Measurement of leptin in dried blood spot samples.

Leptin is important in a wide range of physiological processes, but logistical constraints associated with venipuncture blood collection have limited research on leptin in diverse, community-based settings. The aim of this short report is to present and validate an enzyme immunoassay method for quantifying leptin in samples of capillary whole blood collected from a simple finger prick and dried on filter paper. The method was evaluated through analysis of precision, reliability, stability, and comparisons with matched plasma and blood spot samples. We report acceptable levels of assay precision and reliability, and good agreement between results obtained from matched plasma and blood spot samples (r = 0.976, P < 0.001). Leptin concentrations begin to deteriorate after only 3 days at room temperature. Thus, care should be taken to refrigerate or freeze samples promptly. The relative ease of blood spot sample collection may facilitate research on leptin in a wider range of cultural and ecological settings.     

Diabetes in female rats; changes in liver microsomal aminopyrine N-demethylase and UDP-glucuronyl transferase activities

Short or long term diabetes in female rats produced remarkable activation of aminopyrine N-demethylation, inhibition of oestrone and p-nitrophenol glucuronidation and no changes in morphine UDP-glucuronyltransferase activity in vitro. Km and Vmax for these reactions were determined. Insulin treatment partially antagonized diabetes activation of aminopyrine N-demethylation: it restored decreased UDP-glucuronyltransferase activities for oestrone and p-nitrophenol only in long term and short term diabetes, respectively. Insulin also markedly inhibited morphine glucuronidation. Triton X-100 also displayed a differential pattern of activation for the glucuronidation reactions in liver microsomes of diabetic rats. Results suggest that diabetes in female rats may increase the actual amount of enzyme protein for aminopyrine metabolism and to decrease that for oestrone and p-nitrophenol.     

Abnormal neuromuscular junctions in the lateral rectus muscle of wobbler mice

We have studied the lateral rectus muscles and neuromuscular junctions (NMJs) of abducens motoneurons in wobbler (wr/wr) mutant mice from 26 to 58 days of age. The muscles of wr/wr weighed about 70% of the weight of littermate controls and were composed of fiber types comparable to those of controls, as assayed by succinate dehydrogenase activity. The most obvious difference between wr/wr and control NMJs was a reduction in the length of the postjunctional membrane of wr/wr mice. The mutant muscle endplate membrane was only about 70% (6.58 micron) the length of control muscle regions (9.44 micron). There were no obvious differences at the light microscopic level in the distribution of acetylcholine (ACh) receptors at junctional regions or staining of acetylcholinesterase, as assayed with alpha-bungarotoxin binding or enzyme histochemistry. Indirect immunocytochemical studies using antibodies directed against the subunits of the ACh receptor failed to indicate an abnormal presence of immature receptors clustered at the NMJs of wr/wr mice. Our findings suggest that the formation or maintenance of normal postjunctional folds and the differentiation of receptors at the junctions are under independent control during development. Furthermore, the wobbler mutation may affect muscle cell differentiation as well as neuronal differentiation. This mutant mouse should prove a useful model for study of postjunctional fold formation and function.     

Quality control of perfusion: monitoring venous blood oxygen tension to prevent hypoxic acidosis

The long-held belief that venous oxygen tension mirrored tissue oxygen tension became suspect in the 1960s when new instrumentation consistently showed that tissue oxygen tension was 10 to 30 torr less than venous oxygen tension. Moreover, a countercurrent of oxygen exchange between terminal arteries and veins was shown to exist. Despite this conflict in scientific theory, however, monitoring venous oxygen tension as a means to control hypothermic cardiopulmonary bypass has been repeatedly urged, since myocardial acidosis is clearly extremely detrimental. This study of the relationship between venous oxygen tension during hypothermic bypass and a concurrent increment in lactacidemia yields strong objective evidence to support the use of on-line venous oxygen tension monitoring to guide perfusion. In a random series of 36 patients, venous blood samples were drawn at five preselected intervals during operation and were analyzed for pH, carbon dioxide tension, oxygen tension, lactic acid, hematocrit, and base excess. Analysis of the data revealed that venous pH and base excess showed no correlation to venous oxygen tension. However, lactic acid showed a strong correlation with venous oxygen tension, with a correlation coefficient of 0.4338 at a confidence level of p less than 0.0001. If the patients were divided into three clinically pertinent groups based on the lowest venous oxygen tension recorded, a strong relationship between venous oxygen tension and lactic acid emerged. If the lowest measurement of venous oxygen tension was greater than 35 mm Hg (group A), the mean rise in lactic acid was only 0.12 microns/ml. If the lowest measurement was between 30 and 34 mm Hg (group B), the mean rise was 0.64 microns/ml. Whereas, if any venous oxygen tension value fell below 30 mm Hg (group C), the mean rise in lactic acid was 2.56 microns/ml. Analysis of variance showed that group C values were significantly different from groups A and B values (p less than 0.0002). A scientific hypothesis relating venous oxygen tension to adequate tissue oxygenation is proposed. Use of venous oxygen tension monitoring with the goal to maintain the level above 35 mm Hg is strongly supported by this study.     

Development of specificity in corticospinal connections by axon collaterals branching selectively into appropriate spinal targets

Corticospinal projections in adult rodents arise exlusively from layer V neurons in the sensorimotor cortex. These neurons are topographically organized in their connections to spinal cord targets. Previous studies in rodents have shown that the mature distribution pattern of corticospinal neurons develops during the first 2 weeks postnatal from an initial widespread pattern that includes the visual cortex to a distribution restricted to the sensorimotor cortex. To determine whether specificity in corticospinal connections also emerges from an intially diffuse set of projections, we have studied the outgrowth of corticospinal axons and the formation of terminal arbors in developing hamsters. The sensitive fluorescent tracer 1, 1′, dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchlorat (DiI) was used to label corticospinal axons from the visual cortex or from small regions of the forelimb or hindlimb sensorimotor cortex in living animals at 4–17 days postnatal. Initially axon outgrowth was imprecise. Some visual cortical axons extended transiently beyond their permanent targets in the pontine nuclei, by growing through the pyramidal decussation and in some cases extending as far caudally as the lumbar enlargement. Forelimb sensorimotor axons also extended past their targets in the cervical enlargement, in many cases growing in the corticospinal tract to lumbar levels of the cord. By about 17 days postnatal these misdirected axons or axon segments were withdrawn from the tract. Despite these errors in axon trajectories within the corticospinal tract, terminal arbors branching into targets in the spinal gray matter were topographically appropriate from the earliest stages of innervation. Thus visula cortical axons never formed connections in the spinal cord, forelimb sensorimotor axons arborized only in the cervical enlargement, and hindlimb cortical axons terminated only in the lumbar cord at all stages of development examined. Corticospinal arbors formed from collaterals that extended at right angles from the shafts of primary axons, most likely by the process of interstitial branching after the primary growth cone had extended past the target. Once collaterals extended into the spinal gray matter, highly branched terminal arbors formed within 2–4 days, beginning at about 4 and 8 days postnatal for the cervical and lumbar enlargements, respectively. These results show that specificity in connectivity is achieved by selectivty growth of axon collaterals in to appropriate spinal targets from the beginning and not by the later remodeling of intially diffuse connections. In contrast, errors occur in the initial outgrowth of axons in the corticospinal tract, which are subsequently corrected. Copyright © 1994 Wiley-Liss, Inc.     

Gastrinomas demonstrate amplification of the HER-2/neu proto-oncogene.

OBJECTIVE: This study determined whether genomic amplification of HER-2/neu or mutations of the p53 and ras genes were present in gastrinomas. SUMMARY BACKGROUND DATA: Amplification of HER-2/neu, a proto-oncogene related to the epidermal growth factor receptor, and mutation of the ras proto-oncogene and p53 tumor suppressor gene appear to play a role in the pathogenesis of some human cancers. Little is known about possible molecular alterations in gastrinomas, tumors that may be particularly virulent because of gastrin overproduction, resulting in the severe ulcer diathesis, the Zollinger-Ellison syndrome. METHODS: The differential polymerase chain reaction (PCR) procedure was used to detect amplification of the HER-2/neu gene in DNA samples from the novel human gastrinoma cell line (PT) and from paraffin-embedded samples of gastrinomas. Sequencing techniques were used to determine whether mutations of the p53 or ras (Ha-ras, N-ras, Ki-ras) genes were present. RESULTS: Amplification (> twofold) occurred in all gastrinoma tumor samples. Compared with normal pancreas or ileum, a 4- to 12-fold amplification of HER-2/neu was found in 3 gastrinomas, 3 to 3.3-fold in four samples and 2.1- to 2.4-fold in the remaining five tumors. A heterozygous point mutation in the p53 gene (codon 273) was found in a single sample; none of the gastrinomas contained a mutation of the ras genes. CONCLUSIONS: Amplification of the HER-2/neu gene, but not alterations of either p53 or ras, may be involved in the pathogenesis of gastrinomas. The unique PT cell line will be a useful model to further elucidate the molecular mechanisms that contribute to gastrinoma formation and growth.     

Pilomatrix carcinoma. A new case

We present a new case of Pilomatrix Carcinoma after having review the 22 cases previously published. The tumor arisen in a 74 years old male, in the left preauricular region. The lesion was excised with wide margins. A year after there is not any evidence of recurrence or metastases.     

Simulated implant surgery in rabbit long bones: a descriptive study.

The objectives of this study were: (a) to observe and describe the variability of bone healing in implant receptor sites which were prepared in rabbit femurs by use of different surgical methods; and (b) to determine if the animal model which was used was suitable for the detection of differences in healing reactions in implant receptor sites which were prepared by different surgical methods. Three 3-mm-wide implant receptor sites were prepared in the right and left femurs of four large New Zealand white rabbits. The surgical parameters used in preparation of the different sites included: low speed with no irrigation (LSO); low speed with internal irrigation only (LSI); low speed with external irrigation only (LSE); high speed with no irrigation (HSO); or high speed with external irrigation only (HSE). The sites were randomized so that each animal had one of each type of site in either the right or left femur. A non-treated control site was located in each animal for comparison with experimental sites. The animals were killed at 7, 14, 21, and 28 days post-operatively. The resultant samples were fixed, embedded, sectioned, and stained with basic fuchsin and toluidine blue. The results indicated that this was probably not a suitable animal model, since no discernible differences were detected in the various healed sites.