Age-linked changes in the activity of enzymes of the tricarboxylate cycle and lipid oxidation, and of carnitine content, in muscles of the rat

The activities of citrate synthase, NAD-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase were measured in homogenates of soleus, diaphragm and heart muscles of the rat, in an attempt to define potential tricarboxylate cycle activity and its response to aging. Activities were significantly decreased in 24-month animals versus 6-month controls in every case (except 2-oxoglutarate dehydrogenase in heart muscle). Age-linked decrements were greatest in the soleus and least in heart. Cytochrome oxidase was measured as an index of total respiratory chain activity and decreased significantly in each case, with the smallest decrease in the heart. Acyl-CoA dehydrogenase and 3-hydroxyacyl-Co-A dehydrogenase were measured as an index of beta-oxidative activity; the former decreased in soleus and diaphragm, the latter in soleus and heart, with the decrease in the soleus being the greater. Carnitine acetyl- and palmitoyltransferases were measured, together with the muscle content of carnitine and acylcarnitine, as determining the potential rate of entry of acyl groups into the mitochondria for oxidation. Carnitine acetyltransferase activity was decreased with age in each of the muscles, but to the greatest extent in the heart. Carnitine palmitoyltransferase was decreased in both soleus and diaphragm. Carnitine content was decreased most in the soleus and the heart and to a lesser extent in the diaphragm. It is concluded that there is a generalized decline in oxidative activity in all of these muscles with age, on the basis of wet weight; this occurs to the greatest extent in the soleus and to the least extent in the heart. There is, in addition, a specific deficiency in the ability to oxidize fatty acids, relative to other substrates, in heart muscle.     

Subcellular and regional location of “brain” proteins BASP1 and MARCKS in kidney and testis

Proteins BASP1 and MARCKS are abundant in axonal endings of neurons. Similarly to brain-specific protein GAP-43, BASP1 and MARCKS are reversibly bound to the plasma membrane. These proteins control both actin polymerization and actin cytoskeleton binding to the membrane. Performing these functions, BASP1 and MARCKS take part in growth cone guidance during development and in neurotransmitter secretion in adults. These activities predetermine the pivotal role of BASP1 and MARCKS in learning and memory. BASP1 and MARCKS were also found in non-nerve tissues, in particular, in the kidney and testis. Evidently, the physiological roles of these proteins differ in different tissues. Correspondingly, their intracellular location and activities may not be similar to those in neurons. In this paper, we analyze subcellular fractions (cytoplasm and nuclei) of rat kidney and testis with the purpose of determining the intracellular location of BASP1 and MARCKS. Western blots demonstrated that in these tissues, as in the brain, both proteins are present in the cytoplasm of the cell. According to our immunohistochemical study, BASP1 and MARCKS are specifically distributed in the tissues studied. In kidney, both proteins are present in cells located in glomeruli. In the testicular tubules, BASP1 is mainly expressed at the late stage of spermatogenesis (in spermatids) and is preserved in mature spermatozoa, while MARCKS appears equally during all stages of spermatogenesis. MARCKS is not found in mature spermatozoa. The results indicate that study of functions of BASP1 and MARCKS in the kidney and in the reproduction system holds much promise.     

Morphometric study of ultrastructural changes in oligodendroglial cells in the postmortem brain in endogenous psychoses

A qualitative and quantitative electron microscopic study of oligodendroglial cells was performed in autoptic (4-6.5 hours after death) prefrontal area 10 in 16 cases of schizophrenia, 6 cases of bipolar affective disorder and 16 normal controls, as well as in the caudate nucleus in same schizophrenic and control cases. The signs of reactive, regressive, and progressive changes of oligodendroglia were described in endogenous psychoses. ANOVA demonstrated a significant decrease in the area of the nucleus, in the volume density of euchromatin, in the volume density and count of mitochondria in oligodendroglial cells in the caudate nucleus and prefrontal area. In affective psychosis, there was a significant reduction in the area of the nucleus and in the volume density of euchromatin and slight changes in cellular organelles. No correlation between the changes and the postmortem interval, age, and neuroleptic therapy, as well as the most pronounced changes in oligodendroglial cells in subgroups of continuous schizophrenia and those with predominantly negative symptoms suggest the involvement of abnormal oligodendroglial cells in the pathogenesis of endogenous psychoses.     

Morphofunctional state of the adrenal glands in albino rats under conditions of toxic stress caused by cadmium salt in winter and summer periods

We studied the morphology and function of the adrenal glands in male and female albino rats in cadmium intoxication during winter and summer periods (January and July). In animals of the control group, sex-related differences in the total area of the adrenal glands and in the size of their zones were revealed. In females, zones of adrenal gland were larger than in males. In winter months, these differences were most pronounced. Analysis of seasonal differences in the area of the adrenal glands in males revealed no significant differences in winter and summer months. Irrespective of the season and gender, cadmium chloride treatment led to an increase in the size of the adrenal glands. Cadmium salts caused more pronounced functional strain in males in winter months and in females in summer.     

Distribution of capsaicin-sensitive substance P- and calcitonin gene-related peptide-immunoreactive nerves in bovine respiratory tract

The distribution of nerve fibers immunoreactive for substance P (SP) and calcitonin gene-related peptide (CGRP) was examined by means of immunohistochemical methods in the respiratory tract from nose to lung of normal and capsaicin-treated cattle. SP- and CGRP-immunoreactive (IR) nerve fibers with varicosities were detected in all portions. They were more numerous in calves than in cows. They were abundant in the nasal and laryngeal mucosae and tracheal bronchus, and few in number in the lung. SP- and CGRP-IR nerve fibers were mainly seen in the epithelium, in connective tissue beneath the epithelium and around blood vessels, and in the glands throughout the respiratory tract. In contrast, SP- and CGRP-IR nerve fibers were sparse in the smooth muscle layer. Capsaicin treatment of neonates caused a remarkable reduction in the number of SP- and CGRP-IR nerve fibers in the respiratory tract of calves. Double immunofluorescence experiments showed the colocalization of SP and CGRP in most of the nerve fibers. The present findings suggest that SP- and CGRP-IR nerve fibers are involved in the regulation of the bovine respiratory tract, and that capsaicin-sensitive SP- and CGRP-IR nerve fibers are sensory neurons of the bovine respiratory tract.     

Localization of alkaline and acid phosphatase activity in the intestine of healthy breams (Abramis brama l.) and those infected with plerocercoid of tapeworm Ligula intestinalis (Linné 1758).

The examination included 40 breams 4 to 7 years old (18 infected and 22 uninfected). Alkaline and acid phosphatase activity, as shown by the azo-coupling method, has been localized in the oesophagus and the intestine, being the strongest in the epithelium. It was distinctly less intensive in the Lamina propria mucosae and in the intermuscular connective tissue of the oesophagus, in the submucosa, in the cells of AUERBACH plexus and in the blood vessel walls. Besides only the acid phosphatase activity was noted in single (sometimes rather numerous) spherical cells - visible within the epithelium and Lamina propria mucosae. The cells are known as the components of so called "yellow bodies" (melanine macrophage centers) entering particular numerously in the spleen and in the pronephric kidney of infected breams. The activity of both enzymes in the epithelium was considerably weaker in the last third of the intestine, and none in cloaca and in Tunica muscularis all over the length of the intestine (and oesophagus) except for some cells of the connective tissue separating the layers of muscle fibres. No perceptible differences in the activity and localization of both enzymes in the intestine were observed between infected and uninfected fishes examined in different seasons of the year.     

Biocompatibility and degradation of poly(ether-ester) microspheres: in vitro and in vivo evaluation

Microspheres of a hydrophobic and a hydrophilic poly(ether–ester) copolymer were evaluated for their in vitro and in vivo biocompatibility and degradation. The microspheres prior to and after sterilization were tested for in vitro cytotoxicity. The in vivo biocompatibility of the poly(ethylene glycol) terephthalate and poly(butylene terephthalate) (PEGT/PBT) microspheres was evaluated subcutaneously and intramuscularly for 24 weeks in rabbits. The in vivo degradation of the microspheres was studied microscopically and compared to the in vitro degradation. The in vitro and in vivo studies showed the biocompatibility of the microspheres of both the hydrophobic and the hydrophilic PEGT/PBT copolymer. Extracts of these microspheres showed no cytotoxic reactivity in the in vitro cytotoxicity test. Sterilization of the microspheres by gamma irradiation did not affect the cytotoxicity. PEGT/PBT microspheres injected subcutaneously and intramuscularly in rabbits showed a mild tissue response in vivo, in terms of the inflammatory response, the foreign body reaction and the granulation tissue response. Although an in vitro degradation experiment showed a decrease in molecular weight due to hydrolysis, the in vivo degradation of the microspheres was slower than previously published.     

Alterations in bronchoalveolar lavage constituents, oxidant/antioxidant status, and lung histology following intratracheal instillation of respirable suspended particulate matter.

Urban suspended particulate pollutants differ with place of occurrence, meteorological conditions, physicochemical compositions, and the response of the bronchopulmonary apparatus. Lung injury following intratracheal instillation of respirable suspended particulate matter (RSPM) collected in an urban setting in India was investigated in rats. The animals were killed 15 days after exposure to 2.5, 5.0, and 10.0 mg of RSPM. We examined the changes in lung histology, enzymatic activity in the bronchoalveolar lavage (BAL), and the oxidant/ antioxidant status in lung homogenates.The alterations in these parameters were compared with those in rats instilled with quartz particulates, which were used as positive controls. Exposure to RSPM resulted in an increase in the relative weight of lungs and inflammatory changes evidenced by an increase in the total cellularity of the lungs, predominantly polymorphonuclear cells, demonstrable both in the lungs sections and in the bronchoalveolar lavage of the exposed animals. An increase in the protein content and in the lactate dehydrogenase activity in the BAL was found in the RSPM-exposed rats. A marked increase in the output of lipid peroxides and a dose-dependent increase in the formation of reactive nitrogen species (NO) in lung homogenates and BAL, respectively, was found in the RSPM-exposed rats. A significant decrease in the enzymatic lung antioxidants, superoxide dismutase, and catalase was observed. However, the alterations in the levels of glutathione in the lungs of the RSPM-exposed animals were not significant. The inflammatory reaction, oxidative changes, and enzyme release, were more marked in quartz-exposed animals in comparison to the RSPM-exposed rats.     

The Dopaminergic System of the Telencephalo-Diencephalic Areas of the Vertebrate Brain in the Organization of the Sleep–Waking Cycle

The aim of the present work was to study the involvement of the dopaminergic system of the telencephalic and diencephalic areas of the vertebrate brain in the organization of the sleep–waking cycle in cold-blooded and warm-blooded vertebrates. Immunohistochemical studies of tyrosine hydroxylase content, this being the key enzyme in dopamine synthesis, in the striatum, supraoptic and arcuate nuclei, and zona incerta of the hypothalamus of sturgeon and mammals (rats) of three age groups (14 and 30 days and adults), in conditions of tactile and sleep-deprivation stressors. In fish, transient stress was followed by the detection of tyrosine hydroxylase-immunoreactive cells in all parts of the brain. In prolonged stress, tyrosine hydroxylase-immunoreactive cells and fibers were not found in the forebrain, though they were well represented in the hypothalamic nuclei. In 14-day-old rat pups, 2-h sleep deprivation increased the tyrosine hydroxylase content of fibers in the caudate nucleus and cells in the zona incerta of the hypothalamus, while 30-day-old animals subjected to 6-h sleep deprivation showed increases in tyrosine hydroxylaseimmunoreactive material contents in cells in the paraventricular nucleus and decreases in the quantity in fibers. In adult rats, the arcuate nucleus and zona incerta showed decreases in the content of tyrosine hydroxylase-immunoreactive material on the background of sleep deprivation, with increases during postdeprivation sleep. These data are discussed in the light of the phylo- and ontogenetic development of the neurosecretory and neurotransmitter functions of the dopaminergic system in the evolutionarily ancient diencephalic and evolutionarily young telencephalic areas of the vertebrate brain as major systems triggering and maintaining the functional states of the body during the sleep–waking cycle.     

Immunohistochemical study of fibronectin in experimental myocardial infarction.

Light microscopic immunohistochemical studies were performed to evaluate the distribution of fibronectin in paraffin sections of p-formaldehyde-fixed normal rat hearts and the hearts of rats that had undergone ligation of the left coronary artery. A peroxidase-labeled antibody technique was used, together with appropriate immunohistochemical control procedures, for the localization of fibronectin in normal hearts and in the hearts of sham-operated animals. Fibronectin was localized in the interstitial space between myocytes, and beneath arterial, venous, and capillary endothelium. At 4 hours after coronary ligation, fibronectin was localized in a patchy fashion in the cytoplasm and interstitial space of some of the myocytes in the area supplied by the ligated vessel. At 24 hours, there was more intense, homogeneous staining in necrotic myocytes in the infarcted area and in the capillary endothelium in the border zone. At 48 hours, the intensity of staining for fibronectin was maximal in and between the necrotic myocytes in the center of the infarct and in proliferating and migrating capillaries and fibroblasts in the border zone. Similar patterns of localization were observed at 3 and 7 days after coronary ligation, but with progressive decreases in the intensity of staining. Two sources of fibronectin appeared to have contributed to these changes: plasma fibronectin diffusing through damaged blood vessels would account for the early staining observed in necrotic myocytes in the center of the infarct, whereas de novo synthesis of fibronectin by connective tissue cells and endothelial cells in sprouting capillaries would be responsible for the subsequent staining observed in viable capillaries in the border zone of the infarct. Known properties of fibronectin in vitro, combined with these in vivo observations, indicate that fibronectin may influence the thrombotic, inflammatory, angiogenic, and fibrotic processes involved in infarct healing.     

Change in intramembranous particle distribution in epididymal spermatozoa of the boar

Membranes of boar spermatozoa from different regions of the epididymis and after ejaculation were studied by the freeze-fracture replica technique. The ordered pattern of the intramembranous particles of spermatozoan plasma membranes was different in the five arbitrary zones of the epididymis and in the semen. A distinctive ordered pattern was absent in zone 1, which is the proximal segment of the epididymis. In zone 2, paired parallel rows of the particles were present in the plasma membrane over the acrosomal region. This parallel arrangement was not present in zone 3 spermatozoa. Anterior to the posterior ring, cords formed by packed particles were apparent in zone 2 spermatozoa and reached their maximum prominence in zone 3, and persisted in zones 4 and 5 and in the semen. The plasma membrane over the marginal ridge of the acrosome had a hexagonal array of particles only in zones 4 and 5 spermatozoa. A similar pattern appeared on the post-acrosomal region of spermatozoa in zone 5 and in the semen. The plasma membrane of the middle piece had a rectilinear arrangement of the particles in zone 2 spermatozoa in which the migration of the cytoplasmic droplet was complete. Rudiments of the rectilinear arrangement persisted in spermatozoa in zones 4 and 5 and in the semen. These changes are discussed in relation to sperm maturation in the epididymis. The acrosomal membrane had a hexagonal arrangement of particles in the equatorial segment. The marginal ridge of the outer acrosonal membrane had parallel rows of intramembranous particles. The organization of the acrosomal membrane particles did not change during the epididymal passage of boar spermatozoa.     

食管癌血管内皮生长因子和受体的表达及在癌转移中的作用

目的观察人食管癌组织和淋巴管中血管内皮生长因子(VEGF-C)及其受体-3(VEGFR-3)的表达,探讨食管癌的淋巴道转移机制。方法取临床手术切除的食管癌组织,用免疫组化方法检测人食管癌早期和进展期癌细胞或淋巴管对VEGF-C及其VEGFR-3的表达情况。结果在食管癌的癌细胞中可见VEGF-C阳性表达,阳性颗粒主要定位于肿瘤细胞胞浆内。淋巴管内皮细胞仅见VEGFR-3阳性表达,VEGFR-3在血管和癌细胞中也存在少量阳性表达。进展期食管癌VEGF-C和VEGFR-3的表达率和表达强度均强于早期。结论食管癌癌细胞VEGF-C的表达和淋巴管内皮细胞上VEGFR-3的表达均与肿瘤进展呈正相关,推测VEGF-C通过受体VEGFR-3促进食管癌组织淋巴管生成,从而引起癌淋巴道转移。     

大鼠嗅结节与黑质间的往返投射

将ＨＲＰ或ＷＧＡ－ＨＲＰ注入大白鼠一侧的嗅结节，凡注射涉及嗅结节的多形层者均在注射黑质致密部－Ａ８，Ａ９区出现标记细胞，标记细胞主要位于黑质致密部的内侧，其数量由内向外递减，黑质致密内部的标记细胞以吻侧１／３段最多，中１／３段次之，尾１／３段最少。在ＷＧＡ－ＨＲＰ注射区涉及嗅结节的多形层的各例，除看到黑质致密部中的标记细胞外，还可见密集的成串珠样和点状的标记终末，黑质致密部内的标记终末分布于吻尾方     

Association of Brain Weight Value with Morphometric and Histochemical Properties of Cortical Neurons and Higher Nervous Activity Parameters in Rats in Prepubertal Period

We compared thickness of the neocortex, morphometric and histochemical characteristics of neurons in frontoparietal and parietal lobes and hippocampal field I in animals from great litters with lower brain weight (group 1) and from small (artificially reduced) litters with higher brain weight (group 2). It was found that the thickness of the neocortex in the frontoparietal and parietal lobes does not differ in the compared groups, while the size of neuronal cytoplasm in layer II of the frontoparietal and parietal lobes and in layer V of the frontoparietal lobe in group 2 animals was lower than in group 1. Nuclei of cortical neurons in layer II of the parietal and frontoparietal lobes and in frontoparietal lobe layer V in group 2 animals were smaller than in group 1. Neuronal nucleoli in group 2 animals were also smaller than in group 1 rats. RNA concentration in neuronal cytoplasm in the hippocampus and neocortex of group 2 rats was higher than in group 1 animals. NADPH-dehydrogenase activity in neurons of parietal lobe (layer II) and hippocampus in group 2 rats was lower than in group 1 animals, NADH-dehydrogenase activity was lower in parietal lobe layer II neurons. Group 2 rats demonstrated increased number of hanging down, sniffing, movements, entries into open and closed arms, and lower immobility time in the elevated plus-maze test.     

Characteristics of behavior and stress reactivity of the hypophyseal-adrenocortical system in rats with prenatal inhibition of testosterone metabolism

The effects of administration of the aromatase blocker 1,4,6-androstatrien-3,17-dione (ATD) to female rats in the last third of pregnancy on the stress reactivity of the hypophyseal-adrenocortical system (HACS), behavior in a novel environment (an open field), and anxiety in an elevated cross maze in their adult offspring of both genders were studied. Inhibition of testosterone aromatization in the brain during the prenatal period of development was found to lead to a decrease in the basal activity of the HACS in males and longer-lasting hormonal stress responses in animals of both genders. However, the intergender differences in the nature of the stress reactivity of the system in the experimental animals persisted. Prenatal administration of ATD also induced increases in the levels of anxiety and emotionality and the duration of grooming reactions in males and females and eliminated intergender differences between control males and experimental females in terms of measures of behavior in a new environment such as movement activity, duration of the freezing reaction, and grooming. These data led to the conclusion that impaired testosterone metabolism in the brain during the prenatal period of development induced by administration of the aromatase blocker leads to changes in the nature of the stress response of the HACS in adult male and female rats and impairs the formation of sexual dimorphism in anxiety levels and the extent of behavioral reactions to environmental novelty in females. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 9, pp. 1071–1079, September, 2005.     

Body height and weight of children in Novi Sad

BACKGROUND: Height and weight are the most important measures of growth and development and indirect indicators of living conditions. The socio-economic conditions in Serbia have varied drastically. In 1990, a political and economic crisis started causing a rapid fall of standards of living. AIM: The study aimed to determine the height and weight of children aged 3-11 in the periods between 1971, 1981, 1991 and 2001 in Novi Sad, Serbia. SUBJECTS AND METHODS: This investigation was carried out in the same primary schools and pre-school institutions. The data included children of Serbian nationality born in Novi Sad whose parents were also born there and had the same socio-economic background. RESULTS: Positive changes in height and weight were recorded in the decades 1971-1981 and 1981-1991, except for the height of 8-year-old children. In the period 1991-2001, an increase in height was observed only at the age of 8 in boys and until the age of 9 in girls. As for weight, an increase was recorded in 9-year-old boys, while in girls it was present at all ages except for the ages of 7 and 10. Considering the period 1971-2001, positive changes in height were recorded from the age of 6 in boys and 5 in girls. The changes in weight were positive at all ages except the age of 5 in boys and after the age of 6 in girls. CONCLUSION: Lower values of height and weight recorded in 2001 are probably due to the changes in living conditions or they indicate that the acceleration reached its peak.     

Expression of epithelial growth factor receptor and its two ligands, transforming growth factor-alpha and epithelial growth factor, in normal and neoplastic squamous cells in the vulva: an immunohistochemical study

Epithelial growth factor receptor (EGFR) sends signals to the proliferation signal transduction system, receiving two ligands: epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). This immunohistochemical study examined the roles of EGFR and its ligands in the proliferation of normal and neoplastic vulvar squamous cells in 25 patients with vulvar squamous cell carcinoma (VSCC), 10 patients with vulvar condyloma acuminata (VCA), 15 patients with vulvar intra-epithelial neoplasm I-II or III (VIN I-II or III), and 5 subjects with vulvar normal squamous cells (VNSC). EGFR was detected in a few basal cells in 40% of the VNSC, in highly dysplastic cells in 40% of the VIN III, in many neoplastic cells in 80% of the VCA, and in some malignant cells in 64% of the VSCC. EGF was seen in the cytoplasm in 20% of the VIN I-II, 100% of the VIN III, 100% of the VCA, and 100% of the VSCC. Diffuse TGF-alpha was weakly expressed in the cytoplasm in 100% of the VNSC, more intensely in 100% of the VIN and 100% of the VCA, and intensely in 100% of the VSCC. These findings led to the suggestion that the TGF-alpha-EGFR system maintains the growth of normal squamous cells and, in part, maintains the growth of dysplastic and neoplastic squamous cells in the vulva. EGF expression was an early sign of neoplasia. The expression of EGFR with overexpression of its two ligands contributed to the proliferation of dysplastic and neoplastic squamous cells in VIN III and VCA. EGFR expression appeared to contribute to essential neoplastic abnormalities in 64% of the VSCC.     

Phosphorylation of tau and alpha-synuclein in synaptic-enriched fractions of the frontal cortex in Alzheimer's disease, and in Parkinson's disease and related alpha-synucleinopathies

Phosphorylation of tau and phosphorylation of alpha-synuclein are crucial abnormalities in Alzheimer's disease (AD) and alpha-synucleinopathies (Parkinson's disease: PD, and dementia with Lewy bodies: DLB), respectively. The presence and distribution of phospho-tau were examined by sub-fractionation, gel electrophoresis and Western blotting in the frontal cortex of cases with AD at different stages of disease progression, PD, DLB pure form and common form, and in age-matched controls. Phospho-tauSer396 has been found in synaptic-enriched fractions in AD frontal cortex at entorhinal/transentorhinal, limbic and neocortical stages, thus indicating early tau phosphorylation at the synapses in AD before the occurrence of neurofibrillary tangles in the frontal cortex. Phospho-tauSer396 is also found in synaptic-enriched fractions in the frontal cortex in PD and DLB pure and common forms, thus indicating increased tau phosphorylation at the synapses in these alpha-synucleinopathies. Densitometric studies show between 20% and 40% phospho-tauSer396, in relation with tau-13, in synaptic-enriched fractions of the frontal cortex in AD stages I-III, and in PD and DLB. The percentage reaches about 95% in AD stage V and DLB common form. Yet tau phosphorylation characteristic of neurofibrillary tangles, as revealed with the AT8 antibody, is found in the synaptic fractions of the frontal cortex only at advanced stages of AD. Increased phosphorylated alpha-synucleinSer129 levels are observed in the synaptic-enriched fractions of the frontal cortex in PD and DLB pure and common forms, and in advanced stages of AD. Since tau-hyperphosphorylation has implications in microtubule assembly, and phosphorylation of alpha-synuclein at Ser129 favors alpha-synuclein aggregation, it can be suggested that synapses are targets of abnormal tau and alpha-synuclein phosphorylation in both groups of diseases. Tau phosphorylation at Ser396 has also been found in synaptic-enriched fractions in 12-month-old transgenic mice bearing the A53T alpha-synuclein mutation.     

Different expression of 25-kDa heat-shock protein (Hsp25) in Meckel's cartilage compared with other cartilages in the mouse

The 25-kDa heat-shock protein (Hsp25) is expressed in the cartilage of the growth plate and suggested to function in chondrocyte differentiation and degeneration. Using immunohistochemistry, we examined the temporal and spatial occurrence of Hsp25 in Meckel's cartilage in embryonic mice mandibles, and in other types of cartilage in both embryonic and adult mice. In adults, Hsp25 immunoreactivity was detected in the hypertrophic chondrocytes located in growth plates of long bones and in non-osteogenic laryngeal and tracheal cartilages. No chondrocytes in the resting or proliferating phase exhibited Hsp25 immunoreactivity. In the embryonic mandibles, resting and proliferating chondrocytes in the anterior and intermediate portions of Meckel's cartilage showed Hsp25 immunoreactivity from the 12th day of gestation (E12) through E15, whereas those in the posterior portion showed little or no immunoreactivity. After E16, the overall Hsp25 immunoreactivity in Meckel's cartilage substantially reduced in intensity, and little or no immunoreactivity was detected in the hypertrophic chondrocytes located in the degenerating portions of Meckel's cartilage. The antisense oligonucleotide for Hsp25 mRNA applied to the culture media of the mandibular explants from E10 embryos caused significant inhibition of the development of the anterior and middle portions of Meckel's cartilage. These results suggested that Hsp25 is essential for the development of Meckel's cartilage and plays different roles in Meckel's cartilage from those in the permanent cartilages and the cartilages undergoing endochondral ossification.     

Alterations in subcellular localization of TDP-43 immunoreactivity in the anterior horns in sporadic amyotrophic lateral sclerosis

TDP-43 is ubiquitously expressed in the nucleus of motor neurons and is closely associated with the pathogenesis of amyotrophic lateral sclerosis (ALS). However, little is known about alterations in the subcellular or intracellular localization of TDP-43, either under normal conditions or in ALS. We examined the anterior horn neurons of the spinal cord in patients with sporadic ALS and age-matched controls immunohistochemically and immunoelectron-microscopically using anti-TDP-43 antibody. Immunohistochemically, the present study showed a decrease in TDP-43 immmunoreactivity in the nucleus and, by contrast, an increase in the cytoplasm in ALS patients. Immunoelectron-microscopically, we demonstrated the consistent presence of TDP-43-immunogold-labeled deposits primarily in the nucleus, particularly in the nucleolus, and frequently in the rough endoplasmic reticulum (rER), and, to a lesser extent, in the mitochondria and the synaptic vesicles of the presynaptic terminals on the surface of anterior horn neurons both in controls and ALS subjects. In ALS, a reduced number of TDP-43-immunogold-labeled deposits were observed in the nuclei, particularly in the nucleoli of even normal-looking motor neurons. In contrast, the number of TDP-43-immunogold-labeled deposits in the rER of the normal-appearing motor neurons was significantly larger in ALS than in the controls (p = 0.0036). These findings suggest that TDP-43 is synthesized in the rER and translocates to the nucleus, particularly to the nucleolus, and in ALS, TDP-43 trafficking between the nucleus and the cytoplasm is disturbed, resulting in an accumulation of TDP-43 in the cytoplasm in the form of insoluble aggregates.