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Advances in development of a genetic system for Thermoanaerobacterium spp.: expression of genes encoding hydrolytic enzymes, development of a second shuttle vector, and integration of genes into the chromosome.

Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.

Bacteria, Anaerobic↗

MR imaging of the developing human brain. Part 2. Postnatal development.

In vivo magnetic resonance (MR) images of the brain in neonates and infants up to 24 months of age were retrospectively studied to evaluate normal maturation patterns in the brain. Sequential changes in myelination of various brain structures and development of the corpus callosum were noted. At birth and for the first 4-6 months of life, signal intensities of gray and white matter are the reverse of those seen in an adult brain, with the signal intensity of white matter being lower than that of gray matter on T1-weighted images and higher than that of gray matter on T2-weighted images. With advancing age, white matter shows a progressive increase in signal intensity on T1-weighted images. On T2-weighted images, high-signal-intensity unmyelinated white matter progressively changes to myelinated white matter of a signal intensity lower than that of gray matter. At birth, the corpus callosum is isointense relative to white matter and progressively increases in signal intensity, so that at age 8 months the corpus callosum has an appearance identical to that of an adult. Familiarity with the temporal sequence of normal myelination as seen with MR imaging is helpful in the diagnosis of pathologic processes involving white matter.

Age Factors↗

Report on the Dutch consensus development meeting for implementation and further development of population screening for colorectal cancer based on FOBT.

A consensus development meeting was held to evaluate whether or not in the Netherlands all requirements were fulfilled for implementation of population screening with FOBT for colorectal cancer, or whether consensus was present that fulfilment by additional research or organisational actions could be obtained within 2-3 years. There was consensus that all classical Wilson and Jungner (1968) criteria, and six additional ones added more recently, had already been fulfilled or could be fulfilled within 2-3 years. Consequently, it was concluded that a national population screening for colorectal cancer should be implemented and carried out in the Netherlands in line with current national and European cancer screening programmes. A list of organisational actions to be taken was established. Research that is needed before the actual national launch of the screening within 2-3 years has been defined. Priorities have to be set for research and organisational actions for the coming 2-3 years for the implementation of population screening. In addition, research suggestions have been defined for the next 10-15 years for evaluation and/or improvement of implemented FOBT screening, and for future screening methodology. It was considered essential that infrastructure for future research would be embedded in the screening programme. A project group to arrange this should be formed.

Adenoma↗

Spinal cord stimulation: a quarter century of development and investigation. A review of its development and effectiveness in 1,336 cases.

The past two and a half decades have seen the development of a spinal cord stimulator from the early 2-electrode fixed system to the present multielectrode computerized systems. During these 25 years, spinal cord stimulation has been studied in the treatment of motor disorders. The effectiveness was studied in 1,336 cases, including cerebral palsy (456), dystonia (173), torticollis (90), multiple sclerosis (130), spinocerebellar degeneration (71), spinal cord injury (303) and posttraumatic brain injury (113). It has become increasingly evident that the maximum therapeutic effect is achieved by virtue of the applied field variables of the spinal cord level stimulated, the field configuration, its polarity, and the frequency of the stimulation. These observations have led to investigational corollaries of the therapeutic specificity of the applied field, the neurophysiologic mechanisms of these fields and the underlying abnormal neurophysiologic substrate, which may indeed be secondary to abnormalities in the nerve impulse itself.

Cerebral Palsy↗

Effects of thyroid state on the formation and early morphological development of Bergmann glia in the developing rat cerebellum.

The formation and morphological maturation of Bergmann glia cells, which might play an important role in the histogenesis of the cerebellar cortex, was studied by histological and electron microscopic techniques in the cerebellum of normal young rats and animals made hypothyroid since the 18th day of gestation or hyperthyroid since birth. In comparison with controls, an acceleration in the morphological development of Bergmann glia cells was observed in the hyperthyroid animals; on the contrary, in the thyroid-deficient rats, the formation and morphological maturation of these cells were retarded and their final number was increased.

Animals↗

Incorporation of stearic acid into brain lipids in the developing brain: blood-brain relationships during development.

The blood-brain relationship for stearic acid varies during development. Subcutaneously injected [1-14C]-stearic acid is taken up by brain. Age-related changes in the metabolism of stearic acid have been determined in mouse brain from birth to maturity. Total lipid radioactivity reaches a maximum at 18 days of age and decreases afterwards until adulthood. However, specific radioactivity presents the highest value at 1 day of age and declines from then on. At any age, the injected acid is taken up and partly metabolized in the brain, either by elongation or by degradation in situ and resynthesis of new fatty acids; it is also desaturated, and the oleic acid thus formed is eventually elongated. The labeled stearic acid is incorporated into brain lipids with a different pattern according to the age of the injected animal.

Age Factors↗

Lipid metabolism of developing central nervous tissues in organotypic cultures. II. A comparative study of medium and tissue fatty acids in developing rat cerebral cortex and cerebellum.

Organotypic cultures of rat cerebral cortex and cerebellum have been supplemented with different media. Fatty acid analysis in incubated media and in tissue explants revealed a characteristic differentiation of three periods during maturation: a posttraumatic repair phase, a synaptogenic period and a synaptic stage. Medium fatty acids are biphysically depleted from the nutrient. Both tissues differ in the depletion pattern of individual fatty acids. The development of tissue fatty acids is mainly observed during the synaptogenic period. Turnover studies of individual fatty acids indicate a tissue-specific and time-dependent characteristic.

Animals↗

PYY in developing murine islet cells: comparisons to development of islet hormones, NPY, and BrdU incorporation.

Exhaustive characterizations of antisera to the structurally related peptides pancreatic polypeptide (PP), neuropeptide Y (NPY), and peptide YY (PYY) enabled us to establish the developmental pattern of these peptides in rat and mouse pancreas. PYY was the earliest detectable peptide and was present in all early appearing endocrine cell types. NPY appeared later and occurred exclusively in a subpopulation of insulin cells, whereas PP cells arose latest. At the earliest stage studied, all endocrine cells stored PYY. Most of these cells also contained glucagon. Subsequently, the endocrine cells comprised glucagon+PYY cells and glucagon+PYY+insulin cells. Later, cells storing either only insulin or insulin+PYY appeared. Quantitations of the relative numbers of these cell populations during development were consistent with a precursor role of triple-positive (insulin+glucagon+PYY) cells. Moreover, bromodeoxyuridine (BrdU) injections at E15.5 showed that a large percentage of triple-positive cells were in S-phase and therefore were actively dividing, whereas almost no pure insulin cells or insulin+PYY cells synthesized DNA at this time. These results suggest that PYY-positive endocrine cells may represent precursors for mature islet cells.

Animals↗

Development and validation of assessment measures for a newly developed physical examination simulator.

OBJECTIVE: Define, extract and evaluate potential performance indicators from computer-generated data collected during simulated clinical female pelvic examinations. DESIGN: Qualitative and quantitative study analyzing computer generated simulator data and written clinical assessments collected from medical students who performed physical examinations on three clinically different pelvic simulators. SETTING: Introduction to patient care course at a major United States medical school. PARTICIPANTS: Seventy-three pre-clinical medical students performed 219 simulated pelvic examinations and generated 219 written clinical assessments. MEASUREMENTS: Cronbach's alpha for the newly defined performance indicators, Pearson's correlation of performance indicators with scored written clinical assessments of simulator findings. RESULTS: Four novel performance indicators were defined: time to perform a complete examination, number of critical areas touched during the exam, the maximum pressure used, and the frequency at which these areas were touched. The reliability coefficients (alpha) were time = 0.7240, critical areas = 0.6329, maximum pressure = 0.7701, and frequency = 0.5011. Of the four indicators, three correlated positively and significantly with the written clinical assessment scores: critical areas, p < 0.01; frequency, p < 0.05; and maximum pressure, p < 0.05. CONCLUSION: This study demonstrates a novel method of analyzing raw numerical data generated from a newly developed patient simulator; deriving performance indicators from computer generated simulator data; and assessing validity of those indicators by comparing them with written assessment scores. Results show the new assessment measures provide an objective, reliable, and valid method of assessing students' physical examination techniques on the pelvic exam simulator.

Clinical Competence↗

Ego development and sociometrically evaluated maturity: an investigation of the validity of the Washington University Sentence Completion Test of Ego Development.

The reported study was an attempt to validate the Washington University Sentence Completion Test of Ego Development (WU-SCT), using a broad, sociometric index of maturity as the criterion variable. Sixty college women living in a sorority house completed the WU-SCT and evaluated one another's readiness for mature functioning in each of four adult social roles: career, marriage, parenthood, and community involvement. The reliabilities and the intercorrelations of the ratings suggested that peers distinguished two facets of maturity. While results support the hypothesized relationship between WU-SCT scores and a global sociometric index of maturity, the test's substantial correlations with indices relating to relatively impersonal roles (career and community) and apparent lack of correlation with indices relating to intimate interpersonal roles (marital and parental) suggests that the validity of the WU-SCT may be less broad than Loevinger's theory would imply. Several alternative interpretations of the data are suggested.

Journal Article↗

Postnatal development of beta-endorphin-related peptides in rat anterior and intermediate pituitary lobes: evidence for contrasting development of proopiomelanocortin processing.

The concentration of immunoreactive (ir) beta-endorphin (beta-END) in the neurointermediate pituitary lobe was 15-fold higher in adult than in newborn rats; in contrast, that of ir-beta-END in the anterior lobe was twice as high in newborn as in adult animals. Ir-beta-END in the neurointermediate lobe of newborn rats consisted exclusively of beta-END-sized peptides, indicating that at birth rats are capable of processing the opioid peptide precursor proopiomelanocortin (POMC) to beta-END. Moreover, beta-END-related peptides in the neurointermediate lobe of newborn rats were found to be predominantly alpha-N-acetylated and, therefore, inactivated with respect to their opiate-like properties. Further analysis of these alpha-N-acetylated forms on high performance liquid chromatography indicated that newborn rats predominantly contained alpha-N-acetyl-(Ac-)beta-END-(1-31), whereas the major forms in adult rats were Ac-beta-END-(1-27) and -(1-26). Thus, the C-terminal processing of Ac-beta-END-(1-31) to -(1-27) and -(1-26) may not yet be fully active at birth, in contrast to the processing of POMC to beta-END. In the anterior lobe of newborn rats, however, the ratio of beta-lipotropin/beta-END resembled that of adults, and more than 80% of beta-END-sized ir-material was found to consist of nonacetylated (and therefore opiate-active) beta-END-(1-31), as in adults, suggesting that the enzymatic system responsible for processing of POMC to beta-lipotropin and beta-END is already mature at birth. The high concentrations of beta-END in the anterior lobe of newborn rats suggest a possible role of this opioid peptide in perinatal development and/or parturition.

Animals↗

Storage protein gene expression is localised to regions lacking mitotic activity in developing pea embryos. An analysis of seed development in Pisum sativum XIV.

Storage protein gene expression has been studied in relation to mitotic activity to ascertain whether these processes are linked during embryo development in pea. Sections from immature pea embryos were probed by in situ hybridisation to show the pattern of vicilin storage protein gene expression. In addition, the location of mitotic cells was identified using fluorescence microscopy. Vicilin mRNA was first localised in the parenchyma cells of the upper adaxial region of the cotyledons. As the embryos increased in fresh weight, gene expression spread from this region, in a wave-like manner, down and across the cotyledons. The gene was only expressed in those regions of the embryo that lacked mitotic activity.

Fabaceae↗

Dynamics of the control of body pattern in the development of Xenopus laevis. IV. Timing and pattern in the development of twinned bodies after reorientation of eggs in gravity.

The mesendodermal anatomy of twinned larval axes is described in relation to the normal single pattern, when twinning has been caused by experimental tilting of eggs before first cleavage. The formation of two origins for gastrulation movements (dorsal lips) and their relatively rapid spread and coalescence to give a circular blastopore, is a predictor of twin formation in individual embryos after treatment. The anatomy of twins where development has been disturbed from the outset in this way is appreciably different from that induced by the later operation of second dorsal lip implantation. It is also variable in a systematic way. The total sizes of cellular allocations to individual notochords and prechordal head patterns are enhanced above normal if they arise relatively close together in the tissue, but significantly reduced if they arise far apart. These and other features of twinned patterns due to precleavage disturbance are discussed in terms of what they might indicate about the physicochemical nature of the body positional system. The results confirm that by a variety of rather simple, nonsurgical manipulations the relative amounts of territory in the egg devoted to different parts of the body can be greatly influenced.

Animals↗

Development of the murine periodontium. I. Role of basement membrane in formation of a mineralized tissue on the developing root dentin surface.

The presence of basement membrane components on the forming root surface suggests a role for this structure during cementoblast differentiation. The purpose of this study was to investigate the role of root-associated basement membrane (RBM) in murine cementogenesis using tissue separation and recombination techniques. Root dentin specimens, with or without RBM, were combined with dental sac tissue, cultured for 2 weeks, harvested, and examined by light, immunofluorescence, and electron microscopy. Recombinations in which RBM was preserved on the root dentin surface were characterized by formation of an adherent mineralized tissue resembling acellular cementum; recombinations in which RBM was excluded were characterized by formation of mineralized tissue which did not adhere to the root dentin surface. These results suggest that formation of an adherent mineralized tissue on the developing root dentin surface is dependent upon the presence of indigenous basement membrane components.

Animals↗

Recent developments of the chemistry development kit (CDK) - an open-source java library for chemo- and bioinformatics.

The Chemistry Development Kit (CDK) provides methods for common tasks in molecular informatics, including 2D and 3D rendering of chemical structures, I/O routines, SMILES parsing and generation, ring searches, isomorphism checking, structure diagram generation, etc. Implemented in Java, it is used both for server-side computational services, possibly equipped with a web interface, as well as for applications and client-side applets. This article introduces the CDK's new QSAR capabilities and the recently introduced interface to statistical software.

Computational Biology↗

Histochemical studies of muscle development in decapitated and hypophysectomized pig fetuses: blood vessel development.

In Exp. 1, fetuses were decapitated in utero on d 45 of gestation and examined at 110 d of gestation; in Exp. 2, fetuses were hypophysectomized (hypox) in utero with a cauterizing needle at d 70 of gestation and examined at 110 d of gestation. Semitendinosus muscles were sampled and transverse cryostat sections were cut from the medial portion of the muscle. Quantitative analysis of lectin-stained sections showed that capillary:fiber ratios (C/F) were lower (P less than .01) for sections from decapitated and hypox fetuses than for sections from control fetuses. The superficial and deep aspects of muscle sections from control fetuses had markedly different C/F ratios (deep region, 24.1 +/- 2; superficial region, 11.2 +/- .5). Fetal decapitation and hypophysectomy abolished differences in C/F ratios between deep and superficial regions of muscle sections (hypox fetuses; deep region, 6.9 +/- 1; superficial region, 6.4 +/- .6). Capillaries in sections from control fetuses were reactive for several enzymes, whereas capillaries in sections from experimental fetuses (decapitated and hypox) were not reactive for these enzymes. The number of small arterioles was reduced in sections from experimental fetuses (decapitated and hypox) compared with sections from control fetuses. These studies demonstrate that lowering the levels of pituitary hormones in the fetus retards the development of blood vessels in muscle tissue both quantitatively and qualitatively.

Animals↗

Development of extender and techniques for frozen turkey semen. 1. Development.

An extender for turkey semen with a frozen-thawed recovery of greater than or equal to 50% motile spermatozoa and a vigorous swirl was developed. The effect of glucose, a comparison of 10 different sugars in the extender, the use of sodium acetate and potassium acetate, the ratio of dimethylsulfoxide to ethylene glycol, the percent total cryoprotectant, equilibration time, and osmotic pressure were all tested. Replacement of approximately half of the extender with an iso-osmolar glucose solution yielded higher percentages of motile spermatozoa with both fresh and frozen-thawed semen samples. Replacement of glucose with other carbohydrates did not enhance recovery of frozen-thawed semen. The proportion of sodium and potassium acetates to TESNaK2 in the extender had no effect on motility. Approximately equal proportions of ethylene glycol to dimethylsulfoxide, with a final concentration of 11.2% after dilution, produced at or near optimal recovery of spermatozoal motility after freezing. Twenty to 90 min equilibration times before freezing yielded significantly higher recovery of motile sperm postthaw than shorter periods. A wide range of extender osmotic pressures were compatible with recovery of spermatozoal motility postthaw. The final extender consisted of 4.70 g sodium acetate, 3.39 g potassium acetate 9.23 g TES, .353 g sodium hydroxide, .492 g potassium hydroxide, 32.00 g glucose, H2O q.s. 1000 ml, 370 mOsm/kg, pH 7.2. Ethylene glycol and dimethylsulfoxide were added (1:1 ratio) for a final concentration of 11.2% cryoprotectant after dilution. Semen was held undiluted for 6 min, extended 1:4 at 22 C, and equilibrated for 30 min at 0 C before pellet freezing.

Animals↗

Ultrasonography as a tool for monitoring in ovo chicken development. 2. Effects of eggshell alteration and ultrasonography on embryonic and posthatch development.

The influence of creating and closing acoustic windows on embryonic and posthatch development for the purpose of chicken embryogenesis monitoring by real-time ultrasonography (US) was evaluated at 2, 6, 9, 14, and 17 days of incubation. Acoustic windows were closed using either a porous dialysis film and tape (FM) or an eggshell allograft attached with collodion (CP). Results from eggs closed in each manner with and without concurrent nonsterile US examination were compared with two control groups. Window creation reduced hatchability. The hatchability reduction was caused primarily by bacterial contamination. Contamination was more common in the FM eggs than in the CP eggs and was greater in eggs that also underwent US. Hatchability increased and contamination decreased when US was performed closer to hatch. Egg weight loss was increased after Day 6 of incubation in FM + US eggs treated on Day 2 and after Day 9 in all eggs with windows except CP eggs treated on Day 9 and CP + US eggs treated on Days 6 and 9. Hatch weight decreased in chicks from eggs that had windows, particularly in FM eggs.

Animals↗