Oligonucleotide mapping of picornavirus RNAs by two-dimensional electrophoresis.

Considerable differences have been found in two-dimensional polyacrylamide gel electrophoresis fingerprints of complete ribonuclease T₁ digestion products of the RNAs of representative members of the entero-, cardio-, and foot-and-mouth disease virus subgroups of the picornavirus family. Individual members of the different subgroups, serotypes of a virus, and even subtypes within a serotype can be distinguished by the use of this technique. The method has also facilitated the identification of homopolymeric regions within the different picornavirus genomes, and the presence of a poly(C) tract in the cardio- and foot-and-mouth disease virus subgroups has been confirmed. A poly(A)-rich tract of approx 40–100 nucleotides has been detected in all the picornaviruses studied. Oligonucleotide fragments possibly specific to the enterovirus subgroup were also detected and partially characterised.     

Myosin VIIA gene: heterogeneity of the mutations responsible for Usher syndrome type IB

Usher syndrome is recognized as the most frequent cause of hereditary deaf-blindness. Usher syndrome type I (USH1), the most severe form of the disease, is characterized by profound congenital sensorineural deafness, constant vestibular dysfunction, and retinitis pigmentosa of prepubertal onset. This form is genetically heterogeneous and five loci (USH1A-E) have been mapped thusfar. However, only the gene responsible for USH1 B (which accounts for approximately 75% of USH1 cases) has been characterized. It encodes a long-tailed unconventional myosin, myosin VIIA, with a predicted 2215 amino acid sequence. Primers covering the complete myosin VIIA coding sequence as well as the 3' non coding sequence were designed, allowing direct sequence analysis of each of the 48 coding exons and flanking splice sites in seven patients affected by USH1. Four novel mutations were thereby identified. The possibility should now be considered of a sequence-based prenatal diagnosis in some of the families affected by this very severe form of Usher syndrome.      

Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium

Human Fallopian tubal epithelial cells in culture lose morphological features associated with the epithelium in situ and the extent to which they retain their in-vivo phenotype or function is unknown. In order to address this question, immunocytochemical markers were identified which distinguish secretory (HMFG2+, LhS28-) from ciliated (HMFG2-, LhS28+) epithelial cells in tissue sections of Fallopian tube. These markers were used to analyse the phenotype of tubal cells in vitro. Primary cultures of human tubal epithelial cells were seeded onto glass and grown to confluence before addition of oestradiol-17beta. In the absence of hormone, tubal epithelial cells expressed cytokeratins and nuclear receptors for oestrogen and progesterone and adopted a homogeneous (HMFG2+, LhS28-) secretory cell phenotype. Following the addition of oestradiol-17beta, a proportion of cells became positive for LhS28. The induction of a ciliated epithelial cell phenotype was confirmed by scanning electron microscopy, where on permeable collagen membranes, approximately one-third of tubal epithelial cells became ciliated in the presence of oestradiol-17beta. We suggest that in vitro, tubal epithelial cells adopt an immature secretory-like phenotype and that oestrogen can induce differentiation to a ciliated epithelial cell phenotype.      

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.      

Effects of massage on delayed-onset muscle soreness, swelling, and recovery of muscle function

Context: Delayed-onset muscle soreness (DOMS) describes muscle pain and tenderness that typically develop several hours postexercise and consist of predominantly eccentric muscle actions, especially if the exercise is unfamiliar. Although DOMS is likely a symptom of eccentric-exercise–induced muscle damage, it does not necessarily reflect muscle damage. Some prophylactic or therapeutic modalities may be effective only for alleviating DOMS, whereas others may enhance recovery of muscle function without affecting DOMS.Objective: To test the hypothesis that massage applied after eccentric exercise would effectively alleviate DOMS without affecting muscle function.Design: We used an arm-to-arm comparison model with 2 independent variables (control and massage) and 6 dependent variables (maximal isometric and isokinetic voluntary strength, range of motion, upper arm circumference, plasma creatine kinase activity, and muscle soreness). A 2-way repeated-measures analysis of variance and paired t tests were used to examine differences in changes of the dependent variable over time (before, immediately and 30 minutes after exercise, and 1, 2, 3, 4, 7, 10, and 14 days postexercise) between control and massage conditions.Setting: University laboratory.Patients or Other Participants: Ten healthy subjects (5 men and 5 women) with no history of upper arm injury and no experience in resistance training.Intervention(s): Subjects performed 10 sets of 6 maximal isokinetic (90°·s⁻¹) eccentric actions of the elbow flexors with each arm on a dynamometer, separated by 2 weeks. One arm received 10 minutes of massage 3 hours after eccentric exercise; the contralateral arm received no treatment.Main Outcome Measure(s): Maximal voluntary isometric and isokinetic elbow flexor strength, range of motion, upper arm circumference, plasma creatine kinase activity, and muscle soreness.Results: Delayed-onset muscle soreness was significantly less for the massage condition for peak soreness in extending the elbow joint and palpating the brachioradialis muscle (P < .05). Soreness while flexing the elbow joint (P = .07) and palpating the brachialis muscle (P = .06) was also less with massage. Massage treatment had significant effects on plasma creatine kinase activity, with a significantly lower peak value at 4 days postexercise (P < .05), and upper arm circumference, with a significantly smaller increase than the control at 3 and 4 days postexercise (P < .05). However, no significant effects of massage on recovery of muscle strength and ROM were evident.Conclusions: Massage was effective in alleviating DOMS by approximately 30% and reducing swelling, but it had no effects on muscle function.     

Human monocyte production of interleukin-1: parameters of the induction of interleukin-1 secretion by lipopolysaccharides

The role of lipopolysaccharide (LPS) as an activator of human monocyte interleukin-1 (IL-1) synthesis and secretion has been examined in this study. The results demonstrate that when blood monocytes are prepared under low endotoxin conditions, they do not spontaneously secrete IL-1 activity. When cells are exposed to LPS extracted from different bacterial species, there is variation seen in the potency, with LPS from Salmonella species being the most potent in inducing IL-1 activity from human monocytes. This material is tenfold more potent than LPS obtained from three different strains of Escherichia coli and 10,000-fold more potent than material obtained from two other bacterial species. Detoxified endotoxins are inefficient activators for IL-1 secretion. When monocytes are exposed to LPS, there is a rapid rise in the level of IL-1 activity detected. Activity can be detected in cell lysates after 1 hr with appreciable accumulation seen over the first 6 hr of culture. This is accompanied by IL-1 release into the surrounding medium after 2 hr of culture with subsequent accumulation. Monocyte synthesis of IL-1 activity appears to be sensitive to fg/ml levels of Salmonella minnesota LPS, while appreciable secretion of this activity by monocytes requires pg/ml levels.     

Mutations of ZFPM2/FOG2 gene in sporadic cases of tetralogy of Fallot

Two out of 47 patients with sporadic tetralogy of Fallot (TOF), the most common cyanotic conotruncal heart defect (CTD), showed heterozygous missense mutations of the ZFPM2/FOG2 gene. Knockout mice carrying mutations in the ZFPM2/FOG2 gene have similarly been found to exhibit TOF. While both mutant ZFPM2/FOG2 proteins, E30G (c.88A>G) and S657G (c.1968A>G), retain the ability to bind the partner protein GATA4 and repress GATA4 mediated gene activation, the S657G, but not the E30G, mutation is subtly impaired in this function. ZFPM2/FOG2 gene mutations may contribute to some sporadic cases of TOF.     

Embryo donation: attitudes toward donation procedures and factors predicting willingness to donate

BACKGROUND: The aim of the study was to assess infertile couples' attitudes toward the procedures of embryo donation (ED) and to identify factors predicting interest in donation. METHODS: Fifty-one couples who had received IVF treatment and had subsequently had embryos cryopreserved for >3 years were located and sent written information about the procedures for ED and possible implications of donation. A total of 49 couples agreed to participate in the study with 36 women and 31 men subsequently returning questionnaires describing their reasons for not claiming unused embryos and attitudes towards ED. RESULTS: Patients were supportive of donor screening procedures, but less comfortable sharing non-identifying information. Comfort levels declined as information became increasingly personal. Support for unconditional (i.e. the donation of embryos without conditions attached) and conditional (i.e. where couples could limit the donation of their embryos to persons/couples according to their preferences) models of donation was highly polarized and a substantial minority expressed strong opposition to each model. Willingness to donate was associated with greater comfort about disclosing personal information, a desire to know the outcome of donation and willingness to have future contact with a child, but not with current family size. CONCLUSIONS: Comfort in sharing information with a recipient couple is more important than acceptance of screening procedures, or attainment of family size goals in predicting willingness to donate embryos. Offering the option of conditional donation could increase the acceptability of ED for some patients.     

Impaired antibody responses in the hyperimmunoglobulin E syndrome

Patients with the hyper-IgE (HIE) syndrome have recurrent bacterial infections with Staphylococcus aureus and other polysaccharide encapsulated organisms. To determine whether an impairment of the antibody response to polysaccharide antigens contributes to infections in this syndrome, we measured serum antibody to the teichoic acid of S. aureus and to the capsular polysaccharide of Haemophilus influenzae type b. Compared to control subjects who had no history of S. aureus infections (N = 14), sera from patients with HIE (N = 9) lacked the expected elevation of serum antibody to teichoic acid (p greater than 0.05) and had significantly lower levels of this antibody than sera from 14 patients with atopic dermatitis, complicated by recurrent cutaneous S. aureus infections (p less than 0.01). After immunization with the capsular polysaccharide of Haemophilus influenzae type of vaccine, the antibody response of patients with HIE was significantly impaired compared to that of age-matched control subjects (p = 0.01). Although patients with HIE syndrome had normal total IgG levels, most patients with HIE but not patients with atopic dermatitis had IgG2 subclass deficiency. Defective antibody responses in patients with HIE were not restricted to polysaccharide antigens because the serum levels of antitetanus toxoid antibody in these patients were significantly lower than that of control subjects (p less than 0.001). Impaired antigen-specific antibody responses in patients with HIE syndrome may contribute to their increased susceptibility to infection.