Demonstration of the Microvasculature of the Spinal Cord by Intravenous Injection of the Fluorescent Dye, Thioflavine S

In the past, thioflavine S has been used for visualizing blood vessels and patterns of blood flow (Schlegel 1949; Schlegel and Moses 1950; Oliver et al. 1951). Methods employed have involved an intravenous injection of the dye, immersion of hand-cut sections in glycerol and examination of sections under incident Wood's light. With improved techniques it is possible to obtain microtome-cut sections and to use a more intense light source for enhancing fluorescence and resulting visualization of small vessels. Occlusion of arterioles by undissolved dye particles is prevented by ultracentrifugation of the solution to be injected.     

Comparison of the effect of semisynthetic human insulin and porcine insulin on glucose kinetics, plasma free fatty acid and amino acid levels in man

The effect of semisynthetic human insulin on hepatic glucose output, peripheral glucose clearance, plasma levels of C-Peptide, free fatty acids and amino acids was compared with purified pork insulin using the glucose clamp technique. 8 normal overnight-fasted subjects received intravenous infusions of either human or porcine insulin at 20 mU/m2.min(-1) during 120 min achieving plasma insulin levels of approximately equal to 50 mU/l. Plasma glucose levels were maintained at euglycaemia by variable rates of glucose infusion. Hepatic glucose production measured by continuous infusion of 3-(3) H-glucose was similarly suppressed by both insulins to rates near zero. The metabolic clearance rate of glucose increased during infusion of human insulin by 120%, C-peptide levels decreased by 41% and plasma FFA concentrations fell by 74%. The respective changes during infusion of pork insulin were similar, 118%, 48% and 72%. Both insulins decreased the plasma levels of branched-chain amino acids, tyrosine, phenylalanine, methionine, serine and histidine similarly. Thus, the results demonstrate that semisynthetic human and porcine insulin are aequipotent with respect to suppression of hepatic glucose output, stimulation of glucose clearance, inhibition of insulin secretion, lipolysis and proteolysis.     

Determination of the amino acid change responsible for the nontoxic, cross-reactive exotoxin A protein (CRM 66) of Pseudomonas aeruginosa PAO-PR1.

Analysis of purified exotoxin A from parental Pseudomonas aeruginosa PAO1 and mutant strain PAO-PR1, which produces enzymatically inactive exotoxin A (CRM 66), revealed that CRM 66 lost 90% of parental enzymatic activity. Nucleotide sequence analysis of cloned exotoxin A genes showed a single amino acid substitution in CRM 66. Position 426 in the mature protein of parental (PAO1) exotoxin A is histidine, whereas in CRM 66, it is tyrosine.     

Identification of regB, a gene required for optimal exotoxin A yields in Pseudomonas aeruginosa

The yield of exotoxin A from Pseudomonas aeruginosa has been shown to be strain-dependent. Exotoxin A production requires the presence of the positive regulatory gene, regA. We cloned the regA genetic locus from the prototypical P. aeruginosa strain PAO1 and examined its ability to influence exotoxin A yields compared to the same region cloned from the hypertoxin-producing strain, PA103. The P. aeruginosa regA mutant strain, PA103-29, containing the PAO1 regA locus in trans produced approximately five to seven times less extracellular exotoxin A than PA103-29 containing the regA locus cloned from the hypertoxigenic strain, PA103. Nucleotide sequence analysis of the PAO1 regA locus revealed several differences, the most striking of which was the absence of a second open reading frame that was present in the analogous PA103 DNA. In addition, an amino acid substitution was found at position 144 of RegA (Thr in PAO1 and Ala in PA103). Recombinant molecules were constructed to test the contribution of each of these changes in nucleotide sequence on extracellular exotoxin A yields. The amino acid substitution in the PAO1 RegA protein was found not to affect overall exotoxin A yields. In contrast, the presence of the second open reading frame immediately downstream of the PA103 regA gene was found to influence extracellular exotoxin A yields. This open reading frame encodes a gene which we call regB. Nucleotide sequence analysis indicates that regB is 228 nucleotides in length and encodes a protein of 7527 Daltons. Our data suggest that regB is required for optimal exotoxin A production and its absence in strain PAO1 partially accounts for the difference in yield of extracellular exotoxin A between P. aeruginosa strains PAO1 and PA103.     

Use of a FACS III for fluorescence depolarization with DPH

We have previously demonstrated age-related differences in human lymphocyte membrane fluidity, by use of steady-state polarization measurements on bulk cell suspensions with the fluorescence probe DPH. However, for exact analysis of the possible functional importance of these changes, single-cell measurements were deemed of interest. We have now used an analog division device to measure fluorescence depolarization "p" of DPH in real time with a FACS III flow cytometer. The measurements are reliable, as we have been able to confirm the differences in DPH "p" between monocytes and lymphocytes previously shown in bulk suspension and to demonstrate the expected differences in fluidity of lipid-modulated cells. We also found significant differences in DPH "p" between lymphocytes of young and elderly blood donors. Lymphocyte subsets did not differ in polarization values but did differ in fluorescence intensity with Th less than Ts less than B = NK cells.     

Intragenic TaqI restriction fragment length polymorphism (RFLP) in CICN4, between the loci for X linked ocular albinism (OA1) and microphthalmia with linear skin defects syndrome (MLS)

A TaqI RFLP was detected within the C1CN4 gene, which lies between the loci for OA1 and MLS. There were no observed recombinations between this RFLP and the OA1 mutation in three informative families. Thus, the marker will be useful for genetic counseling in OA1.     

Localization and identification of the compound causing peak ‘X’ in the 31P-NMR spectrum of Saccharomyces cerevisiae

The present study is aimed at identifying the unidentified compound which gives rise to the so-called resonance ‘X’ in the ³¹P-NMR spectra of yeast cells. In addition, it is attempted to determine the localization of X (inside or outside the cell). Enzymic removal of the cell wall causes resonance ‘X’ to disappear in the spectra of the cells. This observation indicates an extracellular localization of X. The ³¹P-NMR spectrum of the phosphomannan extracted from the yeast shows a single resonance at exactly the same position as that of resonance ‘X’. Extraction of the phosphomannan from delipidized cells causes resonance ‘X’ to disappear from the ³¹P-NMR spectrum of the cells. The intensity of resonance ‘X’ in the spectrum of the intact cells can be almost quantitatively attributed to the amount of phosphomannan present in the yeast. The present results demonstrate that the resonance ‘X’ in the ³¹P-NMR spectrum of yeast cells is caused by phosphomannan in the cell wall.     

Potentiation of dietary restriction-induced lifespan extension by polyphenols

Dietary restriction (DR) extends lifespan across multiple species including mouse. Antioxidant plant extracts rich in polyphenols have also been shown to increase lifespan. We hypothesized that polyphenols might potentiate DR-induced lifespan extension. Twenty week old C57BL/6 mice were placed on one of three diets: continuous feeding (control), alternate day chow (Intermittent fed, IF), or IF supplemented with polyphenol antioxidants (PAO) from blueberry, pomegranate, and green tea extracts (IF + PAO). Both IF and IF + PAO groups outlived the control group and the IF + PAO group outlived the IF group (all p < 0.001). In the brain, IF induced the expression of inflammatory genes and p38 MAPK phosphorylation, while the addition of PAO reduced brain inflammatory gene expression and p38 MAPK phosphorylation. Our data indicate that while IF overall promotes longevity, some aspects of IF-induced stress may paradoxically lessen this effect. Polyphenol compounds, in turn, may potentiate IF-induced longevity by minimizing specific components of IF-induced cell stress.