A shared regulatory perspective on deferral from blood donation of men who have sex with men (MSM).

National Regulatory Authorities (NRAs) establish deferral criteria for donors with risk factors for transfusion transmissible infections (TTI). In most jurisdictions, epidemiological data show that men who have sex with men (MSM) have a significantly higher rate of TTI than the general population. Nevertheless, changes from an indefinite donor deferral for MSM have been considered in many countries in response to concerns over a perceived discrimination and questioning of the scientific need. Changes to MSM donor deferral criteria should be based on sound scientific evidence. Safety of transfusion recipients should be the first priority, and stakeholder input should be sought.

Cloning and characterisation of a prenyltransferase from the aphid Myzus persicae with potential involvement in alarm pheromone biosynthesis.

The majority of aphid species release an alarm pheromone with the most common component being the sesquiterpene (E)-beta-farnesene, sometimes accompanied by other sesquiterpenes or monoterpenes. The genes/enzymes involved in the production of these compounds have not been identified in aphids although some components of isoprenoid biosynthesis have been identified in other insect species. Here we report the cloning, expression and characterisation of a prenyltransferase from the aphid Myzus persicae which can act as a farnesyl pyrophosphate synthase or a geranyl pyrophosphate synthase to produce both sesquiterpenes and monoterpenes and hence could be responsible for the biosynthesis of the observed components of the alarm pheromones. In addition, the enzyme can utilise geranyl pyrophosphate to produce farnesyl pyrophosphate showing that the synthesis of the latter involves the sequential condensation of isoprenyl pyrophosphate units.

Prospective randomised trial of amifostine cytoprotection in myeloma patients undergoing high-dose melphalan conditioned autologous stem cell transplantation.

In this prospective multicentre trial, 90 patients undergoing autologous stem cell transplantation (ASCT) were randomised to receive (n=43) or not receive (n=47) amifostine 910 mg/m(2) prior to melphalan 200 mg/m(2). Patients were monitored for regimen-related toxicity, engraftment, supportive care, response and survival. Both groups underwent ASCT at a median of 8 months from diagnosis and were matched for disease characteristics, prior therapy and pre-ASCT disease responsiveness. Amifostine infusional side-effects were frequent, occurring in 65% of patients, but of mild severity. Amifostine use was associated with a reduction in the median grade of oral mucositis (1 vs 2, P=0.01) and the frequency of severe (WHO grades 3 or 4) mucositis (12 vs 33%, P=0.02), but no reduction in the requirement for parenteral nutrition or analgesic use. Conversion to complete remission post-ASCT occurred in 30 and 14% of the amifostine and control groups, respectively (P=0.09). With a median follow-up of 35 months, there was no statistically significant difference between the median progression-free or overall survival times for the two groups. We conclude that amifostine can be safely administered prior to high-dose melphalan and significantly reduces the frequency and severity of therapy-induced oral mucositis.

Hydroxyurea did not enhance the clinical response to vinblastine in patients with anthracycline-resistant metastatic breast cancer.

BACKGROUND: Vinblastine is commonly used in metastatic breast cancer after anthracycline failure. The response rate to vinblastine is approximately 20%, with short duration of response. In vitro studies have shown that the addition of hydroxyurea resulted in increased accumulation of vinblastine in tumor cells and in loss of double minutes. We evaluated the combination of vinblastine and hydroxyurea in patients with anthracycline-resistant metastatic breast cancer. PATIENTS AND METHODS: Fourteen assessable patients with metastatic breast cancer were entered in the study. All patients had progressed on anthracyclines or progressed within 8 months of stopping anthracyclines. Patients received hydroxyurea (500 mg orally) every Monday, Wednesday and Friday starting one week before the first course of chemotherapy and continuing throughout treatment until disease progression. Vinblastine (6 mg/m2) was given intravenously every 21 days, RESULTS: The median number of courses for vinblastine was 3.5 (range, 1-6). Three patients had partial responses in soft tissue metastases (21%). Four patients had stable disease. Four patients had > grade 2 neutropenia, and 1 patient had grade 4 thrombocytopenia. There were 2 cases of grade 3 constipation, 2 of grade 3 nausea, and 1 each of grade 2 neuropathy and myalgia. There was no treatment-related mortality. CONCLUSIONS: Low-dose hydroxyurea in combination with vinblastine has a 21% response rate in metastatic breast cancer after anthracycline failure. Toxicity was mild and generally reversible. At the adopted dose schedule of hydroxyurea, the antitumor activity of vinblastine in anthracycline-resistant metastatic breast cancer did not appear to be enhanced.

Autologous peripheral blood stem cell transplantation with in vivo T-cell depletion for life threatening refractory systemic lupus erythematosus.

We report the first Australian application of autologous haemopoietic stem cell transplantation in a 39-year old woman with severe systemic lupus erythematosus (SLE) and multiple life threatening complications, refractory to conventional therapy including intravenous cyclophosphamide. Our transplant technique, although not unique, differs from most published reports, in which an unmanipulated peripheral stem cell graft was used with in vivo lymphocyte depletion using rabbit antithymocyte globulin (ATG). Successful stem cell mobilization was achieved using granulocytecolony stimulating factor mobilization with methylprednisolone cover, after an initial attempt at mobilization was curtailed by respiratory arrest from upper airway obstruction due to cricoarytenoiditis, requiring tracheostomy. Conditioning regimen for the transplantation was cyclophosphamide 50 mg/kg on days -5 to -2 and rabbit ATG 2.2 mg/kg on days -3 and -2. An unmanipulated autograft was infused, with in vivo T-cell depletion achieved through a further dose of ATG given on day +2 postinfusion. The autologous transplant was well tolerated without fever or other serious complication. At 12 months follow-up post-transplantation, there is an objective evidence of near-complete response with SLE disease activity index scores falling from 40 pretransplant to 2. We conclude that HSCT with unmanipulated peripheral stem cell graft and in vivo lymphocyte depletion with ATG is safe and effective therapy for cyclophosphamide refractory SLE.

Microfibrillar protein from elastic tissue: a critical evaluation.

Many workers have claimed to have isolated proteins which have been derived from the microfibrillar components of elastic tissue. Virtually all of these preparations have been derived from extracts made with strong solutions of guanidinium chloride (GuHCl) under reducing conditions following Ross and Bornstein (1969). The products have ranged from heterogeneous mixtures of proteins to discrete glycoproteins. In no case has identity between an individual protein and the elastin-associated microfibrils been confirmed by immunoelectron microscopy. We have undertaken a detailed re-examination of the extractability of elastin-associated microfibrils and of the composition of the extracts from foetal bovine nuchal ligament. Finely homogenized samples were subjected to a series of extractions (including cyclical treatments with GuHCl and purified bacterial collagenase) in the presence of inhibitors of protease activity. Under these conditions it has been shown that--(i) microfibrils were removed progressively by GuHCl, throughout the extraction schedule, without the need for reduction; (ii) all remaining microfibrils were removed by reductive GuHCl extraction; (iii) the product from this reductive extraction consisted of a heterogeneous mixture of proteins including several glycoproteins; (iv) a major antigenic constituent of the mixture of proteins localized to elastin-associated microfibrils, as shown by immunoelectron microscopy. It is concluded that, while reductive GuHCl extracts do contain components with antigenic activity that is localized on elastin-associated microfibrils, they have many non-microfibrillar components. We stress that claims that a macromolecule is microfibrillar must be substantiated by immunoelectron microscopy.

Possible roles of microfibrils in elastogenesis.

On examination in the electron microscope elastic tissue is seen to consist of an amorphous component surrounded by microfibrillar components. The exact relationship between these components is unknown, although during development the microfibrils appear before the amorphous material. In this report we summarize our recent observations on the microfibrillar material. At high magnification the microfibrils are seen to have a poorly staining central core around the periphery of which are arranged more densely staining filaments which appear to wind around the microfibrils in a spiral fashion. Careful measurements of microfibrillar diameters from the aorta of four large species show that there are significant differences in the mean diameter and population distributions with species. The mean diameter of the microfibrils changes with age during fetal and postnatal development. The results of immunoelectronmicroscopic localization of an antibody to a microfibrillar component are reported and the possible roles of microfibrils in elastic tissue formation are examined.

Missense C168T in the Wiskott--Aldrich Syndrome protein gene is a common mutation in X-linked thrombocytopenia.

We describe a large Syrian--Lebanese family who clinically manifest X-linked thrombocytopenia (XLT). To date, five family members have undergone splenectomy with rapid and sustained normalization of their platelet numbers. Genomic analysis demonstrated that affected men in this cohort had the missense C168T (Thr45Met) mutation in exon 2 of the Wiskott-Aldrich Syndrome protein (WASp) gene. Exon 2 is the commonest site for mutations associated with XLT and mild forms of WAS, and the C168T missense mutation is the most frequent. Detection of this mutation by restriction enzyme digestion provides an efficient screening test for prompt identification and for assessment of female carrier status.

Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase.

The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti-spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase.

Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane.

Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells. A clone which complements this mutation was isolated from a cDNA library prepared from S. cerevisiae poly(A)+ RNA. This clone contains an insert which is 2775 bp in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa. Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms. Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1. When the cDNA for SUL1 was expressed in S. cerevisiae, a high affinity sulphate uptake activity (Km = 7.5 +/- 0.6 microM for SO2-4) was observed. A genomic mutant of S. cerevisiae in which 1096 bp were deleted from the SUL1 coding region was constructed. This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA. We conclude that the SUL1 cDNA encodes a S. cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.

Ethylene-independent and ethylene-dependent biochemical changes in ripening tomatoes.

Fruits of Lycopersicon esculentum Mill cv Sonatine stored in 6% CO(2), 6% O(2), and 88% N(2) for 14 weeks at 12 degrees C, exhibited a temporal separation of certain biochemical events associated with ripening.The specific activity of two citric acid cycle enzymes, citrate synthase and malate dehydrogenase, fell substantially during the first 2 weeks of storage when changes in organic acid concentration also occurred. During this period, lycopene, polygalacturonase, and ethylene were undetectable.When fruit were removed from store, ethylene was evolved and polygalacturonase and invertase activity were rapidly initiated as was synthesis of lycopene.To determine whether the changes in organic acid metabolism were affected by ethylene, fruit was kept at 22 degrees C in either a normal atmosphere or a normal atmosphere supplemented with 27 microliters per liter of ethylene, and it was shown that in both atmospheres similar quantitative changes to those described above occurred in the citric acid cycle enzymes specific activities before any detectable increase in the specific activities of invertase and polygalacturonase. These latter changes, together with pigment changes, occurred between 2 and 3 days earlier in fruit exposed to ethylene, compared with those kept in a normal atmosphere.

Molecular cloning and characterisation of asparagine synthetase from Lotus japonicus: dynamics of asparagine synthesis in N-sufficient conditions.

Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthetases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequences indicted a high level of identity with other plant AS sequences. No other AS genes were detected in the L. japonicus genome. LJAS1 gene expression was found to be root-enhanced and lower levels of transcript were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expression are different from those seen in pea. AS gene expression was monitored throughout a 16 h light/8 h dark day, under nitrate-sufficient conditions. Neither transcript showed the dark-enhanced accumulation patterns previously reported for other plant AS genes. To evaluate AS activity, the molecular dynamics of asparagine synthesis were examined in vivo using 15N-ammonium labelling. A constant rate of asparagine synthesis in the roots was observed. Asparagine was the most predominant amino-component of the xylem sap and became labelled at a slightly slower rate than the asparagine in the roots, indicating that most root asparagine was located in a cytoplasmic 'transport' pool rather than in a vacuolar 'storage' pool. The steady-state mRNA levels and the 15N-labelling data suggest that light regulation of AS gene expression is not a factor controlling N-assimilation in L. japonicus roots during stable growth in N-sufficient conditions.

Rapid disruption of nitrogen metabolism and nitrate transport in spinach plants deprived of sulphate.

Hydroponically grown spinach plants were deprived of an external source of sulphate after an initial period when the S-supply was sufficient. The time-course of events following this treatment was monitored. The first responses were found in the uptake and translocation of NO(3)(-) and the uptake of SO(4)(2-). The former declined by approximately 50%, the effect being most significant at higher [NO(3)(-)](ext.) while the latter increased 6-fold over a 4 d period. Growth in the absence of external SO(4)(2-) resulted in exhaustion of internal SO(4)(2-) pools, the effect being seen first in roots, then in young leaves and, after a marked delay, in mature leaves. In young leaves, there were dramatic increases in the [NO(3)(-)] and the content of arginine in the first 2 d of S-deprivation. The concentration of glutamine, the most abundant amino acid in S-sufficient conditions, also more than doubled in S-deficient young leaves. The changes in arginine levels were also found in older leaves, but the change in glutamine level was not seen. Assays of nitrate reductase activity (NRA) and nitrate reductase (NR) mRNA from young leaves of S-replete and S-deprived plants revealed a divergence in activity and content only late in the experiments (between days 4 and 8) when results were expressed on a unit leaf basis. However, there were also time-dependent changes in the protein content that kept the specific activities (NRA:protein and RNA:protein) more or less unchanged. The results imply that the impact of S-deficiency on N-utilization are more sensitively monitored by simple measurements of the chemical composition of young leaves than by measurements of NRA or NR transcript abundance. They also suggest that protein synthesis in young leaves is strongly dependent on a continuous supply of SO(4)(2-) from outside the plant.

Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization.

In situ hybridization was used to determine the morphologic distribution of tropoelastin and alpha 1(I) procollagen mRNA expression in elastic intralobar arteries from neonatal calves with hypoxic pulmonary hypertension induced by a 15-day exposure to a simulated altitude of 1500 m. In vessels from normotensive control animals, low levels of hybridizable tropoelastin mRNA were detected in smooth muscle cells (SMC) of the inner media associated with large elastic lamellae. Compared to control arteries, vessels from hypertensive animals demonstrated a markedly different pattern of hybridization. In these arteries, strong hybridization signals for tropoelastin mRNA were seen in SMC lying between the elastic lamellae of the outer media, and the density of labeling associated with these medial cells decreased progressively toward the lumen. Endothelial and adventitial cells in both control and hypertensive arteries were negative for tropoelastin mRNA. Type I procollagen mRNA was dispersed through the media of control arteries, and in hypertensive calves, the hybridization signal was more intense and was unevenly distributed through the media similarly to that for tropoelastin mRNA. Adventitial cells were strongly positive for procollagen mRNA, and the signal was equally intense for both control and hypertensive arteries. Cells that had no detectable tropoelastin mRNA were noted in the outer media of both control and hypertensive vessels. These cells occurred as broad circumferential bands in the normotensive artery and as nodular foci in the hypertensive artery. Immunocytochemical studies with antibodies to smooth muscle specific actin, desmin, and vimentin demonstrated that cells within these foci, as well as tropoelastin mRNA-positive cells, were SMC. These studies demonstrate that expression of tropoelastin and procollagen mRNA was differentially stimulated by pulmonary hypertension within specific regions and SMC populations of the vascular wall.

Polyclonal antibodies to tropoelastin and the specific detection and measurement of tropoelastin in vitro.

Because tropoelastin is difficult to purify, most antibodies to elastin are raised against the insoluble form of the molecule. While these antibodies cross-react with tropoelastin, antigenic differences between insoluble and soluble elastin suggest that antibodies raised directly against tropoelastin might provide a more sensitive and specific reagent for evaluating tropoelastin production in elastin-producing systems. Using an improved method for purifying tropoelastin from tissue culture explants, we describe the generation and characterization of an antibody to bovine tropoelastin. This antibody was used to develop a sensitive, direct-binding immunoassay capable of quantifying small levels of tropoelastin in conditioned medium from cultured cells. This assay takes advantage of the propensity of tropoelastin to adsorb to vinyl microtiter plates, even in the presence of serum proteins. This property, in combination with the increased sensitivity obtained using antibodies to tropoelastin, provides for a direct-binding immunoassay that detects nanogram quantities of tropoelastin directly in cell culture medium, avoiding sample preparation steps that result in extensive loss of tropoelastin. In addition, this direct-binding assay is ten- to 30-fold more sensitive than the existing competitive ELISA assays.

Phase I study of oral menogaril administered daily for 14 consecutive days.

Twenty-eight patients were treated with oral menogaril daily x 14 every 4 weeks. Granulocytopenia was dose-limiting at 50-60 mg/m2 per day. Neutropenic fever occurred in one patient. Thrombocytopenia occurred in 3 of 6 patients treated with menogaril 60 mg/m2/day. Nausea and vomiting and other toxic effects were generally mild. No cardiac toxicity was seen. One partial remission and three minor responses were noted among 11 previously treated patients with carcinoma of the bladder. One minor response was noted among 3 patients with colorectal cancer. The dose recommended for phase II studies is 50 mg/m2 per day for 14 days. Phase II studies are recommended in carcinoma of the bladder. Gastrointestinal toxicity is substantially less on this schedule than with oral menogaril administered on a weekly or q4wk schedule, but thrombocytopenia may be more common.

Duration of intravenous antibiotics for patients with neutropenic fever.

BACKGROUND: Standard therapy for febrile neutropenia after chemotherapy has consisted of intravenous antibiotic until resolution of both fever and neutropenia. We attempted to shorten the hospital stay by discontinuing intravenous antibiotics in blood culture negative patients who remained clinically stable and afebrile for 48 hours. PATIENTS AND METHODS: Febrile neutropenic admissions of non-leukemic patients were reviewed. They were divided by three consecutive six month intervals into Group 1 (prior to initiation of the policy), Group 2 (after the policy was instituted), and Group 3 (to monitor the implementation of the policy after the initial six months). RESULTS: There were 134 admissions for neutropenic fever. Median duration of intravenous antibiotic for Group 1 was 7 days (95% Confidence Interval 6-9). It was significantly decreased to 5 days (4-6) and 4 days (3-5) for Groups 2 and 3 respectively (p = 0.004 and p < 0.001). Median duration of hospital stay for Group 1 was 10 days (7-13). It was also significantly decreased to 7 (5-8) and 6 days (5-7) for Groups 2 and 3 respectively (p = 0.04 and p = 0.002). CONCLUSION: Early discontinuation of intravenous antibiotics in patients with negative blood culture who remain afebrile and clinically stable for 48 hours results in shorter duration of hospital stay with potential for reduction in hospital costs.