PAX6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects.

The human eye malformation aniridia results from haploinsufficiency of PAX6, a paired box DNA-binding protein. To study this dosage effect, we characterized two PAX6 mutations in a family segregating aniridia and a milder syndrome consisting of congenital cataracts and late onset corneal dystrophy. The nonsense mutations, at codons 103 and 353, truncate PAX6 within the N-terminal paired and C-terminal PST domains, respectively. The wild-type PST domain activates transcription autonomously and the mutant form has partial activity. A compound heterozygote had severe craniofacial and central nervous system defects and no eyes. The pattern of malformations is similar to that in homozygous Sey mice and suggests a critical role for PAX6 in controlling the migration and differentiation of specific neuronal progenitor cells in the brain.

Antimicrobial resistance of Campylobacter isolates from retail meat in the United States between 2002 and 2007.

The emergence of antimicrobial resistance in Campylobacter spp. has been a growing public health concern globally. The objectives of this study were to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Campylobacter spp. recovered by the National Antimicrobial Resistance Monitoring System (NARMS) retail meat program. Retail meat samples (n = 24,566) from 10 U.S. states collected between 2002 and 2007, consisting of 6,138 chicken breast, 6,109 ground turkey, 6,171 ground beef, and 6,148 pork chop samples, were analyzed. A total of 2,258 Campylobacter jejuni, 925 Campylobacter coli, and 7 Campylobacter lari isolates were identified. Chicken breast samples showed the highest contamination rate (49.9%), followed by ground turkey (1.6%), whereas both pork chops and ground beef had <0.5% contamination. The most common resistance was to doxycycline/tetracycline (46.6%), followed by nalidixic acid (18.5%), ciprofloxacin (17.4%), azithromycin and erythromycin (2.8%), telithromycin (2.4%), clindamycin (2.2%), and gentamicin (<0.1%). In a subset of isolates tested, no resistance to meropenem and florfenicol was seen. C. coli isolates showed higher resistance rates to antimicrobials, with the exception of doxycycline/tetracycline, than those seen for C. jejuni. Pulsed-field gel electrophoresis (PFGE) fingerprinting resulted in 1,226 PFGE profiles among the 2,318 isolates, with many clones being widely dispersed throughout the 6-year sampling period.

The prevalence of BRCA1 mutations among young women with triple-negative breast cancer.

BACKGROUND: Molecular screening for BRCA1 and BRCA2 mutations is now an established component of risk evaluation and management of familial breast cancer. Features of hereditary breast cancer include an early age-of-onset and over-representation of the 'triple-negative' phenotype (negative for estrogen-receptor, progesterone-receptor and HER2). The decision to offer genetic testing to a breast cancer patient is usually based on her family history, but in the absence of a family history of cancer, some women may qualify for testing based on the age-of-onset and/or the pathologic features of the breast cancer. METHODS: We studied 54 women who were diagnosed with high-grade, triple-negative invasive breast cancer at or before age 40. These women were selected for study because they had little or no family history of breast or ovarian cancer and they did not qualify for genetic testing using conventional family history criteria. BRCA1 screening was performed using a combination of fluorescent multiplexed-PCR analysis, BRCA1 exon-13 6 kb duplication screening, the protein truncation test (PTT) and fluorescent multiplexed denaturing gradient gel electrophoresis (DGGE). All coding exons of BRCA1 were screened. The two large exons of BRCA2 were also screened using PTT. All mutations were confirmed with direct sequencing. RESULTS: Five deleterious BRCA1 mutations and one deleterious BRCA2 mutation were identified in the 54 patients with early-onset, triple-negative breast cancer (11%). CONCLUSION: Women with early-onset triple-negative breast cancer are candidates for genetic testing for BRCA1, even in the absence of a family history of breast or ovarian cancer.

Use of high-frequency ultrasound in the assessment of injectable dermal fillers.

BACKGROUND: The use of dermal fillers for enhancing lips and reducing wrinkles is currently one of the fastest growing sectors of the cosmetic surgery market. There are numerous fillers available, some are synthetic others are isolated from biological material. Once injected the fillers have a varied lifespan ranging from months to years depending upon the material, site of injection and individual response. Current assessment techniques of filler performance are mostly limited to evaluations of the skin surface topography, and not to what is happening to the filler beneath the skin surface. The aim of this work was to see if high-frequency ultrasound could be used to image and measure filler dimensions in situ. METHOD: This was a pilot study of six healthy female volunteers aged 36-53 visiting the surgical outpatients department of a hospital in Glasgow. Volunteers had been injected with filler material into their upper lip 6 months before the visit. The patients all had their upper lip scanned using high-frequency ultrasound. The subsequent images were then assessed using the scanner software to assess the dimensions of the filler. RESULTS: The filler material was clearly visible with the ultrasound and subsequently measurable in each scan. Each scan procedure was completed within a short time period meaning quantitative data could be acquired with minimum trauma to the volunteer. The scan images and data also provided valuable information for the volunteers and reinforced their perception of the fillers effect on their features. CONCLUSIONS: High-frequency ultrasound scanning provides a non-invasive, convenient and rapid technique for the assessment of filler performance. This pilot study produced three valuable pieces of information: The ultrasound can image the filler material from which quantitative measurements can be made. The technique is rapid and cost effective ...This investigation helped to reinforce the volunteer's perception of the filler effect.

Resistance to Bacillus thuringiensis toxin Cry2Ab in a strain of Helicoverpa armigera (Lepidoptera: Noctuidae) in Australia.

Transgenic cotton, Gossypium hirsutum L., expressing the crylAc and cry2Ab genes from Bacillus thuringiensis (Bt) Berliner variety kurstaki in a pyramid (Bollgard II) was widely planted for the first time in Australia during the 2004-2005 growing season. Before the first commercial Bollgard II crops, limited amounts of cotton expressing only the crylAc gene (Ingard) was grown for seven seasons. No field failures due to resistance to CrylAc toxin were observed during that period and a monitoring program indicated that the frequency of genes conferring high level resistance to the CrylAc toxin were rare in the major pest of cotton, Helicoverpa armigera (Htibner) (Lepidoptera: Noctuidae). Before the deployment of Bollgard II, an allele conferring resistance to Cry2Ab toxin was detected in field-collected H. armigera. We established a colony (designated SP15) consisting of homozygous resistant individuals and examined their characteristics through comparison with individuals from a Bt-susceptible laboratory colony (GR). Through specific crosses and bioassays, we established that the resistance present in SP15 was due to a single autosomal gene. The resistance was recessive. Homozygotes were highly resistant to Cry2Ab toxin, so much so, that we were unable to induce significant mortality at the maximum concentration of toxin available. Homozygotes also were unaffected when fed leaves of a cotton variety expressing the cry2Ab gene. Although cross-resistant to Cry2Aa toxin, SP15 was susceptible to CrylAc and to the Bt product DiPel.

Alpha fetoprotein: effect of heterologous antiserum on hepatoma cells in vitro.

Hepatoma cells derived from The Jackson Laboratory mouse hepatoma BW7756 synthesized alpha fetoprotein (AFP) in vitro. The AFP was immunologically identical to that circulating in the sera of hepatoma-bearing mice. An in vitro cytotoxic effect of rabbit antiserum to AFP was studied in hepatoma cells obtained both from fresh cell suspensions and short-term cell culture. The use of intact and/or inactivated anti-AFP serum inhibited the growth of the AFP-producing cells. The cytotoxic effects of the antiserum depended on exposure time and serum concentration. The cytotoxicity was complement independent, as demonstrated by studies with heat-deactivated serum devoid of extrinsic complement. The control target cells included fresh cell suspensions of normal mouse liver and mouse muscle fibroblasts grown in short-term culture. Specificity of the antisera for the target cells was demonstrated by absorption with purified mouse AFP. The results could be explained by the presence of AFP on the hepatoma cell surface.

Comparison of the effects of semi-occlusive polyurethane dressings and hydrocolloid dressings on dermal repair: 1. Cellular changes.

The effects on dermal repair of two wound dressings, one the semi-occlusive polyurethane sheet Opsite, the other the hydrocolloid Granuflex, were compared in full-thickness excised lesions on porcine skin during the period from 5 d to 6 months after injury. Quantitative studies were made of changes in the populations of polymorphonuclear leucocytes, macrophages, fibroblasts, and endothelial cells. The progress of repair in the wounds covered with the semi-occlusive dressing showed a decrease in the number of inflammatory cells (polymorphonuclear leukocytes and macrophages) from 5 to 60 d, whereas the number of proliferative phase cells (fibroblasts and endothelial cells) increased from 5 to 7 d. The total cellularity per unit area showed an increase between 5 and 7 d, that is, during the proliferative phase of repair, and then progressively decreased as the proliferative phase was succeeded by the remodeling phase. In contrast, the repair process in the hydrocolloid-dressed wounds was more complex. The number of inflammatory cells remained relatively high throughout and there were consistently fewer endothelial cells present throughout. Fibroblast number showed an initial fall from 5 to 14 d but then started to increase in number from 21 to 60 d. This chronic inflammatory reaction appeared to be in response to particulate matter that had been incorporated into the wound bed and hypodermis, and was still apparent 6 months after injury, when hydrocolloid particles were detectable microscopically in the hypodermis.

Comparison of the effects of moist and dry conditions on the process of angiogenesis during dermal repair.

The effect of moist and dry conditions on the process of angiogenesis during dermal repair was investigated. The moist conditions were achieved by covering excised wounds on porcine flank skin with the adhesive polyurethane dressing Opsite and dry conditions were achieved by exposure to air through dry gauze dressings. Angiogenesis was assessed during the period from 3 to 60 d after injury. Quantitative studies, using computerized image analysis, were carried out on microfocal x-ray images of skin sections whose blood system had been perfused in vivo with a radio-opaque medium. The analytical technique yielded information with regard to vessel number per wound and also the area occupied by blood vessels per unit wound area. Three regions were assessed in each wound bed: upper zone, just below the surface of the wound; the lower zone, just above the base of the wound bed; and the middle zone, midway between the other two zones. The results showed that the wounds maintained in a moist environment revascularized at a greater rate than those maintained in a dry environment. This was apparent in all of the zones of the wound bed examined. The development of new vessels occurred in a more orderly manner in the moist wounds. There was an early increase in vessel number rising to a peak around days 3-5, then a gradual decrease in number starting around day 7. In contrast, in the dry wounds the development of blood vessels was less rapid. Peak vessel number in the upper zone was significantly less than that achieved in the moist wounds, and was not reached until 7 d after injury. The decrease in vessel number from the peak was less rapid in the dry wounds, suggesting that there was a delayed entry into the remodeling phase in comparison with the moist wounds. The results also showed that the total percentage area of the wound bed occupied by blood vessels was greater in the moist wounds than the dry wounds from 3 d after injury until day 7. This level of vascularization was maintained beyond 7 d after injury even when the vessel number in the moist wounds was significantly less than in the dry wounds, suggesting that the vessels in the moist wounds were larger and, presumably, more mature. In general, moist wounds showed a more rapid decline towards uninjured skin levels of vascularization than dry wounds.

Embryogenesis of arborization pattern and topography of individual axons in N. laminaris of the chicken brain stem.

This study examined the development of individual axon terminal fields in n. laminaris (NL) of the chicken brainstem. In their mature form axons from the nucleus magnocellularis (NM), second-order auditory neurons in the chicken brainstem, project bilaterally onto the NL. Axons from the ipsilateral and contralateral NM neurons form spatially segregated, elongated arbors in the dorsal and ventral neuropil of NL, respectively. The long axes of these arbors correspond to physiologically defined isofrequency bands. To assess the development of this stereotyped arborization pattern, 6-17-day embryonic chicken brain stems were maintained in vitro while injecting horseradish peroxidase into small groups of axons. Three-dimensional reconstructions were made from serial sections and projected onto a cartesian plane for quantitative analyses. At embryonic day 6 (E6), the ventral axons already course beneath the recently migrated NL neurons. The arrival of the dorsal NM axon branches is delayed and their paths are indirect. They first loop dorsally into the the ventricular layer, where they seem to make specific connections with migrating NL neurons and use these as guides to their appropriate positions in the NL. During the period from E9 to E17 the dorsal and ventral terminal fields become similar, each adopting properties of the other's initial pattern. The dorsal terminal fields extend to form bands similar to the early ventral terminal fields, while the ventral terminal fields narrow and appear to shift position in order to achieve the tonotopic specificity characteristic of the early dorsal terminal fields. The results show that a complex, mature pattern of neuronal connections can be formed during development by the combination and reorganization of two simple patterns--each shaped, in turn, by its respective axonal trajectory.

The detection of Mycobacterium leprae protein and carbohydrate antigens in skin and nerve from leprosy patients with type 1 (reversal) reactions.

Type 1 (reversal) reactions are the most common immunological complications of leprosy. These episodes of delayed hypersensitivity produce severe local immunopathology and ultimately nerve damage. To date, the Mycobacterium leprae antigens associated with type 1 reactions have not been identified. Using monoclonal antibodies to defined protein and carbohydrate M. leprae epitopes (65, 35 and 28 kd and lipoarabinomannan [LAM]) in a two-step immunoperoxidase staining technique, M. leprae antigens were demonstrated in skin and nerve biopsies from patients in reversal reaction. Antigen presence and staining patterns were similar in skin and nerve lesions, implying that the pathological processes are similar in the two sites. Antigens were present both in macrophages and Schwann cells but also as a diffuse extracellular infiltrate associated with the inflammatory infiltrate. The 28-kd antigen was present most strongly and may be a potential candidate antigen for initiating type 1 reactions. LAM also stained strongly and persisted after treatment. The possible roles of LAM and 65 kd in the cellular events of type 1 reactions are discussed.

High time resolution fluorescence imaging with a CCD camera.

We have built a high speed, sensitive camera system capable of capturing sequences of low-light level images synchronized with recordings of membrane potential. The camera system is based on a cooled, scientific grade CCD camera controlled by a PC/AT computer. It can take 100 frames/sec of 18 X 18 element images and 40 frames/sec of 50 X 50 element images with no lag in response to step changes in light intensity. High accuracy and dynamic range of the measurements result from the fact that light levels of the picture elements are digitized with 12 bit accuracy with intrinsic camera noise levels typically less than 1/10,000 of the maximum detectable light level. We have used this system to record calcium dependent fura-2 fluorescence transients in the dendrites of cerebellar Purkinje cells and from different regions of leech neurons in segmental ganglia or isolated in culture.

Long-term maintenance of mature hippocampal slices in vitro.

Cultures of primary neurons or thin brain slices are typically prepared from immature animals. We introduce a method to prepare hippocampal slice cultures from mature rats aged 20-30 days. Mature slice cultures retain hippocampal cytoarchitecture and synaptic connections up to 3 months in vitro. Spontaneous epileptiform activity is rarely observed suggesting long-term retention of normal neuronal excitability and of excitatory and inhibitory synaptic networks. Picrotoxin, a GABAergic Cl(-) channel antagonist, induced characteristic interictal-like bursts that originated in the CA3 region, but not in the CA1 region. These data suggest that mature slice cultures displayed long-term retention of GABAergic inhibitory synapses that effectively suppressed synchronized burst activity via recurrent excitatory synapses of CA3 pyramidal cells. Mature slice cultures lack the reactive synaptogenesis, spontaneous epileptiform activity, and short life span that limit the use of slice cultures isolated from immature rats. Mature slice cultures are anticipated to be a useful addition for the in vitro study of normal and pathological hippocampal function.

The retinal binocular field of the pigeon (Columba livia: English racing homer).

An ophthalmoscopic reflex technique has shown that in sedated pigeons maximum retinal binocular field width occurs approximately 20 degrees above the bill. The binocular field has a maximum width of 27 degrees and extends vertically by 130 degrees (90 degrees above the bill, 40 degrees below it). Both the bill and cere intrude into the binocular field. Maximum optical binocularity also occurs approximately 20 degrees above the bill. The plane containing the optic axes of each eye coincides with the bill.

Optics of retinal oil droplets: a model of light collection and polarization detection in the avian retina.

A wave optical model was used to analyse the scattering properties of avian retinal oil droplets. Computations for the near field region showed that oil droplets perform significant light collection in cone photoreceptors and so enhance outer segment photon capture rates. Scattering by the oil droplet of the principal cone of a double cone pair, combined with accessory cone dichroic absorption under conditions of transverse illumination, may mediate avian polarization sensitivity.

The significance of membrane changes in the safe and effective use of therapeutic and diagnostic ultrasound.

The cellular changes, such as alterations in motility and the stimulation of synthesis and secretion, induced by relatively low intensities of therapeutic ultrasound (e.g. 500 mW cm-2, SAPA; 100 mW cm-2 SATA) are primarily non-thermal in origin. They appear to be associated with changes in the permeability of the cell (plasma) membrane and in the transport of ions and molecules across it, effects which have been demonstrated in cells irradiated in suspension. In epithelial tissues, both in vitro and in vivo, it has been demonstrated that not only the cellular membrane transport pathways but also the paracellular or intercellular pathways are affected. Although membrane-mediated effects can be of value therapeutically, they could produce adverse effects if they were to occur during development, for the reception and transmission by the membrane of environmental signals are involved in determination of the fate of each cell. Determination is followed by selective gene expression and differentiation, that is, by the progressive increase in structural complexity brought about by the acquisition of specialised characteristics by various cell groups. Most cells of early embryos are ionically coupled via gap junctions which provide an intercellular pathway for electrochemical signalling and the maintenance of the concentration gradients which provide the cells with positional information. Differentiation of the cells varies according to their location with respect to these gradients. Increase in the intracellular concentration of calcium ions, which has been shown to occur after exposure to therapeutic levels of ultrasound, can decrease the permeability of gap junctions and uncouple cells, in the manner which occurs when they differentiate. Ultrasonically induced increases in calcium ion concentration are thus of considerable clinical significance, since they could affect differentiation and consequently histogenesis. Modification of plasma membrane permeability and transport properties, resulting in changes in the availability and activity of second messengers such as free calcium ions, can have profound effects on cell behaviour. Calcium channels appear to be the first channels to develop in the cell membranes of embryos, and internal calcium ion concentration is known to affect the synthesis of fetal proteins. Although generally reversible at intensities of less than 500 mW cm-2, changes in membrane permeability, particularly to calcium ions, could, if prolonged, have undesirable side effects not only on embryogenesis but on late prenatal and postnatal development. It is therefore recommended that the environmental conditions, thresholds, and mechanisms involved in the production of such changes be determined, so that they can be avoided when ultrasound is used diagnostically on sensitive targets such as embryos and fetuses.

Elevated blood serotonin in autistic probands and their first-degree relatives.

Whole blood serotonin levels and platelet counts were studied in 14 families, representing 57 family members and 15 probands who met DSM III criteria for infantile autism. High serotonin appeared to segregate in families. When two parents had high serotonin, the serotonin level in their offspring was twice the parental level. When one parent had high serotonin, the serotonin level in the offspring approximated the level of serotonin in either the high serotonin parent or the low serotonin parent. For the case where both parents had low serotonin, in one family the children had low serotonin and in a second family, high serotonin levels were present in the autistic proband, and a sibling with severe mental retardation. Mean serotonin levels were higher for both male and female, autistics and family members, in the four black families than in the 10 Caucasian families.

Developing genetic privacy legislation: the South Carolina experience.

The availability of presymptomatic and predisposition genetic testing has spawned the need for legislation prohibiting health insurance discrimination on the basis of genetic information. The federal effort, the Health Insurance Portability and Accountability Act (HIPAA) of 1996, falls short by protecting only those who access insurance through group plans. A committee of University of South Carolina professionals convened in 1996 to develop legislation in support of genetic privacy for the state of South Carolina. The legislation prevents health insurance companies from denying coverage or setting insurance rates on the basis of genetic information. It also protects the privacy of genetic information and prohibits performance of genetic tests without specific informed consent. In preparing the bill, genetic privacy laws from other states were reviewed, and a modified version of the Virginia law adopted. The South Carolina Committee for the Protection of Genetic Privacy version went a step further by including enforcement language and excluding Virginia's sunset clause. The definition of genetic information encompassed genetic test results, and importantly, includes family history of genetic disease. Our experience in navigating through the state legislature and working through opposition from the health insurance lobby is detailed herein.

The effect of therapeutic ultrasound on angiogenesis.

The effect of therapeutic ultrasound on the formation of new blood vessels in full-thickness excised lesions in the flank skin of adult rats was assessed quantitatively using microfocal x-ray techniques. Wounds were either sham-treated (control group) or exposed to ultrasound for 5 min daily at an intensity of 0.1 W/cm2 SATA (frequency either 0.75 MHz or 3.0 MHz). By 5 days after injury there were more blood vessels in equivalent regions of the granulation tissue of the ultrasound-treated wounds than in the control wounds. This suggested that the ultrasound-treated wounds were at a more advanced stage in the repair process. By 7 days after injury there was no significant difference in blood vessel number between the three groups.

Macrophage responsiveness to therapeutic ultrasound.

Macrophages are a source of many important growth factors which can act as wound mediators during tissue repair. The aim of this work was to find out if levels of ultrasound which accelerate repair could stimulate the release of fibroblast mitogenic factors from an established macrophage-like cell line (U937). The U937 cells were exposed in vitro to continuous ultrasound at a space average, temporal average intensity of 0.5 W/cm2 at either 0.75 MHz or 3.0 MHz, for 5 min. The macrophage-conditioned medium was removed either 30 min or 12 h after exposure, and placed on 3T3 fibroblast cultures. Fibroblast proliferation (defined here as increase in cell number) was assessed over a 5-day period. The results showed that 0.75 MHz ultrasound appeared to be effective in liberating preformed fibroblast affecting substances from the U937 cells, possibly by producing permeability changes, whereas 3.0 MHz ultrasound appeared to stimulate the cell's ability to synthesize and secrete fibroblast mitogenic factors.

Gene Amplification of c-erbB-2 Detected by FISH.

In 1981, a novel transforming gene called neu, related to, but distinct from, the c-erbB protooncogene, was identified (1-3). In 1985, two groups independently isolated identical erbB-related genes from human DNA that they called HER-2 (4) and c-erbB-2 (5) located at chromosome band 17q11.2 and encoding a 185,000 Dalton tyrosine kinase (2,4). Studies conducted on human breast cancers showed 28% to have amplified HER2/neu present (7) and it was suggested that amplification or increased protein expression might confer a selective advantage (6). Over the last ten years, many studies have confirmed the value of using overexpression or amplification of the HER2/neu gene as a predictor of poor outcome in cases of breast cancer (7-9). Clinical trials are currently underway using anti-HER2/neu in an attempt to destroy malignant breast cells (10,11).