Changes in the Microvasculature and Interstitium of Aged Human Mandibles and Maxillae1

The present study describes the age changes to the microvasculature and connective tissue interstitium of the osteons and periosteums of aged human mandibles and maxillae. The mandibles and maxillae obtained from 14 and 19 year old males, respectively, were also studied. In the nutrient canals of the aged osteons, the walls of the arterioles and venules stained intensely PAS positive, and alcian blue negative. The walls of the blood capillaries were thick and strongly PAS positive. There was a deposition of PAS positive material in the connective tissue stroma of the nutrient canals which progressed to the obliteration of the canal space. Many of the nutrient canals exhibited diffuse calcification within the connective tissue interstitium localized around the blood vessels. The lacunae and canaliculi of those osteons in which the nutrient canals were partially or completely obliterated were filled with PAS material. None of these histochemical changes were seen in the osteons of young individuals. The microvasculature of the aged periosteum showed similar changes. The periosteal tissue consisted of thick collagenous bundles and few osteogenic cells. There was a thin darkly stained amorphous calcified layer forming the bone surface.     

Morphometry of cat's pulmonary arterial tree

Morphometic data of the pulmonary artery in the cat's right lung are presented. Silicone elastomer casts of cat's right lung were made, and measured, counted and analyzed. The Strahler system is used to describe the branching pattern of the arterial vascular tree. These data are needed for any quantitative approach to the study of the pulmonary circulation. For all the pulmonary blood vessels of the cat lying between the main pulmonary artery and the capillary beds, there are a total of 10 orders of vessels in the right upper lobe, 9 orders of vessels in the right middle lobe and 11 orders of vessels in the right lower lobe. The ratio of the number of branches in successive orders of vessels or the branching ratio, is 3.58. The corresponding average diameter ratio is 1.72, whereas the average length ratio is 1.81.     

Thirtyfold multiplex genotyping of the p53 gene using solid phase capturable dideoxynucleotides and mass spectrometry

A mass spectrometry (MS) based multiplex genotyping method using solid phase capturable (SPC) dideoxynucleotides and single base extension (SBE), named the SPC-SBE, has been developed for mutation detection. We report here the simultaneous genotyping of 30 potential point mutation sites in exons 5, 7, and 8 of the human p53 gene in one tube using the SPC-SBE method. The 30 mutation sites, including the most frequently mutated p53 codons, were chosen to explore the high multiplexing scope of the SPC-SBE method. Thirty primers specific to each potential mutation site were designed to yield SBE products with sufficient mass differences. This was achieved by tuning the mass of some primers using modified nucleotides. Genomic DNA was amplified by multiplex PCR to produce amplicons of the three p53 exons. The 30 primers were combined with the PCR products and biotinylated dideoxynucleotides for SBE to generate 3'-biotinylated extension DNA products. These products were then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction were washed away. The pure extension DNA products were subsequently released from the solid phase and analyzed with MS. We simultaneously genotyped 30 potential mutation sites in the p53 gene from Wilms' tumor, head and neck tumor, and colorectal tumor. Both homozygous and heterozygous genotypes were accurately determined with digital resolution. This is the highest level of multiplex genotyping reported thus far using MS, indicating that the approach might be applicable to screening a repertoire of genotypes in candidate genes as potential disease markers.     

Fine needle aspiration of gastrointestinal stromal tumors

OBJECTIVE: Gastrointestinal stromal tumors (GISTs) are uncommon mesenchymal tumors of the gastrointestinal tract. Fine needle aspiration (FNA) is one option for diagnosing GISTs before surgery. This study was designed to evaluate the clinical utility of FNA in the diagnosis of GISTs. STUDY DESIGN: FNAs from 19 GISTs originating in the stomach, small bowel and colon obtained from 1988 to 1998 were studied. Immunocytochemistry was performed on 12 cases. The GISTs were classified as benign, borderline and malignant, according to location, size, mitotic activity and clinical outcome. RESULTS: Benign (three) and borderline (five) GISTs were all spindle cell type; malignant GISTs included five spindle cell type and six epithelioid type. Most smears contained abundant cellular material. Benign and borderline GISTs of spindle cell type tended to have cells arranged in tightly cohesive clusters, while malignant GISTs were more likely to exhibit loosely cohesive groups with many single cells, occasional nuclear pleomorphism, hyperchromasia and irregular nuclear contours. Epithelioid-type GISTs mimicked adenocarcinoma. Mitoses were seldom observed in either type. CD117 (KIT protein product) was demonstrated by immunocytochemistry in 9 cases, CD34 in 11, desmin in 3, S-100 protein in 2 and smooth muscle actin in 6 cases. CONCLUSION: FNA can be used to diagnose GISTs as spindle cell and epithelioid types, but cytomorphology alone cannot be used to assess malignant potential. Immunocytochemical staining for CD117 is helpful in confirming the diagnosis. Care must be taken to differentiate epithelioid-type GISTs from adenocarcinoma.     

Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry

We report here the design, synthesis and evaluation of a novel photocleavable (PC) biotinylated nucleotide analog, dUTP-PC-Biotin, for DNA polymerase extension reaction to isolate DNA products for mass spectrometry (MS) analysis. This nucleotide analog has a biotin moiety attached to the 5-position of 2′-deoxyribouridine 5′-triphosphate via a photocleavable 2-nitrobenzyl linker. We have demonstrated that dUTP-PC-Biotin can be faithfully incorporated by the DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA polymerase extension reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. Therefore, the DNA extension fragments generated by using the dUTP-PC-Biotin can be efficiently isolated by a streptavidin-coated surface and recovered by near-UV light irradiation at room temperature in mild condition for further analysis without using any chemicals or heat. Single and multiple primer extension reactions were performed using the dUTP-PC-Biotin to generate DNA products for MALDI-TOF MS analysis. Such nucleotide analogs that carry a biotin and a photocleavable linker will allow the isolation and purification of DNA products under mild conditions for MS-based genetic analysis by DNA sequencing or multiplex single nucleotide polymorphism (SNP) detection. Furthermore, these nucleotide analogs should also be useful in isolating DNA–protein complexes under non-denaturing conditions.     

Collagen and elastin fibers in human pulmonary alveolar walls

The morphology and morphometric data of collagen and elastin fibers in the pulmonary alveolar walls are presented. Specimens were obtained from postmortem lungs quick-frozen at specified transpulmonary pressures. Collagen was stained by silver, and elastin was stained by orcein. Photomicrographs were composed by computer. Young lungs typically show small collagen fibers that radiate from the "posts," whereas larger fiber bundles traverse the septum irrespective of capillary blood vessels. In older lungs, rings of collagen around the posts appear enlarged. Elastin bundles do not show obvious variation in pattern with age and inflation pressure. Statistical frequency distributions of the fiber width and curvature are both skewed, but the square root of the width and the cube root of the curvature have approximate normal distributions. Typically, for young lungs at transpulmonary pressure of 4 cmH2O, the mean of (width)1/2 (in micron1/2) for collagen fibers is 0.952 +/- 0.242 (SD), that of (curvature)1/3 (in micron-1/3) is 0.349 +/- 0.094. The corresponding values for elastin are 0.986 +/- 0.255 and 0.395 +/- 0.094.     

Elasticity of arterioles and venules in postmortem human lungs

The elasticity and branching order of noncapillary microscopic blood vessels less than 100 microns diam were studied in human lungs obtained 7-30 h postmortem, using a silicone elastomer method that selectively filled pulmonary arterioles or venules. The lungs were inflated to 10 cmH2O pressure and a gradient of transmural vascular pressure of 0-17 cm H2O, from lobe base to apex, was established in the silicone-filled vascular system. Histological materials were obtained after airway fixation by formaldehyde solution and analyzed for vessel diameter in the branching order of 1, 2, and 3, with the smallest noncapillary vessel designated as order 1, in accord with the Strahler system. The change in vessel diameter within a branching order at different levels of transmural pressure is a derived measure of vascular elasticity expressed as compliance coefficient alpha, alpha Values are 0.128, 0.164, and 0.210 micron/cmH2O or 0.682, 0.472, and 0.354%/cmH2O, respectively, of orders 1-3 for arterioles and 0.187, 0.215, and 0.250 micron/cmH2O or 0.992, 0.612, and 0.424%/cmH2O, respectively, of orders 1-3 for venules. The percent is normalized with D0, which is the value of diameter (D) when the transmural pressure is zero. These data are compared with those for the cat where alpha = 0.274 for similar juxta-alveolar vessels.     

The identification, and optimisation of hERG selectivity, of a mixed NET/SERT re-uptake inhibitor for the treatment of pain

Hit compound 1, a selective noradrenaline re-uptake transporter (NET) inhibitor was optimised to build in potency at the serotonin re-uptake transporter (SERT) whilst maintaining selectivity against the dopamine re-uptake transporter (DAT). During the optimisation of 1 it became clear that selectivity against the Kv11.1 potassium ion channel (hERG) was also a parameter for optimisation within the series. Discrete structural changes to the molecule as well as a lowering of global cLogP successfully increased the hERG selectivity to afford compound 11m, which was efficacious in a mouse model of inflammatory pain, complete Freund’s adjuvant (CFA) induced thermal hyperalgesia and a rat model of neuropathic pain, spinal nerve ligation (SNL) induced mechanical allodynia.