Congenital anomalies concomitant with persistent primary congenital hypothyroidism.

The Israeli national neonatal screening program for congenital hypothyroidism (CH) was initiated in May 1978. The overall incidence of persistent primary congenital hypothyroidism (PPCH) during the first 10 years of screening was 1:2,950 live births. The purpose of this study was to ascertain the incidence of congenital extrathyroid anomalies (ETAs) among the infants with PPCH and to compare it with the Israeli Birth Defect Monitoring System data. Among 243 PPCH infants on whom adequate data were available, 38 infants (15.6%) had associated congenital anomalies. Fourteen infants had congenital cardiac anomalies (5.8%): VSD (n = 7), PDA (n = 3), PS (n = 2), one mitral insufficiency, and one congenital atrial flutter. Eight children (3.3%) had congenital dislocation of the hip; their M:F ratio was 3:5 similar to the M:F ratio in CH (unlike the ratio in the general population). Some additional anomalies were considerably more common than in the general population. It is reasonable to assume that teratogenic effects active during organogenesis may affect simultaneously many organs, including the developing thyroid, causing a relatively high percentage of CH infants with congenital ETA.


Xp22.3 microdeletion syndrome with microphthalmia, sclerocornea, linear skin defects, and congenital heart defects.

We report on an infant girl with congenital erythematous, linear skin lesions on face and neck, bilateral microphthalmia, sclerocornea, cataracts, and a complex cardiac anomaly including atrial septal and ventricular septal defects. This patient had an Xp22.3 microdeletion and a chromosome satellite on the abnormal X p-arm. The abnormal X chromosome was late replicating in peripheral blood lymphocytes and cultured skin fibroblasts. Four other patients with similar congenital anomalies and Xp deficiency have been reported previously and are compared with this patient. One patient had an interstitial or terminal deletion, but in others the material translocated to Xp22.3 was variable (Yq material in two patients, and Yp material and an unidentifiable satellite in one patient each). Several mechanisms are suggested by which this chromosome abnormality might produce this phenotype in these patients. Our patient is the first with this syndrome to have a congenital heart defect.


Anencephaly and limb deficiencies.

Limb deficiencies (LDs) are rarely reported in anencephalic infants. A review of 662 patients in the literature on non-neural defects in anencephaly only showed five patients with LDs. We report on eight patients with various LDs from the records of 141 necropsies of the anencephalic infants found among 495,830 births. Compared with another group of anencephalic infants reported in the literature, the patients in this group of anencephalic infants with LDs were predominantly male, their mean gestational age was younger by approximately 5 weeks, their mean birth-weight was approximately 1,400 g less, and they presented with a higher incidence of polyhydramnios during gestational development. The association of this pair of anomalies, which was 100 times more frequent than expected, seems not due to chance. Since all eight patients had other multiple congenital anomalies (MCA), in addition to anencephaly and LDs, the postmortem study should be mandatory in anencephalic infants with LDs. The most common associated anomalies were cardiovascular and renal defects. Oral clefts, diaphragmatic hernia, esophageal atresia, and imperforate anus were also observed in these infants. The recognition of LDs in anencephalic infants indicates severe and extensive disturbance of the early embryogenesis (blastogenesis), which affects the midline of the embryo.


Double mosaic aneuploidy: 45,X/47,XY,+8 in a male infant.

We report on a 13-month-old boy with abnormalities consistent with mosaic trisomy 8 syndrome and male genitalia with partial penoscrotal transposition without hypospadias, a retractile left testis in inguinal canal, and an absent right testis. A voiding cystourethrogram showed an outpouching close to the lower right side of the bladder (utriculum) and bilateral hydronephrosis secondary to vesicoureteral reflux. Peripheral blood karyotype was 45,X/47,XY,+8. The karyotype of cultured skin fibroblasts was 47,XY,+8 with no 45,X cells detected among 20 cells counted. Tissues removed during surgery documented a 45,X/47,XY,+8 complement in the left testicle and utriculum, but only a 45,X line among 20 cells counted from vas deferens tissue. A possible mechanism for the origin of this previously unreported mosaicism might be an abnormal zygote with a 47,XY,+8 complement with subsequent simultaneous loss of chromosome Y and 8 in a cell at a very early embryonic stage.


Assessment of body composition in the Prader-Willi syndrome using bioelectrical impedance.

The accurate assessment of body composition is of importance in the Prader-Willi syndrome. Many techniques are not applicable due to ethical or practical reasons. However, the bioelectrical impedance technique is a rapid, painless, noninvasive method of estimating total body water and hence, fat-free mass in obese children and adolescents. We have compared estimates of total body water derived from bioelectrical impedance with actual measurements taken, using H2 18O dilution, in 14 children with Prader-Willi syndrome. Existing equations for predicting total body water from impedance showed a bias to underestimate actual measures of body water. There were positive correlations between the degree of underestimation with age and body fatness. It is possible that changes in body shapes influence bioelectrical impedance measurements in obese individuals, and that a prediction equation based upon a normal population will not be applicable to obese individuals.


Beare-Stevenson cutis gyrata syndrome.

Beare-Stevenson cutis gyrata syndrome consists of skin furrows of corrugated appearance, acanthosis nigricans, craniofacial anomalies, particularly craniosynostosis and ear defects, anogenital anomalies, skin tags, and prominent umbilical stump. Four cases of this striking syndrome are reported. Together with two previously reported cases, the syndrome is delineated from the six known cases. Cutis gyrata variably affects the scalp, forehead, face, preauricular area, neck, trunk, hands, and feet. Craniosynostosis is present in four cases, with cloverleaf skull in three of these. Intrauterine growth has been normal in all cases. Performance and life expectation appear to be related to the presence or absence of cloverleaf skull. All cases observed to date have been sporadic. Increased paternal age suggests the possibility of an autosomal dominant mutation.


Upper and lower airway compromise in the Apert syndrome.

Both upper and lower airway compromise may be responsible for early death in some patients with the Apert syndrome. We report on two and review six cases with complete or partial cartilage sleeve abnormalities of the trachea. Possible mechanisms include tracheal stenosis and/or lack of tracheal distensibility which may result in respiratory inefficiency, inability to clear secretions, and/or increased liability to surface injury from tracheal suctioning. Upper airway compromise, consisting of obstructive sleep apnea and cor pulmonale, may result from reduced nasopharyngeal and oropharyngeal dimensions in the Apert craniofacial configuration.


Expression of multi-cytokine resistance and multi-growth factor independence in advanced stage metastatic cancer. Malignant melanoma as a paradigm.

Evidence is briefly reviewed, primarily in the context of human melanoma, which suggests that acquisition of resistance to multiple, structurally diverse, growth inhibitory cytokines occurs during malignant tumor progression. A similar pleiotrophy appears to occur with respect to growth factor independence. Also discussed is the notion that growth factors that behave as inhibitors for early-stage, benign tumor cells can 'switch' so as to function as mitogens for tumor cells from more advanced stages of disease progression, i.e., when the cells acquire competence for metastasis. The combined phenotypes of 'multicytokine resistance' and 'multi-growth factor independence' help provide tumor cells with the competence to grow in the mesenchymes of the primary organ site and in distant organs.


Serum amyloid A gene transcription in synovial cells during retroviral arthritis.

In situ hybridization was used to demonstrate serum amyloid A (SAA) gene expression in arthritic joint tissue from lentivirus-infected sheep and goats. SAA gene transcription occurs in isolated individual synovial cells and occasional giant cells but not in uninfected or uninflamed synovial tissue. These findings support the notion (derived from in vitro observations) that at least one member of the SAA gene/protein family may function as an autocrine collagenase inducer in inflammatory arthritis. Since we used heterologous DNA probes derived from human clones, our findings also support the growing evidence for interspecies conservation of SAA genes.


In situ hybridization and immunohistochemistry of p53 tumor suppressor gene in human esophageal carcinoma.

We have studied expression of p53, a tumor suppressor gene, by using both immunohistochemistry and in situ hybridization in 20 cases of squamous cell carcinoma of the esophagus. Immunohistochemical analysis was performed by using monoclonal antibody pAb1801. Immunoreactive p53 was observed in the nuclei of the tumor cells in 17 cases. We used 35S-labeled anti-sense single-stranded synthetic oligonucleotide probe ON102, which hybridized with DNA sequence near the 5' end of p53, for in situ hybridization. In all the cases of invasive squamous cell carcinoma studied, no significant accumulation of p53 hybridization signals was observed in carcinoma cells. This result indicates that overexpression of p53 observed by immunohistochemical staining is not due to an increase in the steady-state level of p53 mRNA in frank carcinoma cells. In six cases of morphologically normal esophageal mucosa distant from carcinoma, accumulation of hybridization signals was observed in basal and parabasal cells of the mucosa. The mucosa of these cases was negative for p53 immunoreactivity except for one case showing sporadic positivity. Accumulation of hybridization signals was observed in foci of squamous dysplasia not associated with invasion in three cases.


Relationship between interleukin-1 beta and platelet-activating factor in the pathogenesis of acute immune complex alveolitis in the rat.

Intrapulmonary interleukin-1 beta (IL-1 beta) participates in the pathogenesis of acute IgG immune-complex alveolitis through a mechanism involving neutrophil recruitment. We have examined the relationship between intrapulmonary IL-1 beta and locally produced platelet-activating factor (PAF) in the development of acute alveolitis. Instillation of IgG anti-bovine albumin into the lungs of rats, followed immediately by intravenous infusion of bovine serum albumin (BSA), resulted in acute neutrophil-mediated lung injury. Development of IgG immune-complex lung injury was accompanied by three- and five-fold increases in bronchoalveolar lavage (BAL) fluid and whole lung PAF levels, respectively. Intratracheal administration of the PAF antagonists, WEB-2086 (Boehringer) or L-652,731 (Merck, Sharpe, and Dohme, Rahway, NJ), reduced pulmonary vascular leakage. Neutralization of intrapulmonary IL-1 activity with anti-IL-1 beta antibodies reduced pulmonary vascular permeability and whole lung PAF levels. Morphometric analysis and whole lung myeloperoxidase measurements revealed a differential effect between the PAF antagonists and anti-IL-1 beta with respect to pulmonary neutrophil recruitment. Intratracheal instillation of anti-IL-1 beta retarded net pulmonary neutrophil recruitment while the PAF antagonists retarded migration of neutrophils from the interstitial/vascular compartments into the alveolar compartment. Intratracheal instillation of anti-IL-1 beta plus L-652,731 resulted in reduction in lung vascular permeability and retarded net pulmonary neutrophil recruitment. No additive effect was observed. Stimulation of isolated mouse alveolar macrophages with recombinant murine IL-1 beta or IL-1 alpha resulted in rapid, dose-dependent, and cell concentration-dependent increases in PAF secretion. These data suggest that intrapulmonary IL-1 beta amplifies local PAF production and that IL-1 beta and PAF modulate different aspects of pulmonary neutrophil recruitment.


Influence of genetics on the nephritogenic potential of proteoglycans.

Nephritogenic potential of antibodies directed against one of the glomerular basement membrane (GBM) components, i.e., heparan sulfate-proteoglycan (HS-PG), was investigated in different strain of rats, i.e., Brown Norway, Lewis, Long Evans, and Sprague-Dawley. The rats were given two intravenous injections of anti-HS-PG antibody on days 1 and 3, and killed 2 to 8 weeks later. Before killing, blood and urine were collected for determination of anti-rabbit IgG levels and excretion of proteins, respectively. In addition, the right kidney was perfused with 125I-anti-rat IgG to quantitate the amount of immune-complexes present within the GBM. The tissues were processed for morphologic, autoradiographic, and immunofluorescent studies. The anti-HS-PG antibody was seen uniformly bound to GBM equally in all strains of rats. However, the protein-uric response was as follows: Brown Norway much much greater than Lewis much greater than Long Evans greater than Sprague Dawley. Also, the glomerular cells, monocytes in the glomerular capillaries, immunoreactivity of rat IgG and C3 frequency of subepithelial immune deposits, serum levels of anti-rabbit IgG, and the amount of 125I-anti-rat IgG bound to the GBM were proportionately increased among different strains of rats. The data suggest that the sustained presence of anti-HS-PG antibodies in the subepithelial aspect of the GBM with differential humoral response in the production of the antibody by the host most likely attributed to the variable glomerular damage in different strains of rats. Thus, it seems that the genetic makeup of a given strain of rat heavily influences the nephritogenic potential of an antibody and consequentially the outcome of the immune complex-mediated glomerular injury.


Nonradioactive in situ hybridization using digoxigenin-labeled oligonucleotides. Applications to musculoskeletal tissues.

We have optimized a technique for in situ localization of specific mRNAs using digoxigenin-11-dUTP-labeled oligonucleotide probes. DNA probes were synthesized for type I and type II collagen as well as transforming growth factor-beta 1 and 2 (TGF beta 1 and TGF beta 2). Control experiments, such as competitive inhibition, nonsense sequence hybridization, and RNAse digestion all indicated that the technique was highly sensitive and specific. In sections of growth plate, type II collagen mRNA was predominantly expressed in the lower proliferative and upper hypertrophic zone, whereas chondrocytes in articular cartilage stained equally. These techniques then were applied to sections cut from archival pathology specimens of musculoskeletal tissues. Primitive chondrocytes in a chondrosarcoma expressed type I and type II collagen mRNA, but did not stain with the nonsense probe. Sections from an osteosarcoma, an aneurysmal bone cyst, and a neurofibroma also were investigated. The ability to use chemically synthesized oligonucleotide probes, the high resolution, and the short development times possible with this in situ procedure makes this technique appealing for applied research into the gene expression of normal and pathologic cellular events.


Coexpression of type I and type II human macrophage scavenger receptors in macrophages of various organs and foam cells in atherosclerotic lesions.

Macrophage scavenger receptors are trimeric membrane glycoproteins implicated in the pathologic deposition of cholesterol in arterial walls during atherogenesis. Two types of cDNAs for functional human receptors have been cloned, but their physiologic roles remain obscure. To study the expression of these receptors, the authors generated antibodies against scavenger receptor type-specific synthetic peptide. Immunohistochemical examination using these antibodies and other anti-human receptor antibodies shows that type I and type II receptor proteins can be detected in foam cells in various stages of atherosclerosis, most evidently in fatty streaks. Co-expression of the two types of receptor protein was also detected in macrophages of various organs. Both types of the protein were detected on the surface and the membrane of endosomes in macrophages. These results indicate that both type I and type II scavenger receptors are expressed and functionally active in physiologic and pathologic conditions.


Interaction of human malignant melanoma (ST-ML-12) tumor spheroids with endothelial cell monolayers. Damage to endothelium by oxygen-derived free radicals.

Clinical and experimental observations suggest that tumor-induced endothelial cell injury may be one of several initial events in the establishment of tumor metastases. To test this hypothesis, the authors have analyzed the interaction of malignant melanoma (ST-ML-12) multicenter tumor spheroids with endothelial cell monolayers in a three-dimensional coculture system. After 1.5 hours of interaction, the authors observed a toxic effect on endothelial cells in the perispheroid region. The latter was demonstrated by testing membrane integrity with the fluorescent probes acridine orange/ethidium bromide and resulted in sensitivity to shear stress of the damaged cells. The endothelium then underwent a regenerative cycle to replace the denuded halo. Addition of the oxygen radical-scavenging enzyme superoxide dismutase to the culture medium prevented this endothelial cell damage in a dose-dependent manner for up to 12 hours. By contrast, catalase, deferoxamine mesylate, allopurinol, and the proteinase inhibitors soybean trypsin inhibitor and aprotinin were not protective under the same conditions. The endothelial damage was dependent on the attachment of the spheroids. Medium conditioned by ST-ML-12-spheroids proved to be ineffective. A similar, but less prominent, deleterious effect was seen when human peritoneal mesothelial cells were used in place of the human umbilical vein endothelial cells. Spheroids of the uroepithelial cell line HU-609 were used as control. No toxicity was observed in these cocultures. Melanin biosynthesis is associated with the production of oxygen-derived free radicals. The results suggest a possible implication of these free radicals in metastasis formation of malignant melanoma.


Production of matrix metalloproteinases 2 and 3 (stromelysin) by stromal cells of giant cell tumor of bone.

Matrix metalloproteinases play a central role in the catabolism of extracellular matrix macromolecules. Here the authors report that giant cell tumor of bone (GCT) produces two matrix metalloproteinases (MMPs) in zymogen form, which have been identified as proMMP-2 (also known as "72-kDa-progelatinase/type IV procollagenase") and proMMP-3 (prostromelysin). Giant cell tumor is known to consist of two major cell populations, multinucleated giant cells and stromal cells. On several passages of the tumor cells in culture, only stromal cells proliferated. These stromal cells produced proMMP-2 but not proMMP-3. Addition of the conditioned medium of primary GCT culture or human macrophage-conditioned medium to the passaged stromal cells induced the production of proMMP-3. The production of proMMP-3 was also induced by interleukin 1 (IL-1), but not by tumor necrosis factor alpha (TNF alpha). ProMMP-1 (tissue procollagenase) was not detected even after treatment with these stimuli. Immunohistochemical studies have demonstrated that multinucleated giant cells in GCT both produce IL-1 and TNF alpha, suggesting that IL-1 secreted by multinucleated giant cells may be responsible for in vivo production of proMMP-3 by the stromal cells. The authors propose that GCT has a self-stimulatory system for the production of matrix-degrading proteinases and that the ability of the passaged stromal cells to synthesize and secrete proMMP-3 with appropriate stimuli may contribute the malignant behavior of GCT.


Localization of transforming growth factor-alpha in human appendageal tumors.

Transforming growth factor-alpha (TGF alpha) is a potent mitogen for epithelial cells that has been localized to normal human appendageal epithelia. To further understand the role of TGF alpha in human appendages, we examined TGF alpha expression immunohistochemically in 17 types of human appendageal tumors differentiating toward hair follicles, eccrine, apocrine, and sebaceous glands. In order of decreasing degrees of differentiation, tumors could be divided into hyperplasias, adenomas, benign epitheliomas, and primordial epitheliomas. Using an antibody that recognizes primarily the 6-kd and 13-kd forms of TGF alpha, TGF alpha immunostaining in 16 of 17 tumor types analyzed was found to follow a similar pattern, with expression in hyperplasias greater than adenomas greater than benign epitheliomas greater than primordial epitheliomas. Within a given tumor, TGF alpha expression also correlated well with the known differentiation state of the tumor cell types. The results suggest that TGF alpha expression is directly correlated with the differentiation state of hair follicle, eccrine, apocrine, and sebaceous tumors in human skin, and raises the possibility that TGF alpha may play a role in the differentiation of appendageal epithelia.


Localization of SIV in the genital tract of chronically infected female rhesus macaques.

Despite the fact that human immunodeficiency virus (HIV) is transmitted primarily by sexual contact, the biology of the sexual transmission of HIV is poorly understood. Simian immunodeficiency virus (SIV) can be transmitted to female rhesus macaques by placing cell-free virus into the vaginal canal, and SIV can be isolated from the vaginal secretions of infected rhesus macaques. The authors examined the genital tracts from 16 chronically infected female rhesus macaques and localized SIV-infected cells using in situ hybridization and immunohistochemistry. SIV-infected cells were found in the genital tract of 13 of the 16 animals examined, and in most cases the SIV-infected cells were located in the submucosa of the cervix and vagina. However, SIV-infected cells were also found in the vaginal epithelium. SIV-infected cells were more common in sites of inflammation than in normal areas. These findings suggest that SIV gains access to genital tract secretions from the cervix and vaginal epithelium.


Antigenic heterogeneity of vascular endothelium.

The antigenic status of vascular endothelium from different sites of the normal adult and fetal human cardiovascular system was investigated. Tissues included aorta (n = 9), pulmonary artery (n = 8), coronary artery (n = 6), ventricle/atrium (n = greater than 10), lymph node (n = 2), fetal whole heart (n = 3), and umbilical cord (n = 7). Frozen sections were studied using monoclonal antibodies recognizing endothelial markers (EN4, vWf, Pal-E, and 44G4), vascular adhesion molecules (ICAM-1, ELAM, VCAM, and PECAM), the monocyte/endothelial marker (OKM5), and major histocompatibility complex (MHC) molecules (class I and class II). Results demonstrate that capillary endothelium is phenotypically different from endothelial cells (EC) lining large vessels. Capillary EC strongly express MHC classes I and II, ICAM, and OKM5, which are variably weak to undetectable on large vessels. In contrast, the large vessels strongly express vWf and appear to constitutively express ELAM-1. This suggests that the capillary EC may be more efficient at antigen presentation or more susceptible to immune attack in vivo. Interestingly, normal coronary arteries, unlike all other large vessels, express MHC class II and VCAM molecules. Future studies should concentrate on comparative functional studies between capillary, coronary, and large vessel EC.


Platelet morphologic changes and fibrinogen receptor localization. Initial responses in ADP-activated human platelets.

Platelet exposure to agonists results in rapid morphologic changes paralleled by fibrinogen binding and platelet aggregation. The current study used standardized stereology in conjunction with immunogold electron microscopy to correlate the initial morphologic changes with fibrinogen receptor localization on the surfaces of ADP-activated human platelets. A 45% increase in platelet circumference was observed after 3 seconds of activation (P = 0.001). Virtually all of this increase was due to a 13-fold increase in projection membrane, and the projections observed by stereo microscopy at this time were mostly blunt. Both blunt and long projections also accounted for the increase in platelet-platelet contacts at 10 seconds of activation. Immunogold electron microscopy using the monoclonal antibodies P2 and AP-2 against the fibrinogen receptor, glycoprotein IIb/IIIa (GP IIb/IIIa), showed relatively equivalent immunogold densities on projections compared with cell body during 30 seconds of activation. The activation-dependent anti-GP IIb/IIIa monoclonal antibody, 7E3, showed an immunogold density 37% greater on projections compared with cell body (P = 0.0001). Colocalization studies using 7E3 with a polyclonal antifibrinogen antibody showed bound fibrinogen in close proximity to the GP IIb/IIIa localized by 7E3 on projections. These studies support an important role for platelet projections during the earliest stages of fibrinogen binding and ADP-induced aggregation.


Helicobacter pylori. Its role in the pathogenesis of peptic ulcer disease in a new animal model.

The association and causative role of Helicobacter pylori infection of the stomach with gastric ulcer, duodenal ulcer, non-ulcer dyspepsia, and gastritis has remained controversial. The authors studied the effects of daily intragastric administration of H. pylori suspension in saline (10(8) CFU/ml) and bacteria-free filtrates of saline H. pylori suspensions in 85 Sprague-Dawley rats (weight, 150 to 200 g) with normal mucosa and with surgically produced experimental gastric ulcers. Group I rats (n = 30) with pre-existent experimental gastric ulcers received H. pylori suspension (ATCC 43504, 10(8) CFU/ml); Group II rats (n = 20) with experimental gastric ulcers received bacteria-free H. pylori filtrates; Group III rats with ulcers (n = 20) received saline alone; and Group IV control rats (n = 15) without ulcers received intact H. pylori organisms in suspension (ATCC 43504, 10(8) CFU/ml). At death, ulcer surface areas were measured with a dissecting microscope. Full-thickness sections were obtained for quantitative and qualitative histologic parameters, including the area of remaining mucosal necrosis; characteristics and cellular composition of restored mucosal architectures; and presence or absence of inflammation including counts of neutrophils and lymphocytes. H. pylori organisms were identified within the surface mucus and crypts using routine, special, and immunohistochemical stains. Our results indicate that the continued presence of either intact H. pylori organisms or bacteria-free H. pylori filtrates in the stomachs of rats with pre-existent gastric ulcers resulted in delayed healing of the ulcers and persistence of chronic active inflammation. Daily administration of suspensions of H. pylori organisms to sham-operated rats with intact gastric mucosa, however, resulted in no ulceration or inflammation despite identification of surface H. pylori organisms at death. The authors conclude that H. pylori alone causes little or no effect on an intact gastric mucosa in the rat, that either intact organisms or bacteria-free filtrates cause similar prolongation and delayed healing of pre-existing ulcers with active chronic inflammation, and that the presence of predisposing factors leading to disruption of gastric mucosal integrity may be required for the H. pylori enhancement of inflammation and tissue damage in the stomach.


Pregnanolone: a new steroid intravenous anaesthetic. Dose-finding study.

The intravenous steroid anaesthetic pregnanolone has been investigated as an induction agent in 60 fit adults premedicated with morphine and atropine. The drug was given in a stepwise fashion starting with 0.5 mg.kg-1; following a successful induction, the next patient received 15% less, while the subsequent patient received 15% more if anaesthesia was not achieved. Taking loss of eyelash reflex as the end point, 31 patients were satisfactorily induced; the AD50 in these patients was 0.44 mg.kg-1 (95% CI 0.41-0.47). A further 23 patients were induced satisfactorily as assessed by cessation of counting. Induction was trouble free with minimal changes in heart rate and arterial blood pressure, a low incidence of apnoea and few involuntary movements; pain on injection was not a feature. Loss of eyelash reflex is not a good end point for assessment of loss of consciousness following pregnanolone.


Tramadol: pain relief by an opioid without depression of respiration.

Two independent clinical trials were conducted simultaneously. In one, tramadol and pethidine were compared in 30 patients by patient-controlled analgesia during the first 24 h following abdominal surgery. The mean 24 h consumption of tramadol and pethidine was 642 mg and 606 mg respectively, giving a potency estimate of tramadol relative to pethidine of 0.94 (95% confidence interval 0.72-1.17). In the second trial, the effect on respiration of three doses of tramadol (0.5, 1.0, and 2.0 mg.kg-1) was compared with that of morphine sulphate (0.143 mg.kg-1) by intravenous injection during stable halothane anaesthesia. At approximately 1.5 times the equipotent dose, as estimated from the first trial, tramadol transiently depressed the rate of respiration but had no effect on end-tidal carbon dioxide tension. Morphine caused apnoea or considerable depression of ventilation. The results suggest that mechanisms other than opioid receptor activity play a significant role in the analgesia produced by tramadol.


Gastric emptying after minor gynaecological surgery. The effect of anaesthetic technique.

Gastric emptying was measured using the paracetamol absorption method in 30 patients immediately after a general anaesthetic for minor gynaecological surgery and in 10 female controls. Anaesthesia was induced with either propofol alone, propofol and alfentanil (4.5 micrograms.kg-1) or propofol and fentanyl (1.4 micrograms.kg-1) and maintained with intermittent propofol and 66% nitrous oxide in oxygen. Gastric emptying was delayed significantly in all patient groups when compared with volunteers. However, the delay in gastric emptying was similar in the three patient groups.


Cardiovascular responses to insertion of the laryngeal mask.

We have compared, in 40 healthy patients, the cardiovascular responses induced by laryngoscopy and intubation with those produced by insertion of a laryngeal mask. Anaesthesia was induced with thiopentone and maintained with enflurane and nitrous oxide in oxygen; vecuronium was used for muscle relaxation. Arterial pressure was measured with a Finapres monitor. The mean maximum increase in systolic arterial pressure after laryngoscopy and tracheal intubation was 51.3% compared with 22.9% for laryngeal mask insertion (p less than 0.01). Increases in maximum heart rate were similar, (26.6% v 25.7%) although heart rate remained elevated for longer after tracheal intubation. We conclude that insertion of the laryngeal mask airway is accompanied by smaller cardiovascular responses than those after laryngoscopy and intubation and that its use may be indicated in those patients in whom a marked pressor response would be deleterious.


The relationship between the concentration of temazepam in cerebrospinal fluid and sedation in man.

Twenty-six patients received oral temazepam and subsequently spinal anaesthesia. Blood and lumbar cerebrospinal fluid temazepam levels were measured together with the degree of sedation. The plasma and cerebrospinal fluid concentrations correlated well with the temazepam dose but even better with the weight standardised dose (r = 0.65, p = 0.0003 and r = 0.75, p = 0.00001 respectively). Both the plasma and cerebrospinal fluid concentrations of temazepam were correlated with the patient's sedation (r = 0.42 p = 0.037, and r = 0.46 p = 0.021 respectively), but neither was strong. Thus, although the drug concentration at the receptor may be a major factor in producing sedation, other factors, possibly the receptor population or their responsiveness, are also important contributors.


Peroperative arterial hypoxaemia. The interaction between intrapulmonary shunt and cardiac output. A computer model.

A computer model of respiratory physiology was used to investigate the interaction between cardiac output and intrapulmonary shunting during anaesthesia. Significant degrees of arterial desaturation were demonstrated as the cardiac output was reduced. We suggest that volume expansion may correct some episodes of desaturation observed during anaesthesia.


A nasal CPAP system. Description and comparison with facemask CPAP.

Nasopharyngeal pressures were compared in eight subjects breathing through either a nasal CPAP system or facemask CPAP system at a fresh gas flow of 50, 75 and 100 l.min-1. During nose breathing there was no significant difference in nasopharyngeal pressure between the two systems. During mouth breathing pressures were significantly lower with nasal CPAP. During nose breathing at 75 l.min-1 the mean inspiratory and expiratory pressures in cmH2O (SD) were 3.4 (0.68) and 5.9 (0.55) for nasal CPAP and 3.3 (0.71) and 6.3 (0.73) for facemask CPAP. The respective pressures during mouth breathing were 0.3 (0.73) and 2.9 (1.74) for nasal CPAP and 3.9 (0.73) and 5.8 (0.82) for facemask CPAP.


Nasal CPAP after coronary artery surgery.

Two groups of 14 patients were compared after coronary artery bypass surgery where the left internal mammary artery had been used as a conduit. One group received nasal continuous positive airway pressure for 1 h, the other group acted as a control. Mean pulmonary shunt fraction was 16.3% before, 12.6% during and 15.7% after continuous positive airways pressure. In the control group the shunt fraction fell from 17.3% to 16.8%. The reduction in shunt fraction was significantly greater with nasal continuous positive airways pressure than in the control group (p = 0.016). There was a significant reduction (p = 0.025) in respiratory rate from 18.3 to 16.7 breath.min-1 during continuous positive airway pressure. Other measured cardiorespiratory variables did not differ significantly between the groups. Visual analogue scores showed no significant difference in chest pain or mask comfort between the groups. The ease of breathing score was, however, significantly better in the continuous positive airways pressure group, 7.5 (SD 1.8) cm and control 5.6 (SD 2.6) cm.


The effect of nitrous oxide on laryngeal mask cuff pressure. In vitro and in vivo studies.

We have studied the effect of nitrous oxide on the cuff pressure of a laryngeal mask both in vitro and in vivo. In laboratory tests, we showed that nitrous oxide and carbon dioxide diffuse across the cuff wall much more rapidly than nitrogen and oxygen. Differing partial pressures of these gases across the cuff wall therefore give rise to changes in volume and pressure within the cuff. We then studied 18 patients undergoing general anaesthesia with nitrous oxide, and found a consistent and linear increase in cuff pressure in all patients. After 30 min, the mean pressure had increased by 30 mmHg, and there was approximately 10% nitrous oxide in the cuff. It is difficult to relate these findings to pressure on pharyngeal structures, but methods of limiting the rise in intracuff pressure are discussed.


Topical nonsteroidal anti-inflammatory gel for the prevention of peripheral vein thrombophlebitis. A double-blind, randomised, placebo-controlled trial in normal subjects.

A double-blind, randomised, placebo-controlled study was undertaken to determine whether topical application of a nonsteroidal anti-inflammatory gel to skin overlying peripheral vein cannula sites has a role in reducing the incidence or delaying the onset of peripheral vein thrombophlebitis. Fifty normal subjects had intravenous cannulae placed in right and left arms. Subjects were randomised to receive twice daily application of either active nonsteroidal anti-inflammatory gel or placebo gel to each cannula site. Cannula sites were observed and signs and symptoms of inflammation recorded up to 108 h. If any site had signs extending beyond 2 cm then the cannula was removed. Cannula sites that had 'active' gel applied had half the incidence of marked signs at 108 h (44% vs 22%, p less than 0.05). These results suggest that local application of topical nonsteroidal anti-inflammatory gel to cannula sites may have a significant role to play in the prevention of peripheral vein thrombophlebitis.


Preparation of the critically ill for interhospital transfer.

Changes in monitoring and therapy during the preparation of 100 critically ill patients for interhospital transfer by a specialist team were documented prospectively with the aim of providing guidelines for nonspecialists. Severity of illness scores were recorded before and after preparation. Median duration of preparation for ambulance journeys was 50 min and for aeroplane journeys was 82 min. During preparation, a portable electrocardiogram and pulse oximeter were attached to 21 and 76 patients respectively and intra-arterial pressure monitoring was continued or instituted in 88 patients. Supplemental oxygen and intravenous fluids were the therapies most commonly increased or instituted by the transport team; mechanical ventilation, positive end-expiratory pressure and inotropic drugs were increased or instituted less frequently. Median therapeutic intervention scores before and after preparation were 21 and 23 respectively, highlighting the need to increase rather than withdraw support for transfer.


A clinical evaluation of the 'Rapitrach'. A bedside percutaneous tracheostomy technique.

A number of percutaneous tracheostomy devices are now available commercially. We report a preliminary clinical study of the use of the 'Rapitrach' kit (Fresenius, Runcorn, Cheshire, UK). Twenty critically ill adult patients underwent tracheostomy using the kit. Nineteen of the procedures were carried out at the bedside in the intensive therapy unit. Correct tube placement was achieved in all cases.


Nosocomial sinusitis in ventilated patients. Nasotracheal versus orotracheal intubation.

A total of 68 postoperative patients whose lungs were ventilated for more than 4 days were studied prospectively during a one-year study period to investigate the effect of the mode of intubation on the paranasal sinuses. After an initial X ray of the skull showing no pathological findings, patients were assigned randomly to one of the study groups; the lungs of patients in group A were ventilated via an orotracheal tube (n = 32), and patients in group B via a nasotracheal tube (n = 36). X ray examinations of the sinuses were performed at regular intervals. Diagnosis of sinusitis was confirmed by transantral needle puncture and culture of fluids obtained. Antibiotic regimens were altered according to laboratory testing. Two patients in group A developed signs of sinusitis in comparison to 15 patients in group B (p less than 0.01). However, there were significantly more airway complications in the orotracheal group, particularly during the period of weaning from ventilation. We conclude that orotracheal intubation should be preferred as the routine route of intubation.


Resistance and additional inspiratory work imposed by the laryngeal mask airway. A comparison with tracheal tubes.

Laryngeal mask airways and tracheal tubes were studied to determine both their resistance to constant gas flows and additional inspiratory work during simulated inspiration. Laryngeal mask airways imposed less resistance and required lower additional inspiratory work compared with the corresponding sized tracheal tubes. If inspiratory loading during anaesthesia is an important consideration, then the laryngeal mask airway may be preferable to a tracheal tube.


Measurement of forces during laryngoscopy.

The forces transmitted by the laryngoscope blade onto the base of the tongue are assumed to be a major cardiovascular stimulus. This study investigates the various forces applied onto a Macintosh laryngoscope during laryngoscopy. The forces acting along the axis of the handle are described, as well as the forces exerted by the anaesthetist to prevent the laryngoscope from turning. Based on a relatively simple technique, a modified laryngoscope has been designed to measure these forces. Four different laryngoscopic parameters are determined: (1) the duration of laryngoscopy, (2) the maximally applied force, (3) the mean force and (4) the integral of the force over the time (area under the curve). The force measurements of 49 anatomically normal patients undergoing uncomplicated intubation are included in the study. The duration of laryngoscopy was 16.3s (SD 11.8), the applied peak force was 35N (SD 12) and mean force necessary was 20N (SD 6) while the force-time integral was 324N (SD 194).


Low dose amitriptyline in the treatment of chronic pain.

The analgesic efficacy of amitriptyline 25 mg was compared with placebo in 41 patients with chronic (more than 3 months) nonmalignant pain, using a double-blind randomised multiple-dose 3-week treatment period crossover design. Amitriptyline 25 mg provided significantly greater efficacy than placebo, with significant differences evident within the first week. There was no significant difference on mood scores between amitriptyline and placebo. The results suggest that surprisingly low doses of amitriptyline may be effective without substantial adverse effects, that the effect is evident early, and that the effect is distinguishable from any effect of the amitriptyline on mood.


Phenytoin reduces suxamethonium-induced myalgia.

A prospective, randomised trial was undertaken in 60 healthy adults to determine the efficacy of intravenously administered phenytoin in doses of 5 mg.kg-1 for the prevention of suxamethonium-induced fasciculations, a rise in serum K+ and myalgia. This was compared with tubocurarine pretreatment and no pretreatment (control group). Phenytoin pretreatment significantly reduced myalgia from 45% (nine patients) in the control group to 10% (two patients) (p less than 0.05). It also decreased the duration and mean intensity of fasciculations. Incidentally, phenytoin was also found to decrease significantly mean serum Na+ levels (p less than 0.001) both at 5 and 20 min after administration. Tubocurarine pretreatment (3 mg) resulted in a significant decrease in fasciculations, but myalgia, which occurred in five patients, remained the same. No significant correlation was found between muscle fasciculations, postoperative myalgia and K+ changes, but patients with myalgia had a significant decrease in mean serum Na+ levels at 5 and 20 min after suxamethonium (p less than 0.01).


A comparison of laryngeal mask airway with tracheal tube for intra-ocular ophthalmic surgery.

The use of the laryngeal mask was compared with tracheal intubation in 30 patients who underwent intra-ocular ophthalmic surgery and who received intravenous anaesthesia with propofol. Changes in intra-ocular pressure, heart rate and mean arterial pressure after the insertion of the laryngeal mask airway or the tracheal tube were not significantly different. However, at the end of the procedure, a significantly higher percentage of patients with a tracheal tube coughed, reacted to head movement and suffered breath-holding. In addition, significantly more patients in this group complained of a sore throat (p less than 0.05). During intravenous propofol anaesthesia, the laryngeal mask airway does not offer any advantage over tracheal intubation in the control of intra-ocular pressure for intra-ocular ophthalmic surgery. However, there were fewer complications immediately following surgery in the laryngeal mask group.


A multiple dose comparison of combinations of ibuprofen and codeine and paracetamol, codeine and caffeine after third molar surgery.

In a randomised, double-blind, double-dummy, multiple dose, crossover study in 30 patients we compared an ibuprofen/codeine combination (400 mg ibuprofen/25.6 mg codeine phosphate) with a paracetamol/codeine/caffeine combination (1 g paracetamol/16 mg codeine phosphate/60 mg caffeine) for pain relief over 6 days after two-stage bilateral lower third molar removal. The ibuprofen combination produced significantly greater analgesia than the paracetamol combination, both on single-dose analysis of the first and second days and on multiple-dose measures for days 1, 2, 3 and 4. The mean incidence of adverse effects over the 6 days was 20% for both combinations. This trial design (crossover with multiple dosing in outpatients) is a sensitive way of testing for analgesia, and is potentially more predictive of adverse effect problems than single-dose studies. It confirms that multiple dosing may show increased efficacy.


Upper respiratory tract infections and general anaesthesia in children. Peri-operative complications and oxygen saturation.

Conflicting reports regarding the hazards of anaesthesia in children presenting for surgery with an upper respiratory tract infection have appeared in the literature. In the present study 130 children undergoing general anaesthesia with face mask for myringotomy and grommet insertion were graded as having either an acute or recent upper respiratory tract infection or were asymptomatic according to predetermined clinical symptoms and signs. The severity of respiratory and related complications were scored during induction, emergence and recovery. The peripheral oxygen saturation was recorded during induction, emergence, transfer to the recovery ward and in the recovery ward itself. There were no significant differences (p greater than 0.05) in the complication scores between the three groups of children. However, the incidence of hypoxaemia (oxygen saturation less than or equal to 93%) was significantly greater during transfer in the acute infection group (p = 0.001) and the recent infection group (p = 0.02), as well as during recovery in the acute group (p = 0.03) compared with asymptomatic children.


Patient-controlled sedation using propofol in day surgery.

Patient-controlled sedation using propofol was studied in 30 unselected patients aged 26-72 years undergoing day case general surgical or orthopaedic procedures under local or regional anaesthesia. Using a fixed propofol dose of 0.7 mg.kg-1 body weight, the technique provided anxiolysis and sedation with few side effects and a high degree of patient satisfaction. Increasing age was significantly correlated with a deeper maximum level of sedation (p less than 0.001), indicating a need for a lower bolus dose in the higher age groups. Interpatient variability in propofol requirement was reflected in the lack of significant correlation between propofol dose and procedure duration (p = 0.095) and patient age (p = 0.72).


The laryngeal mask airway. Two cases of prehospital trauma care.

Two cases of emergency prehospital airway control using the laryngeal mask are described. The patients were trapped following road traffic accidents and limited access prevented tracheal intubation. The laryngeal mask airway may be a useful alternative to tracheal intubation in some cases of prehospital trauma care.


Failed extradural anaesthesia for caesarean section. Complication of subsequent spinal block.

Two cases of unexpected high spinal anaesthesia following failed extradural anaesthesia for Caesarean section are described. In both cases rapid and unexpected advance of blockade, after the subarachnoid injection of moderate doses of local anaesthetic, required tracheal intubation. In one of the cases 15 ml of 0.9% saline, but no local anaesthetic, had been injected into the extradural space, suggesting that the mechanism involved is the cephalad displacement of the cerebrospinal fluid by extradural fluid, and not leakage of extradural anaesthetic solution into the subarachnoid space.


An automated system for testing the accuracy of patient-controlled analgesia devices.

A system was developed to test the accuracy of patient-controlled analgesia devices in situations simulating clinical use. Bolus requests are made automatically at predetermined intervals, and the infusate delivered is measured and recorded without the need for operator presence. To ensure clinical relevance, the bolus request times used in this study corresponded to a pattern typical of those requested by patients on the ward. Graseby, Abbott Provider 5500 and IVAC patient-controlled analgesia devices were tested and found to deliver reasonably accurately over a 24 h period. However, when an infusion was started in an unprimed system or after a period of no bolus requests in a bolus-only mode the Graseby and IVAC machines under-delivered. This system provides a means of testing patient-controlled analgesia devices operating in any delivery mode.


Laryngeal mask and anaesthetic waste gas exposure.

In recent years there has been a growing awareness of the possible hazards caused by anaesthetic gases in operating theatres. The laryngeal mask airway provides an alternative both to tracheal intubation and the face mask although the implications for operating theatre contamination have not been quantified. This paper describes the incidence and magnitude of exposure of theatre personnel to waste anaesthetic gases during laryngeal mask airway anaesthesia. The leakage of anaesthetic gases to the anaesthetist's breathing zone was monitored using a Bruel & Kjaer Multi Gas Monitor, Type 1302 during 50 general anaesthetics employing either spontaneous (n = 24) or controlled (n = 26) ventilation. All patients were anaesthetised with propofol, alfentanil and nitrous oxide. There was no statistically significant association between the amount of anaesthetic gas leakage and ventilation method. The laryngeal mask airway meets occupational safety requirements on nitrous oxide concentrations in the operating theatre environment.


The upgraded Finapres 2300e. A clinical evaluation of a continuous noninvasive blood pressure monitor.

Measurements from the upgraded Finapres 2300e continuous noninvasive blood pressure monitor, the Finapres 2300 and Colin oscillometric noninvasive blood pressure monitor were compared with invasive arterial line blood pressure readings. Fifteen young Chinese patients undergoing elective spinal surgery of more than 2 h duration had contemporaneous blood pressure measurements digitally recorded every minute. Data were analysed using methods described by Bland and Altman to assess agreement of noninvasive devices with an arterial line. Results showed that although the Finapres 2300e was significantly more accurate than the Finapres 2300 and Colin noninvasive blood pressure monitors it could not be recommended as a substitute for continuous arterial line blood pressure monitoring. Both Finapres devices demonstrated reductions in accuracy related to time (drift) and over-read diastolic and mean pressures by 5-8 mmHg throughout the range of mean arterial line pressures (bias). The Colin was consistently less accurate than the Finapress monitors and performed worst at low mean arterial line pressures.


Light anaesthesia with propofol for paediatric MRI.

Anaesthetic techniques and monitoring equipment may interfere with the technical demands of magnetic resonance imaging. The purpose of this study was to evaluate the safety and efficacy of a light anaesthetic technique with intravenous propofol in nonintubated children. In 20 neuropaediatric patients sedation was induced with propofol 1 mg.kg-1, followed by a continuous infusion titrated to produce adequate immobilisation. Oxygen, 4 l.min-1, was administrated by paediatric face mask. Respiratory rate, end-tidal carbon dioxide tension and oxygen saturation were continuously monitored. In 10 patients capillary blood gas tensions were determined 3 and 20 min after the procedure. Data are reported as mean (SD) and the mean (SD) total propofol dose was 5 (2) mg.kg-1.h-1. Oxygen saturation remained constantly higher than 96% in all patients. End-tidal carbon dioxide tension varied between 35 (7) mmHg 3 min after induction, and 41 (6) mmHg 30 min after the start of the procedure. End-tidal to capillary PCO2 difference was 4 (3) mmHg. Within 20 min after the end of the procedure all patients were fit for dismissal to the ward. One imaging sequence out of 49 was repeated because of movement artefacts. In conclusion, intravenous propofol sedation appears to be a safe and reliable technique for paediatric sedation during magnetic resonance imaging.


Dermal patch anaesthesia for venous cannulation with 10% lignocaine gel containing glycyrrhetinic acid monohemiphthalate disodium as an absorption promoter.

The clinical efficacy of transdermal 10% lignocaine gel mixture containing 3% w/w glycyrrhetinic acid monohemiphthalate disodium as an absorption promoter was evaluated at venous cannulation in 17 paediatric (6-11 years) and 17 adult (29-65 years) patients. After about 60 min of occlusive application, the mean (SD) pinprick pain score was 0.7 (0.7) in the paediatric group, compared with 1.4 (1.3) in the adults (p less than 0.05). Twenty-five patients (14 children and 11 adults) who had a pinprick score of 0 or 1 underwent venous cannulation without intradermal local anaesthetic. The mean (SD) pain scores at venepuncture showed no significant differences between children and adults.


Enzyme-catalyzed oligosaccharide synthesis.

Cell-surface carbohydrates and their conjugates are involved in many types of molecular recognition. This review describes recent developments in enzyme-catalyzed oligosaccharide synthesis, with particular focus on glycosyltransferase and glycosidase reactions. With the increasing availability of glycosyltransferases via recombinant DNA technology, glycosyltransferase-catalyzed glycosylation with in situ regeneration of sugar nucleotides appears to be the most effective method for large-scale stereocontrolled oligosaccharide synthesis.


Determination of the intravesicular ionized sodium concentration in a cell-free brain membrane vesicle preparation using the fluorescent indicator, SBFI.

The intravesicular ionized Na concentration (Nai) was measured using the fluorescent Na indicator, SBFI, in microsacs, a cell-free brain vesicle preparation. SBFI fluorescence was monitored by a dual excitation-wavelength method at the same wavelengths commonly employed for Fura-2 determination of intracellular ionized calcium concentrations (Cai). Calibration of SBFI fluorescence was reliably performed in brain microsacs in situ. Resting Nai was dependent on the extravesicular Na concentration (Nao) and was about 36 mM in the presence of 120 mM extracellular Nao. In the presence of ouabain, an inhibitor of the plasma membrane Na/K-ATPase, Nai increased by 27 mM over 60 s. Nai was also increased by resuspension of microsacs in buffers of low free Ca concentrations (0 to 0.8 mM), indicating that the extravesicular Ca concentration (Cao) is an important regulator of Nai. Alkaloids active at voltage-sensitive Na channels, veratridine and aconitine, also increased Nai. These results demonstrate the presence of homeostatic mechanisms for neuronal Nai regulation and show that Nai can be measured in a cell-free brain vesicle preparation using SBFI.


Determination of the molar absorptivities of phenothiazine cation radicals generated by oxidation with hydrogen peroxide/peroxidase.

Phenothiazines are used as antipsychotic drugs and as reagents to determine microamounts of hemoglobin in biological fluids and tissues. Several agents cause the oxidation of phenothiazines to chromophoric cation radicals, whose stability may be related with their biological action. Enzymes and proteins with peroxidase activity catalyze the oxidation by H2O2 of phenothiazines to their corresponding cation radicals, which suffer a nonenzymatic breakdown. The instability of these cation radicals makes the determination of their respective molar absorptivities very difficult. These properties, however, have been determined for a few phenothiazine cation radicals by cumbersome or unreliable procedures. In this paper a new method is proposed and applied to six different phenothiazines oxidized with H2O2/peroxidase. The method involves the stoichiometric exhaustion of H2O2, under assay conditions which yield a fast enzymatic formation of phenothiazine cation radicals and which slow down their nonenzymatic breakdown. This method may be useful for quantitative studies on the enzymatic activity and the reaction mechanism of the oxidation of a number of phenothiazines catalyzed by different types of peroxidase, as well as by proteins with peroxidase activity, such as hemoglobin.


Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylenediaminetetraacetic acid.

The thiobarbituric acid (TBA) reactivity of rat liver and brain homogenates was characterized to elucidate what kinds of aldehyde species contributed to the reactivity. Characteristic pH dependence of the reactivity with a maximum at around pH 3 and marked enhancement of the reactivity by t-butyl hydroperoxide (t-BuOOH) and ferric ion were similar to those of alkadienals. The amounts of aldehyde species, including alkadienals determined as 2,4-dinitrophenylhydrazones, were high enough to account for the enhanced reactivity. The reactivity was inhibited by ethylenediaminetetraacetic acid (EDTA) but not completely, suggesting the presence of malonaldehyde whose reactivity was not affected by EDTA. The amounts of malonaldehyde determined as 1-(2,4-dinitrophenyl)pyrazole could account for a part of the reactivity in the presence of EDTA. Hence, the TBA reactivity of liver and brain homogenates at around pH 3 in the presence of t-BuOOH and ferric ion may be accounted for by alkadienals and malonaldehyde and that in the presence of EDTA by malonaldehyde.


An assay for cocaethylene and other cocaine metabolites in liver using high-performance liquid chromatography.

Cocaethylene (benzoylecgonine ethyl ester or ethyl cocaine) is a transesterification product of cocaine and ethanol that has been observed in the urine of individuals using these drugs in combination. There is evidence that cocaethylene is pharmacologically active, and its formation in vivo may contribute to the toxicity of cocaine. A new method is presented here which enables the quantification of cocaethylene and cocaine, as well as the cocaine metabolites benzoylecgonine and norcocaine in liver tissue. This method utilizes high-performance liquid chromatography with uv detection (235 nm), and the propyl ester of cocaine is used as an internal standard. Liver homogenates are first buffered with 0.1 N dibasic potassium phosphate (pH 9.1) and then extracted with methylene chloride:isopropanol (9:1). Extraction efficiencies were approximately 75-85% for the compounds of interest. The coefficient of variation for replicate determinations (N = 10) of cocaethylene concentration was 5.75%, with comparable values obtained for cocaine, norcocaine, and benzoylecgonine. The detection limit for cocaethylene, based on a peak height threefold greater than background noise, was approximately 1.7 ng of injected compound. Using this method, it was demonstrated that cocaethylene is present in mouse liver following cocaine and ethanol administration, with an apparent rapid rate of formation and elimination.


Rapid robust separation of hydroxyproline and proline.

The presence of hydroxyproline, and the determination of the ratio of the secondary amino acids proline to hydroxyproline, in amino acid hydrolysates specifically identifies collagen and collagen peptides. o-Phthalaldehyde, and then 9-fluorenylmethyl chloroformate, were used to carry out sequential prederivatization of amino acid hydrolysates in an in-line high-performance liquid chromatography sample loop. After derivatization, reversed-phase high-performance liquid chromatography with a C-18 ODS Hypersil cartridge column was used to resolve the hydroxyproline and proline from all primary amino acids, with resolution and detection of hydroxyproline and proline within 2.0 and 2.8 min, respectively, at concentrations in the range of picomoles per microliter of derivatized amino acid. The assay has a turnaround time of 10.75 min.


Assessment study on the high-performance liquid chromatography-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate.

The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant. A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration. When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins. Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution. When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite. On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS. When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides. The mechanism of binding of the protein polypeptides was discussed on the basis of these results. The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite.


Computer-assisted tracking of actin filament motility.

In vitro motility assays, in which fluorescently labeled actin filaments are propelled by myosin molecules adhered to a glass coverslip, require that actin filament velocity be determined. We have developed a computer-assisted filament tracking system that reduced the analysis time, minimized investigator bias, and provided greater accuracy in locating actin filaments in video images. The tracking routine successfully tracked filaments under experimental conditions where filament density, size, and extent of photobleaching varied dramatically. Videotaped images of actin filament motility were digitized and processed to enhance filament image contrast relative to background. Once processed, filament images were cross correlated between frames and a filament path was determined. The changes in filament centroid or center position between video frames were then used to calculate filament velocity. The tracking routine performance was evaluated and the sources of noise that contributed to errors in velocity were identified and quantified. Errors originated in algorithms for filament centroid determination and in the choice of sampling interval between video frames. With knowledge of these error sources, the investigator can maximize the accuracy of the velocity calculation through access to user-definable computer program parameters.


Spectrofluorometric assay for the quantitation of cell-tissue electrofusion.

Fluorometric assay for quantitating electrofusion, FAQE, was developed to measure the number of cells fused to intact tissue. The fluorescent vital dye hydroethidine is used in this method. The fluorescent intensity detected in the cell tissue hybrids is proportional to the number of individual cells fused. The number of cells fused was determined after fusion by lysing the epithelial layer with 0.2% sodium dodecyl sulfate and the supernatant fluids were measured in a spectrofluorometer and compared to established standard curves. The mean number of cells fused, in five separate experiments, was determined to be approximately 5000. All the experimental corneas had approximately the same number of fused cells with less than 10% variation. In addition, the technique was used to demonstrate an increase in the number of cells fused when multiple fusions were applied to the cell-tissue electrofusion system. These results demonstrate that FAQE can be utilized to quantitatively analyze the fusion yields.


New vectors for high level expression of recombinant proteins in bacteria.

A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.


Solid-phase extraction of urinary 11-dehydrothromboxane B2 for reliable determination with radioimmunoassay.

In this paper we elaborate a one-step procedure for the selective extraction of urinary 11-dehydrothromboxane B2 on octylsilyl silica cartridges for reliable determination with radioimmunoassay. The immunoreactivity profile of nonselectively extracted urine after HPLC separation showed that as much as 70% of the total 11-dehydrothromboxane B2 immunoreactivity comigrates with polar interfering material. Its amount could be considerably decreased using acetonitrile:water (18:82, v/v) as wash solvent before elution of 11-dehydrothromboxane B2 from the cartridge. Alternatively, very high immunoreactive purity was achieved without the preceding wash step by selective elution of the analyte with dichloromethane:hexane (70:30). After both optimized steps in the extraction procedure were combined, immunoreactivity was found only in HPLC fractions corresponding to the retention volume of authentic 11-dehydrothromboxane B2. The homogeneity of this immunoreactivity was confirmed by two-step HPLC separation. A significant correlation of values was observed between samples measured after extraction and those measured after subsequent HPLC purification. A high correlation was also found with concentrations determined by radioimmunoassay using four different antisera. The values of 24 h excretion of 11-dehydrothromboxane B2 in 10 male volunteers (595 +/- 114 ng/g creatinine, mean +/- SD) as well as the inhibitory effect of acetylsalicylic acid (80 +/- 13%) closely correspond with those reported in the literature. This selective extraction procedure provides a high validity in radioimmunoassay without requiring any further purification step.


Improvement in the method of sample stacking for gravity injection in capillary zone electrophoresis.

A method that allows capillary electrophoresis to be used as a microconcentrating technique is presented. An 85-fold improvement in the amount of material that can be injected into a capillary column without loss of resolution is shown. The method can be used for negative- and positive-charged species but it cannot be used for both species simultaneously.


A cell-based assay for evaluating the interaction of heparin-like molecules and basic fibroblast growth factor.

A simple panning procedure that allows for the evaluation of interactions between various heparin-like molecules and basic FGF has been developed. This assay measures the ability of compounds to inhibit the interaction of transfected human lymphoblastoid cells, UC 729-6 (UC cells), expressing hamster syndecan and basic FGF-coated plastic plates. The transfected cells bind rapidly to basic FGF-coated plates while the control cells do not bind well. Binding of the transfected cells to basic FGF was inhibited by heparin and heparin sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. There was little inhibition of binding by chemically modified heparin such as completely desulfated, N-acetylated heparin, completely desulfated, N-sulfated heparin, and N-desulfated, N-acetylated heparin. These results suggested that both the N-sulfate and O-sulfate groups of heparin are required for binding to basic FGF. In addition, inhibition by oligosaccharides derived from depolymerized heparin increased with fragment size; partial inhibition was observed with oligosaccharides as small as hexamers. The biochemical basis for the binding of transfected cells to basic FGF was established by showing a significant increase of 35SO4 incorporation into HS. In particular, the level of 35SO4-HS in the trypsin-releasable (cell surface) pool increased fivefold. This increase was accounted for by demonstration of the presence of HS on immunoprecipitated syndecan from the transfected cells.


Fluorescence lifetime imaging.

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.


The use of a layering technique for enhancing stability of lyophilized reagents.

An enzyme-mediated assay has been developed for the measurement of salicylate using salicylate monooxygenase purified from Pseudomonas cepacia ATCC 29351. Two assay formulations were produced, based on either a multiple-reagent or a single-reagent formulation, to allow sufficient flexibility for automated use. The multiple-reagent formulation was especially suited to diagnostic laboratories performing infrequent manual salicylate estimation where stability of the reconstituted reagent is of paramount importance. This was achieved by preparing the enzyme and color reagents in separate vials, so keeping the enzyme at a stable pH. For more frequent assay use where a reconstituted reagent shelf life was less important, the single-reagent system offers advantages of convenience. However, the working reagent required a pH of 10.0 upon reconstitution. Although the enzyme was sufficiently active at this pH to give a reliable assay, its storage stability was poor at pH 10.0, preventing lyophilization of the reagent at a pH suitable for immediate use on reconstitution. This incompatibility was overcome by use of a layering technique. The enzyme was separated from the buffering solution in the same vial by freezing the buffering solution and then overlayering with the enzyme reagent prior to a second freezing cycle and subsequent freeze drying.


Determination of evolved 14CO2 in decarboxylase reactions with application to measurement of [14C]oxalic acid.

An assay is described for the determination of the radioactive purity of [14C]oxalic acid preparations and the quantity of [14C]oxalic acid in biological samples. In this method oxalate decarboxylase is used to convert oxalate to formate and CO2. The entire procedure is carried out in a scintillation vial. The 14CO2 released in the enzymic reaction is allowed to diffuse off in a fume hood following acidification. Scintillation fluid is added to reacted and unreacted vials and the radioactivity measured. The loss of radioactivity from the reacted versus the unreacted vials provides the quantity of evolved 14CO2. This value is equal to 50% of the [14C]-oxalate (dpm) present. The radioactive purity of four preparations of [U-14C]oxalic acid was 99.0% while a fifth batch had a purity of 88%. A single batch of [U-14C]oxalic acid had a radioactive purity of 99.0% following storage of an aqueous solution, at -20 degrees C for 7 years. Recovery of [14C]oxalic acid from rat fecal extracts was 101.3%. Eight replicate analyses of a [U-14C]oxalic acid preparation gave a coefficient of variation of 0.3%. Following subcutaneous infusion of [U-14C]oxalic acid to rats, 100.2 +/- 2.9%, mean +/- SD, of the 14C in fecal extracts was present as [14C]oxalic acid (n = 10). The procedure provides a rapid, sensitive, and specific method to determine [14C]oxalic acid. It avoids the time consuming and inconvenient procedure for trapping and counting the evolved 14CO2. The approach used to determine the evolved 14CO2 may find application in other radiochemical methods that require its measurement.


High-resolution one-dimensional polyacrylamide gel isoelectric focusing of various forms of elongation factor-2.

A system for analyzing covalent modifications of elongation factor-2 (eEF-2) by one-dimensional isoelectric focusing in slab polyacrylamide gels is described. Depending on the degree of phosphorylation, four species of eEF-2 could be resolved corresponding to the un-, mono-, bis-, and trisphosphorylated factor. Furthermore, the degree of ADP-ribosylation of the protein could also be assessed by this method. It was also shown that an acidic isoform of eEF-2 exists which appears not to be artifactual and that the relative level of this isoform appeared to vary between different cell types. By Western blotting the gels and using an antibody against eEF-2 it is possible to assess the state of phosphorylation of the factor in cells.


Enzyme-linked immunosorbent assay for proteolytically inactivated antithrombin-III: use of sodium dodecyl sulfate to eliminate signal due to intact antithrombin-III.

Antithrombin III (AT-III) is a serine protease inhibitor (serpin) that can be catalytically inactivated by human neutrophil elastase (HNE) without inhibiting HNE activity. As with catalytic inactivation of most serpins, the cleaved form of the inhibitor is difficult to measure in the presence of active inhibitor. One major difference between the cleaved and intact forms of AT-III is that the cleaved form adopts a more stable conformation. Using sodium dodecyl sulfate (SDS), we were able to devise an enzyme-linked immunosorbent assay (ELISA) capable of detecting cleaved AT-III in the presence of intact AT-III. It seems likely that the SDS alters the intact AT-III so that it is not detected in the ELISA. As little as 5 micrograms/ml HNE-cleaved AT-III could be detected when spiked into human plasma; HNE-cleaved AT-III spiked into human plasma at different levels was recovered as expected. Thrombin-cleaved AT-III was also detected using this ELISA. The generation of cleaved AT-III in human plasma by HNE in the presence of heparin could be monitored as well. The cleaved AT-III ELISA is a novel, yet simple way to measure proteolytically inactivated AT-III in the presence of intact AT-III and should be useful for studying the role of proteolytic inactivation of serpins such as AT-III in vivo.


Calibration of a Clark-Type oxygen electrode by tyrosinase-catalyzed oxidation of 4-tert-butylcatechol.

A procedure for calibrating a Clark-type oxygen electrode is described. This method is based on the oxidation of 4-tert-butylcatechol (TBC) by O2 catalyzed by tyrosinase, to yield 4-tert-butyl-o-benzoquinone (TBCQ). This reaction consumes known amounts of oxygen in accordance with the stoichiometry: 2TBC + O2----2TBCQ + 2H2O and can be used to determine the relation between the oxygen concentration and the oxygen electrode response. TBCQ is very stable in the reaction medium for more than 30 min and shows no significant breakdown, which makes the calibration possible. A kinetic study of the oxidation of 3,4-dihydroxyphenylalanine by tyrosinase using the oxygen electrode is shown to confirm the validity of the calibration method.


Determination of selenium in urine by isotope dilution gas chromatography-mass spectrometry using 4-nitro-o-phenylenediamine, 3,5-dibromo-o-phenylenediamine, and 4-trifluoromethyl-o-phenylenediamine as derivatizing reagents.

An isotope dilution gas chromatography-mass spectrometry method for selenium determination in urine using 76Se as an internal standard is described. Three different derivatizing reagents, 4-nitro-o-phenylenediamine (NPD), 3,5-dibromo-o-phenylenediamine (DBPD), and 4-trifluoromethyl-o-phenylenediamine (TFMPD) were investigated for their gas chromatographic behavior including memory and precision and accuracy of isotope ratio measurements. By these criteria, the performance of these reagents was TFMPD greater than DBPD greater than NPD. Overall precision values of 1 to 7% were observed in determining Se isotope ratios at the 10-ng level, with no significant difference in using any of the three reagents. Memory effect was observed in the order NPD greater than DBPD greater than TFMPD with TFMPD showing no measurable memory effect. Accuracy of the GC-MS method was verified by quantitation of selenium in the NIST freeze-dried urine reference material SRM 2670.


Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein.

A simple and sensitive method for the direct measurement of lipid peroxides in lipoprotein and liposomes is described. The method is based on the principle of the rapid peroxide-mediated oxidation of Fe2+ to Fe3+ under acidic conditions. The latter, in the presence of xylenol orange, forms a Fe(3+)-xylenol orange complex which can be measured spectrophotometrically at 560 nm. Calibration with standard peroxides, such as hydrogen peroxide, linoleic hydroperoxide, t-butyl hydroperoxide, and cumene hydroperoxide gives a mean apparent extinction coefficient of 4.52 x 10(4) M-1 cm-1 consistent with a chain length of approximately 3 for ferrous ion oxidation by hydroperoxides. Endoperoxides are less reactive or unreactive in the assay. The assay has been validated in the study of lipid peroxidation of low density lipoprotein and phosphatidyl choline liposomes. By pretreatment with enzymes known to metabolize peroxides, we have shown that the assay measures lipid hydroperoxides specifically. Other methods for measuring peroxidation, such as the assessment of conjugated diene, thiobarbituric acid reactive substances and an iodometric assay have been compared with the ferrous oxidation-xylenol orange assay.


Photochemical amplification for horseradish peroxidase-mediated immunosorbent assay.

A new method for lowering the detection limit for a horseradish peroxidase (HRP) label in an enzyme-linked immunosorbent assay (ELISA) is proposed. The method is based on the use of a photochemical reaction of o-phenylenediamine (o-PD) autosensitized oxidation as an enhancement step in ELISA. The assay consists of two successive steps. The first step is a conventional HRP-mediated ELISA, using high-purity o-PD as a substrate. At this step, an o-PD oxidation product, 2,3-diaminophenazine (DAP), is formed in the dark. At the second step, the sample is illuminated at 400-500 nm for several minutes. Under illumination the concentration of DAP is greatly increased, depending on the duration and intensity of irradiation. Providing that the irradiation conditions are standardized, the final DAP concentration is proportional to the concentration of DAP formed by HRP. An ELISA for human carcinoembryonic antigen has demonstrated that the photochemical amplification method allows the detection limit of an assayed antigen to be lowered and the consumption of antibodies to be reduced. At the second step of this assay, the DAP concentration has been increased 50-fold under 4 min of irradiation.


A method for directly determining intact recombinant insulin fusion proteins in crude fermentation extracts.

In evaluating the purification of genetically engineered human insulin there is no plausible correlation between the yield of expression as determined by SDS-PAGE (taking into account all normally occurring losses during the several purification steps) and the actual yield, i.e., the final pure product. The aim of our work was to develop a fast, accurate, and reproducible method for the quantification of the initial yield of the intact insulin fusion protein directly after fermentation and without prior purification of the fermentation product. Therefore, a protocol for efficient tryptic digestion of protease-resistant inclusion bodies was established. The resulting crude peptide mixture was oxidized by performic acid and the insulin A-chain, which contains no cleavage side for trypsin, was quantified by HPLC in the form of a tetrasulfonate derivative to reduce artefacts due to free -SH groups. Compared with SDS-PAGE, this procedure allows sensitive monitoring of possible degradation at the C-terminus. Furthermore, quantification of expression products on the basis of the present method will provide better correlation between initial and actual yield.


Analysis of degradation of proteins labeled with fluorescein isothiocyanate by Sephadex G-25 affinity chromatography.

Fluorescein isothiocyanate (FITC) has strong affinity to Sephadex G-25 gel. Amino acids labeled with FITC were found to have affinity to the gel, their elution from a Sephadex G-25 column being markedly retarded. On the other hand, proteins labeled with FITC had no affinity to the gel and were eluted in the void volume of the column. The affinity of FITC-labeled compounds to a Sephadex G-25 column decreased with increase in their molecular mass. On the basis of these findings, the degradations of various FITC-labeled proteins by disrupted lysosomes in vitro were analyzed using a small Sephadex G-25 column. FITC-labeled degradation products were easily separated from the substrate proteins, and their production was shown to increase with the incubation time and to be suppressed by the proteinase inhibitor leupeptin. This procedure is a simple method for assay of protein degradation and should be useful in analyses of the roles of proteinases in protein degradation.


Chromosome variability and germ cell development in the house mouse.

Structural heterozygosities of the karyotype have detrimental effects on the meiotic process, resulting very often in impairment of fertility in the carriers. Both male and female germ cell development are affected by chromosomal variability although spermatogenesis seems particularly prone to be affected, probably because of the intrinsic characteristics of the male germ cell cytodifferentiation process (i.e. the histological architecture of the seminiferous epithelium). However, euploid and aneuploid sperm do not seem to differ in the molecular organization of the genome they carry, thus explaining the almost regular capacity to accomplish the first zygotic developmental stages by the aneuploid sperm (aneuploid both for gametogenic genes and for entire chromosomal arms). A survey of the molecular and morphological data available on germ cell development in conditions of chromosomal rearrangement leads to the conclusion that the current hypotheses accounting for this phenomenon can only partly explain it. A working hypothesis is proposed which considers the three-dimensional changes (produced by structural heterozygosity) in the spatial order of chromosomes within the nucleus as the primary cause potentially able to trigger distorted functioning of the germ cells.


The role of unpaired sex chromosomes in spermatogenic failure.

In 1974 Miklos reviewed evidence suggesting an association between sex chromosome pairing failure and spermatogenic arrest. He proposed that distributed over all the chromosomes there are 'meiotic pairing sites' which must be 'saturated' by homologous pairing during pachytene. In unpaired regions these sites are 'activated', and set in motion a process which leads ultimately to the death of the cell. In the present 'extended abstract' we summarize studies we have carried out on XSxraO male mice, that substantiate the main tenets of Miklos' model. Miklos' model is then used as a basis for explaining data we have collected on a large series of XYY male mice.


Drosophila spermatogenesis as a model system.

Spermatogenesis is very similar throughout the animal kingdom and is probably based on very old evolutionarily principles. Drosophila can serve as a suitable model system to understand the underlying processes. The molecular and ultrastructural data obtained for Drosophila germ cell development can be applied to understanding spermatogenesis in other organisms, including humans. Various methods used in studies of Drosophila spermatogenesis are presented together with observations which exemplify this conclusion.


Localization of intrinsic and extrinsic SVS II immunoreactivity in rat spermatozoa.

A glycoprotein, designated SVS II, is secreted in an androgen-dependent manner from lateral prostate and seminal vesicles of the rat. The pI of the protein is 10.5 and it has a molecular mass of 49 kDa. F-actin isolated from skeletal and heart muscle is precipitated at a ratio of 2:1 by SVS II. Using a polyclonal rabbit antibody against SVS II, we found an immunoreaction at the head region of rat spermatozoa removed from the vas deferens. In addition, immunoreactive material was observed in the principal piece of the sperm tail in those spermatozoa. We have studied the distribution of SVS II-immunoreactive material in spermatozoa isolated from seminiferous tubules, proximal (efferent ductules), and distal (caudal epididymal duct) epididymis, both in sexually active and inactive rats and found a differential reactivity pattern. Immunoreactivity observed in the principal piece of the sperm tail develops only immediately before spermiation and does not change during the epididymal transit of the spermatozoa. Immunolabelling seen in the head portion is first observed in spermatozoa from proximal epididymis. Simultaneous with its appearance, rhodamine-labelled phalloidin, indicating the presence of F-actin, no longer binds to that region. While the immunoreaction of the sperm head is attributed to extrinsic SVS II, added to the sperm head in proximal epididymis, the immunoreactivity of the sperm tail seems to result from a cross reactive intrinsic sperm tail protein that achieves its final structure briefly prior to the onset of spermiation.(ABSTRACT TRUNCATED AT 250 WORDS)


Progesterone induces capacitation in human spermatozoa.

Mammalian sperm acrosome reaction is a prerequisite for oocyte fertilization, and to date the mechanisms regulating this event are still unclear. Recent studies have demonstrated that human follicular fluid is able to induce an influx of extracellular calcium and acrosome reaction in capacitated spermatozoa. More recently these effects have been attributed to progesterone. The results of our study demonstrated that progesterone is able to trigger the capacitation, acrosome reaction, and fertilizability in human spermatozoa through a calcium mediated mechanism, suggesting a clinical use of this steroid in the treatment of spermatozoa for the different techniques of assisted fertilization.


Effect of nitrous oxide on intracranial pressure after cranial-dural closure in patients undergoing craniotomy.

After cranial-dural closure, nitrous oxide (N2O) may diffuse into and expand an entrapped volume of intracranial air, thereby increasing intracranial pressure (ICP). We performed a prospective clinical study to determine the effect of continuation of N2O after dural closure on ICP in patients undergoing craniotomies. Patients were randomly assigned in a 1.5:1 ratio into a group in which N2O was continued after dural closure (N2O, n = 15) or a group in which N2O was discontinued and replaced with nitrogen (N2, n = 9) at the time of dural closure. PaCO2 was normal prior to closure, and end-tidal PCO2 was kept constant after dural closure. Ipsilateral ICP was recorded at 5-min intervals after dural closure until completion of skin closure and immediately postoperatively. Presence of intracranial air was determined by head computed tomography scan within the first postoperative hour. ICP at the time of dural closure did not differ between the groups (N2O: 3 +/- 2 mmHg vs. N2:5 +/- 1 mmHg). Intraoperatively, ICP did not change after dural closure, regardless of whether N2O was continued or discontinued. Postoperatively, ICP was reduced, with a significant decrease in ICP (P less than 0.01) observed only in the N2O group. Postoperative computed tomography scans demonstrated the presence of intracranial air in all patients, with most exhibiting a mild to moderate degree of pneumocephalus. We conclude that continuation of N2O after dural closure did not affect ICP during the craniotomy closure. These results suggest that it is not necessary to discontinue N2O prior to dural closure for reasons of avoiding expansion of intracranial air and increasing ICP.


Partial characterization of a proacrosin binding protein.

All of the acid (pH 4.0) extracted proacrosin from porcine epididymal spermatozoa was found to be tightly associated with a specific protein referred to as the binding protein. A combination of gel filterations and gel electrophoresis revealed that the binding protein is composed of a major 28 kd and a minor 29 kd protein. Both of the proteins were shown to be nonproteolytic by gelatin SDS-PAGE analysis and the amino acid composition analysis of the purified 28 kd protein revealed that it is not related to the proteolytic component of the proacrosinacrosin system.


Human follicular fluid stimulates motility and velocity of washed human sperm in vitro.

Human follicular fluid collected by laparoscopic oocyte pick-up during IVF was studied with a computer-assisted semen analyser to evaluate the effect of hFF on human sperm motility and velocity. Freshly ejaculated human sperm were washed with phosphate buffered saline and mixed with either PBS or hFF. At various incubation periods of time, hFF increased both sperm motility and velocity as compared with control (P less than 0.01). After incubation of sperm with hFF at 37 degrees C and 5% CO2 in air for 0, 1, 3, 6, and 12 h, the amplitude increase of motility were 49%, 77%, 330%, 2020%, and 3340% when individual control motility was considered to be 100%. The amplitude increase of curvilinear velocity were 43%, 51%, 67%, 152%, 278%, respectively. Comparison of the motility and velocity of the sperm treated with hFF between 0 and 12 h, showed that hFF preserved both motility and velocity in vitro (P less than 0.01). The stimulatory effect of hFF was retained after boiling at 100 degrees C for 30 min, or after being filtered through Amicon membrane cones, but it disappeared if the hFF had been pre-treated with chymotrypsin. However, hFF did not stimulate the motility and velocity of unwashed sperm in freshly ejaculated human semen. A non-dialyzable and heat-stable factor(s) with a molecular weight below 50,000 in hFF may improve and maintain the motility and velocity of washed human sperm. Whether this factor could be used to improve pregnancy rate in assisted reproduction awaits further investigation.


The effect of dobutamine on hepatic blood flow and oxygen supply-uptake ratio during enflurane nitrous oxide anesthesia in humans undergoing liver resection.

Liver surgery is often accompanied by hepatic hypoperfusion and hypoxia, and it is controversial whether catecholamines increase hepatic blood flow and oxygen supply. The effects of 3 micrograms.kg-1.min-1 dobutamine on hepatic circulation and oxygen balance were examined in patients anesthetized with enflurane, nitrous oxide, and oxygen for liver surgery. Dobutamine did not cause a significant increase in hepatic arterial blood flow. However, total hepatic blood flow and portal venous blood flow were increased, resulting in an increase in hepatic oxygen delivery (HDO2). The increase in HDO2 was not associated with an improvement of hepatic oxygen supply-uptake ratio, since hepatic oxygen uptake (HVO2) also increased. After hepatectomy, the increases in portal venous blood flow and HDO2 were not accompanied by an increase in HVO2. The stimulation of hepatocellular oxygen metabolism by dobutamine and depressed responsiveness of adrenoceptors on hepatocytes in which metabolism was already augmented are the likely explanation for the different reactions before and after hepatectomy.


Histological and morphometric studies on the kinetics of germ cells and immature Sertoli cells during human prespermatogenesis.

Human prespermatogenesis between the 8th week of pregnancy and six months after birth was studied in testis material of 28 male foetuses from spontaneous abortions and 81 infants who died from sudden infant death. The foetuses and infants were grouped in 10 age groups. A first steep raise in the numbers of germ cells per 20 tubular cross sections from 22.3 in the first group up to 69.5 in group 3 was observed, i.e. up to the end of the 22nd week of pregnancy. Thereafter, a continuous decrease could be observed modulated by a second slighter increase during the first 4 months after birth. The ratio of germ cells and immature Sertoli cells improves from about 1:20 at the beginning to 1:8 in group 3; afterwards it changes in favour of the immature Sertoli cells down to 1:140 at the end of the study. The initial augmentation of germ cells is interpreted as the effect of a first proliferation wave comparable to that of M-prospermatogonia in other species. The decrease of germ cells is due to the stop of germ cell proliferation and simultaneous high proliferative activity of the immature Sertoli cells.


Preemptive analgesia. Clinical evidence of neuroplasticity contributing to postoperative pain.

Recent evidence suggests that surgical incision and other noxious perioperative events may induce prolonged changes in central neural function that later contribute to postoperative pain. The present study tested the hypothesis that patients receiving epidural fentanyl before incision would have less pain and need fewer analgesics post-operatively than patients receiving the same dose of epidural fentanyl after incision. Thirty patients (ASA physical status 2) scheduled for elective thoracic surgery through a posterolateral thoracotomy incision were randomized to one of two groups of equal size and prospectively studied in a double-blind manner. Epidural catheters were placed via the L2-L3 or L3-L4 interspaces preoperatively, and the position was confirmed with lidocaine. Group 1 received epidural fentanyl (4 micrograms/kg, in 20 ml normal saline) before surgical incision, followed by epidural normal saline (20 ml) infused 15 min after incision. Group 2 received epidural normal saline (20 ml) before surgical incision, followed by epidural fentanyl (4 micrograms/kg, in 20 ml normal saline) infused 15 min after incision. No additional analgesics were used before or during the operation. Anesthesia was induced with thiopental (3-5 mg/kg) and maintained with N2O/O2 and isoflurane. Paralysis was achieved with pancuronium (0.1 mg/kg). Postoperative analgesia consisted of patient-controlled intravenous morphine. Visual analogue scale pain scores were significantly less in group 1 (2.6 +/- 0.44) than in group 2 (4.7 +/- 0.58) 6 h after surgery (P less than 0.05), by which time plasma fentanyl concentrations had decreased to subtherapeutic levels (less than 0.15 ng/ml) in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)