Microbial populations in Antarctic permafrost: biodiversity, state, age, and implication for astrobiology.

Antarctic permafrost soils have not received as much geocryological and biological study as has been devoted to the ice sheet, though the permafrost is more stable and older and inhabited by more microbes. This makes these soils potentially more informative and a more significant microbial repository than ice sheets. Due to the stability of the subsurface physicochemical regime, Antarctic permafrost is not an extreme environment but a balanced natural one. Up to 10(4) viable cells/g, whose age presumably corresponds to the longevity of the permanently frozen state of the sediments, have been isolated from Antarctic permafrost. Along with the microbes, metabolic by-products are preserved. This presumed natural cryopreservation makes it possible to observe what may be the oldest microbial communities on Earth. Here, we describe the Antarctic permafrost habitat and biodiversity and provide a model for martian ecosystems.

Iron-thiolate induced oxidation of methionine to methionine sulfoxide in small model peptides. Intramolecular catalysis by histidine.

Peptides containing either glycine and methionine, or glycine, methionine and histidine at various locations were oxidized by the dithiothreitol/ferric chloride system in phosphate buffer. The yields of peptide degradation and sulfoxide formation were measured as a function of peptide sequence and pH. In general little change of the final yields of peptide degradation is observed whereas the final yields of sulfoxide formation progressively decrease on going from pH 6.0 to 8.0. The pH profiles vary with the structure of the respective peptide. Efficient sulfoxide formation occurred when histidine and methionine were present within the same peptides sequence, and particularly when methionine was located at the C-terminus of the peptide. Added superoxide dismutase, catalase, and methanol did neither promote nor inhibit both the degradation of peptide and the formation of sulfoxide excluding free superoxide, hydrogen peroxide, and hydroxyl radicals as responsible reactive oxygen species. The observations are rationalized by invoking a pH-dependent conversion of an efficiently sulfoxide yielding oxidant into another oxidant which still degrades peptides but does not form methionine sulfoxide. The first might be a metal-bound peroxide or peroxyl species which converts into a metal-bound or 'complexed' hydroxyl radical.

Solid phase synthesis of bifunctional antibodies.

Bifunctional antibodies were prepared using the principle of solid-phase synthesis. The two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')2 fragments from intact IgG were prepared using an immobilized pepsin column. Goat, mouse and human antibodies were digested completely within 4 h. The F(ab')2 fragments thus produced did not contain any IgG impurities. The Fab' fragments were produced by reducing the inter-heavy chain disulfide bonds using 2-mercaptoethylamine. The use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of the bifunctional antibody preparation and the rapid optimization of reaction conditions.

Multiple epitope interactions in the two-step sandwich immunoassay.

The 'hook' effect as related to the two-step sandwich immunoassay has been investigated experimentally and theoretically. The multiple epitope interactions between the analyte and the labeled antibody cause a 'hook' in the two-step sandwich immunoassay. Three different analytes and monoclonal antibodies were chosen to carefully demonstrate the effect of the analyte characteristics on this immunoassay. Two monoclonal antibodies against two different epitopes of biosynthetic human growth hormone (hGH) was the simplest model for this study. The sandwich immunoassay for hGH shows no 'hook' effect. The non-covalent dimeric form of hGH (D-hGH) possesses two repeating epitopes which is the simplest model for an analyte having a discrete number of repeating epitopes. The D-hGH assay demonstrated a 'hook' effect in the two-step sandwich immunoassay if the labeled antibody was allowed to interact with more than one epitope. In a third system multiple epitope interactions with the labeled antibody were observed using ferritin. The effect of the analyte concentration and the liquid-phase antibody have been examined to elucidate the nature of these various interactions. The cause of the 'hook' effect in the two-step sandwich immunoassay is attributed to the desorption of the bound analyte due to a conformational change after the labeled antibody interacts with several epitopes of the adsorbed analyte.

Studies of the 'hook' effect in the one-step sandwich immunoassay.

The one-step sandwich immunoassay is increasingly replacing the traditional two-step immunoassay due to obvious advantages such as assay speed. However, the one-step sandwich immunoassay suffers from the 'hook' effect irrespective of the analyte characteristics. The 'hook' effect is dependent primarily on the analyte concentration. Three different model analytes, human growth hormone (hGH), the dimeric form of hGH (D-hGH, having a discrete number of repeating epitopes) and ferritin (multiple epitopes) having different immunological properties have been employed in studies of the one-step sandwich immunoassay. The characteristics of each of the model analytes offer new insights into general guidelines for assay procedures. These guidelines permit rapid optimization of assay conditions for an immunoassay without a priori knowledge of the immunological characteristics of the antibody or antigen. Both experimental and theoretical data show several instances where high capacity solid-phase antibodies can effectively shift the 'hook' to relatively higher analyte concentrations. The effect of the concentration of labeled antibody on assay response was examined theoretically.

Studies of the low dose 'hook' effect in a competitive homogeneous immunoassay.

The interactions of two monoclonal antibodies with human growth hormone (hGH) have been investigated. The individual antibodies showed normal behavior in a competitive binding assay, but mixtures of the antibodies demonstrated a 'hook' attributable to cooperative interactions. Cooperativity was observed in titrations which preceded the competitive binding assay. Size exclusion chromatographic data suggest that the cooperativity is explained by the formation of higher molecular weight complexes (up to 700 kDa). The major complex is probably linear, consisting of three antibody molecules. Circular and linear complexes with four antibody molecules (octameric complexes) are also possible. Theoretical models also support the formation of cyclic complexes in a competitive binding assay.

Enhanced immunogenicity of leucine enkephalin following coupling to anti-immunoglobulin and anti-CD3 antibodies.

Leucine enkephalin (Leu-enk) was coupled to both T and B cell antibodies in order to investigate the possibility of enhanced immunogenicity via targeted immunization. The two antibodies used were Hm x Mo CD3 and Gt x Mo Ig, respectively. The data indicate that while both antibody carriers enhanced the immunogenicity of Leu-enk, the use of the Hm x Mo CD3 antibody resulted in a greater number of mice with positive Leu-enk specific serum titers. 12 Leu-enk cell lines were produced and one, LE4H8, was chosen for characterization.

The development of preschool children of heroin-addicted mothers: a controlled study.

Disturbances of growth and behavior in infants and toddlers of women addicted to heroin during pregnancy have been reported in uncontrolled studies. In this study, 3- to 6-year-old children of heroin-addicted mothers were compared to three other groups matched for age, race, sex, birth weight, and socioeconomic status. Heroin-exposed children weighed less and were shorter than those in the comparison groups; 14% had a head circumference below the third percentile. Heroin-exposed children were rated by parents as less well adjusted than control children and they differed significantly in perceptual measures and on subtests of the Illinois Test of Psycholinguistic Abilities and McCarthy Scales of Children's Abilities relating to the process of organization. These findings suggest that chronic intrauterine exposure to heroin may affect growth and behavior as well as perceptual and learning processes in preschool children.

Factors affecting head growth and intellectual function in children of drug addicts.

The effect of maternal heroin and methadone use on head growth and neurodevelopmental performance was studied in preschool children of untreated heroin addicts (n = 25), women receiving methadone therapy (n = 26), and a drug-free comparison group (n = 41) who had been followed from birth. The mean birth head circumference of both groups of drug-exposed infants was significantly below that of the comparison group; however, the only factors determined by multiple regression analysis as associated with head size at birth were maternal nutritional status and birth weight. By preschool age, head size did not differ significantly among groups. The factors associated with postnatal head growth were birth weight, intrapartum risk score, and race. Data show an increased incidence of low-average and mildly retarded intellectual performance in the drug-exposed children. Regression analyses demonstrated that amount of prenatal care, prenatal risk score, and home environment were most predictive of intellectual performance and that the degree of maternal narcotic use was not a significant factor.

Nonhandicapped very-low-birth-weight infants at one year of age: developmental profile.

The developmental profile of 61 very-low-birth-weight infants without major cognitive, motor, or sensory deficits was compared with that of 28 term infants at 1 year chronologic age. The groups significantly differed in two ways on the Revised Gesell Developmental Schedules. First, very-low-birth-weight infants were more likely than term infants to have significant discrepancies between either their fine motor or language abilities and their early problem-solving skills as measured by the Adaptive scale of the Gesell. Second, across all fields of behavior (adaptive, gross motor, fine motor, language, and personal/social), very-low-birth-weight infants scored significantly below term infants. The very-low-birth-weight infant's motor performance significantly correlated with bronchopulmonary dysplasia, intracranial hemorrhage, and number of days spent in the hospital. Language performance significantly correlated with intracranial hemorrhage, birth weight, and sex. These findings underscore the limitations of global developmental scores to describe adequately the developmental performance of very-low-birth-weight infants. Instead, a comprehensive assessment of all fields of behavior is necessary to provide an accurate profile of this high-risk group.

Ultrastructural effects of sodium chloride on the corneal epithelium.

Ten excised rabbit corneas were bathed posteriorly with glutathione bicarbonate Ringers solution (GBR), while anteriorly the bathing solution was either GBR or sodium chloride solution (NaCl). All solutions had an osmolarity of 305 +/- 2 mOsm/kg. The corneas were fixed after 150 min exposure to the solutions, and prepared for electron microscopy. Morphometric analysis of the electron micrographs was conducted by an observer unaware of the anterior bathing solution. In each case, the epithelium was examined along a 1500 micron stretch of the basement membrane. Cells were categorized as normal, abnormal, and sloughing. Abnormal cells showed cytoplasmic and nuclear pallor, and disrupted cell membranes. Sloughing cells showed partial separation from the underlying epithelium. Corneas exposed to NaCl showed statistically significant differences from those exposed to GBR; the differences occurred in both the number of abnormal cells, and the number of sloughing cells. All observed ultrastructural changes were limited to the surface region of the epithelium. It is concluded that sodium chloride solution is inadequate at maintaining the epithelial surface.

Polarography: an oxygen survey correlating oxygen transmissibility to "EOP" values.

A polarographic oxygen electrode was used to measure the oxygen transmissibility of seven contact lenses of varying water content and center thickness. A similar electrode was used to measure the oxygen uptake following wear of these same contact lenses for both the open- and closed-eyelid conditions on five young healthy subjects. Linear regression revealed a strong correlation between oxygen transmissibility and equivalent oxygen percentage (EOP) values for both the open- and closed-eyelid conditions. This strong correlation between these two oxygen parameters shows both are useful in predicting the oxygen tensions across the tear-epithelial interface during contact lens wear.

The thickness of the human precorneal tear film: evidence from reflection spectra.

PURPOSE: Interferometric methods have considerable potential for studying the thickness of layers of the human tear film and cornea because of their ability to make noninvasive, accurate, and rapid measurements. However, previous interferometric studies by Prydal and Danjo yielded tear thickness values near 40 and 11 microm, respectively, considerably greater than estimates made by invasive methods of 4 to 8 microm. Using a modified version of Danjo's method, interference effects from the tear film and cornea were studied, with the aim of correlation with known structure and optical properties of the cornea and hence determining the most probable value of tear film thickness. METHODS: Reflectance spectra from the human cornea were measured at normal incidence. These spectra show oscillations whose maxima correspond to constructive interference between light reflected from the air surface and from some deeper surface. The frequency of these spectral oscillations is proportional to the thickness of the layer between the air surface and the second surface. Therefore, Fourier analysis of reflectance spectra can be used to determine the thickness of layers of the tear film and cornea. In the main experiment, 36 low-resolution spectra were obtained from six normal eyes for measuring thickness up to 100 microm. Control experiments included measurements of the time course of thickness changes and high-resolution spectra for measuring thickness up to 1000 microm. RESULTS: For the main experiment, in the thickness range 1 to 100 microm, the strongest peak in the Fourier transform was near 3 microm (range, 1.5-4.7 microm) beneath the air surface. In the range 20 to 100 microm, the strongest peak was near 55 microm (range, 50-59 microm) for all 36 spectra; none were in Prydal's range near 40 microm. This 55-microm peak is consistent with a reflection from the basement membrane of the epithelium. Time course measurements after a blink show that the 3-microm peak is not an artifact. High-resolution spectra gave a peak near 510 microm, corresponding to the complete thickness of the cornea (plus tear film). This peak had a contrast similar to that of the 3-microm peak. CONCLUSIONS: These studies did not confirm Prydal's estimate of approximately 40 microm. Nor were there prominent peaks near Danjo's value of approximately 11 microm, except in cases of probable reflex tears. Because the reflection at the aqueous-mucus boundary would be expected to be weaker than that from the epithelial surface, the 3-microm peak is unlikely to correspond to the aqueous layer (rather than the complete tear film). The proposal that the 3-microm peak corresponds to a reflection from the front of the cornea is supported by the demonstration of a peak of similar contrast from the back of the cornea. Thus, the current evidence consistently supports a value of approximately 3 microm for the thickness of the human precorneal tear film.

A novel high-dose chemotherapy protocol with autologous hematopoietic rescue in patients with metastatic breast cancer or recurrent non-Hodgkin's lymphoma.

In this phase II trial, we used a double dose-intensive chemotherapy and stem cell rescue protocol to treat breast cancer (BCA) patients or non-Hodgkin's lymphoma patients (NHL). The first cycle consisted of high-dose melphalan followed by ABMT. The second cycle used a novel chemotherapy combination; thiotepa, etoposide, carboplatin and cyclophosphamide (TECC) followed by ABMT. We treated 12 patients in total, nine with BCA, three with NHL. All nine BCA patients were treated with the two cycle protocol. The three NHL patients were treated with the second cycle only. Bone marrow (BM, 1 patient), peripheral blood stem cells (PBSC, 10 patients) or both (1 patient) were reinfused 60-72 h after completion of each cycle of chemotherapy. Recovery was rapid; the ANC rose to greater than 500/microl on day +11 (+8 to + 20) and the platelet count to greater than 20000/microl on day +12 (+6 to +20). The toxicities included the expected neutropenic fevers, severe mucositis, diarrhea, and a low incidence of mild renal insufficiency. No patients developed veno-occlusive disease, hemorrhagic cystitis or overt bleeding. With a mean follow-up of 37 months, 83.3% of the patients are alive. Six patients are in complete remission; one patient with BCA relapsed and expired; one patient with NHL is in CR now over 18 months after relapse and subsequent treatment with interferon; one patient is too early to evaluate. Progression-free survival overall is 75%, which is at least equivalent to many other recent studies using similar regimens. In addition, we have also found that delayed addition of G-CSF during the mobilization of PBSC was feasible and resulted in excellent CD34+ cell counts and engraftments, and reduced treatment costs. These results indicate that this chemotherapy is effective with good remission rates and high progression-free survival rates. It is also well tolerated with acceptable toxicities that are manageable. Long-term follow-up of a larger cohort of patients will be needed to ascertain the overall efficacy of this type of therapy.

Continuous glucose monitoring in the free-moving rat.

The aim of this work was to set up an experimental model of glycemic fluctuations for assessing in the conscious freely moving rat, the performance of a continuous glucose-monitoring system, using a pocket-calculator-size electronic control unit and a miniaturized subcutaneous glucose sensor. The well-known triphasic glycemic pattern following streptozotocin injection (initial peak and secondary hypoglycemia preceding the establishment of permanent hyperglycemia) was used as a way to obtain spontaneous changes in blood glucose level over a wide concentration range. This report demonstrates that streptozotocin injection produced highly reproducible changes in the current generated by the sensor: an initial peak and a secondary nadir, during which blood sampling provided the evidence of hyperglycemia associated with immunoreactive hypoinsulinemia, and of hypoglycemia associated with hyperinsulinemia, respectively. This reproducible experimental model should be valuable for the assessment of a continuous glucose-monitoring system.

Evaluation of bone scan in preoperative clinical staging of breast cancer.

Two hundred and thirty-five patients underwent a bone scan prior to, or within two weeks of, instituting surgical treatment for cancer of the breast. These patients were staged clinically according to the TNM classification. There were three patients with abnormal bone scans of a total of 173 patients in clinical stages I and II; none of 86 patients had stage I disease and only three of 87 patients had stage II disease. The conclusion from the study is that routine preoperative bone scans are of no value for staging purposes in patients with clinical stage I disease. In clinical stage II, bone scans are difficult to justify, considering the low yield (3.4%) and the cost per abnormal scan.

Needle localization of nonpalpable breast masses.

A series of 146 women underwent 150 preoperative localizations of mammographically suspicious but nonpalpable breast lesions. The lesions were localized using the hook-wire method of Frank in 133 of these patients. Carcinoma was discovered in 24 (16%) of the women; 18 (75%) of these women had invasive and six women (25%) had noninvasive carcinomas. Sixty-seven patients demonstrated calcification, and of these, 16 patients (24%) turned out to have malignancies. Eighty percent of the cancers were less than 1 cm in diameter, and 38% met the criteria of minimal carcinoma as described by Gallagher and Martin in 1969. Fourteen percent of the patients with carcinoma had lymph node metastases. We conclude that this is a safe, rapid, and accurate method for localizing small, potentially highly curable breast cancers with minimal sacrifice of breast tissue.

Application of cell culture toxicity tests to the development of implantable biosensors.

Cell culture toxicity testing methods were modified and applied to the development of implantable glucose microsensors, and positive and negative control materials suitable for the microsensor assessment were established. The location, source and degree of the toxic effect in a multi-component biosensor was spatially visualized with cell monolayers. A freshly prepared sensor showed moderate toxicity, mainly as a result of the presence of glutaraldehyde and the residual solvents in the polymer layers. However, it was possible to reduce the toxicity by removing the leachable toxic substances through extraction in phosphate buffer, and a non-toxic sensor was readily obtained.

Electrochemical biosensors: recommended definitions and classification.

Two Divisions of the International Union of Pure and Applied Chemistry (IUPAC), namely Physical Chemistry (Commission 1.7 on Biophysical Chemistry formerly Steering Committee on Biophysical Chemistry) and Analytical Chemistry (Commission V.5 on Electroanalytical Chemistry) have prepared recommendations on the definition, classification and nomenclature related to electrochemical biosensors: these recommendations could, in the future, be extended to other types of biosensors. An electrochemical biosensor is a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element. Because of their ability to be repeatedly calibrated, we recommend that a biosensor should be clearly distinguished from a bioanalytical system, which requires additional processing steps, such as reagent addition. A device that is both disposable after one measurement, i.e. single use, and unable to monitor the analyte concentration continuously or after rapid and reproducible regeneration, should be designated a single use biosensor. Biosensors may be classified according to the biological specificity-conferring mechanism or, alternatively, to the mode of physico-chemical signal transduction. The biological recognition element may be based on a chemical reaction catalysed by, or on an equilibrium reaction with macromolecules that have been isolated, engineered or present in their original biological environment. In the latter cases. equilibrium is generally reached and there is no further, if any, net consumption of analyte(s) by the immobilized biocomplexing agent incorporated into the sensor. Biosensors may be further classified according to the analytes or reactions that they monitor: direct monitoring of analyte concentration or of reactions producing or consuming such analytes; alternatively, an indirect monitoring of inhibitor or activator of the biological recognition element (biochemical receptor) may be achieved. A rapid proliferation of biosensors and their diversity has led to a lack of rigour in defining their performance criteria. Although each biosensor can only truly be evaluated for a particular application, it is still useful to examine how standard protocols for performance criteria may be defined in accordance with standard IUPAC protocols or definitions. These criteria are recommended for authors. referees and educators and include calibration characteristics (sensitivity, operational and linear concentration range, detection and quantitative determination limits), selectivity, steady-state and transient response times, sample throughput, reproducibility, stability and lifetime.

Calibration of a subcutaneous amperometric glucose sensor. Part 1. Effect of measurement uncertainties on the determination of sensor sensitivity and background current.

The calibration of a continuous glucose monitoring system, i.e. the transformation of the signal I(t) generated by the glucose sensor at time (t) into an estimation of glucose concentration G(t), represents a key issue. The two-point calibration procedure consists of the determination of a sensor sensitivity S and of a background current I(o) by plotting two values of the sensor signal versus the concomitant blood glucose concentrations. The estimation of G(t) is subsequently given by G(t) = (I(t)-I(o))/S. A glucose sensor was implanted in the subcutaneous tissue of nine type 1 diabetic patients during 3 (n = 2) and 7 days (n = 7). For each individual trial, S and I(o) were determined by taking into account the values of two sets of sensor output and blood glucose concentration distant by at least 1 h, the procedure being repeated for each consecutive set of values. S and I(o) were found to be negatively correlated, the value of I(o) being sometimes negative. Theoretical analysis demonstrates that this phenomenon can be explained by the effect of measurement uncertainties on the determination of capillary glucose concentration and of sensor output.