, indicating that LTD is reversible and not due to deterioration of individual synapses. Induction of homosynaptic LTD therefore requires an NMDA receptor-dependent change in postsynaptic Ca2+ which may be distinct from that required for long-term potentiation.


Responses of retinal axons in vivo and in vitro to position-encoding molecules in the embryonic superior colliculus.

We show that rat retinal ganglion cell axons exhibit no topographic specificity in growth along the rostral-caudal axis of the embryonic superior colliculus (SC). Position-related, morphological differences are not found between temporal and nasal axon growth cones. However, embryonic retinal axons respond in vitro to a position-dependent molecular property of SC membranes. In vivo, regional specificity in side branching is the earliest indication that axons make topographic distinctions along the rostral-caudal SC axis. Our contrasting in vivo and in vitro results indicate that molecules encoding rostral-caudal position in the SC neither guide nor restrict retinal axon growth, but may promote the development of topographic connections by controlling specificity in the extension or stabilization of branches.


Effects of three anesthetic induction techniques on heart rate variability.

To investigate the effects of different clinical induction techniques on heart rate variability (HRV). Two studies are reported. Study 1 prospectively compared the effects of two induction techniques (etomidate vs. thiopental sodium) known to have widely disparate effects on cardiovascular reflexes. Study 2 specifically investigated whether the vagotonic effects of sufentanil cause an increase in vagally mediated HRV. Elective surgery in a university-affiliated hospital. Study 1: 18 ASA physical status I patients having minor surgery; Study 2: 10 ASA physical status III and IV patients having cardiac surgery. In Study 1, anesthesia was induced with either etomidate 0.3 mg/kg or thiopental sodium 4 mg/kg with 60% nitrous oxide in oxygen. In Study 2, anesthesia was induced with a sufentanil infusion (total dose 2.9 +/- 0.2 micrograms/kg). The electrocardiogram-derived heart rate signal was subjected to power spectral analysis (similar to electroencephalographic analysis) to obtain measurements of (1) absolute HRV power [units of (beats per minute)2] within defined frequency ranges (HRVLO = power between 0 and 0.125 Hz; HRVHI = power between 0.126 and 0.5 Hz; HRVTOT = HRVLO + HRVHI) and (2) normalized HRV power (the percentage of total power) within these same frequency ranges [e.g., %HRVHI = (HRVHI/HRVTOT) x 100%]. In Study 1, both techniques caused large reductions in HRVTOT. The reduction caused by the thiopental sodium technique (-89% +/- 2%) significantly exceeded that caused by the etomidate technique (-58% +/- 13%, p less than 0.02). In Study 2, sufentanil decreased absolute power measurements of vagally mediated HRV (-69 +/- 12 change in HRVHI) but increased corresponding normalized measurements of vagally mediated HRV (90% +/- 30% increase in %HRVHI). In Study 1, the greater reduction in HRV with the thiopental sodium technique provides evidence that the depressant effects of anesthetics on HRV are related in part to their effects on cardiovascular reflexes. However, the significant depression in HRV caused by the etomidate technique suggests that mechanisms other than baroreflex depression (e.g., impaired consciousness) also are important in these depressant effects. In Study 2, the decrease in HRVHI caused by sufentanil documents that absolute power measurements of vagally mediated HRV are not correlated with changes in parasympathetic tone during a potent opioid induction. This lack of a correlation may result from the decrease in total HRV observed with loss of consciousness. The increase in %HRVHI suggests that normalized measurements of HRV may still provide an index of changes in sympathetic-parasympathetic balance, even when total HRV is decreased following anesthetic administration.


Epidural opioids during laminectomy surgery for postoperative pain.

To determine whether morphine applied directly to the dura during laminectomy surgery provides superior postoperative analgesia during the first 24 hours. Randomized, double-blind study. A university-affiliated hospital. Twenty ASA physical status I and II patients ages 18 to 60 years. Simultaneous topical dural application and intramuscular (IM) injection of unknown solutions of saline and morphine 3 mg. Postoperative analgesia was assessed using the visual analog scale (VAS), a modified McGill-Melzack pain questionnaire, subjective nursing evaluations, and the amount of supplemental analgesic medication used. Patients were observed for complications and side effects. Compared with the patients who received epidural saline and IM morphine, the patients who received epidural morphine and IM saline had less postoperative pain as determined by VAS scores, nursing evaluations, and amount of supplemental opioid analgesic doses (1.6 +/- 1.2 vs. 4.1 +/- 1.2 analgesic doses per patient; p less than 0.05) required in the first 24 hours. Minor side effects were similar for the two groups. No patient developed respiratory depression. Morphine 3 mg applied topically to the dura at the end of laminectomy surgery is a simple, safe, and effective way of providing improved postoperative analgesia.


The efficacy of intramuscular ketorolac in combination with intravenous PCA morphine for postoperative pain relief.

To examine the efficacy of intramuscular (IM) ketorolac used in combination with intravenous (IV) patient-controlled analgesia (PCA) morphine for postoperative pain relief following intra-abdominal gynecologic surgery. Randomized, double-blind, placebo-controlled study. Patient care unit at a university medical center. Thirty-five healthy women undergoing intra-abdominal gynecologic surgery who requested postoperative PCA. Postoperatively, all patients received IV PCA morphine, with the PCA device programmed to deliver a maximum of 1 mg every 6 minutes (maximum of 30 mg over 4 hours). In addition, patients received one of three regimens: (1) IM saline every 6 hours; (2) IM ketorolac 30 mg while in the postanesthesia care unit (PACU), followed by 15 mg every 6 hours; or (3) IM ketorolac 60 mg while in the PACU, followed by 30 mg every 6 hours. Patients were assessed at regular intervals. Visual analog scale (VAS) scores were used to assess analgesia and patient satisfaction with therapy. Data on morphine usage were obtained from the PCA device, and the frequency and severity of adverse effects were assessed for the presence or absence of side effects. Cumulative morphine dosages were lower (p less than 0.05) in both ketorolac groups at 12, 18, and 24 hours. VAS scores and the frequency of side effects did not differ significantly among groups. IM ketorolac significantly decreased PCA morphine requirements. The analgesic effects of the two drugs appear to be additive.


Fentanyl-induced muscle rigidity as affected by pretreatment with amantadine hydrochloride.

To determine whether the degree of muscle rigidity in humans caused by the administration of high-dose fentanyl is affected by pretreatment with amantadine hydrochloride, a drug that stimulates the release of dopamine in the basal ganglia. Randomized, observer-blinded comparison of regimes. Inpatient surgery at a university-affiliated teaching hospital. Sixteen patients scheduled for elective surgery in whom the administration of high-dose fentanyl was felt to be appropriate. Eight patients in the control group were given diazepam 5 mg orally 1 hour prior to induction of anesthesia. Eight patients in an experimental group were given the same dose of diazepam and also were treated with amantadine 1 g orally in divided doses over a period of 25 hours prior to their scheduled surgery. At the time of surgery, all patients were given fentanyl 50 micrograms/kg intravenously at a rate of 1 mg/min and were clinically evaluated for the degree of muscle rigidity of the chest wall, abdomen, and extremities. A single observer, blinded to the technique, evaluated and recorded the degree of muscle rigidity present in the chest wall, abdomen, and upper extremities immediately after administration of the fentanyl and 3 minutes later. The observer was the same individual in all instances. In no case did the muscle rigidity compromise our ability to adequately oxygenate the patient. Muscle rigidity associated with the administration of high-dose fentanyl is not attenuated by prior administration of amantadine in the dose range studied.


Anesthesia rebreathing bags: physical characteristics and use as portable oxygen reservoirs.

To examine the physical characteristics of 3-liter anesthesia rebreathing bags and assess their use for oxygen (O2) storage and delivery during transport of patients from the operating room (OR) to the recovery area. Anesthesia laboratory and pulmonary function laboratory. Five anesthesia rebreathing bags were inflated in 2-liter increments with 50 liters of air and then deflated in 2-liter increments. A sixth bag was inflated twice in 1-liter increments. Five bags were inflated with 60 liters of gas and allowed to deflate through a cannula functioning as a flow restrictor. Five bags were inflated with 100 liters of gas and checked for damage. Pressure measurements done at 2-liter increments during inflation of the bags to 50 liters and deflation showed a consistent pattern of pressure-volume relationships. Assessment of gas flow during deflation through the cannula showed flow rates ranging from 3.1 to 3.8 L/min after 5 minutes of continuous flow and from 2.8 to 3.8 L/min after 10 minutes. No weakness or damage was apparent in bags inflated with 100 liters of gas. It is feasible for anesthesia rebreathing bags distended with O2 to serve as lightweight, inexpensive, and easily monitored alternatives to O2 tanks for O2 delivery during transport of patients from the OR to the recovery area.