Shotgun Glycomics Identifies Tumor-Associated Glycan Ligands Bound by an Ovarian Carcinoma-Specific Monoclonal Antibody.

Cancers display distinctive carbohydrate molecules (glycans) on their surface proteins and lipids. mAb A4, an in-house generated monoclonal IgM antibody, is capable of distinguishing malignant ovarian carcinoma cells from benign ovarian epithelia by binding specifically to cancer cell-associated glycans. However, the structural details of the glycan targets of mAb A4 have been elusive. Here we developed a novel approach of isolating and fractionating glycan molecules released from glycoproteins in cancer cell lysates using HILIC-UPLC, and used them as probes on a microarray for affinity-based identification of the binding targets, allowing full-size, difficult to synthesize, cancer-associated glycans to be directly studied. As a result of this "shotgun" glycomics approach, we corroborate the previously assigned specificity of mAb A4 by showing that mAb A4 binds primarily to large (>15 glucose units), sialylated N-glycans containing the H-type 1 antigen (Fuc-α1,2-Gal-ß1,3-GlcNAc). Although mAb A4 was also capable of directly binding to type 1 N-acetyl-lactosamine, this epitope was mostly shielded by sialylation and thus relatively inaccessible to binding. Knowledge of the structure of mAb A4 antigen will facilitate its clinical development as well as its use as a diagnostic biomarker.

Innovative combination of therapeutic mammoplasty and expandable-implant breast augmentation for immediate partial breast reconstruction.

INTRODUCTION: Therapeutic mammoplasty is used in the treatment of suitably-sized and appropriately-located breast cancers to achieve adequate cancer excision, resulting in well-shaped but smaller breasts. In patients wishing to maintain or increase their breast size, simultaneous augmentation will be required. PRESENTATION OF CASE: A 48-year-old female underwent an "augmentation-therapeutic mastopexy". She required mastectomy for a multifocal cancer of the right breast and breast conservation for a unifocal localised cancer in the upper part of the left breast. She requested right immediate breast reconstruction and hoped for larger breasts than she had. Due to complications during neoadjuvant chemotherapy, the right reconstruction plan was changed from a deep inferior epigastric perforator (DIEP) flap to an implant-based technique. On the left, an extended superomedial pedicle therapeutic mammoplasty was combined with a subpectoral augmentation using an expandable-implant. DISCUSSION: The use of expandable-implants for reconstruction of partial mastectomy defects in combination with therapeutic mammoplasty has not been reported. This case report shows that such "augmentation-therapeutic mastopexy" is feasible. CONCLUSION: A "novel" oncoplastic technique herein termed "augmentation-therapeutic mastopexy" is described for partial breast reconstruction during the treatment of a patient with bilateral breast cancer. It enabled adequate treatment of her cancer while reshaping the breast and achieving the desired larger breast size. It should be considered in selected breast-conservation patients who wish to maintain or increase their breast size.

Chromosomal instability causes sensitivity to metabolic stress.

Chromosomal INstability (CIN), a hallmark of cancer, refers to cells with an increased rate of gain or loss of whole chromosomes or chromosome parts. CIN is linked to the progression of tumors with poor clinical outcomes such as drug resistance. CIN can give tumors the diversity to resist therapy, but it comes at the cost of significant stress to tumor cells. To tolerate this, cancer cells must modify their energy use to provide adaptation against genetic changes as well as to promote their survival and growth. In this study, we have demonstrated that CIN induction causes sensitivity to metabolic stress. We show that mild metabolic disruption that does not affect normal cells, can lead to high levels of oxidative stress and subsequent cell death in CIN cells because they are already managing elevated stress levels. Altered metabolism is a differential characteristic of cancer cells, so our identification of key regulators that can exploit these changes to cause cell death may provide cancer-specific potential drug targets, especially for advanced cancers that exhibit CIN.

Small CGG repeat expansion alleles of FMR1 gene are associated with parkinsonism.

Fragile X-associated tremor/ataxia syndrome (FXTAS) affects older males carrying premutation, that is, expansions of the CGG repeat (in the 55-200 range), in the FMR1 gene. The neurological changes are linked to the excessive FMR1 messenger RNA (mRNA), becoming toxic through a 'gain-of-function'. Because elevated levels of this mRNA are also found in carriers of the smaller expansion (grey zone) alleles, ranging from 40 to 54 CGGs, we tested for a possible role of these alleles in the origin of movement disorders associated with tremor. We screened 228 Australian males affected with idiopathic Parkinson's disease and other causes of parkinsonism recruited from Victoria and Tasmania for premutation and grey zone alleles. The frequencies of either of these alleles were compared with the frequencies in a population-based sample of 578 Guthrie spots from consecutive Tasmanian male newborns (controls). There was a significant excess of premutation carriers (Fisher's exact test p = 0.006). There was also a more than twofold increase in grey zone carriers in the combined sample of the Victorian and Tasmanian cases, with odds ratio (OR ) = 2.36, and 95% confidence intervals (CI): 1.20-4.63, as well as in Tasmanian cases only (OR = 2.33, 95% CI: 1.06-5.13), compared with controls. The results suggest that the FMR1 grey zone alleles, as well as premutation alleles, might contribute to the aetiology of disorders associated with parkinsonism.

Accuracy of digitization of bony landmarks for measuring change in scapular attitude.

Digitizing bony landmarks is a common technique used to measure scapular position, but it has not been validated against a gold standard. The aim of this study was to determine the accuracy of this technique for four physiological arm movements using optoelectronic markers mounted on scapular bone pins as a gold standard. Eight subjects had bone pins inserted into their lateral scapular spine. Three points were digitized on the scapula with an optoelectronic probe: the medial root of the scapular spine, the posterolateral corner of the acromion, and the inferior angle of the scapula. The four active movements tested in this study were glenohumeral abduction, glenohumeral horizontal adduction, hand behind back, and forward reaching. The three bony landmarks were digitized six times in three different positions for each movement. Data from one subject were rejected secondary to pin loosening. The overall position-specific r.m.s. errors ranged from 2.0 degrees to 12.5 degrees. The full abduction position had considerably higher r.m.s. errors than the other positions (posterior tipping, 12.5 degrees; upward rotation, 7.3 degrees; internal rotation, 12.0 degrees). It appears that the digitization of bony landmarks may be a valid method for measuring changes in scapular attitude with the following caveats: the full abduction position has a high r.m.s. error, and small scapular motions have high percentage errors.

Human immunodeficiency virus type 1 (HIV-1) Vpr-regulated cell death: insights into mechanism.

The destruction of CD4(+) T cells and eventual induction of immunodeficiency is a hallmark of the human immunodeficiency virus type 1 infection (HIV-1). However, the mechanism of this destruction remains unresolved. Several auxiliary proteins have been proposed to play a role in this aspect of HIV pathogenesis including a 14 kDa protein named viral protein R (Vpr). Vpr has been implicated in the regulation of various cellular functions including apoptosis, cell cycle arrest, differentiation, and immune suppression. However, the mechanism(s) involved in Vpr-mediated apoptosis remains unresolved, and several proposed mechanisms for these effects are under investigation. In this review, we discuss the possibility that some of these proposed pathways might converge to modulate Vpr's behavior. Further, we also discuss caveats and future directions for investigation of the interesting biology of this HIV accessory gene.

Oral hygiene measures and promotion: review and considerations.

Current mechanical and chemotherapeutic approaches to oral hygiene aim to modify the oral microflora to promote healthy periodontal and dental tissues. Current oral hygiene measures, appropriately used and in conjunction with regular professional care, are capable of virtually preventing caries and most periodontal disease and maintaining oral health. Toothbrushing and flossing are most commonly used, although interdental brushes and wooden sticks can offer advantages in periodontally involved dentitions. Chewing sugar-free gums as a salivary stimulant is a promising caries-preventive measure. Despite new products and design modifications, mechanical measures require manual dexterity and cognitive ability. Chemotherapeutic supplementation of mechanical measures using dentifrices, mouthrinses, gels and chewing gums as delivery vehicles can improve oral hygiene. The list includes anticalculus, antibacterial and cariostatic agents. For the population at large to make effective use of these oral hygiene measures, oral hygiene promotion needs to be implemented. Considerations include the role of parents, school and the media for children and the workplace, social environments. nursing homes and trained carers for adults and the elderly. Community oral hygiene promotion must attempt to maximise opportunities for oral health for all and reduce inequalities by removing financial and other barriers. Oral health approaches should be tailored to lifestyles and abilities of children, adults and the elderly in order to enable them to make decisions to improve personal oral hygiene and oral health.

Induction of potent Th1-type immune responses from a novel DNA vaccine for West Nile virus New York isolate (WNV-NY1999).

West Nile virus (WNV) is a vectorborne pathogen that induces brain inflammation and death. Recently, confirmed cases of infection and deaths have occurred in the United States Mid-Atlantic region. In this study, a DNA vaccine encoding the WNV capsid protein was constructed, and the in vivo immune responses generated were investigated in DNA vaccine-immunized mice. Antigen-specific humoral and cellular immune responses were observed, including a potent induction of antigen-specific Th1 and cytotoxic T lymphocyte responses. Strong induction of Th1-type immune responses included high levels of antigen-specific elaboration of the Th1-type cytokines interferon-gamma and interleukin-2 and beta-chemokines RANTES (regulated upon activation, normal T cell-expressed and secreted) and macrophage inflammatory protein-1beta. Dramatic infiltration of CD4 and CD8 T cells and macrophages also was observed at the muscle injection site. These results support the potential utility of this method as a tool for developing immunization strategies for WNV and other emerging pathogens.

Parameters influencing the expression of mature glial-cell-line-derived neurotrophic factor in Escherichia coli.

Factors governing expression in Escherichia coli, namely promoters, fusion partners, targeting signals, host strains, growth temperature of cultures and inducer concentrations, were investigated to elucidate their influence on the accumulation of mature glial-cell line-derived neurotrophic factor (GDNF). The present study provided evidence indicating that translational and/or post-translational events were more important in determining overall accumulation of the target protein than was transcription. Under the control of the strong inducible tac or T7 promoter, no direct correlation between transcript abundance and final yield of recombinant protein was observed. GDNF was also recalcitrant to being produced in a soluble form in E. coli. Direct expression resulted in the exclusive localization of GDNF in inclusion bodies, regardless of whether the protein was produced in the cytoplasm or targeted to the periplasm. The fusion approach was found to be the most efficient method, as it resulted not only in the highest level of GDNF produced, albeit primarily in inclusion bodies, but also in the accumulation of small amounts of soluble proteins. Using different host strains, low inducer concentration or sub-optimal growth temperature did not result in any detectable shift towards solubility. Persistent localization in inclusion bodies, low levels of expression as a native protein and in vivo proteolysis of soluble fusion forms appeared to be influenced by structural features located at the N-terminus of GDNF. Deletion of this region was found to result in substantial alleviation of these problems.

Reduced retention of cadmium in the liver of metallothionein-null mice.

The distribution of cadmium (Cd) in the organs of mice was studied using metallothionein (MT)-null transgenic mice. When mice were administered with Cd chloride at a single subcutaneous dose of 1.0 mg Cd/kg body weight, Cd accumulated mainly in the liver and kidney by 6 h after injection without any significant difference between the MT-null mice and control (C57BL/6J) mice. MT was not detected in these organs of MT-null mice both before and after Cd administration whereas MT was induced mainly in these organs of the control mice. There was a marked elimination of Cd from the liver of MT-null mice by 21 days after administration, compared with a relatively slow Cd elimination in the C57BL/6J mice. Under the condition that no significant liver or kidney damage was observed, MT was considered to play a significant role in the retention of Cd in the liver but not in the uptake of this metal.

Intracellular responses of productive hybridomas subjected to high osmotic pressure.

It has previously been found that hybridoma cells under hyperosmotic stress produce higher amounts of antibody. This study identified the cellular processes and mechanisms that occur during this event. In studies of hybridomas adapted to osmolarities ranging between 300 and 450 mOsm (using NaCI), antibody production increased to a saturation level while cell growth decreased progressively. At 500 mOsm, lower cell numbers and markedly decreased productivity resulted. Sucrose and KCI were found to induce similar trends, except to different extents. Several important changes in cellular responses were observed. Elevation of osmolarity with NaCl from 300 to 350 mOsm causes an increase of zwitterionic amino acid uptake, which occurred via Na+-dependent transport systems. In particular, system A was enhanced by 1.86-fold, but no enhancement was observed for Na+- independent transport systems. In addition, amino acids reactive with Na +-dependent transport systems were observed to be abundant within osmotically stressed hybridomas in the middle and late exponential stages. Sucrose and KCI caused similar uptake effects, but to a lesser degree, as long as sodium ions were present in solution. Specific consumption rates of glucose and glutamine increase by 19% and 20%. respectively, under high osmolarity treatment. These increases were confirmed by the 5% to 10% increase in cellular metabolic activity. At 350 mOsm, growth rate was slower compared with the 300-mOsm culture, which was reflected by the lower DNA concentration. Stressed cultures contained enhanced levels of total RNA, of which -80% is ribosomal RNA (rRNA). Higher rRNA content could in turn increase the translation rates of proteins. This was reflected in the accumulation of both dry cell weight and total cellular protein at linear rates of 0.42 pg/106 cells/mOsm and 0.21 pg/106 cells/mOsm, respectively, with increasing osmolarity between 300 and 450 mOsm. Overall, hybridomas increased their metabolic activities and amino acids uptake via the Na+-dependent symports to compensate for the osmotically elevated external environment. These effects contribute directly and indirectly to the increased cell mass consisting of a larger pool of amino acids, RNA, cellular proteins, and secreted antibody product

Suicide in Singapore, 1986.

Three hundred and forty-two cases of suicide in Singapore (1986) were studied. The rates were highest among Indians males. Jumping from a height was the method used by 60% of the subjects, and the predominant associated factor as ascertained by the coroner was illness (51%), both physical and mental. About 40% had seen a doctor in the year prior to their death, and 20% had a history of a previous suicide attempt. The majority of cases killed themselves during the daylight hours.

Suicide and parasuicide in Singapore (1986).

Suicide and parasuicide rates have increased in Singapore since 1980 and in 1986 were estimated to be 13.1 per 100,000 and 92 per 100,000 respectively. Those most prone to suicidal behaviour are the Indians and the aged above 70 years. Jumping from a height is still the commonest method of suicide and self-poisoning is the most popular method of parasuicide.

Biofeedback device for patients on axillary crutches.

The axillary crutch is commonly prescribed as an ambulatory aid to patients with temporal or permanent disability in the lower extremity. When fitting the axillary crutch, it is important that the user be instructed not to bear excessive weight on the axillary bar. Excessive weight bearing on the axillary bar can result in a sevenfold increase in the reaction force under the armpit. This force may be a contributory factor to crutch paralysis or thrombosis of the axillobrachial artery. In order to prevent this occurrence an electronic biofeedback device was designed and developed for use in the training of 3-point swing-through axillary crutch ambulation. It detects excessive weight bearing on the axillary bar during crutch ambulation and produces an audible signal which prompts the patient to make necessary adjustment to relieve load bearing on the axillary bar. The design and development of the biofeedback device is discussed in this paper.

Interanimal transferability of taste aversion learning for 0.1% saccharin.

Three experiments were performed to assess the interanimal transferability of conditioned taste aversion to 0.1% saccharin. Two experiments used an intracerebrospinal fluid (subdural) route for administering brain extracts and a third used an intraperitoneal (IP) route. As assessed by repeated measurements ANOVA, saccharin consumption was significantly lower during extinction of conditioned aversion for experimental recipients (ER) receiving extracts from aversively conditioned donors, than that of control recipients (CR), receiving extracts from unconditioned donors in one subdural experiment, F(21, 189) = 1.61, p less than 0.05. In the IP experiment the results were in the same direction, though not significant, F(34, 238) = 1.39, p less than 0.1. Results of the other subdural experiment are discussed. It is concluded that these experiments with the conditioned taste aversion paradigm have potential as a model for investigations of behavioral interanimal transfer (BIT) and for neuromolecular research aimed at identification of associated putative neurochemical(s) and the elaboration of their mechanism of action.

Noradrenaline and cyclic AMP--independent growth stimulation in newt limb blastemata.

Adult newts regenerate functional limbs after amputation. This process normally depends on the trophic influence of nerves on the regenerating limbs, particularly in the early stages before differentiation of the regeneration blastema, when it stimulates growth by maintaining high rates of macromolecular synthesis. The sequence of biochemical events involved is unknown, but it has been suggested that intracellular cyclic AMP may be a second messenger within the blastema. Many studies have indicated that the neural agent(s) involved might be protein. The recent finding that blastemata contain high levels of catecholamines, however, has implicated noradrenaline (NA) as the neurotrophic agent, and suggested that it works via stimulation of beta-adrenergic receptors on the blastemal cells, thereby raising the intracellular concentrations of cyclic AMP. To test this hypothesis we studied the ability of NA alone and in combination with alpha-and beta-adrenergic antagonists to increase cyclic AMP levels and to mimic the effects of nerves by maintaining high rates of protein synthesis and high mitotic indices (MI) in denervated blastemata in organ culture. We find that although NA raises cyclic AMP levels through a beta-adrenergic effect, it does not maintain high rates of protein synthesis or high MI in cultured blastemata. It is unlikely therefore, that this hypothesis applies.

Nerve trophic effects: an in vitro assay for factors involved in regulation of protein synthesis in regenerating amphibian limbs.

We have developed a rapid sensitive test for factors that mimic the trophic effects of nerves by maintaining normal rates of protein synthesis in denervated forelimb blastemata of adult newts (Notophthalmus viridescens). Rates of protein synthesis in secondary blastemata are similar. However, after they are denervated and explanted into organ culture the rates of protein synthesis first increase and later fall below control values. Similar changes occur after denervation in vivo. The alterations in the rates of protein synthesis are prevented by adding to the culture medium aqueous extracts from brains of adult newts or chicken embryos. The active material is either a peptide or a protein.

Aminoacyl-tRNA synthetases from calf liver: optimized assay conditions and kinetic properties.

Kinetic studies have been performed on the "family" of aminoacyl synthetases from calf liver. All assays were based on the esterification of amino acids to tRNA. Optimized reaction conditions for each synthetase are reported. Most of the synthetases show hyperbolic kinetics with respect to both amino acid and tRNA concentration, however a few show sigmoidal kinetics with respect to one substrate. Arginine, methionine and proline synthetases show sigmoidal kinetics with respect to mixed tRNA solutions and have Hill coefficients of 1.30, 1.10 and 1.20 respectively. Alanine and isoleucine synthetases show sigmoidal kinetics with respect to amino acid concentration and have Hill coefficients of 1.21 and 1.40 respectively.