Sequence analysis of Lys49 phospholipase A2 myotoxins: a highly conserved class of proteins

A cDNA clone coding for a putative Lys49 phospholipase A2 myotoxin (ACL myotoxin) from Agkistrodon contortrix laticinctus was isolated from a venom gland library and sequenced. The sequence of the first 40 amino acid residues of the predicted protein matches exactly with the N-terminal sequence of the purified myotoxin. Sequence comparison of the predicted sequence of ACL myotoxin and other Lys49 and Asp49 phospholipase A2 enzymes shows that the Lys49 phospholipase toxins form a highly conserved protein family. In addition to the change at position 49, Lys49 myotoxins have several invariant residues not found in the Asp49 group, like Lys7, Glu12, Thr13, Lys16, Lys78, Lys80, Lys115, and Lys116. There are also some conserved residues in the Asp49 group that are not conserved in the Lys49 group: Tyr28, Gly32, Gly33. Gly53. Lys49 myotoxins also have a Lys-rich region in the C-terminus, which is not present in the Asp49 group. These differences clearly indicate that Lys49 myotoxins comprise a conserved and distinct class of phospholipase A2 enzymes.     

Myocyte adaptation to chronic hypoxia and development of tolerance to subsequent acute severe hypoxia

Studies in animal models and humans suggest that myocardium may adapt to chronic or intermittent prolonged episodes of reduced coronary perfusion. Stable maintenance of partial flow reduction is difficult to achieve in experimental models; thus, in vitro cellular models may be useful for establishing the mechanisms of adaptation. Since moderate hypoxia is likely to be an important component of the low-flow state, isolated adult rat cardiac myocytes were exposed to 1% O2 for 48 hours to study chronic hypoxic adaptation. Hypoxic culture did not reduce cell viability relative to normoxic controls but did enhance glucose utilization and lactate production, which is consistent with an anaerobic pattern of metabolism. Lactate production remained transiently increased after restoration of normal O2 tension. Myocyte contractility was reduced (video-edge analysis), as was the amplitude of the intracellular Ca2+ transient (indo 1 fluorescence) in hypoxic cells. Relaxation was slowed and was accompanied by a slowed decay of the Ca2+ transient. These changes were not due to alterations in the action potential. Tolerance to subsequent acute severe hypoxia occurred in cells cultured in 1% O2 and was manifested as a delay in the time to full ATP-depletion rigor contracture during severe hypoxia and enhanced morphological recovery of myocytes at reoxygenation. The latter was still seen after normalization of the data for the prolonged time to rigor, suggesting a multifactorial basis for tolerance. An intervening period of normoxic exposure before subsequent acute severe hypoxia did not result in loss of tolerance but rather increased the delay to subsequent ATP depletion rigor. Cellular glycogen was preserved during chronic hypoxic exposure and increased after the restoration of normal O2 tension. As mitochondrial cytochromes should be fully oxygenated at levels well below 1% O2, hypoxic adaptation may be mediated by a low-affinity O2-sensing process. Thus, adaptations that occur during prolonged periods of moderate hypoxia are proposed to poise the myocyte in a better position to tolerate impending episodes of severe O2 deprivation.     

Intrathecal 131I-labeled antitenascin monoclonal antibody 81C6 treatment of patients with leptomeningeal neoplasms or primary brain tumor resection cavities with subarachnoid communication: phase I trial results

We aimed to determine the maximum tolerated dose (MTD) of 131I-labeled 81C6 in patients with leptomeningeal neoplasms or brain tumor resection cavities with subarachnoid communication and to identify any objective responses. 81C6 is a murine IgG monoclonal antibody that reacts with tenascin in gliomas/carcinomas but does not react with normal adult brain. 131I-labeled 81C6 delivers intrathecal (IT) radiation to these neoplasms. This study was a Phase I trial in which patients were treated with a single IT dose of 131I-labeled 81C6. Cohorts of three to six patients were treated with escalating doses of 131I (starting dose, 40 mCi; 20 mCi escalations) on 10 mg 81C6. MTD is defined as the highest dose resulting in serious toxicity in no more than two of six patients. Serious toxicity is defined as grade III/IV nonhematological toxicity or major hematological toxicity. We treated 31 patients (8 pediatric and 23 adult). Eighteen had glioblastoma multiforme. Patients were treated with 131I doses from 40 to 100 mCi. Hematological toxicity was dose limiting and correlated with the administered 131I dose. No grade III/IV nonhematological toxicities were encountered. A partial response occurred in 1 patient and disease stabilization occurred in 13 (42%) of 31 patients. Twelve patients are alive (median follow-up, > 320 days); five are progression free >409 days median posttreatment. The MTD of a single IT administration of 131I-labeled 81C6 in adults is 80 mCi 131I-labeled 81C6. The MTD in pediatric patients was not reached at 131I doses up to 40 mCi normalized for body surface area.     

Stereotactic fine needle aspiration in the management of mammographic abnormalities detected in breast screening

BACKGROUND: Fine needle aspiration (FNA) is used routinely in the management of palpable breast lumps; with the implementation of the stereotactic technique its use has been extended to the investigation of mammographic abnormalities. The advent of breast screening means that many mammographic abnormalities will be detected; because routine open biopsy is impractical and undesirable, stereotactic fine needle aspiration becomes the method of choice for investigation. METHOD: Within a 28 month interval, 81 Chinese women underwent stereotactic FNA in Kwong Wah Hospital, Hong Kong. RESULTS: Fifty-one women (62.9%) had a benign cytology result and no further investigations were required. Inconclusive results were obtained in 18.6% of the aspirates. Open biopsy was required in 16 women (19.8%). CONCLUSION: Stereotactic FNA is very useful in the exclusion of malignancy and the avoidance of open biopsy in asymptomatic women who have mammographic abnormalities.     

Sagittal abdominal diameter as a practical predictor of visceral fat

OBJECTIVE: To evaluate the relationships between the supine sagittal abdominal diameter (SAD) and visceral fat, as well as to evaluate intra- and inter-observer reliability of sagittal diameter measurement. PATIENTS: Twenty-eight women ranging in age from 27-78 y with a body mass index (BMI) ranging from 16.9-48.1 kg/m2 and 23 men ranging in age from 32-75 y with BMI ranging from 20-41.6 kg/m2. MEASUREMENT: Body fat distribution was measured by waist circumference, waist to hip ratio (WHR), SAD, anthropometrically assessed and a single slice of computed tomography (CT) at the L4-L5 level. RESULTS: In both genders, a significant association was found between visceral adipose tissue (AT) and SAD, as evaluated by CT (women r = 0.80; men r = 0.83, P < 0.001), and SAD by anthropometry (women r = 0.76; men r = 0.82, P < 0.001), as well as between visceral AT and waist circumference (women r = 0.76, men r = 0.86, P < 0.001) and WHR (women r = 0.57, P < 0.01, men r = 0.80, P < 0.001). A significant association was also found between subcutaneous AT and SAD by anthropometry (women r = 0.79, men r = 0.74, P < 0.001). After adjusting for BMI, the association between subcutaneous AT and SAD was no longer significant in men and only moderately significant in women (r = 0.42, P < 0.05), while the association between visceral AT and SAD by anthropometry remained significant in both genders (women r = 0.63, P < 0.001; men r = 0.66, P < 0.001). When the subjects were divided into two groups according to BMI (lean to moderately overweight women with BMI < 28 and men with BMI < 30 and obese women with BMI > 28 and men with BMI > 30) we found that the relationships between SAD by anthropometry, as well as SAD by CT and visceral AT, were higher in lean to moderately overweight subjects than in those who were obese. High inter-observer correlation was found concerning SAD measurement (r = 0.99, P < 0.001). Intra- and inter-observer precision as evaluated by coefficient of variation and intraclass correlation coefficient for SAD measurement was very high. CONCLUSION: Our study shows the usefulness of SAD by anthropometry to predict visceral fat and its very high inter- and intra-observer precision.     

Neurturin exerts potent actions on survival and function of midbrain dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) exhibits potent effects on survival and function of midbrain dopaminergic (DA) neurons in a variety of models. Although other growth factors expressed in the vicinity of developing DA neurons have been reported to support survival of DA neurons in vitro, to date none of these factors duplicate the potent and selective actions of GDNF in vivo. We report here that neurturin (NTN), a homolog of GDNF, is expressed in the nigrostriatal system, and that NTN exerts potent effects on survival and function of midbrain DA neurons. Our findings indicate that NTN mRNA is sequentially expressed in the ventral midbrain and striatum during development and that NTN exhibits survival-promoting actions on both developing and mature DA neurons. In vitro, NTN supports survival of embryonic DA neurons, and in vivo, direct injection of NTN into the substantia nigra protects mature DA neurons from cell death induced by 6-OHDA. Furthermore, administration of NTN into the striatum of intact adult animals induces behavioral and biochemical changes associated with functional upregulation of nigral DA neurons. The similarity in potency and efficacy of NTN and GDNF on DA neurons in several paradigms stands in contrast to the differential distribution of the receptor components GDNF Family Receptor alpha1 (GFRalpha1) and GFRalpha2 within the ventral mesencephalon. These results suggest that NTN is an endogenous trophic factor for midbrain DA neurons and point to the possibility that GDNF and NTN may exert redundant trophic influences on nigral DA neurons acting via a receptor complex that includes GFRalpha1.     

Progress on development of the Nimbus-University of Pittsburgh axial flow left ventricular assist system

Nimbus Inc. (Rancho Cordova, CA) and the University of Pittsburgh have completed the second year of development of a totally implanted axial flow blood pump under the National Institutes of Health Innovative Ventricular Assist System Program. The focus this year has been on completing pump hydraulic development and addressing the development of the other key system components. Having demonstrated satisfactory pump hydraulic and biocompatibility performance, pump development has focused on design features that improve pump manufacturability. A controller featuring full redundancy has been designed and is in the breadboard test phase. Initial printed circuit layout of this circuit has shown it to be appropriately sized at 5 x 6 cm to be compatible with implantation. A completely implantable system requires the use of a transcutaneous energy transformer system (TETS) and a diagnostic telemetry system. The TETS power circuitry has been redesigned incorporating an improved, more reliable operating topography. A telemetry circuit is undergoing characterization testing. Closed loop speed control algorithms are being tested in vitro and in vivo with good success. Eleven in vivo tests were conducted with durations from 1 to 195 days. Endurance pumps have passed the 6 month interval with minimal bearing wear. All aspects of the program continue to function under formal quality assurance.     

A phase II trial and pharmacokinetic analysis of 96-hour infusional paclitaxel in patients with metastatic colorectal cancer

AIMS: To carry out a retrospective study of male breast cancer over a 22-year experience. METHODS: Data from 121 male patients with breast cancer treated between the years 1972 and 1994 at the Surgical Clinic of Ankara Oncology Hospital were reviewed. Distribution of cases according to stage was: 2.5% stage I, 28.9% stage II, 55.4% stage III and 13.2% stage IV (AJCC staging method). The surgical treatment for 23 of the patients (19%) was Halsted's radical mastectomy or modified radical mastectomy. Seventy-three cases (60.3%) had total mastectomy without axillary node dissection and 25 (20.7%) had local tumour excision only. Seventy-two of 121 patients had adjuvant treatment. RESULTS: In general the prognosis of men with breast cancer was worse than for women. In the analysis of patients in stages I, II and III-A (operable disease group), the 5-year survival rates were 73% in axillary node-negative patients and 77% in those with tumours sized under 5 cm (P<0.001). In these patients, univariate analysis demonstrated that axillary status (relative risk of death in positive status vs. negative=3.6), tumour size (relative risk in T3 vs. T1-2=2), surgical treatment type (relative risk in simple mastectomy vs. radical mastectomy=1.9) and adjuvant chemotherapy (relative risk if no chemotherapy=1.4) were statistically significant factors associated with survival. CONCLUSIONS: Cox's regression model revealed that axillary status, tumour size and type of surgical treatment were the most important independent prognostic factors (P<0.001).     

C-terminal fragment of amyloid precursor protein inhibits calcium uptake into rat brain microsomes by Mg2+-Ca2+ ATPase

Numerous lines of evidence suggest that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid beta peptide (A beta). However, the possible role of other cleaved products of APP is less clear. In this study, the effects of a recombinant carboxy terminal 105 amino acid (CT105) fragment of APP on the calcium uptake by endoplasmic reticulum Mg2+-Ca2+ ATPase, the major mechanism for sequestering calcium in this organelle, were investigated. We found that CT 105 is a potent inhibitor of Mg2+-Ca2+ ATPase of endoplasmic reticulum, whereas A beta shows no effect. These results demonstrate that CT 105 inhibits the ability of brain microsomes to sequester calcium and suggest that this inhibitory effect of CT 105 may contribute to disruption of intracellular calcium concentration, possibly being involved in inducing the neural toxicity characteristic of AD.