Metabolic balance studies in premature infants

Many investigators over the past three decades have successfully conducted traditional metabolic balance studies in efforts to determine the retention rates of key nutrients important to the optimal growth of preterm infants. Differences in methodologies discussed in this review may explain the inconsistent results of balance studies reported for some nutrients, particularly calcium. These methodologic differences include (1) variability in nutrient intake and nutritional course prior to the balance period, (2) differences in the method of stool collection (with and without markers), (3) use of single versus repeated balance periods, and (4) different durations of balance periods. The data presented here suggest that the variability of net fat absorptions among VLBW infants was decreased when an acclimation period of nutrient intake supportive of growth was provided prior to a metabolic balance study. In addition, the use of markers affected the estimates of net calcium absorption but not fat absorption. Additional factors that may influence net calcium absorption and methods for the estimation of calcium absorption in VLBW infants warrant further investigation. This review describes the methods of specimen collection for metabolic balance studies in VLBW preterm infants that demonstrated reproducible data. The recent application of stable isotope methodology to metabolic balance studies can be extremely advantageous in identifying the rates of nutrient absorption versus endogenous secretion in the GI tract.     

BCL-2 overexpression attenuates cortical cell loss after traumatic brain injury in transgenic mice

The proto-oncogene, BCL-2, has been suggested to participate in cell survival during development of, and after injury to, the CNS. Transgenic (TG) mice overexpressing human Bcl-2 (n = 21) and their wild-type (WT) littermates (n = 18) were subjected to lateral controlled cortical impact brain injury. Lateral controlled cortical impact brain injury resulted in the formation of a contusion in the injured cortex at 2 days, which developed into a well-defined cavity by 7 days in both WT and TG mice. At 7 days after injury, brain-injured TG mice had a significantly reduced cortical lesion (volume = 1.99 mm3) compared with that of the injured WT mice (volume = 5.1 mm3, P < 0.01). In contrast, overexpression of BCL-2 did not affect the extent of hippocampal cell death after lateral controlled cortical impact brain injury. Analysis of motor function revealed that both brain-injured WT and TG mice exhibited significant right-sided deficits at 2 and 7 days after injury (P < 0.05 compared with the uninjured controls). Although composite neuroscores (sum of scores from forelimb and hind limb flexion, lateral pulsion, and inclined plane tests) were not different between WT and TG brain-injured mice, TG mice had a slightly but significantly reduced deficit in the inclined plane test (P < 0.05 compared to the WT mice). These data suggest that the cell death regulatory gene, BCL-2, may play a protective role in the pathophysiology of traumatic brain injury.     

Immunological regulation of Angiostrongylus cantonensis infections in rats: modulation of population density and enhanced parasite growth following one or two superimposed infections

Rats acquired a degree of protective immunity to reinfection with Angiostrongylus cantonensis after a single infection with 50 infective larvae. Infected rats resisted the establishment of most challenging larvae and protective immunity increased with subsequent reinfections. Part of the primary infection was lost after a superimposed second and also following a superimposed third infection, but the total size of the concurrent adult worm populations remained the same as that from a primary infection. Worms surviving from the primary infection showed enhanced growth after each reinfection but their fecundity was impaired.     

Human MDR 1 protein overexpression delays the apoptotic cascade in Chinese hamster ovary fibroblasts

Several laboratories have reported that overexpression of the multidrug resistance (MDR) protein is associated with intracellular alkalinization, and several investigators have reported that cells induced to undergo programmed cell death (apoptosis) acidify quite significantly. Because it is difficult to fully explain the resistance to apoptosis-inducing chemotherapeutic drugs that is exhibited by MDR tumor cells solely via altered drug transport alone [Hoffman et al. (1996) J. Gen. Physiol. 108, 295-313], we have investigated whether overexpression of the hu MDR 1 protein alters progression of the apoptotic cascade. LR73 fibroblasts induced to undergo apoptosis either via treatment with the chemotherapeutic drug colchicine or by serum withdrawal exhibit cellular volume changes, intracellular acidification, nuclear condensation, and chromosomal digestion ("ladder formation"), characteristic of apoptosis, in a temporally well-defined pattern. However, multidrug resistant LR73/20E or LR73/27 hu MDR 1 transfectants recently created in our laboratory without selection on chemotherapeutic drug are significantly delayed in the onset of apoptosis as defined by the above criteria, regardless of whether apoptosis is induced by colchicine treatment or by serum withdrawal. Thus, the delay cannot simply be due to the well-known ability of MDR protein overexpression to lower chemotherapeutic drug accumulation in MDR cells. LR73/27V500 "selectants", exhibiting similar levels of MDR protein overexpression but higher multidrug resistance due to selection with the chemotherapeutic drug vincristine, exhibit a slightly longer delay in the progression of apoptosis. Normal apoptotic cascade kinetics are partially restored by pre-treatment of the MDR cells with the MDR protein inhibitor verapamil. Untransfected LR73 cells not expressing MDR protein but elevated in pHi via manipulation of CO2/HCO3- as described [Hoffman et al. (1996) J. Gen. Physiol. 108, 295-313] are inhibited in DNA ladder formation, similar to LR73/hu MDR 1 transfectants. These results uncover an additional mechanism whereby MDR protein overexpression may promote the survival of tumor cells and further support the notion that in some systems intracellular acidification may be either causal or permissive for proper progression of the apoptotic cascade.     

Identification and localization of an immunoreactive AMPA-type glutamate receptor subunit (GluR4) with respect to identified photoreceptor synapses in the outer plexiform layer of goldfish retina

L-glutamate, the main excitatory synaptic transmitter in the retina, is released from photoreceptors and evokes responses in second-order retinal neurons (horizontal, bipolar cells) which utilize both ionotropic and metabotropic types of glutamate receptors. In the present study, to elucidate the functional roles of glutamate receptors in synaptic transmission, we have identified a specific ionotropic receptor subunit (GluR4) and determined its localization with respect to photoreceptor cells in the outer plexiform layer of the goldfish retina by light and pre-embedding electron-microscopical immunocytochemistry. We screened antisera to mammalian AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)-preferring ionotropic glutamate receptors (GluR 1-4) of goldfish retina by light- and electron-microscopical immunocytochemistry. Only immunoreactive (IR) GluR4 was found in discrete clusters in the outer plexiform layer. The cones contacted in this manner were identified as long-wavelength ("red") and intermediate-wavelength ("green") cones, which were strongly immunoreactive to monoclonal antibody FRet 43 and antisera to goldfish red and green-cone opsins; and short-wavelength ("blue") cones, which were weakly immunoreactive to FRet 43 but strongly immunoreactive with antiserum to blue-cone opsin. Immunoblots of goldfish retinal homogenate with anti-GluR4 revealed a single protein at M(r) = 110 kDa. Preadsorption of GluR4 antiserum with either the immunizing rat peptide, or its goldfish homolog, reduced or abolished staining in retinal sections and blots. Therefore, we have detected and localized genuine goldfish GluR4 in the outer plexiform layer of the goldfish retina. We characterized contacts between photoreceptor cells and GluR4-IR second-order neurons in the electron microscope. IR-GluR4 was localized to invaginating central dendrites of triads in ribbon synapses of red cones, semi-invaginating dendrites in other cones and rods, and dendrites making wide-cleft basal junctions in rods and cones; the GluR4-IR structures are best identified as dendrites of OFF-bipolar cells. The results of our studies indicate that in goldfish retina GluR4-expressing neurons are postsynaptic to all types of photoreceptors and that transmission from photoreceptors to OFF-bipolars is mediated at least in part by AMPA-sensitive receptors containing GluR4 subunits.