Preventive management of hypoglycemia in very low-birthweight infants following indomethacin therapy for patent ductus arteriosus.

BACKGROUND: To evaluate the effects of an increase in glucose infusion rate of 2 mg/kg per min from the basal infusion rate on the prevention of hypoglycemia in very low-birthweight (VLBW) infants, following indomethacin therapy for patent ductus arteriosus (PDA). METHODS: Forty VLBW infants with PDA were given indomethacin 0.2 mg/kg intravenously up to three doses. In 15 of the 40 infants (supplemented group: between April 1995 and March 1996) the glucose infusion rate was increased in 2 mg/kg per min increments from the basal rate just before the initial indomethacin administration, compared with 25 historical control infants who received a fixed glucose infusion rate during the first 12 h after the initial dose. We evaluated the changes in blood glucose levels and glucose infusion rates in both groups. RESULTS: In the control group 11 of 25 (44%) infants had a blood glucose value below 40 mg/dL between 12 and 60 h (mean 32.7 h). In contrast only two out of 15 infants in the supplemented group reached the glucose level below 40 mg/dL between 72 and 96 h but both two were light-for-dates infants (defined as birthweight below the 10th percentile for gestational age on the standard intrauterine growth curve). Blood glucose values in the supplemented group were significantly higher than those in the control group between 12 and 96 h. However, glucose infusion rates were similar before and between 72 and 96 h. CONCLUSIONS: This retrospective study shows that an increase in glucose infusion rate of 2 mg/kg per min, in addition to the pre-existing stable maintenance glucose intake, might prevent against the occurrence of unexpected hypoglycemia in VLBW infants following indomethacin therapy.

Increased expression of cathepsins E and D in neurons of the aged rat brain and their colocalization with lipofuscin and carboxy-terminal fragments of Alzheimer amyloid precursor protein.

Age-related changes in the expression and localization of two distinct intracellular aspartic proteinases, cathepsin E (CE) and cathepsin D (CD), were investigated in the rat cerebral cortex and the brainstem by immunocytochemical and quantitative methods using discriminative antibodies specific for each enzyme. Nonlysosomal CE was barely detectable in these two brain tissues in the embryonic stages, whereas relatively high expression of lysosomal CD was observed in embryonic tissues. After birth, CE was increasingly expressed in these tissues with aging to attain maximal levels at 30 months of age. Western blot analyses revealed that CE existed predominantly as the mature enzyme at 2 and 17 months of age, whereas it was present as not only the mature enzyme but also the proenzyme at 30 months of age. On the other hand, CD was mainly present in the mature form throughout development, although its level in these tissues was also significantly increased with aging. The CE-positive cortical and brainstem neurons of the aged rat corresponded well with cells emitting autofluorescence for lipopigments. By the double-staining technique, most of the CE-positive cortical and brainstem neurons of the aged rat were also positive for antibody to the carboxyl-terminal fragments of amyloid precursor protein (APP634-695), intracellular accumulation of which is thought to be associated with age-related changes in the endosome/lysosome system. It is important that electron microscopy revealed that CE in brainstem neurons of the aged rat colocalized with CD in the lipofuscin-containing lysosomes. These results indicate that aging results in the increased expression and lysosomal localization of CE in cortical and brainstem neurons and changes in the endosomal/lysosomal proteolytic system, which may be related to lipofuscinogenesis and altered intracellular APP metabolism.

Detection of Staphylococci in mouse phagocytic cells by in situ hybridization using biotinylated DNA probes.

In situ hybridization was used to detect intracellular Staphylococcus aureus and S. epidermidis in mouse phagocytic cells after experimental infection of C3H mice with Staphylococci via abdominal or intravenous injection. Isolated ascites or whole blood were tested by the phagocyte smear technique, using bacteriolytic enzymes to preserve phagocytic cell morphology. The exposed bacterial DNA was visualized as intracellular hybridized signals by use of biotinylated DNA probes and by immunocytochemistry using streptavidin-alkaline phosphatase conjugates as detector molecules. These DNA probes, prepared from randomly cloned genomic DNA fragments of S. aureus and S. epidermidis, were strain-specific and did not cross-hybridize either in situ or on dot-blot hybridization. This technique of in situ hybridization with phagocyte smears is useful for detection and diagnosis of intracellular bacteria regardless of viability.

Binding of streptavidin to bacteria or fungi and its applications in detecting these microbes.

We have investigated the characteristics and utilities of streptavidin-binding to gram-negative and gram-positive bacteria and Candida spp. The pre-treatment of these microbes with chemical reagents such as CHCl3, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte-smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi-digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte-smears).

Cryogenic preservation of low-quality human semen.

The article describes the technical strategies for clinical cryogenic preservation of low-quality human semen. To compensate for sperm dilution resulting from the use of a cryogenic medium, ejaculated semen was concentrated before freezing by means of continuous-step density gradient centrifugation. Freezing was simplified by employing the carbon dioxide pellet method with KS-II cryogenic medium, which contains Pluronic F-68, a nonionic detergent, to solubilize egg yolk (a major cryogenic protectant). Semen of less than 50% motility (n = 23) was processed and then cryogenically preserved. Sperm concentration was increased by a factor of 1.8 +/- 0.96 (n = 23). Sperm motility was improved from 35.9% +/- 13.9% to 69.4% +/- 10.8%. Even after thawing 59.4% +/- 17.5% motility remained, with a mean survival rate of 85% +/- 14%. The concentration of sperm and improved sperm motility by the use of the continuous-step density gradient and the high survival rate ensured by the carbon dioxide pellet method with KS-II cryogenic medium compensated for the lowering of sperm quality during cryogenic preservation.

Efferent tectal cells of crucian carp: physiology and morphology.

Tectal cells of the crucian carp (Carassius ararssius) showing antidromic responses evoked by rhombencephalic electrical stimulation were physiologically studied and subsequently stained with Lucifer Yellow CH. The stained efferent tectal cells were fusiform, horizontal, and multipolar. The main axon of these efferent tectal cells descended along the wall of the deep tegmentum and could be traced to the motor area below the cerebellum. The axons gave off their collaterals in several brain areas: 1) descending collaterals in the torus semicircularis, dorso-lateral tegmental area and mesencephalic reticular formation and 2) an ascending collateral in the area between the hypothalamus and tegmentum. Fifty percent of the efferent cells were unresponsive to visual stimuli, but some of these cells were activated by visual or tactile stimulation in conjunction with rhombencephalic electrical stimulation. On the other hand, most of the visually active cells were On-transient and movement sensitive with habituation and some were bimodal.

An extensive projection of fish dorsolateral tegmental cells to the optic tectum revealed by intra-axonal dye marking.

The cells of the dorsolateral tegmental (DLT) nucleus in the crucian carp (Carassius carassius) were physiologically identified and marked with Lucifer dye. All the identified DLT cells receive both visual and rhombencephalic inputs. These cells project their axons into the contralateral tectum via the tectal commissure as well as into the ipsilateral tectum. The most striking characteristic of the DLT cells was the wide distribution of their axonal branching in the ipsilateral tectum.