Effects of β-escin and saponin on the transverse-tubular system and sarcoplasmic reticulum membranes of rat and toad skeletal muscle

 Mechanically skinned skeletal muscle fibres from rat and toad were exposed to the permeabilizing agents β-escin and saponin. The effects of these agents on the sealed transverse tubular system (t-system) and sarcoplasmic reticulum (SR) were examined by looking at changes in the magnitude of the force responses to t-system depolarization, the time course of the fluorescence of fura-2 trapped in the sealed t-system, and changes in the magnitude of caffeine-induced contractures following SR loading with Ca²⁺ under defined conditions. In the presence of 2 μg ml^–1β-escin and saponin, the response to t-system depolarization was not completely abolished, decreasing to a plateau, and a large proportion of fura-2 remained in the sealed t-system. At 10 μg ml^–1, both agents abolished the ability of both rat and toad preparations to respond to t-system depolarization after 3 min of exposure, but a significant amount of fura-2 remained in sealed t-tubules even after exposure to 100 μg ml^–1β-escin and saponin for 10 min. β-Escin took longer than saponin to reduce the t-system depolarizations and fura-2 content of the sealed t-system to a similar level. The ability of the SR to load Ca²⁺ was reduced to a lower level after treatment with β-escin than saponin. This direct effect on the SR occurred at much lower concentrations for rat (2 μg ml^–1β-escin and 10 μg ml^–1 saponin) than toad (10 μg ml^–1β-escin and 150 μg ml^–1 saponin). The reverse order in sensitivities to β-escin and saponin of t-system and SR membranes indicates that the mechanisms of action of β-escin and saponin are different in the two types of membrane. In conclusion, this study shows that: (1) β-escin has a milder action on the surface membrane than saponin; (2) β-escin is a more potent modifier of SR function; (3) simple permeabilization of membranes is not sufficient to explain the effects of β-escin and saponin on muscle membranes; and (4) the t-system network within muscle fibres is not a homogeneous compartment. Received: 18 November 1998 / Received after revision: 6 January 1999 / Accepted: 7 January 1999     

Genetic Vaccination of Mice with Plasmids Encoding the NS1 Non-structural Protein from Tick-borne Encephalitis Virus and Dengue 2 Virus

Although there is a safe, inexpensive and efficacious vaccine against yellow fever, vaccination against other flavivirus diseases is less successful. There is no licensed vaccine against dengue fever and current vaccines against tick-borne encephalitis (TBE) and Japanese encephalitis are expensive and require several injections. Furthermore novel vaccines containing only virus envelope proteins may raise fears over antibody mediated enhancement (ADE) of disease. Here we report the successful use of genetic vaccination against TBE in an experimental animal model using a plasmid containing the coding sequence of a non-structural protein (NS1). Such vaccines would provide inexpensive protection against disease, without raising concerns over inducing ADE on subsequent exposure to heterotypic infectious virus. Attempts to generate chaemeric plasmids to protect against both TBE and dengue fever were less successful. Although these chaemeric plasmids directed the synthesis and secretion of the virus NS1 protein normally, no protection was observed against either TBE or dengue fever.     

Multiomics uncovers developing immunological lineages in human

The development of the human immune system during embryonic and fetal life has historically been difficult to research due to limited access to human tissue. Experimental animal models have been widely used to study development but cellular and molecular programmes may not be conserved across species. The advent of multiomic single-cell technologies and an increase in human developmental tissue biobank resources have facilitated single-cell multiomic studies focused on human immune development. A critical question in the near future is "How do we best reconcile scientific findings across multiple omic modalities, developmental time, and organismic space?" In this review, we discuss the application of single-cell multiomic technologies to unravel the major cellular lineages in the prenatal human immune system. We also identify key areas where the combined power of multiomics technologies can be leveraged to address specific immunological gaps in our current knowledge and explore new research horizons in human development.     

The findings reported in patients undergoing excretory urography for recurrent urinary tract infection

There was little difference between the proportion of lesions found by excretion urography in patients with recurrent urinary tract infections referred by general practitioners or hospital staff, or between Horton General Hospital which is a district general hospital, and the United Oxford Hospitals, which is a teaching group. Many patients with urinary tract infections may be saved an unnecessary visit to the hospital consultant if this is first investigated by the general practitioner.

About one fifth of all patients had significant lesions shown by excretory urography which suggests that the investigation is worthwhile in patients with recurrent urinary tract infections.

     

Synthetic amyloid beta-protein fails to produce specific neurotoxicity in monkey cerebral cortex.

Because progressive amyloid beta-protein (A beta P) deposition and surrounding neuritic dystrophy occur spontaneously in primates, we evaluated the in vivo effects of synthetic A beta P in monkey cortex. Experimental and control A beta P were stereotactically injected into multiple neocortical sites of adult rhesus monkeys in a vehicle of either artificial cerebrospinal fluid or acetonitrile. After 2 weeks or 3 months, injection sites were identified and characterized histologically and immunocytochemically. A beta P antibodies specifically detected the injected A beta P1-40 peptide. Serial sections stained with silver and antineurofilament protein demonstrated comparable degrees of degenerating neurons, dystrophic neurites, and axonal spheroids associated with both experimental and control peptide injections. Alz 50 staining was sparse or absent in all sites. We conclude that specific cellular changes closely resembling AD pathology were not detected in these experiments, and that control and experimental A beta P peptides produced indistinguishable effects. Methodological concerns regarding the in vivo modeling of A beta P bioactivity are discussed.     

C-myc oncogene product P62c-myc in ovarian mucinous neoplasms: immunohistochemical study correlated with malignancy.

The monoclonal antibody Myc 1-6E10 was used to determine the cellular distribution of the c-myc oncogene product p62c-myc in 60 mucinous ovarian tumours. Three patterns of immunostaining were apparent: (i) nuclear staining alone; (ii) staining of the nucleus and basal cytoplasm; and (iii) staining of the entire cell. Of the 21 cases of mucinous cystadenoma, 11 showed nuclear staining alone, and a further case showed additional weak staining of the basal cytoplasm. Nuclear staining alone was not present in any of the 17 borderline mucinous tumours examined. Strong staining of the nucleus and basal cytoplasm was seen in 16 of these borderline cases, six of which also showed focal staining of the apical cytoplasm. All 22 cases of mucinous cystadenocarcinoma showed staining of the cell nucleus and entire cell cytoplasm. Focal staining of the apical cytoplasm in six of 17 borderline mucinous tumours produced a pattern of c-myc immunostaining similar to that of cystadenocarcinoma. Retrospective analysis of the clinical data showed that no significant differences between patients with borderline tumours of these two categories could be defined. Although immunostaining with Myc 1-6E10 can be used in the categorisation of mucinous ovarian tumours, it is concluded that standard histological criteria are more accurate indicators of tumour behaviour than is an assessment of c-myc expression.     

SKALP/elafin gene polymorphisms are not associated with pustular forms of psoriasis

Psoriasis is a multifactorial skin disease characterised by epidermal abnormalities and infiltration by lymphocytes and polymorphonuclear leukocytes (PMN). Skin-derived antileukoproteinase (SKALP), also known as elafin, is a potent inhibitor of human leukocyte elastase and proteinase 3, two PMN-derived proteinases implicated in tissue destruction and leukocyte migration. We have shown that, at least at the protein level, SKALP is significantly decreased in lesional skin of patients with pustular psoriasis compared with plaque-type psoriasis. This finding raised the possibility that SKALP could be one of the candidate genes for pustular forms of psoriasis. We therefore performed single strand conformation polymorphism (SSCP) analysis on the SKALP gene to screen for mutations/polymorphisms in the exons of 30 patients with plaque-type psoriasis, 15 patients with pustular psoriasis and 48 healthy controls. In exon 1 a polymorphism was detected at position + 43 relative to the translation start site, resulting in a substitution of threonine for alanine in the signal peptide. In the promoter region a dinucleotide repeat polymorphism was identified. Both polymorphisms were not associated with pustular psoriasis, or psoriasis in general. Our data indicate that the decrease in SKALP activity in pustular psoriasis is not caused by mutations in the coding region of the gene, and that there is no allelic association between pustular psoriasis and SKALP gene polymorphisms.     

Detection of a 15q deletion in a child with Angelman syndrome by cytogenetic analysis and flow cytometry

A proximal 15q deletion, del(15) (q11:q13), was detected in a child with Angelman syndrome by cytogenetic analysis of peripheral lymphocytes. The chromosomes of both parents appeared normal. Flow karyotype analysis carried out on lymphoblastoid cell lines derived from the child and her parents confirmed the presence of a de novo 15 deletion. The estimated size of the deleted segment ranged from 6.1-9.5% of chromosome 15 (approximately 6-9.3 million base pairs). The parental origin of the deleted chromosome could not be resolved by flow cytometry, but cytogenetic evidence suggested that it was derived from the smaller chromosome 15 homologue in the mother.     

Assessing the conditions forin vivo electrical virtual biopsies in Barrett's oesophagus

It has previously been shown that it is possible to differentiate between squamous and columnar epithelia in rat and resected human tissues using an impedance probe to makein vitro measurements. This probe can be passed down an endoscope allowing measurements to be made in patients. However, the probe emerges parallel to the oesophageal wall, with little room to manoeuvre. The conditions of control required to give reliable readings have been investigated. The importance of pressure applied and the angle of approach to the oesophagus was assessed. Pressures in the range 26.6 Pa to 46.3 kPa and angles in the range 15–90 degrees were considered. Inin vitro studies it was observed that it was possible to obtain consistent readings with pressures greater than 2.9 kPa and with angles greater than 15 degrees between the probe and the oesophagus. These conditions can be achievedin vivo, and readings obtained from twelve patients are shown (45 readings on normal squamous, 34 on Barrett's oesophagus and 22 on stomach). At low frequencies (9.6–153.2 kHz), a Mann-Whitney test shows a significant difference (p<0.001) when comparing the means from squamous and columnar, and also when readings from Barrett's and normal gastric epithelia are compared (p<0.001).     

Effects of "Below-Zero" Habituation on the Electrodermal Orienting Response to a Test Stimulus

Two experiments were designed to investigate the effect of below-zero habituation training on skin conductance response (SCR) amplitude to a change in auditory stimulus frequency. In both experiments, subjects were trained with a 1000 Hz tone until zero responding and then received 5, 10, or IS further training trials. In Experiment 1 (N=45), subjects then received 1 presentation of a test stimulus of 1400 Hz, while in Experiment 2 (N=45), the test stimulus was a tone of 670 Hz. On the basis of dual-process theory, it was hypothesized that response amplitude to the test stimulus would be inversely related to amount of below-zero training. However, the results of both experiments indicate that SCR amplitude was positively related to amount of below-zero training. These results suggest that in situations of extended habituation training, an expectancy or subjective probability of stimulus occurrence gradient is important in determining response amplitude to a test stimulus.