Brown Reaction Product Research Articles

Immunolocalization of putative neurotransmitters innervating autonomic regulating neurones of cat ventral medulla

This study investigated possible sites of contact of nerve fibers containing a range of putative neurotransmitter substances onto neurons in the cat ventral medulla oblongata concerned with autonomic, particularly cardiovascular, regulation. The parasympathetic preganglionic neurons of the nucleus ambiguus were identified by retrograde horseradish peroxldase tracing from the vagus nerve, and the groups of neurons in the Al and C1 cell areas and the raphe nucleus by cafscholamine enzyme or 5-hydroxytryptamins (5-HT) inurnxuohistoduemistry, respectively. Immunoreactive (-ir) nerve fibers and terminals in the vicinity of these neurons were visualized by subjecting the sections to a dual-staining technique using a brown peromddase-diaminobenzidine reaction product and a blue alkaline phosphatese-Fast blue reaction product: By employing monochrome photography with combinations of blue and orange-red filters, it was possible to discriminate neural elements displaying one or the other reaction product, or colocalization of reaction products. The results revealed the presence of calcitonin gene-related peptide (CORP) and galanin (GAL)ir in some motonsurons of the nucleus ambiguus, but not in those innervating the heart via the cardiac vagus nerve. The latter group of parasympathetic efferent neurons were found to be densely innervated by fibers innnrunoreactive for dopamine β-hydroxylase (DBH, indicating noradrenalineL ghrcine (GLY), γ-aminobutyric acid (GABA), 5-HT, enkephalin (ENK, neuropeptide Y (NPY), substance P (SP), and i hormone (TRK and, to a lesser extent, by other netropep ide-ir fibers. The came cells of the rostal C1 and caudal A1 groups showed a broadly similar pattern of innervation, most noticeably by fibers immurwreactive for DBH, GABA, 5-HT, cholecystokinin (CCIQ, CGRP, ENK, GAL, NPY, and SP. The 5-HT-ir neurons of the raphe nucleus, some also containing SP, TRH, ENK, or aorticotropin-releasing factor (CRF)-ir, were most prominently innervated by terminals containing DBH, GABA, CCK, ENK, NPY, TRH, somatostatin (SRIF), and vasoactive intestinal polypeptide (VIP)-ir. Although the proof that these groups of neurons receive functional synaptic contacts from the immunoreactive fibers awaits furtis ultrastructural studies, the results do suggest that a wide range of putative transmitters may influence the activity of efferent neurons in the cat medulla controlling autonomic functions.

Estrogen receptor-immunoreactive neurons contain calcitonin gene-related peptide, methionine-enkephalin or tyrosine hydroxylase in the female rat preoptic area

We have shown in our previous studies that estrogen treatment selectively influences calcitonin gene-related peptide (CGRP)-, methionine-enkephalin (Met-Enk)- and tyrosine hydroxylase (TH)-immunoreactive (IR) intensities in the neurons of the periventricular preoptic nucleus (PPN) and the medial preoptic area (MPA) of the female rat. In the present study, we examined whether estrogen receptor (ER)-IR neurons in the PPN and MPA contain CGRP, Met-Enk, or TH using a double-labeling immunohistochemical method and investigated changes in the number of double-labeling cells upon treatment with estrogen. Brain sections of ovariectomized rats and ovariectomized and estrogen-treated rats were stained using the avidin-biotin-peroxidase complex method followed by the peroxidase-anti-peroxidase method. The sections were first incubated with an anti-ER antibody in conjunction with nickel diaminobenzidine which produces a dark blue reaction product in the nucleus. Subsequently, CGRP, Met-Enk or TH antisera were applied to these sections and the resulting brown diaminobenzidine reaction product in the cytoplasm was examined. Neurons that were double-labeled for ER and CGRP, Met-Enk or TH were investigated in the PPN and MPA. The number of doubly labeled ER/CGRP- and ER/TH-IR neurons was large, whereas the number of ER/Met-Enk-IR neurons was small. These results suggest that ER in the PPN and MPA may be more closely related to the mechanism of changes in CGRP- and TH-IR intensities upon estrogen treatment than that in Met-Enk-IR intensity.

Localization of androgen receptor within peptidergic neurons of the rat forebrain

This study tested for the presence of androgen receptor-immunoreactivity in somatostatin, galanin, vasopressin, corticotropin-releasing hormone, and oxytocin neurons in the rat forebrain. The brains of adult male Sprague-Dawley rats were fixed with 4% paraformaldehyde. Androgen receptor was visualized in coronal sections using nickel intensification of diaminobenzidine, and the neuropeptides were identified using a brown diaminobenzidine reaction product. Androgen receptor was localized to the nuclei of neurons in the septum, amygdala, cortex, hippocampus, and hypothalamus. The majority of somatostatin-containing neurons in the periventricular hypothalamic nucleus also contained androgen receptor. Androgen receptor was also found within galanin-expressing cells in the bed nucleus of the stria terminalis and in the amygdala. Androgen receptor was not observed in corticotropin-releasing hormone, vasopressin, or oxytocin neurons in all areas examined. The data suggest that androgens may be capable of directly regulating somatostatin-expressing neurons of the periventricular nucleus of the hypothalamus and galanin-containing neurons of the bed nucleus of the stria terminalis and amygdala.

Early Detection and Measurement of Experiment Myocardial Infarcts with Horseradish Peroxidase

Current methods of experimental infarct size measurement require tisue analysis several hours after the ischemic event. Electron microscopy identifies irreversibly injured myocytes early after ischemia, but cannot be used to measure myocardial infarct size. Horseradish peroxidase (HRP), a tracer protein that permeates the disrupted sarcolemma of injured myocytes, was used to determine infarct size. New Zealand White rabbits ( n = 15) were subjected to 30 min coronary artery occlusion and 24 h reperfusion. Prior to euthanasia, HRP was administered intravenously. The hearts were excised, perfused with triphenyl tetrazolium chloride (TTC), and perfusion fixed. A frozen section was cut from left ventricular slices, and the brown HRP reaction product was developed with 3,3′-diaminobenzidine tetrahydrochloride and H 2O 2. The areas delineated by intramyocyte HRP (as a percent of the left ventricle) closely correlated with infarct size determined by conventional hematoxylin-eosin (H&E) stain and TTC (means ± S.E.M.): 29.5 ± 3.0.% vs 27.6 ± 2.9% vs 28.6 ± 2.6%, respectively. The coefficient of correlation between HRP and H&E was 0.94. This method was tested for early infarct detection in rabbits subjected to 30 min coronary occlusion followed by intravenous injection of HRP and sacrifice. HRP-delineated infarcts measured 21.4 ± 3.7% of the left ventricle, and electron microscopic examination from ischemic and non-ischemic areas was used to confirm that cells containing HRP were irreversibly injured. Thus, HRP can be used to accurately measure myocardial infarct size in experimental coronary artery occlusion and reperfusion in infarcts as early as 30 min after coronary occlusion or following 24 h of reperfusion.

Neurogenesis and differentiation of sympathetic B and C cells in the bullfrog tadpole

The relation between birthdates of sympathetic neurons and their subsequent differentiation into cutaneous B cells and vasomotor C cells was examined in paravertebral ganglia 9 and 10 of the bullfrog tadpole. Neurons undergoing terminal cell division were identified by injecting tadpoles repeatedly with 5'-bromodeoxyuridine (BRDU) for one to six developmental stages between III and XXI. After allowing the tadpoles to enter late metamorphic stages (XX-XXV), the ganglia were double immunostained for BRDU and neuropeptide Y (NPY). NPY is a marker for mature C-type neurons (Horn et al., 1987). Double-labeled neurons were readily discerned through use of distinct black and brown HRP reaction products and also because immunoreactivity for BRDU was localized in nuclei while that for NPY was localized in perinuclear cytoplasm. Counts of labeled cells showed that neurogenesis occurs throughout limb bud and paddle stages, and that it ceases during early foot stages (XII-XIV), a time coinciding with the onset of NPY expression. By contrast, the labeling of non-neuronal satellite cells with BRDU was most common weeks later during metamorphic stages. Irrespective of their birthdates, about half of the BRDU-labeled neurons were also positive for NPY immunoreactivity. This proportion of NPY-positive cells is indistinguishable from that in the entire adult ganglia (Horn et al., 1987). In addition to establishing that neurogenesis and gliogenesis occurs during tadpole stages, the results indicate that the onset of NPY expression by vasomotor C neurons is unrelated to their time of origin. In other words, the last wave of neurogenesis in sympathetic ganglia does not give rise to a specific subclass of sympathetic neurons.

Colocalization of progestin receptors with serotonin in raphe neurons of macaque.

Progesterone stimulates prolactin secretion in estrogen-primed women and monkeys. We hypothesize that this effect is neurally mediated since pituitary lactotropes do not contain progestin receptors (PR). In rodents, progesterone enhances hypothalamic serotonin (5HT) content, and both progesterone and 5HT stimulate prolactin and LH secretion. However, it was not known whether progesterone acts directly on 5HT neurons or through other neurons. Using a double immunocytochemical procedure, we show that 5HT neurons in macaque contain PR and thus are a progestin target system. Midbrain tissue blocks were obtained from two female monkeys and immersion-fixed prior to freezing and sectioning. PR was detected with a monoclonal antibody against human PR (B39) bridged to horseradish peroxidase and developed in diaminobenzidine. PR immunoreactivity appeared as a brown reaction product which localized in the nuclei of individual neurons. 5HT was detected with an antiserum generated against a conjugate of 5HT and BSA bridged to alkaline phosphatase. 5HT immunoreactivity appeared as a blue reaction product in the cytoplasm and axons of the pontine raphe nucleus. Neurons containing both nuclear reaction product for PR and cytoplasmic reaction product for 5HT were observed in both the dorsal and ventral aspects of the midbrain raphe nucleus as well as the raphe magnus. In summary, progesterone can have a direct action on 5HT neuronal function and thereby influence those endocrine and affective systems under serotonergic control.

Beta-endorphin innervation of dopamine neurons in the rat hypothalamus: a light and electron microscopic double immunostaining study.

Pharmacological data suggest that opiates, acting indirectly via the catecholaminergic system, are involved in the inhibition of LH release and the stimulation of PRL secretion. The aim of this study was to demonstrate on the ultrastructural level whether beta-endorphin-immunoreactive fibers form synaptic contacts with hypothalamic dopaminergic neurons. Light and electron microscopic double immunostaining experiments were performed on vibratome sections prepared from the hypothalamus of acrolein-fixed female rat brains. Immunoreactivity for beta-endorphin was visualized by a dark blue to black nickel ammonium sulfate-intensified diaminobenzidine reaction, and in a consecutive immunostaining procedure, the tyrosine hydroxylase-immunoreactive dopamine cells were labeled with the brown diaminobenzidine reaction product. Under the light microscope, beta-endorphin axon terminals were found to contact dopamine cell bodies and dendrites throughout the hypothalamus. The majority of opiate target dopamine neurons were found in the periventricular area, retrochiasmatic area, and lateral part of the zona incerta. A much smaller number was observed in the dorsomedial hypothalamic nucleus and the anterior hypothalamus, and only a very few dopamine cells could be detected in contact with beta-endorphin axons in the arcuate nucleus (particularly in the posterior part where the beta-endorphin cells are located) and the medial part of the zona incerta. After light microscopic examination and color photography, the double immunostained sections were embedded for correlated electron microscopy to verify and characterize the putative synaptic connections. Electron microscopy revealed symmetric synaptic connections between beta-endorphin-immunoreactive boutons and tyrosine hydroxylase-immunopositive cell bodies and dendrites. These results together with the observation of dopamine innervation of LHRH-producing neurons and progesterone receptor-containing cells indicate that neurons of the hypothalamic dopaminergic system probably mediate opiate effects on hypophyseal hormone secretion.

Beta-endorphin innervation of dopamine neurons in the rat hypothalamus: a light and electron microscopic double immunostaining study

Pharmacological data suggest that opiates, acting indirectly via the catecholaminergic system, are involved in the inhibition of LH release and the stimulation of PRL secretion. The aim of this study was to demonstrate on the ultrastructural level whether beta-endorphin-immunoreactive fibers form synaptic contacts with hypothalamic dopaminergic neurons. Light and electron microscopic double immunostaining experiments were performed on vibratome sections prepared from the hypothalamus of acrolein-fixed female rat brains. Immunoreactivity for beta-endorphin was visualized by a dark blue to black nickel ammonium sulfate-intensified diaminobenzidine reaction, and in a consecutive immunostaining procedure, the tyrosine hydroxylase-immunoreactive dopamine cells were labeled with the brown diaminobenzidine reaction product. Under the light microscope, beta-endorphin axon terminals were found to contact dopamine cell bodies and dendrites throughout the hypothalamus. The majority of opiate target dopamine neurons were found in the periventricular area, retrochiasmatic area, and lateral part of the zona incerta. A much smaller number was observed in the dorsomedial hypothalamic nucleus and the anterior hypothalamus, and only a very few dopamine cells could be detected in contact with beta-endorphin axons in the arcuate nucleus (particularly in the posterior part where the beta-endorphin cells are located) and the medial part of the zona incerta. After light microscopic examination and color photography, the double immunostained sections were embedded for correlated electron microscopy to verify and characterize the putative synaptic connections. Electron microscopy revealed symmetric synaptic connections between beta-endorphin-immunoreactive boutons and tyrosine hydroxylase-immunopositive cell bodies and dendrites. These results together with the observation of dopamine innervation of LHRH-producing neurons and progesterone receptor-containing cells indicate that neurons of the hypothalamic dopaminergic system probably mediate opiate effects on hypophyseal hormone secretion.

Proliferative activity of colonic mucosa at different distances from primary adenocarcinoma as determined by the presence of statin: a nonproliferation-specific nuclear protein.

The field change is one hypothesis concerning the development of colorectal carcinoma. Removal of a carcinoma without its entire surrounding altered mucosa may result in the development of a recurrence. S44, a monoclonal antibody directed against statin, a nuclear protein expressed in nonproliferating cells in either a quiescent or senescent state, was used to determine the rate of cell growth in colorectal mucosa at different distances from carcinomas. The specimens of 18 patients undergoing resection of a colorectal carcinoma were immediately opened after operation, and strips of mucosa were taken at distances of 1 cm, 5 cm, and 10 cm from the carcinoma. For each location, 10 longitudinally oriented crypts were evaluated for statin-positive cells identified by the presence of a dark brown peroxidase-conjugated antibody reaction product. The average percentage of statin-positive cells per crypt was significantly lower at a 1-cm distance from the carcinoma compared with the mucosa located 5 and 10 cm from the carcinoma (20.89 +/- 4.33 at 1 cm, 32.41 +/- 5.27 at 5 cm, and 34.23 +/- 6.45 at 10 cm). None of the calculated parameters showed any significant difference between the 5-cm and 10-cm locations. The fact that the proliferation rate of the mucosal cells returns to the normal level at 5 cm from the margin of the carcinoma suggests that cells located within this distance still retain proliferative potential even though they are morphologically indistinguishable from their normal counterparts. We conclude that failure to remove this transitional, potentially proliferative mucosa may result in subsequent development of anastomotic or perianastomotic recurrences.

Immunohistochemical evidence of endothelin-1 in human choroid plexus.

An immunohistochemical investigation was carried out on 17 specimens of human choroid plexus obtained post mortem, 1 biopsy of normal choroid plexus including part of the lateral ventricle and 1 papilloma of the choroid plexus removed surgically. The material was fixed in formalin. Paraffin and cryostat sections were used. A polyclonal antiserum to endothelin-1 served as a primary antibody. The avidin-biotin-peroxidase method was applied to demonstrate the immunoreaction. The epithelial cells of the choroid plexuses, the choroid papilloma and most ependymal cells of the lateral ventricle showed a distinct brown reaction product in their cytoplasm indicating antigenic sites to endothelin-1. The reaction was of lesser intensity in the ependymal cells. The connective tissue in choroid plexus was unstained. A positive immunoreaction was present in the walls of some vessels in the choroid plexus in cryostat sections. This is the first report on the presence of antigenic sites to endothelin-1 in the epithelial cells of the human choroid plexus. The role of endothelin in these cells should be investigated to ascertain if the cells synthesize this biologically active peptide or if it is merely bound to receptors in them.

Simultaneous characterization of efferent and afferent connectivity, neuroactive substances, and morphology of neurons.

We present a method for establishing in a single experiment four characteristics of individual neurons: the efferent and afferent connectivity, the morphology, and the content of a particular neuroactive substance. The connectivity of the neurons is determined by retrograde fluorescent tracing with Fast Blue and anterograde tracing with the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). After fixation, the brain is cut into 300-micron thick slices. Neurons containing retrogradely transported Fast Blue are intracellularly injected with the fluorescent dye Lucifer Yellow to fill their dendritic trees. The slices are then resectioned at 20-40 microns. One section through the soma of a Lucifer Yellow-filled neuron is selected for the detection of a neuroactive substance contained by this cell [immunofluorescence, secondary antiserum conjugated to tetramethylrhodamine (TRITC)]. Using appropriate filtering, it can be determined in the fluorescence microscope whether a Lucifer Yellow-containing cell body has also been labeled with TRITC, i.e., whether it is immunoreactive for this neuroactive substance. The adjacent sections are subjected to dual peroxidase immunocytochemistry with different chromogens to visualize the PHA-L-labeled afferent fibers (nickel-enhanced diaminobenzidine, blue-black reaction product) and to stabilize the Lucifer Yellow (diaminobenzidine, brown reaction product) in the dendrites of the intracellular injected cells. The other sections are used for electron microscopic visualization of the transported PHA-L. The relationships between the PHA-L-labeled afferent fibers (blue color) and the dendrites of the intracellularly Lucifer Yellow-injected, retrogradely Fast Blue-labeled cells (brown color) are studied by light microscopy. The electron microscope supplies ultrastructural data on the PHA-L-labeled axon terminals.

Antioxidative activity of browning reaction products isolated from storage-aged orange juice

The antioxidative activity of methanol extracts of browning reaction products isolated from stored orange juice on the oxidation of linoleic acid was investigated at pH 8.0. The antioxidative activity was assessed by conjugated diene formation from peroxidation of linoleic acid at 40 o C. Quantitative changes in linoleic acid were also measured by reversed-phase HPLC. Browning reaction products were found to possess potent antioxidant activity as measured by peroxidation of linoleic acid

Immunocytochemical localization of estrogen receptors within neurotensin cells in the rostral preoptic area of the rat hypothalamus

In situ hybridization procedures indicate that estrogen selectively increases neurotensin and neuromedin (NT/N) mRNA levels in the rostral preoptic area of the rat hypothalamus (RPH). Using the co-localization procedures of Axelson and Van Leeuwen. J. Neuroendocrinol., 2 (1990) 209–216, the present study examined whether NT cells in the RPH contained estrogen receptors (ER). Vibratome sections of brains from adult ovariectomized, colchicine-treated rats were first incubated with estrogen receptor antibody and stained with diaminobenzidine (DAB)-Ni + producing a blue-black nucleus. Subsequently, NT antisera were used to provide a brown reaction product with DAB as chromogen. Approximately 25% of the NT cells in the RPH contained ER. These data support the hypothesis that NT cells in the RPH that play a role in luteinizing hormone release from the pituitary are, in part, influenced directly by estrogen feedback via nuclear ER and may act as interneurons controlling luteinizing hormone releasing hormone turnover.

Effect of furfural on ethanol production by S. cerevisiae in a cross-linked immobilized cell reactor

Furfural, a browning reaction product, inhibits yeast ( Saccharomyces cerevisiae) growth and metabolism at low concentration levels in batch culture. The performance of an immobilized cell reactor (ICR) in the presence of 0–2.0 g 1- −t of furfural was examined. Cell growth in the ICR, with and without furfural in the media, indicated that either furfural did not impair glucose utilization, or that the negative effects of furfural were negated by increasing cell density in the reactor. Ethanol yields were constant at 0.48 g ethanol per g glucose regardless of the furfural concentration in the media. Although the specific productivity in the ICR decreased with furfural concentration, the productivity based on liquid hold-up remained constant. Furfural was depleted in the ICR during the experimental operation. Thus, furfural levels of 2.0 g I −1 or less can be tolerated by the yeast for ethanol production in the ICR without negatively affecting reactor performance.

The effects of furfural on ethanol production by saccharomyces cereyisiae in batch culture

Browning reaction products such as furfural and 5-hydroxy-methyl-furfural (HMF) have been shown to inhibit microbial growth and metabolism in ethanol fermentations using Saccharomyces cerevisiae. This paper quantifies the extent of furfural inhibition on yeast growth, glucose utilization, and ethanol production as a function of inoculum size (0.1–9 gl −1. Batch culture experiments were conducted using furfural concentrations in the range of 0 to 2.0 gl −1. and mathematical correlations were proposed and tested. The results indicate that the specific growth rate decreased with increasing furfural concentration and inoculum size, while the maintenance coefficients were unaffected. The apparent and true cell yield coefficients on glucose were depressed with the addition of furfural. Specific production rates were unaffected at the furfural levels used but ethanol inhibition was apparent. The specific production rate was less inhibited by ethanol at higher inoculum sizes. Global specific productivities were not affected by the presence of furfural. At a 0.1 gl −1. inoculum size, furfural depletion was complete within 15–20 h, depending upon the furfural concentration employed. At higher inoculum levels (2–9 gl −1. all furfural was depleted in less than 5 h.

Ultrastructural relationships between choline acetyltransferase- and neuropeptide Y-containing neurons in the rat striatum

The relationships between cholinergic and neuropeptide Y-containing neuronal systems in the rat striatum were examined using a dual immunoperoxidase labelling method. These neurons were identified by their immunoreactivity to choline acetyltransferase and neuropeptide Y, respectively, and were visualized on the same sections using 3,3′-diaminobenzidine and benzidine dihydrochloride as distinct chromogens under two conditions: (i) neuropeptide Y detection by the 3,3′-diaminobenzidine diffuse brown reaction product and choline acetyltransferase detection by the benzidine dihydrochloride blue, granular reaction product. (ii) choline acetyltransferase detection by 3,3′-diaminobenzidine and neuropeptide Y detection by benzidine dihydrochloride. Although both neuropeptide Y- and choline acetyl-transferase-immunoreactive cell bodies were simultaneously detected and were easily distinguishable whatever the conditions used, neuropeptide Y- and choline acetyltransferase-immunoreactive dendrites and axons could not be visualized on the same sections, since only the diaminobenzidine-labelled processes were detectable. Light microscopic observations on sections dual labelled with either method confirmed that choline acetyltransferase and neuropeptide Y immunoreactivities were localized in morphologically different populations of striatal neurons scattered throughout the striatum, choline acetyltransferase immunoreactivity being associated with large neurons and neuropeptide Y immunoreactivity with medium-sized neurons. In addition, the choline acetyltransferase-immunoreactive neurons were found to be more numerous than the neuropeptide Y-immunoreactive neurons and to be prevalent in the dorsolateral areas of the striatum, whereas neuropeptide Y-immunoreactive neurons were preferentially found in the ventromedial areas of this structure. Electron microscopic observations on sections processed under either condition revealed that choline acetyltransferase-positive terminals form synaptic contacts of the symmetrical type with neuropeptide Y-positive somata and proximal dendrites and that choline acetyltransferase-positive neurons are contacted by neuropeptide Y-positive terminals. These data show that the striatal neuropeptide Y- and choline acetyltransferase-containing neuronal systems have reciprocal synaptic interactions and provide morphological support for the hypothesis that striatal cholinergic and neuropeptide Y interneuron activities may be functionally linked.

Further immunohistochemical study of crooke's hyaline change.

In a previous study, we immunohistochemically investigated Crooke's hyaline change and concluded that it was composed of cytokeratin. The present study was undertaken to further identify the cytokeratin subfamily by immunohistochemistry. Twenty-eight postmortem, routinely processed pituitary glands revealing unequivocal Crooke's hyaline change were selected. To demonstrate Crooke's cellsand cytokeratin subfamilies simultaneously, serial hori zontal sections were sliced. Using an avidin-biotin peroxidase complex method, one was stained with a monoclonal antibody against synthesized adrenocorticotropic hormone (ACTH) 1-24, and the adjacent ones were stained with one of eight test monoclonal antibodies against cytokeratin subfamilies (containing cytokeratins 1, 3, 7, 8, 13, 18, and 19, and one containing 1,5, 10, and 11, respectively). A different antibody for each type of cytokeratin was applied. Crooke's cells showed a variable intensity of cytoplasmic stainingfor antibodiesagainst cytokeratins 8 and 18 (molecular weight 52.5 and 45 kD, respectively), from focal to more even and intense staining, revealing a characteristic wide brown ring around the nucleus or under the plasmalemma. The most severely affected cells were totally replaced by dark brown reaction products with no secretory granules detectable in the cytoplasm. However, Crooke's cells did not react with other test anticytokeratin antibodies. Thus far, it can be concluded that Crooke's hyaline change was composed of low-molecular-weight cytokeratin subfamilies 8 and 18, which are found in pairs in normal ACTH cells.

Role of Glycation in Modification of Lens Crystallins in Diabetic and Nondiabetic Senile Cataracts

To assess the significance of glycation, nonenzymatic browning, and oxidation of lens crystallins in cataract formation in elderly diabetic patients, we measured three distinct products of glycation, browning, and oxidation reactions in cataractous lens crystallins from 29 diabetic patients (mean +/- SD age 72.8 +/- 8.8 yr) and 24 nondiabetic patients (age 73.5 +/- 8.3 yr). Compounds measured included 1) fructoselysine (FL), the first stable product of glycation; 2) pentosidine, a fluorescent, carbohydrate-derived protein cross-link between lysine and arginine residues formed during nonenzymatic browning; and 3) N epsilon-(carboxymethyl)lysine (CML), a product of autoxidation of sugar adducts to protein. In diabetic compared with nondiabetic patients, there were significant increases (P less than 0.001) in HbA1 (10.2 +/- 3.1 vs. 7.1 +/- 0.7%), FL (7.6 +/- 5.4 vs. 1.7 +/- 1.2 mmol/mol lysine), and pentosidine (6.3 +/- 2.8 vs. 3.8 +/- 1.9 mumol/mol lysine). The disproportionate elevation of FL compared with HbA1 suggests a breakdown in the lens barrier to glucose in diabetes, whereas the increase in pentosidine is indicative of accelerated nonenzymatic browning of diabetic lens crystallins. CML levels were similar in the two groups (7.1 +/- 2.4 vs. 6.8 +/- 3.0 mmol/mol lysine), providing no evidence for increased oxidative stress in the diabetic cataract. Thus, although the modification of lens crystallins by autoxidation reactions was not increased in diabetes, the increase in glycation and nonenzymatic browning suggests that these processes may acclerate the development of cataracts in diabetic patients.

Immunohistochemical study of crooke’s hyaline change

Crooke's hyaline change of the human pituitary gland appears as an intracytoplasmic accumulation of fine filaments under electron microscopy. This study was attempted to identify the fine filaments by immunohistochemical methods. Twenty-eight postmortem, formalin-fixed or chrome-alum-fixed, paraffin-embedded pituitary glands revealing unequivocal Crooke's hyaline change on hematoxylin and eosin stain were selected for this study. To demonstrate Crooke's cells and fine filaments simultaneously, mirror image sections were sliced and stained with the following monoclonal antibodies using an avidin-biotin-peroxidase complex method: an antibody against synthesized adrenocorticotropic hormone 1-24, human cytokeratins (55-57 kilodalton [kd] and 68 kd), porcine vimentin (57 kd), porcine desmin (53 kd), bovine neurofilaments (70, 160, and 210 kd), human glial fibrilfary acidic protein (GFAP) (56 kd), and chicken actin. Crooke's cells showed a variable intensity of cytoplasmic staining for 55- to 57-kd cytokeratins, from focal to more even and intense staining revealing a characteristic wide brown ring around the nucleus or beneath the cell membrane. The most severely affected cells were totally replaced by dark brown reaction products with no secretory granules detectable in the cytoplasm. However, 68-kd cytokeratin could not be unequivocally demonstrated. Crooke's cells were all negative for vimentin, desmin, neurofilaments, GFAP, and actin. Thus far, it could be concluded that Crooke's hyaline change was composed of intermediate-subunit molecular weight cytokeratins that are normal constituents of the ACTH cell.

Aspartate-like immunoreactivity in vestibulospinal neurons in gerbils

In an effort to further characterize vestibulospinal pathways in the gerbil, immunocytochemistry was combined with retrograde identification of neurons. Vestibulospinal neurons were retrogradely labeled following injections of horseradish peroxidase into the cervical cord of anesthetized gerbils. Sections were reacted with nickel acetate-diaminobenzidine for horseradish peroxidase, giving a black reaction product. Sections were incubated in polyclonal antisera to aspartate, incubated in an avidin-biotin-peroxidase procedure, and reacted to give a brown reaction product. Alternatively, fluoro-gold was used as a retrograde tracer and aspartate-like immunoreactivity was demonstrated with avidin conjugated to Texas red. Cells stained with aspartate-like immunoreactivity, were located in all vestibular nuclei. Double-labeled cells were located in the medial nucleus and in the lateral vestibular nucleus where many of the large cells were double labeled.