Lymphangiogenesis in abdominal aortic aneurysm.

BACKGROUND: Ongoing angiogenesis is implicated in the inflammatory environment that characterizes abdominal aortic aneurysm (AAA). Although lymphangiogenesis has been associated with chronic inflammatory conditions, it has yet to be demonstrated in AAA. The aim was to determine the presence of lymphangiogenesis and to delineate the relationship between inflammation and neovascularization in AAA tissue. METHODS: AAA samples and preoperative computed tomography images were obtained from patients undergoing elective AAA repair. Control samples were age-matched abdominal aortic tissue. Specific immunostains for blood vessels (CD31, CD105), lymphatic vessels (D2-40), vascular endothelial growth factor (VEGF) A and VEGF receptor (VEGFR) 3 allowed characterization and quantitation of vasculature. RESULTS: The AAA wall contained high levels of inflammatory infiltrate; microvascular densities of blood (P < 0·001) and lymphatic (P = 0·003) vessels were significantly increased in AAA samples compared with controls. Maximal AAA vascularity was observed in inflammatory areas, with vessels that stained positively for CD31 (ρ = 0·625, P = 0·017), CD105 (ρ = 0·692, P = 0·009) and D2-40 (ρ = 0·675, P = 0·008) correlating positively with the extent of inflammation. Increased VEGFR-3 and VEGF-A expression was also evident within inflammatory AAA areas. CONCLUSION: These findings demonstrated lymphatic vessel involvement in end-stage AAA disease, which was associated with the degree of inflammation, and confirmed the involvement of neovascularization.

Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease.

Alexander disease is a rare disorder of the central nervous system of unknown etiology. Infants with Alexander disease develop a leukoencephalopathy with macrocephaly, seizures and psychomotor retardation, leading to death usually within the first decade; patients with juvenile or adult forms typically experience ataxia, bulbar signs and spasticity, and a more slowly progressive course. The pathological hallmark of all forms of Alexander disease is the presence of Rosenthal fibers, cytoplasmic inclusions in astrocytes that contain the intermediate filament protein GFAP in association with small heat-shock proteins. We previously found that overexpression of human GFAP in astrocytes of transgenic mice is fatal and accompanied by the presence of inclusion bodies indistinguishable from human Rosenthal fibers. These results suggested that a primary alteration in GFAP may be responsible for Alexander disease. Sequence analysis of DNA samples from patients representing different Alexander disease phenotypes revealed that most cases are associated with non-conservative mutations in the coding region of GFAP. Alexander disease therefore represents the first example of a primary genetic disorder of astrocytes, one of the major cell types in the vertebrate CNS.

The effects of a quillaja and yucca combination on performance and carcass traits of coccidia-vaccinated broilers exposed to an enteric disease challenge.

A series of 6 floor pen trials was conducted to determine the effects of a quillaja and yucca combination product on the performance and carcass traits of growing broiler chickens vaccinated for coccidiosis at the hatchery. In each of the trials graded levels (0, 250, and 500 ppm) of a quillaja and yucca combination (QY) were fed to Ross 708 broilers for the duration of each 42 d test. Trials were arranged in completely randomized block designs involving a minimum of 11 blocks per trial. At the start of each trial, pens contained 55 broilers. In order to provide each bird with an enteric disease challenge, 5 kg commercial broiler litter containing 104 CFU Clostridium perfringens per gram was placed in each pen. In addition, the sporulated oocysts of Eimeria acervulina and E. maxima were added to each pen at the outset of each test. At d 21 of the trials, coccidial lesion scores, mortality and performance were determined; final performance and total mortality were assessed at 42 d. At the completion of each test, 10 birds of average body weight per pen were selected for carcass evaluations; whole and chilled carcass yield were determined, and pre- and post-chill breast measurements were made. A combined analysis of the results of the 6 trials (75 replications per treatment) was used to determine treatment effects and each variable was assessed by linear regression analysis. Results indicated that QY significantly reduced mortality and coccidial lesions scores at d 21 (P < 0.05). Performance was significantly improved by both levels of QY at 21 and 42 d, and significant linear effects were observed for these variables (P < 0.05). All carcass characteristics were significantly improved by QY administration and significant linear responses were observed for each carcass variable (P < 0.05). These results indicate that by reducing intestinal disease challenge, QY provided linear improvements in performance. In addition, QY positively affected carcass parameters as each variable responded linearly to QY feeding (P < 0.05).

The Effects of a Combination of Quillaja saponaria and Yucca schidigera on Eimeria spp. in Broiler Chickens.

A series of studies was carried out to determine the anticoccidial effects of a product derived from plant material sourced from Quillaja saponaria and Yucca schidigera. These plants are known to contain high concentrations of triterpenoid and steroidal saponins, substances that are known to display an array of biological effects. Battery tests involving individual Eimeria acervulina, Eimeria maxima, and Eimeria tenella infections and graded levels of a quillaja/yucca combination (QY) (0, 200, 250, and 300 ppm) were conducted. Body weight gain, coccidial lesion scores, and total oocysts per gram of feces (OPG) were used to evaluate anticoccidial effects. In addition, three floor pen trials evaluated the effects of 250 ppm QY in the control coccidial infections. The first pen trial measured the effects of 250 ppm QY, both alone and in combination with 66 ppm salinomycin (Sal), in a 2 3 2 factorial treatment arrangement. Two additional 42-day pen studies assessed the effects 250 ppm QY in birds vaccinated for coccidiosis. Data from the three battery trials indicated that at doses of 250 ppm QY or more, weight gain was improved, E. acervulina and E. tenella lesion scores were reduced, and OPG was lowered. In general, OPG was reduced by about 50% across all species by 250 and 300 ppm QY. Results of the pen study indicated that 250 ppm QY and Sal, when fed individually, reduced OPG and lesion scores and improved final performance. However, when QY and Sal were administered concurrently, further significant reductions in OPG occurred. The final performance of broilers vaccinated for coccidiosis was also improved at 250 ppm QY, as was OPG at both 21 and 28 days. Thus, at QY doses of 250 ppm or more, anticoccidial activity was evident but lacked the potency exhibited by many standard anticoccidials. When combined with either Sal or a live coccidiosis vaccine, QY improved the anticoccidial effects and performance of these anticoccidial methods.

Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome.

The cerebro-hepato-renal syndrome is a rare familial malady with cerebral, renal, and skeletal abnormalities, severe hypotonia, cirrhosis, iron and lipid storage, and death within 6 months. Correlated electron microscopic, histochemical, and biochemical studies demonstrate defects in two oxidative organelles. Peroxisomes cannot be found in hepatocytes and renal proximal tubules. In hepatocytes and cortical astrocytes, mitochondria are distorted in their appearance and glycogen stores are increased. Oxygen consumnption of brain and liver mitochondrial preparations with succinate and with substrates reducing nicotinamide adenine dinucleotide is markedly diminished, but the consumption is normal with ascorbate and tetramethylphenylenediamine, which suggests a defect in electron transport prior to the cytochromes. Histochemical studies of mitochondrial oxidation point to a defect between the succinate dehydrogenase flavoprotein and coenzyme Q, possibly in the region of nonheme iron protein.

Knowledge, attitude and perception of adults on childhood hypertension in a rural community in Nigeria.

BACKGROUND: Childhood hypertension is less common and not readily recognised compared to adult hypertension. It is being missed because of lack of routine blood pressure measurements in some health facilities in the developing countries. This setback is likely to affect the knowledge, attitude and perception of parents and caregivers to the disease. OBJECTIVE: To assess the knowledge, attitude and perception of childhood hypertension by adult respondents in a rural community in Nigeria. METHOD: The study was carried out with the aid of pre-tested questionnaires, which sought information on wide ranging issues bothering on knowledge, attitude and perception of childhood hypertension. RESULTS: Fifty-four 54/62 (89%) knew that hypertension implied high blood pressure. Fifty-eight 58/61(95%) also indicated that hypertension is detected by measuring the blood pressure. However 37/60 (62%) respondents did not think that childhood hypertension exist, while 53/59 (90%) respondents were not aware of any child diagnosed with hypertension. All our 62 respondents claimed to have seen 9 children with hypertension compared to 81 adults some of whom were related to them. CONCLUSIONS: We conclude that there is still a low awareness about the existence of childhood hypertension by the rural populace. While this problem is not a priority health problem in the community, an increased awareness of it is crucial the successful management of any child that develops it. Periodic community survey for childhood hypertension and mandatory routine blood pressure measurement in all children presenting for consultation is suggested.

Genomic alterations in cervical carcinoma: losses of chromosome heterozygosity and human papilloma virus tumor status.

Specific human papilloma virus (HPV) types appear to be necessary etiological factors for most cervical cancers, yet additional genetic alterations seem to be required for their development and progression. The aim of this study is to determine the likely chromosomes location of tumorigenicity suppressor-like genes, the loss of function of which might be important in the origin or progression of cervical carcinomas. PCR with primers for 75 highly polymorphic microsatellite loci located on the major autosome arms were used to estimate the incidence of loss of heterozygosity (LOH) in 38 tumors. The HPV status of the tumors was also determined. LOH was found to involve 19 chromosome arms in 20-43% of the tumors. Chromosome arms 6p, 3p, and 18q are most frequently involved in LOH in 43, 39, and 35% of the informative carcinomas, respectively. The respective regions involved are 6p21.1-23, 3p13-25.3, and 18q12.2-21.2. LOH is generally limited to specific band segments within these regions. Similar high incidences of LOH of the same 3p segments have been reported in cervical carcinomas from different parts of the world. The same 3p and 6p segments are involved in many types of common cancers, whereas 18q changes are less frequent in other cancers. Chromosome arms 1q, 2q, 3q, 4p, 4q, 5p, 5q, 6q, 7q, 8p, 8q, 11q, 13q, 16p, 18p, and 19p are involved in LOH in 20-33% of the cervical tumors. Chromosome 11 alterations are among the most frequently found in many different types of neoplasias. In this study, 11p was involved in 16% of the tumors, and 11q was involved in 22%. Chromosome 17 alterations are found in more cancers than those of any other chromosome, frequently involving the p53 gene on 17p. LOH of 17p was found in 5 (15%) cervical tumors; 2 of these were HPV negative and expressed mutant p53. In such HPV-negative tumors, direct mutation of the wild-type p53 appears to replace the inactivation of the p53 product by oncogenic HPV types. Tumors with LOH at many loci were, on the average, at more advanced stages, as were tumors with mutant p53. The higher overall incidence of LOH in cervical carcinomas as compared to other cancers, and the diversity of LOH patterns found, suggest that different cervical carcinomas probably arise and/or progress, in part, because of the loss of function of different yet finite sets of tumorigenicity suppressor genes and genes that are involved in tumor progression and metastasis. The findings also indicate that certain chromosome segments that are often altered in cervical carcinomas are also frequently altered in several other types of cancers. It remains to be determined whether the same or different genes located within these segments are involved in the different cancer types.

Mitochondrial abnormalities in Alzheimer's disease.

The finding that oxidative damage, including that to nucleic acids, in Alzheimer's disease is primarily limited to the cytoplasm of susceptible neuronal populations suggests that mitochondrial abnormalities might be part of the spectrum of chronic oxidative stress of Alzheimer's disease. In this study, we used in situ hybridization to mitochondrial DNA (mtDNA), immunocytochemistry of cytochrome oxidase, and morphometry of electron micrographs of biopsy specimens to determine whether there are mitochondrial abnormalities in Alzheimer's disease and their relationship to oxidative damage marked by 8-hydroxyguanosine and nitrotyrosine. We found that the same neurons showing increased oxidative damage in Alzheimer's disease have a striking and significant increase in mtDNA and cytochrome oxidase. Surprisingly, much of the mtDNA and cytochrome oxidase is found in the neuronal cytoplasm and in the case of mtDNA, the vacuoles associated with lipofuscin. Morphometric analysis showed that mitochondria are significantly reduced in Alzheimer's disease. The relationship shown here between the site and extent of mitochondrial abnormalities and oxidative damage suggests an intimate and early association between these features in Alzheimer's disease.

Abnormal activation of glycogen synthesis in fibroblasts from NIDDM subjects. Evidence for an abnormality specific to glucose metabolism.

To determine whether the tendency for NIDDM to run in families could relate to genetically determined defects in insulin stimulation of glycogen synthesis, skin fibroblasts from subjects with a strong family history of NIDDM were studied. Fibroblasts from nondiabetic subjects without any family history of NIDDM were studied as control subjects. The cells were studied after 7-16 passages in culture. Rates of glycogen synthesis were lower in fibroblasts from NIDDM subjects both basally and with maximal insulin stimulation (0.77 +/- 0.11 vs. 0.46 +/- 0.04 pmol.well-1 x h-1 [P < 0.02] and 1.49 +/- 0.26 vs. 0.69 +/- 0.05 pmol.well-1 x h-1 +adP < 0.01]). Rates of glycogen synthesis were stimulated 1.9 +/- 0.2-fold above basal in the control cells and 1.5 +/- 0.1-fold above basal in the NIDDM cells (P < 0.02). Rates of [3H]thymidine uptake were similar in control and NIDDM fibroblasts (basal, 28.3 +/- 2.8 vs. 39.2 +/- 8.0; maximum, 50.9 +/- 7.2 vs. 69.3 +/- 16.9 dpm x 10(-3), respectively). Rates of uptake increased similarly in control and NIDDM cells by 1.8 +/- 0.1- and 1.7 +/- 0.1-fold above basal. Maximum specific fibroblast insulin binding was similar for control and NIDDM subjects (194.0 +/- 29.2 vs. 176.1 +/- 24.9 fmol 125I-labeled insulin bound/mg protein respectively). The tyrosine kinase activity of insulin receptors isolated from the control and NIDDM fibroblasts was similar (basal, 135 +/- 30 vs. 149 +/- 33; submaximal, 153 +/- 28 vs. 155 +/- 30; and maximal insulin, 191 +/- 45 vs. 213 +/- 48 dpm.mg protein-1 x min-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Does suppression of postprandial blood glucose excursions by the alpha-glucosidase inhibitor miglitol improve insulin sensitivity in diet-treated type II diabetic patients?

OBJECTIVE: Insulin sensitivity is impaired in patients with type II diabetes and is exacerbated by high mean blood glucose (BG). Potentially, large postprandial swings in BG could result in further decrements of insulin sensitivity. Because alpha-glucosidase inhibitors cause a marked reduction in the amplitude of BG changes, the aim of this study was to determine if such a BG-smoothing effect improves insulin sensitivity in well-controlled type II diabetic subjects treated with diet alone. RESEARCH DESIGN AND METHODS: Patients received either miglitol (BAY m 1099) (50 mg three times daily) or placebo for 8 weeks in a randomized double-blind parallel study. The miglitol (9 men, 2 women) and placebo (7 men, 3 women) groups were well matched (mean +/- SD) for age, weight, and blood glucose control (fasting BG, 6.4 +/- 1.0 vs. 6.9 +/- 1.6 mmol/l; HbA1, 7.7 +/- 1.0 vs. 7.9 +/- 0.4%; fructosamine, 0.99 +/- 0.08 vs. 1.07 +/- 0.17 mmol/l). The glucose metabolic clearance rate was calculated during the last 30 min of a 150 min glucose/insulin sensitivity test (glucose, 6 mg . kg-1 . min-1; insulin, 0.5 U . kg-1 . min-1). RESULTS: There was no significant improvement in metabolic clearance rate (0.21 +/- 0.27 vs. 0.16 +/- 0.35 l . kg-1 . min-1) for the miglitol- and placebo-treated groups, respectively. There were no statistically significant differences between miglitol and placebo for changes from baseline in BG (0.1 +/- 0.1 vs. -0.1 +/- 0.2 mmol/l), HbA1 (0.1 +/- 0.1 vs. 0.3 +/- 0.1%), and fructosamine (-0.06 +/- 0.02 vs. -0.03 +/- 0.02 mmol/l). CONCLUSIONS: Alpha-glucosidase-induced improvement in postprandial hyperglycemia does not result in increased insulin sensitivity.

Effect of raising injection-site skin temperature on isophane (NPH) insulin crystal dissociation.

In eight healthy subjects, skin temperature at the injection site was raised from mean +/- SD 31.7 +/- 0.5 to 40.8 +/- 0.9 degrees C 180 min after injection of 0.25 U/kg isophane (NPH; Human Insulatard) insulin and maintained for 180 min. On the control day, skin temperature was kept constant. On warming of the injection site, serum insulin concentration rose from mean +/- SE 14.4 +/- 2.5 to 17.7 +/- 3.1 mU/L after 40 min (P less than .01) but did not change on the control day over the same period. The change in insulin concentration from the prewarming hour was higher on the warming day than control day in the 1st h (123 +/- 8 vs. 93 +/- 7%, P less than .01), 2nd h (115 +/- 14 vs. 83 +/- 9%, P less than .05), and 3rd h (113 +/- 17 vs. 80 +/- 10%, P less than .05) of warming, providing evidence for both early increased absorption of the free-insulin pool surrounding the protamine-insulin complexes and continuing increased dissociation of the complexes.

Aspirin therapy is associated with less compact fibrin networks and enhanced fibrinolysis in patients with abdominal aortic aneurysm.

OBJECTIVE: Thrombotic changes in fibrin networks contribute to increased cardiovascular risk in patients with abdominal aortic aneurysm (AAA). Given that aspirin modulates the fibrin network, we aimed to determine if aspirin therapy is associated with changes in ex-vivo fibrin clot characteristics in AAA patients and also conducted an exploratory analysis of 5-year mortality in these individuals. METHODS: We recruited 145 male patients, divided into controls (aortic diameter < 3 cm, n = 49), AAA not taking aspirin (AAA-Asp, n = 50) and AAA on 75 mg day(-1) aspirin (AAA+Asp, n = 46), matched for aneurysm size. Characteristics of clots made from plasma and plasma-purified fibrinogen were investigated using turbidimetric analysis, permeation studies, and confocal and electron microscopy. Plasma fibrinogen, D-dimer and inflammatory marker levels were also measured. RESULTS: Maximum absorbance (MA) of plasma clots from controls was lower than that of AAA patients not on aspirin (AAA-Asp) at 0.30 ± 0.01 and 0.38 ± 0.02 au, respectively (P = 0.002), whereas aspirin-treated subjects had MA similar to controls (0.31 ± 0.02 P = 0.9). Plasma clot lysis time displayed an identical pattern at 482 ± 15, 597 ± 24 and 517 ± 27 s for control, AAA-Asp and AAA+Asp (P = 0.001 and P = 0.8). The lysis time of clots made from purified fibrinogen of AAA-Asp was longer than that of AAA+Asp patients (756 ± 47 and 592 ± 52 s, respectively; P = 0.041). Permeation studies and confocal and electron microscopy showed increased clot density in AAA-Asp compared with the AAA+Asp group. Mortality in AAA-Asp and AAA+Asp was similar, despite increased cardiovascular risk in the latter group, and both exhibited higher mortality than controls. CONCLUSION: Aspirin improves fibrin clot characteristics in patients with AAA, which may have important clinical implications.

Alexander disease: new insights from genetics.

Prior to finding that GFAP mutations underlie many cases of Alexander disease, it was unclear whether the disease originated in astrocytes or if the formation of Rosenthal fibers was a response to an external insult. It was also unclear whether the etiology of the disease was environmental or genetic. For many cases of Alexander disease, these questions have now been answered. An immediate clinical benefit of this discovery is the possibility of diagnosing most cases of Alexander disease through analysis of patient DNA samples, rather than resorting to brain biopsy. In addition, fetal testing is now an option for parents who have had an Alexander disease child with an identified mutation and who wish to have additional children. For the future, these mutations should provide a unique window for illuminating the mechanism of the disease.

Mucolipidosis IV. Clinical, ultrastructural, histochemical, and chemical studies of a case, including a brain biopsy.

A 7-year-old Ashkenazi Jewish boy with normal early development started to regress at 8 months of age and made no further developmental progress. Corneal clouding was noted at age 10 months. Corneal and conjunctival biopsy at 14 months, cerebral biopsy at 24 months, and fibroblast cultures at 32 months showed lysosomal inclusions, suggesting the storage of lipid-like and mucopolysaccharide-like material. In the brain, dense fluorescent inclusions resembled those in ceroid-lipofuscinosis. Total ganglioside content of white matter was raised, but the pattern was normal. The level of nonlipid hexosamine in the brain was normal. The cornea and conjunctiva contained electronlucent vacuoles resembling those in the mucopolysaccharidoses. Cornea, brain, and lymphocytes contained concentric membranous lamellar structures reminiscent of those in the gangliosidoses. The clinical picture and ultrastructural findings support the impression that this case belongs to a new variant of the mucolipidoses, mucolipidosis IV.

Molecular findings in symptomatic and pre-symptomatic Alexander disease patients.

BACKGROUND AND OBJECTIVE: Alexander disease is a slowly progressive CNS disorder that most commonly occurs in children. Until recently, the diagnosis could only be established by the histologic finding of Rosenthal fibers in brain specimens. Mutations in the glial fibrillary acidic protein (GFAP) gene have now been shown in a number of biopsy- or autopsy-proven patients with Alexander disease. A prospective study on patients suspected to have Alexander disease was conducted to determine the extent to which clinical and MRI criteria could accurately diagnose affected individuals, using GFAP gene sequencing as the confirmatory assay. METHODS: Patients who showed MRI white matter abnormalities consistent with Alexander disease, unremarkable family history, normal karyotype, and normal metabolic screening were included in this study. Genomic DNA from patients was screened for mutations in the entire coding region, including the exon-intron boundaries, of the GFAP gene. RESULTS: Twelve of 13 patients (approximately 90%) were found to have mutations in GFAP. Seven of those 12 patients presented in infancy with seizures and megalencephaly. Five were juvenile-onset patients with more variable symptoms. Two patients in the latter group were asymptomatic or minimally affected at the time of their initial MRI scan. The mutations were distributed throughout the gene, and all involved sporadic single amino acid heterozygous changes that changed the charge of the mutant protein. Four of the nine changes were novel mutations. CONCLUSIONS: In symptomatic and asymptomatic patients with a predominantly frontal leukoencephalopathy by MRI, GFAP gene mutation analysis should be included in the initial diagnostic evaluation process for Alexander disease.

Commercial horseradish peroxidase degrades myelin encephalitogenic protein during coupling for immunohistochemical studies.

Conjugates of myelin encephalitogenic basic protein (EP) and commercial horseradish peroxidase (HRP) have been used for immunohistochemical demonstrations of anti-EP antibody in animals with experimental allergic encephalomyelitis. We performed gel electrophoresis studies on EP-HRP conjugates prepared with glutaraldehyde and on mixtures of EP and HRP incubated without glutaraldehyde. The results show that under conditions of one-and two-step coupling HRP causes rapid loss of the native EP band, apparently due to EP degradation. The EP-HRP mixtures are not encephalitogenic in rabbits, or encephalitogenic activity is lost during processing. The immunohistochemical reactivity of conjugates, however, signals some preservation of antibody-combining sites. The mechanism of the HRP effect on EP is unknown. The possibilities of a contaminating proteinase or direct peroxidatic attack are suggested. Until this action of HRP can be overcome, the effect of coupling procedures on the biological activities of EP will be difficult to assess, and EP-HRP conjugates cannot be expected to reveal sites that may bind encephalitogenic portions of the EP molecule.

Ultrastructural immunogold labeling of glial filaments in osmicated and unosmicated epoxy-embedded tissue.

On-grid (post-embedding) immunolabeling methods with epoxy resins have been difficult to apply to thin structures such as intermediate filaments, which may remain inaccessible within the plastic. In this study, glial fibrillary acidic protein (GFAP), the major protein of astrocyte intermediate filaments, was localized with a post-embedding immunogold method, using both unosmicated and osmicated material embedded in epoxy resin. The tissue studied was from a diagnostic brain biopsy on a child with Alexander's disease. This disorder is characterized by proliferation of astrocyte intermediate filaments and formation of Rosenthal fibers. With unosmicated tissue, as in a previous study, extensive labeling of the glial filaments was achieved only when ultra-thin sections were pre-treated with dilute sodium ethoxide, an agent that dissolves plastic. Fifteen-nm gold could be used. With osmicated tissue, localization to glial filaments required pre-treatment with sodium ethoxide and with the oxidizing agent sodium metaperiodate, followed by the use of small (5 nm) colloidal gold. That 5-nm gold was required for labeling filaments in osmicated material suggested that osmication increases problems of penetrability and antigen accessibility within ultra-thin sections. The large Rosenthal fibers were labeled by 15-nm gold in both unosmicated and osmicated material. The methods employed may be useful for electron immunolocalizations to other thin structures in material embedded in epoxy resin.

Histochemical characteristics of the striated inclusions of adrenoleukodystrophy.

The straited accumulations in adrenal cortical cells and brain macrophages that are characteristic of adrenoleukodystrophy have been studied histochemically in cryostat sections to seek leads for the biochemical identification of the striated material. It stained pale pink with oil red O and did not stain with the Schultz cholesterol procedure or periodic acid-Schiff technique. By utilizing the birefringence of the accumulations as a marker, it was determined that, unlike natural cholesterol and cholesterol esters, the striated material was resistant to acetone and ethanol extraction. It was readily soluble, however, in nonpolar solvents such as n-hexane and chloroform. These findings indicated that the material was most probably a lipid, and they suggested that sequential extraction of adrenoleukodystrophy adrenal and brain with acetone and then n-hexane could be used to isolate this material in relatively pure form. Based on this lead, biochemical studies have just revealed a fatty acid abnormality in adrenoleukodystrophy which appears to be unique to this genetic disease.

Immunocytochemical localization of hepatic legandin and Z protein utilizing frozen sections for light and electron microscopy.

Ligandin (glutathione-s-transferase) and Z protein are soluble hepatocellular proteins that are involved in the transfer of organic ions, including bilirubin and some hormones and carcinogens from the plasma to the liver. The intracellular distribution of ligandin and Z protein was studied by applying the peroxidase-antiperoxidase procedure of L. A. Sternberger (Immunocytochemistry, Prentice Hall Inc., 1974) to paraffin sections and free-floating 10-micrometers frozen sections that were processed for both light and electron microscopy. Ligandin and Z protein were localized to the cytosol of hepatocytes in association with smooth endoplasmic reticulum (SER), but no reaction product was present between cisternae of rough endoplasmic reticulum. Penetration of reagents was enhanced in 10-micrometers frozen sections and the preservation of subcellular structures was equivalent to thicker, unfrozen sections.