Prevalence and audiological profiles of GJB2 mutations in a large collective of hearing impaired patients.

Mutations in the GJB2 gene are known to represent the commonest cause of hereditary and congenital hearing loss. In this study, a complete sequencing of the GJB2 gene in a cohort of 506 patients from a single, large cochlear implant program in Europe was performed. Audiological testing for those patients who could actively participate was performed using pure tone audiometry (PTA). Those unable to undergo PTA were measured using click-auditory brainstem response (ABR). Data analysis was performed to determine genotype-phenotype correlations of the mutational status vs. audiological profiles and vs. age at the time of presentation. An overall prevalence of biallelic mutations of 13.4% was found for the total collective. When subsets of younger patients were examined, the prevalence increased to 27% of those up to age 18 and 35% of those up to age 5 at the time of testing, respectively. This increase was found to be highly significant (p < 0.001). Analysis of the mean PTA thresholds revealed a strong correlation between allele combination status and mean PTA (p = 0.021). The prevalence of simple heterozygotes was found to be approximately 10.1%, which is around 3.3 times the value expected in the general population. As GJB2 follows a recessive pattern of inheritance, the question arises as to why such a large fraction of simple heterozygotes was observed among the hearing impaired patients included in this study.

[Hereditary hearing loss: Part 2: Syndromic forms of hearing loss]. / Hereditäre Schwerhörigkeit Teil 2: Syndromale Formen der Schwerhörigkeit.

Syndromic hearing loss is responsible for approximately 30% of cases of inherited hearing loss. The syndromic form can be differentiated from nonsyndromic hearing loss by the presence of associated symptoms in other organ systems. While for many forms of syndromic hearing loss the individual genes responsible have been identified, the etiology of other associated symptoms remains unclear. The role of the ENT physician is to select appropriate clinical and genetic diagnostic tools based on the presentation of the patient and to subsequently initiate and perform the required hearing loss therapy.

[Hereditary hearing loss: Part 1: diagnostic overview and practical advice]. / Hereditäre Schwerhörigkeit: Teil 1: Überblick und praktische Hinweise zur Diagnostik.

Hearing loss is the most frequently occurring congenital sensory defect in humans. It is believed that between one and five of every 1000 children born suffers from hearing loss of at least 40 dB. The economic consequences of deafness are staggering and affect not only the individual patient but also society as a whole. A genetic cause is suspected in 50 %-70 % of cases of congenital hearing loss. To date over 130 loci have been associated with genetic hearing loss, with some loci containing more than one gene and others containing as yet unidentified genes. The present article is intended to provide some insight into the complex background issues involved and offer guidance on appropriate decision-making with regard to genetic testing in affected patients. Part 1 is concerned with non-syndromic hearing loss, while part 2 deals with syndromic hearing loss.

Countertransference and the multiple amputee patient: pitfalls and opportunities in rehabilitation medicine.

Current psychoanalytic literature on countertransference broadly defines the term as a helping professional's overall response to an individual patient. Reactions of rehabilitation professional to their traumatically injured patients can significantly impact on the patient's treatment as well as the individual therapist's and entire rehabilitation team's effectiveness. In this paper, a case is presented illustrating a process of understanding countertransference toward a multiple amputee patient. Implications for the rehabilitation team are also described. The analysis of a single case demonstrates how countertransference interpretation might be used as a vehicle to enhance understanding the patient and promote team effectiveness.

Plasmid stability in Bacillus sphaericus 2362 during recycling in mosquito larvae.

Bacillus sphaericus 2362 strains transformed with the plasmid pUB110 (4.5 kb) and plasmids derived from it, pLDT103 (7.6 kb) and pLDT117 (9.3 kb), were able to recycle (spore germination, vegetative growth, sporulation) in larvae of Culex quinquefasciatus. During the course of recycling, the pUB110 vector and the recombinant plasmid pLDT103 were stable (100 and 99.2%, respectively). However, the recombinant plasmid pLDT117 exhibited 23% segregational instability. Isolates which lost pLDT117 during recycling retained the one large plasmid native to B. sphaericus 2362.

Construction of tracer plasmids for Bacillus sphaericus 1593 utilizing the xylE gene from Pseudomonas putida.

Genetically engineered microorganisms (GEMS) released into the environment must be traceable in order to accurately assess their impact on the area of release. Tracer genes other than those that introduce antibiotic resistance are preferred for use in identifying genetically engineered strains. In this study, we describe the construction of a series of tracer plasmids for use in Bacillus sphaericus using the xylE gene from the Pseudomonas putida TOL plasmid. This gene codes for the enzyme catechol 2,3-dioxygenase which converts the colorless substance catechol to 2-hydroxymuconic semialdehyde, a yellow product which is easily detected. Colonies of cells which express the xylE gene turn yellow shortly after being exposed with a solution of catechol.

Modification of the drop dialysis technique for use with larger volume samples.

Desalting of nucleic acids by the drop dialysis method is limited by the fact that only small volume samples can be used due to the lack of sample containment on the membrane filters. A specially modified Styrafoam cup can be used as a membrane filter holder which serves to contain the sample, thus permitting dialysis of larger sample volumes.

Carrier RNA enhancement of recovery of DNA from dilute solutions.

A study of the use of carrier RNA to improve precipitation of DNA from dilute solutions was conducted to define the conditions which optimize DNA recovery. Replicate samples containing labeled pBR322 and increasing concentrations of commercially-available Torula yeast RNA were ethanol precipitated at -20 degrees C for 1 h in microfuge tubes obtained from various manufacturers. Nucleic acids were pelleted by centrifugation for either 5 or 30 min, dried and resuspended. Although recovery was not identical in each type of microfuge tube, in all cases the percent recovery increased when carrier was added. In most cases, extending centrifugation to 30 min did not significantly increase recovery. Recovery of unlabeled DNA's of heterogeneous molecular weight and conformation was also enhanced by the addition of carrier RNA. DNA's recovered by this method can be successfully digested with BamHI and ligated with T4 DNA ligase.

Construction and characterization of plasmid vectors for cloning in the entomocidal organism Bacillus sphaericus 1593.

A plasmid vector for cloning in Bacillus sphaericus 1593 was constructed in B. subtilis from two parent plasmids, pBC16 and pBD64. When characterized, the 3.9-MDa chimeric plasmid pNN101 was found to consist of the MspI fragment containing the chloramphenicol acetyltransferase (CAT) gene from pBD64 inserted into an MspI site in pBC16. pNN101 was shown to replicate, express, and be stably maintained in B. sphaericus 1593 without affecting the mosquito larvicidal activity of this organism. A derivative of this plasmid, pNN302, was constructed in which a unique HindIII site was introduced into the CAT gene without loss of chloramphenicol resistance.

Plasmid transformation of Bacillus sphaericus 1593.

Plasmids pUB110 and pBC16 were introduced by protoplast transformation into the entomocidal bacterium Bacillus sphaericus 1593. Transformants expressed the antibiotic resistance determinants present on the plasmid and exhibited sporulation frequencies and larvicidal toxicities which were equivalent to those characteristic of the parent strain. These transformations constitute the first report of genetic transfer in B. sphaericus.

Effect of UV Light on Spore Viability and Mosquito Larvicidal Activity of Bacillus sphaericus 1593.

UV light from a germicidal lamp rapidly reduced the viability of Bacillus sphaericus 1593 spores, but insecticidal activity was resistant to inactivation by continuous exposure to UV light for 4 h.

Bacillus subtilis extracellular nuclease production associated with the spoOH sporulation locus.

A number of nuclease-deficient mutants of Bacillus subtilis were isolated and found to be concurrently asporogenous. The nuclease-deficient phenotype appeared to be associated with the spoOH sporulation locus. Spontaneously occurring sporogenous revertants concomitantly recovered the ability to produce extracellular nuclease activity. The position of the ncl mutation was determined using transformation and PBS1 transduction and found to map in the same site as spoOH. The map order of ncl and markers in the vicinity was determined to be purA-cysA-ncl(spoOH)-strA.

Extracellular exonuclease as a stage 0 biochemical marker in Bacillus subtilis sporulation.

Calcium-dependent exonuclease activity was produced by sporulating cells of Bacillus subtilis 168. Nuclease activity was released into the culture medium at approximately the same time as sporulation proteases, and production of these enzymes was tied to DNA replication. Results suggest that nuclease production is a function of the spo0H locus.

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis.

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.

Construction and characterization of the host component of the Bacillus subtilis HV2 cloning system.

A strain of Bacillus subtilis has been developed and tested for use as the host component of the B. subtilis HV2 system. The strain was constructed incorporating the following mutations: thyA, thyB, uvr-1, dal, spoOA delta 677, citD delta 20 (uvrC), strd. The strain is unable to grow in the absence of thymine, D-alanine, or streptomycin; unable to sporulate; and sensitive to UV radiation. Survival of the attenuated strain was severely restricted in nonpermissive environments, and the organism suffered a five-log decrease in viability within 24 h when dried at room temperature. The strain could be transformed with plasmid DNA using the protoplast transformation system and was transducible with PBS1. The ability of the strain to serve as host for the temperate phages phi 3T and rho 11 and for the plasmid vectors pUB110 and pBD64 was unimpaired.

Ultraviolet sensitivity ofBacillus subtilis citD mutants.

Cells ofBacillus subtilis 168 with deletions in thecitD locus were found to be sensitive to irradiation with ultraviolet light and to mitomycin C but were able to repair DNA damage induced by methyl methanesulfonate. The recombination abilities of these cells, as determined by transformation and PBS1-mediated transduction experiments, were unaffected by the deletion. These phenotypic characteristics do not result from a metabolic imbalance caused by the deficiency of a functional α-ketoglutarate dehydrogenase complex, but most likely are a consequence of a genetic locus involved in ultraviolet repair, which is deleted together with thecitK gene when the deletion is formed.

Selective inhibition of Bacillus subtilis sporulation by acridine orange and promethazine.

Two structurally similar compounds were found to inhibit sporulation in Bacillus subtilis 168. A dye, acridine orange, and an antischizophrenic drug, promethazine, blocked spore formation at concentrations subinhibitory to vegetative growth, while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to promethazine through T2, whereas acridine orange was inhibitory until T4. The drug-treated cells were able to support the replication of phages phie and phi29, although the lytic cycles were altered slightly. The selective inhibition of sporulation by these compounds may be related to the affinity of some sporulation-specific genes to intercalating compounds.

Isolation, characterization, and activation of the magnesium dependent endodeoxyribonuclease from Bacillus subtilis.

A major endodeoxyribonulcease was isolated from a mutant of the transformable Bacillus subtilis 168. The magnesium-dependent endonuclease was purified approximately 750-fold to electrophoretic homogeneity. The enzyme had a molecular weight of about 31 000, as determined by gel filtration and polyacrylamide gel electrophoresis. The protein appears to be composed of two subunits. The nuclease was dependent on magnesium or maganese ions for hydrolytic activity. The purified nuclease degraded DNA from several species of Bacillus, as well as Escherichia coli DNA, alkylated, depurinated, and thymine-dimer containing B. subtilis DNA, and hydroxymethyluracil-containing phage DNA. The enzyme also hydrolyzed single-stranded DNA, although native DNA was the preferred substrate. However, the nuclease was unable to degrade ribosomal RNA. The cleavage products of the DNA hydrolysis have 5'-phosphate and 3'-hydroxyl ends. The enzyme could be activated in crude extracts by heat treatment or treatment with guanidine hydrochloride. The nuclease activity was inhibited by phosphate and by high concentrations of NaCl. A possible function for this endonuclease in bacterial transformation is discussed.