Determination of egg proteins in food products by enzyme immunoassay.

An enzyme-linked immunosorbent assay (ELISA) was developed to determine the presence of egg proteins in foods. The polyclonal antibodies developed were specific to whole egg proteins and did not cross-react with any of the 38 nuts, legumes, or other common food ingredients tested. The concentrations of egg proteins that will inhibit 50% of antibody-antigen binding, IC50, were 3-7 ng/mL, and the linear range was 0.5-62.5 ng/mL. The detection limit was 0.2 ppm for various foods. Recoveries ranged from 67 to 96%. The intra- and inter-assay coefficients of variation in this procedure were 10-13% for ice cream spiked at 0.8 and 1.6 ppm. The ELISA has been applied to ice creams, noodles, pasta, and breads. Egg proteins were identified in all declared egg products, and no false positives were found.

The insulin receptor tyrosine kinase substrate p58/53 and the insulin receptor are components of CNS synapses.

The synapse is the primary locus of cell-cell communication in the nervous system. It is now clear that the synapse incorporates diverse cell signaling modalities in addition to classical neurotransmission. Here we show that two components of the insulin pathway are localized at CNS synapses, where they are components of the postsynaptic density (PSD). An immunochemical screen revealed that polypeptides of 58 and 53 kDa (p58/53) were highly enriched in PSD fractions from rat cerebral cortex, hippocampus, and cerebellum. These polypeptides were purified and microsequenced, revealing that p58/53 is identical to the insulin receptor tyrosine kinase substrate p58/53 (IRSp53). Our analysis of IRSp58/53 mRNA suggests that within rat brain there is one coding region for IRSp58 and IRSp53; we find no evidence of alternative splicing. We demonstrate that IRSp58/53 is expressed in the synapse-rich molecular layer of the cerebellum and is highly concentrated at the synapses of cultured hippocampal neurons, where it co-localizes with the insulin receptor. Together, these data suggest that insulin signaling may play a role at CNS synapses.

CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses.

Long-term changes in synaptic efficacy may require the regulated translation of dendritic mRNAs. While the basis of such regulation is unknown, it seemed possible that some features of translational control in development could be recapitulated in neurons. Polyadenylation-induced translation of oocyte mRNAs requires the cis-acting CPE sequence and the CPE-binding protein CPEB. CPEB is also present in the dendritic layers of the hippocampus, at synapses in cultured neurons, and in postsynaptic densities of adult brain. alpha-CaMKII mRNA, which is localized in dendrites and is necessary for synaptic plasticity and LTP, contains two CPEs. These CPEs are bound by CPEB and mediate polyadenylation-induced translation in injected Xenopus oocytes. In the intact brain, visual experience induces alpha-CaMKII mRNA polyadenylation and translation, suggesting that this process likely occurs at synapses.

Intra-ocular pressure during cardiopulmonary bypass--a comparison of crystalloid and colloid priming solutions.

Raised intra-ocular pressure secondary to alterations in plasma oncotic pressure has been implicated in the development of optic neuropathy after cardiopulmonary bypass. Patients presenting for open heart surgery received either crystalloid (n = 9) or colloid (n = 10) priming solutions for cardiopulmonary bypass. No differences in intra-ocular pressure or plasma oncotic pressure occurred between the groups before the onset of cardiopulmonary bypass. Five minutes after the start of bypass the median intra-ocular pressure increased to 31 mmHg in the crystalloid group compared with 13 mmHg in the colloid group (p < 0.05). At the same time plasma oncotic pressure decreased from approximately 20 mmHg in both groups to 10.6 mmHg with crystalloid and 15.7 mmHg with colloid primed cardiopulmonary bypass solutions (p < 0.05). Over the following hour of cardiopulmonary bypass, intra-ocular pressure and plasma oncotic pressure tended to return towards their pre-cardiopulmonary bypass values. Changes in plasma oncotic pressure, through fluid shifts, may have contributed towards this unexpected increase in intra-ocular pressure with crystalloid primed cardiopulmonary bypass.

HPV-16-related DNA sequences in Kaposi's sarcoma.

In the USA, Kaposi's sarcoma associated with the acquired immunodeficiency syndrome (AIDS-KS) is ten times more common in homosexual or bisexual men than in heterosexual men with AIDS. One explanation for this finding is that AIDS-KS may be caused by an infectious agent. Because there is a high incidence of human papillomavirus (HPV) infection, especially HPV-16, in homosexual men, we have sought HPV DNA sequences in Kaposi's sarcoma. We used the polymerase chain reaction with a primer pair specific for the highly conserved E6 region of HPV-16 to detect HPV-16 homologous DNA fragments in tumour tissues from 97 patients with KS and in KS-derived cell cultures. HPV DNA sequences were found in 11 of 69 KS skin tumours from homosexual men with AIDS-KS, in 3 of 11 KS biopsy specimens from homosexual men who had no clinical or laboratory evidence of HIV-infection, and in 5 of 17 KS skin lesions from HIV-1-negative elderly men and women with classic KS. The same primer pair amplified HPV-16 homologous fragments from two different continuous cell cultures derived from pleural effusion fluid of patients with pulmonary AIDS-KS and two continuous cell cultures derived from KS skin lesions. The findings suggest that HPV-16-related DNA sequences are associated with different forms of KS and may have a role in the pathogenesis of this neoplasm.

Detection of anti-HTLV-I Tax antibodies in HTLV-I enzyme-linked immunosorbent assay-negative individuals.

The HTLV-I tax gene protein (Tax) is not packaged within the mature viral particle from which the proteins for the commercially available enzyme-linked immunosorbent assay (ELISA) are derived. Screening of 162 individuals within a cohort of white intravenous (IV) drug abusers, previously identified as having an increased incidence of HTLV-I infection, demonstrated that seven of them had antibodies to the HTLV-I Tax protein but tested negative in HTLV-I ELISAs and Western blots prepared from purified virion proteins. Three out of 35 individuals in other behaviorally defined high-risk groups also displayed this limited pattern of reactivity to HTLV-I proteins. The presence of the anti-HTLV-I p40/Tax antibodies was determined by radioimmunoprecipitation assay (RIPA), which also revealed low levels of anti-env reactivity. The specificity of the anti-p40 reactivity was confirmed on specific Tax ELISAs and Western blots prepared from recombinantly produced Tax. In vitro gene amplification by the polymerase chain reaction (PCR) was used to establish the presence of sequences homologous to HTLV-I proviral DNA in four/four of these HTLV-I ELISA negative, Tax ELISA/Tax western blot/RIPA positive individuals. These data suggest that the true incidence of HTLV-I infection within high-risk cohorts is greater than previously reported.

Detection of sequences homologous to human retroviral DNA in multiple sclerosis by gene amplification.

Twenty-one patients with multiple sclerosis, chronic progressive type, were examined for DNA sequences homologous to a human retrovirus. Genomic DNA from peripheral blood mononuclear cells was analyzed for the presence of homologous sequences to the human T-cell leukemia/lymphoma virus type I (HTLV-I) long terminal repeat, 3' gag, pol, and env domains by the enzymatic in vitro gene amplification technique, polymerase chain reaction. Positive identification of homologous pol sequences was made in the amplified DNA from six of these patients (29%). Three of these six patients (14%) also tested positive for the env region, but not for the other regions tested. In contrast, none of the samples from 35 normal individuals studied was positive when amplified and tested with the same primers and probes. Comparison of patterns obtained from controls and from patients with adult T-cell leukemia or tropical spastic paraparesis suggests that the DNA sequences identified are exogenous to the human genome and may correspond to a human retroviral species. The data support the detection of a human retroviral agent in some patients with multiple sclerosis.

Comparison of methohexital and propofol for electroconvulsive therapy: effects on hemodynamic responses and seizure duration.

Electroconvulsive therapy can produce severe disturbances in the cardiovascular system, most commonly a transient period of hypertension. This study was designed to determine whether propofol, in comparison with methohexital, would attenuate this hypertensive response. Fifteen patients were studied during courses of six ECT administrations, each patient receiving propofol or methohexital on different occasions. Arterial pressure, heart rate, and cardiac rhythm were recorded. The induction doses were 1.08 +/- 0.03 mg.kg-1 of methohexital, and 1.60 +/- 0.04 mg.kg-1 of propofol. Systolic pressure, diastolic pressure, and heart rate were consistently lower following propofol than methohexital (P less than 0.005). The mean maximum increase over baseline systolic pressure was 2.1 +/- 2.9 mmHg with propofol, and 26.7 +/- 4.5 mmHg with methohexital (P less than 0.001). Cardiac rhythm abnormalities were infrequent, and their incidence did not differ significantly between the two induction agents (P greater than 0.3). The duration of seizures, as measured clinically, was reduced with propofol (17.9 +/- 2.5 s) in comparison with methohexital (30.9 +/- 2.8 s) (P less than 0.001). Recovery times were similar for the two agents. Since the role of seizure duration in the therapeutic efficacy of ECT remains controversial, propofol may be a useful induction agent for this procedure.

Enzymatic gene amplification: qualitative and quantitative methods for detecting proviral DNA amplified in vitro.

We evaluated various detection methods to identify amplified human retroviral sequences after Thermus aquaticus-directed polymerase chain reaction (PCR). A combination of hybridization formats and direct incorporation assays provided the most information. This multiphasic approach enabled us to detect specific human T cell leukemia virus type I (HTLV-I)-homologous regions in several HTLV-I-seronegative patients with T cell lymphoma, as well as variants of HTLV-I and human immunodeficiency virus type 1 in patients with prototype disease. In all diagnostic assays designed to detect a particular retrovirus, it was necessary to include a hybridization step, because sequences (endogenous or exogenous) homologous to certain primers were present in most human DNA preparations and yielded discrete products, sometimes of the predicted molecular weight, after amplification. These products could be discriminated by hybridization from amplified prototype proviral sequences. The intensity of the signal generated after hybridization was proportional to input target DNA, an observation making it feasible to quantitatively measure the proviral load in a DNA sample.

The control of intra-ocular pressure during the induction of anaesthesia for emergency eye surgery. A high-dose vecuronium technique.

A technique of rapid sequence induction of anaesthesia for use in patients with penetrating eye injuries is described. This utilises a three maximal breaths method of pre-oxygenation, the intravenous injection of thiopentone and high dose vecuronium (0.2 mg/kg). Using the loss of eyelash reflex as the starting point for timing, all patients were intubated after 60 seconds without coughing and bucking. No postintubation increases in intra-ocular pressure were seen in 70% of patients and in no patient did the increase in intra-ocular pressure exceed 5 mmHg. After 3 minutes of apnoea, the minimum haemoglobin oxygen saturation was 94% with a mean value of 97.6%.

Anaesthesia for extracorporeal shock wave lithotripsy.

The anaesthetic considerations of patients presenting for extracorporeal shock wave lithotripsy are described. Regional anaesthesia with sedation may be preferable to general anaesthesia for patients undergoing this form of therapy. If regional anaesthesia is contra-indicated, general anaesthesia using controlled ventilation with muscle relaxation, supplemented with a narcotic and a low concentration of volatile anaesthetic has been found to be a suitable alternative. The additional epidural preparation time has to be balanced against the benefits of easier patient transfer, especially during multi-stage procedures, and better postoperative analgesia. The epidural catheter can be left in situ in patients who require multiple treatments or who may experience severe ureteric pain as the resulting 'sand mass' is passed. Epidural space localisation using a 'loss of resistance to saline' technique is recommended, in order to avoid the possible risk of damage to the spinal cord and emerging nerves (due to the presence of an air-water interface). Patients with cardiac insufficiency need special consideration, in view of the effects of immersion on right and left heart filling pressures.