Kinetics of Regional Blood-Brain Barrier Glucose Transport and Cerebral Blood Flow Determined with the Carotid Injection Technique in Conscious Rats

Anesthetics, particularly barbiturates, have depressive effects on cerebral blood flow and metabolism and likely have similar effects on blood-brain barrier (BBB) transport. In previous studies utilizing the carotid injection technique, it was necessary to anesthetize the animals prior to performing the experiment. The carotid injection technique was modified by catheter implantation in the external carotid artery at the bifurcation of the common carotid artery. The technique was used to determine cerebral blood flow, the Km, Vmax, and KD of glucose transport in hippocampus, caudate, cortex, and thalamus-hypothalamus in conscious rats. Blood flow increased two to three times from that seen in the anesthetized rat. The Km in the four regions ranged between 6.5 and 9.2 mM, the Vmax ranged between 1.15 and 2.07 mumol/min/g, and the KD ranged between 0.015 and 0.035 ml/min/g. The Km and KD in the conscious rat did not differ from the values seen in the barbiturate anesthetized rat. The Vmax, on the other hand, increased two- to three-fold from that seen in the anesthetized rat and was nearly proportional to the increase in blood flow seen in the conscious rat. The development of the external carotid catheter technique now allows for determination of BBB substrate transport in conscious animals.     

Covalent coupling of 4-thiouridine in the initiator methionine tRNA to specific lysine residues in Escherichia coli methionyl-tRNA synthetase

A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNAfMet). Incubation of the affinity-labeling tRNAfMet derivative with E. coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme. A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated. Cross-linking was effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAPhe. The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography. The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography. Two major peptide products were isolated plus several minor peptides. N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS. Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins.     

Male and female genotype mediate pheromonal regulation of the mouse estrous cycle

Estrous cyclicity was studied to examine the possibility that strain differences in the regularity of the mouse estrous cycle are the result of different olfactory signals produced by the male. Females with regular estrous cycles (lines E and S1) were housed in the olfactory presence of males from a line with irregular cycles (line CN-) or in the presence of males of their own line (used as a control). Females with irregular cycles (line CN-) were housed in the presence of males from a line with regular cycles (line E) or were exposed to males of their own line. The regularity of the estrous cycle decreased in line E females (regular cycles) when exposed to line CN- males (irregular cycles). The decreased regularity of line E cyclicity resulted from an increased period of diestrus, i.e., lengthening of the cycle. In contrast, line S1 females (regular cycles) did not show any change in estrous cyclicity when exposed to line CN- males. The period of diestrus increased in line CN- females when they were exposed to line E males. These results provide evidence that 1) the genotype of the male can influence the regularity of the estrous cycle, and 2) the genotype of the female regulates her responsiveness to environmental factors (e.g., male odor).     

Peptides at the tRNA binding site of the crystallizable monomeric form of E. coli methionyl-tRNA synthetase.

A protein affinity labeling derivative of E. coli tRNA(fMet) carrying lysine-reactive cross-linking groups has been covalently coupled to monomeric trypsin-modified E. coli methionyl-tRNA synthetase. The cross-linked tRNA-synthetase complex has been isolated by gel filtration, digested with trypsin, and the tRNA-bound peptides separated from the bulk of the free tryptic peptides by anion exchange chromatography. The bound peptides were released from the tRNA by cleavage of the disulfide bond of the cross-linker and purified by reverse-phase high-pressure liquid chromatography, yielding three major peptides. These peptides were found to cochromatograph with three peptides of known sequence previously cross-linked to native methionyl-tRNA synthetase through lysine residues 402, 439 and 465. These results show that identical lysine residues are in close proximity to tRNA(fMet) bound to native dimeric methionyl-tRNA synthetase and to the crystallizable monomeric form of the enzyme, and indicate that cross-linking to the dimeric protein occurs on the occupied subunit of the 1:1 tRNA-synthetase complex.     

Anomalous origin of the left coronary artery from the pulmonary artery: Scintigraphic diagnosis with Tc 99m lung scan

A case of anomalous origin of the left coronary artery from the pulmonary artery is reported. The diagnosis was made on the basis of characteristic Tc 99m lung scan findings and was confirmed by subsequent angiocardiography and surgery.     

Variation in S phase in synchronous human cell lines

Summary Growth parameters of diploid and trisomic human fibroblasts were determined. The rate of growth of both classes of cells was examined in asynchronous cultures, and diploid and trisomic cells had similar growth rates. Synchronous cultures were developed using simple mitotic selection. The patterns and length of the DNA synthetic period (S phase) were found to be altered in trisomy 21 cells when compared to diploid human or to heteroploid HeLa cells. Early S-phase synthesis was absent or reduced and the overall length of the S phase was extended. However, the trisomic cells have apparently normal rates of DNA chain elongation and normal replicon sizes. This work was supported by the United States Department of Energy and the National Institutes of Health.     

Development of floating rafts after the rewetting of cut-over bogs: the importance of peat quality

The usual method of restoring cut-over bogs is to rewet the peat surface, but this often leads to the remaining peat layers being deeply inundated. For Sphagnum-dominated vegetation to develop at deeply inundated locations, it is important for floating rafts of buoyant residual peat to develop. In this study, the chemical and physical characteristics of buoyant and inundated peat collected from rewetted cut-over bog were compared. In general, buoyant peat was poorly humified; high methane (CH₄) production rates (2 µmol g ^–1 DW day ^–1) were important to ensure buoyancy. Although the peat water CH₄ concentrations increased with depth, the CH₄ production rates were higher in the uppermost peat layers. High CH₄ production rates were related positively with P concentrations and negatively with lignin concentrations. The pH to bulk density ratio (0.05) also appeared to be a good indicator of CH₄ production rates, providing an easy and cheap way to measure the variable for restoration practitioners. Our results indicated that analysing certain simple characteristics of the residual peat can greatly improve the success of the rewetting measures taken in cut-over bogs. If the analysis reveals that the residual peat is unsuitable for floating raft formation, deep inundation is inappropriate unless suitable peat from other locations can be introduced.     

Patterns of variability at the major histocompatibility class II alpha locus in Atlantic salmon contrast with those at the class I locus

In order to investigate the mechanisms creating and maintaining variability at the major histocompatibility (MH) class II alpha (DAA) locus we examined patterns of polymorphism in two isolated Atlantic salmon populations which share a common post-glacial origin. As expected from their common origin, but contrary to the observation at the MH class I locus, these populations shared the majority of DAA alleles: out of 17 sequences observed, 11 were common to both populations. Recombination seems to play a more important role in the origin of new alleles at the class II alpha locus than at the class I locus. A greater than expected proportion of sites inferred to be positively selected (potentially peptide binding residues, PBRs) were found to be involved in recombination events, suggesting a mechanism for increasing MH variability through an interaction between recombination and natural selection. Thus it appears that although selection and recombination are important mechanisms for the evolution of both class II alpha and class I loci in the Atlantic salmon, the pattern of variability differs markedly between these classes of MH loci.     

UV inactivation of Cryptosporidium hominis as measured in cell culture

The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.