Facial Aesthetics in Young Adults after Cleft Lip and Palate Treatment over Five Decades.

Cleft Lip and Palate (CLP) - a common facial malformation in newborns - is typically corrected by surgical intervention to allow for normal speech development, psychosocial adjustment, and facial attractiveness. The long term treatment outcome can be evaluated after a number of years, possibly in adulthood. We investigated the aesthetics of the nasolabial region by subjective ratings. To compare various surgical approaches we recruited 12 raters to evaluate 429 patients. Expert and lay raters judged photographs from patients, who have completed treatment with one of three different surgical strategies performed in our institution over 50 years. Facial photographs were cropped, presented to the raters in a randomized sequence, and judged by the raters on a 5 point Likert scale. The subjective ratings between the raters revealed a fair to substantial inter-rater reliability. The average ratings of the surgical outcome improved continuously over the investigated 5 decades. Despite possible differences between raters and rater groups this overall result was consistently seen in the gender groups (male/female), or expertise related groups (expert/lay). Our analysis revealed that patients with bilateral CLP scored worse than patients with unilateral CLP when treated in the fifties; more recently treated patients of both groups scored similarly.

Nanoparticle-mediated treatment of pulmonary arterial hypertension.

Nanomedicine is an emerging field with great opportunities to improve the treatment of diseases which are currently not curable. Pulmonary arterial hypertension (PAH) is one of these diseases treatable by inhalation of medicines that provide novel depots for drugs with short pharmacological half-lives to improve the quality of life for patients. In this context, nanostructured drug delivery systems such as liposomes and polymeric nanoparticles are depot forms that can also act as penetration enhancers and solubilizers of drugs. The pulmonary use of these drug carriers will improve the therapeutic effect of potent drugs that are currently not fully applicable. This review focuses on the design and characterization of drug delivery systems with the potential to improve the treatment options for hypertonic conditions (like PAH). Liposomes as well as polymeric nanoparticles based on lactic acid, proticles and nanocrystalline drugs have good potential to be developed toward clinical use. Preparation methods and characterization techniques of nanoparticles such as light scattering or microscopy are provided.

Pulmonary and systemic effects of inhaled PACAP38 in healthy male subjects.

BACKGROUND: Pituitary adenylate cyclase activating polypeptide 1-38 (PACAP38) displays biological activities (e.g. bronchodilatory, pulmonary vasodilatory and anti-inflammatory properties) that are relevant in several pulmonary diseases. The aim of this study was to assess the safety and tolerability and the pulmonary and systemic effects of inhaled PACAP38 in humans. MATERIALS AND METHODS: Twelve healthy male subjects (mean age 28) were studied in a randomized, double-blind, placebo-controlled dose escalation trial with inhalation of PACAP38 to a cumulative dose of 480 microg. Lung function was measured by body plethysmography. Systemic absorption was evaluated by plasma levels, skin blood flux (estimated by laser Doppler imager fluxmetry) and systemic haemodynamics. RESULTS: Inhalation of PACAP38 did not cause relevant adverse reactions or an increase of PACAP38 plasma levels. No statistically significant changes in lung function tests and no systemic effects (blood pressure, pulse rate or skin blood flux) occurred. CONCLUSION: Inhaled PACAP38 was well tolerated without systemic side-effects in healthy male subjects.

Long-term sequelae of perinatal asphyxia in the aging rat.

Information on the consequences of perinatal asphyxia (PA) on brain morphology and function in the aging rat is missing although several groups have hypothesized that PA may be responsible for neurological and psychiatric deficits in the adult. We therefore decided to study the effects of PA on the central nervous system (CNS) in terms of morphology, immunohistochemistry, neurology and behavior in the aging animal. Hippocampus and cerebellum were evaluated morphologically by histological, immunohistochemical and magnetic resonance imaging and cerebellum also by stereological tests. Neurological function was tested by an observational test battery and rota rod test. Cognitive functions were examined by multiple-T-maze and the Morris water maze (MWM). Increased serotonin transporter (SERT) immunoreactivity in the CA2 region of the hippocampus and a significant difference in the escape latency, when the platform of the MWM was moved to a new location, were observed in asphyxiated rats. We showed that deteriorated cognitive functions accompanied by aberrant expression of hippocampal SERT and impaired relearning are long-term sequelae of perinatal asphyxia, a finding that may form the basis for understanding CNS pathology in the aging subject, animal or human.

Aerosolised iloprost improves pulmonary haemodynamics in patients with primary pulmonary hypertension receiving continuous epoprostenol treatment.

BACKGROUND: Continuous intravenous treatment with epoprostenol significantly improves pulmonary haemodynamics and survival in patients with primary pulmonary hypertension (PPH). Its beneficial effect, however, may be blunted due to adverse effects such as catheter sepsis and systemic hypotension. Recent investigations have shown that inhaled iloprost is effective in the treatment of PPH. Based on their different pharmacokinetics, we hypothesised that the combination of intravenous epoprostenol and inhaled iloprost would be more efficacious than epoprostenol alone during acute testing in patients with PPH. METHODS: The effect of a single dose of inhaled iloprost (30 microg total over 15 minutes) on pulmonary haemodynamics was examined in eight patients with PPH (initial non-responders to nitric oxide) who had considerable adverse effects during treatment with epoprostenol. RESULTS: The combination of inhaled iloprost and intravenous epoprostenol significantly improved mean pulmonary artery pressure (MPAP), cardiac index (CI), mixed venous oxygen saturation (SvO2), and systemic arterial oxygen pressure (PaO2) compared with epoprostenol treatment alone. Mean systemic arterial pressure (MSAP) and pulmonary capillary wedge pressure (PCWP) remained unchanged. CONCLUSIONS: The pulmonary vasoreactivity shown by additional iloprost inhalation during effective epoprostenol treatment suggests that an improvement of treatment for pulmonary hypertension may be possible by combining vasoactive substances.

Nuclear Ras: unexpected subcellular distribution of oncogenic forms.

The Harvey-ras gene encodes small guanine nucleotide binding proteins, mutant forms of which are associated with a number of human malignancies. Based on studies with truncated forms of the protein it is known that correct post-translational processing of Ras is essential for cytoplasmic membrane localization and function. Surprisingly, immunofluorescence analysis provided evidence that in addition to its cytosolic localization, activated H-Ras(Val 12) was also localized in the nuclei of transformed cells both in vitro and in vivo. Immunoblot analysis of nuclear fractions was consistent with results found by immunohistochemistry. Moreover, inhibition of protein farnesylation prevented the nuclear targeting of activated H-Ras(Val 12) and NFkappaB. Alterations in subcellular distribution pattern and phosphorylation of the cell cycle inhibitor p27, which is involved in Ras driven tumor growth, coincided with nuclear localization of H-Ras(Val 12). Proteins are often not functional until they are transported to their final destination. Indeed, Ras was found to complex with NTF2 a factor involved in nuclear protein import and export. Therefore it is suggested that NTF2 is the actual carrier for oncogenic Ras. In view of these observations the question arises whether the nuclear localization of H-Ras(Val 12) in tumors is important in oncogenic activation or whether it is a response to apoptosis. J. Cell. Biochem. Suppl. 36: 1-11, 2001.

Arrangement of ribosomal genes in nucleolar domains revealed by detection of "Christmas tree" components.

We investigated how the transcribing ribosomal genes ("Christmas trees") of HeLa cells are arranged in the nucleolus. Hypotonic conditions let the granular component disperse, while fibrillar centres and parts of the dense fibrillar component were resistant to low ionic strength conditions. Both remained within the former nucleolar territory. We used immunocytochemistry and in situ hybridisation at the light microscopic and ultrastructural level for the analysis of the internal nucleolar structures. The 5' ends of ribosomal RNA and ribosomal DNA sequences were found associated with the periphery of fibrillar centres. The hypotony-resistant parts of the dense fibrillar component did not contain the 5' end of the transcript or the gene. The downstream ribosomal DNA sequences were found in the nucleolar territory but not associated with any hypotony-resistant structures. The downstream ribosomal RNA revealed a similar distribution. We show that transcription initiation and transcript elongation occur in different molecular and structural environments. Transcription initiation is located at the periphery of fibrillar centres. Evidently the dense fibrillar component is non-homogeneous in molecular composition. Transcript elongation is continued in a part of the dense fibrillar component which is dissolved under intermediate hypotonic conditions. A structural model of nucleolar transcription is suggested.

Long term neurological and behavioral effects of graded perinatal asphyxia in the rat.

Perinatal hypoxic-ischemic states can cause irreversible damage to the brain, ranging from minimal brain dysfunction to death. Only few studies have been reported describing neurological, cognitive and behavioral deficits following perinatal asphyxia. We therefore decided to study long term effects of perinatal asphyxia in a well-documented animal model resembling the clinical situation. Caeserean section in rats was performed and the pups, still in the uterus horns, were placed into a water bath at 37 degrees C for periods of 5-20 min; pups were then given to surrogate mothers and examined at three month of age. Examinations consisted of a battery of motor and reflex tests, Morris water maze, multiple T-maze, elevated plus maze and open field studies. No abnormalities were found in rats even with long periods of perinatal asphyxia by neurological examination, in the open field and in mazes. Interestingly, in the elevated plus maze rats with long lasting exposure to hypoxia (15 and 20 min of asphyxia) showed reduced anxiety-related behavior. This finding may be relevant for the explanation of anxiety related disorders in adulthood with a tentative history in the perinatal period.

Brain RNA polymerase and nucleolar structure in perinatal asphyxia of the rat.

Ribosomes are integral constitutens of the protein synthesis machinery. Polymerase I (POL I) is located in the nucleolus and transcribes the large ribosomal genes. POL I activity is decreased in ischemia but nothing is known so far on POL I in perinatal asphyxia. We investigated the involvement of POL I in a well-documented model of graded systemic asphyxia at the level of activity, mRNA, protein, and morphology. Caeserean section was performed at the 21st day of gestation. Rat pups still in the uterus horns were immerged in a water bath for asphyctic periods from 5-20 min. Brain was taken for measurement of pH, nuclear POL I activity, and mRNA steady state, and protein levels of RPA40, an essential subunit of POL I and III. Silver staining and transmission electron microscopy with morphometry when appropriate were used to examine the nucleolus. Brain pH and nuclear POL I activity decreased with the length of the asphyctic period while POL-I mRNA and protein levels were unchanged. Accompanying the decrease in brain pH we found significant changes of nucleolar structure in the course of perinatal asphyxia at the light and electron microscopic level. As early as ten min following the asphyctic insult, morphological disintegration of the nucleolus was observed. The changes became more dramatic with longer duration of perinatal asphyxia. We conclude that severe acidosis may be responsible for decreased POL activity and for disintegration of nucleoli in neurons. This condition may lower the ribosome content in neonatal neurons and impair protein synthesis.

Ribosome biogenesis in man: current views on nucleolar structures and function.

Nucleoli develop when preribosomes are synthesized at the chromosomal nucleolar organizer regions. Typically they consist of at least three nucleolar subcompartments, the fibrillar center (FC), the dense fibrillar component (DF), and the granular component (GC). The understanding of the functional arrangements of these subcompartments relates to aspects in cell biology, pathology, and virus research. In the present review morphological studies are discussed in the light of molecular findings. The available data confirm the hypothesis that rDNA transcription is connected with the DF but not necessarily with the presence of an FC. Within the DF, rDNA transcription is restricted to foci, possibly representing single transcribing genes. FCs may serve to store inactive transcription factors, to initiate rDNA transcription, and may provide structural support for transcription. The GC can be interpreted as a collection of maturing preribosomes. More recently the nucleolar subcompartments were focused on in the context of virus research and tumor biology.

Cytokine expression in bronchial biopsies of cystic fibrosis patients with and without acute exacerbation.

In patients with cystic fibrosis (CF), the progression of pulmonary disease differs considerably, even in identical cystic fibrosis transmembrane conductance regulator-genotypes which could reflect an additional influence of the host's immune response. This study therefore measured cytokine expression patterns in CF patients with different clinical presentation. Expression of interleukin (IL)-8, interferon gamma (IFN-gamma), IL-4, IL-10, and transforming growth factor (TGF)beta(I) was assessed in bronchial mucosal biopsies of eight CF patients with acute exacerbation (age 6.0-14.2 yrs), eight CF patients with chronic stable disease (age 7.3-17.4 yrs), and in five normal control subjects by semiquantitative and quantitative reverse transcriptase polymerase chain reaction combined with histopathological assessment and immunohistochemical staining. All CF patients expressed IL-8. In acute exacerbation, expression of TGF-beta1 and IFN-gamma was either absent or extremely low. In contrast, all patients with stable disease strongly expressed TGF-beta1. The highest expression of TGF-beta1 and IFN-gamma was found in CF patients with mild disease and a history of infrequent exacerbations. No correlation was found between the expression of IL-4 and IL-10 and patient history. In normal control subjects, only a weak expression of TGF-beta1 was observed. These results show a remarkable correlation between cytokine pattern and the clinical course of cystic fibrosis. High expression of transforming growth factor-beta1 and interferon gamma was associated with mild disease, whereas no or very weak expression of these cytokines was typical for patients with acute disease and frequent exacerbations suggesting a contribution of the immune response to the progression of pulmonary disease in cystic fibrosis.

Cholinergic, monoaminergic and glutamatergic changes following perinatal asphyxia in the rat.

Perinatal asphyxia (PA) is considered to lead to a variety of brain disorders including spasticity, epilepsy, mental retardation, and minimal brain disorder syndromes and may form the basis for psychiatric and neurodegenerative diseases later in life. We examined markers for neuronal transmission involved in the pathomechanisms of PA and candidates as mediators for long-term sequelae. We tested tyrosine hydroxylase (TH) and the vesicular monoamine transporter (VMAT) representing the monoaminergic system, the vesicular acetylcholine transporter (VAChT), and the excitatory amino acid carrier 1 (EAAC1), a neuronal subtype of the glutamate transporter, using immunohistochemistry on brain sections of rats subjected to graded PA. Three months following the asphyxiant insult immunoreactive (IR)-TH was decreased in striatum, hippocampus, thalamus, frontal cortex, and cerebellum; IR-VMAT was increased, and IR-VAChT was decreased in striatum. IR-EAAC1 glutamate transporter was increased in frontal cortex. The cholinergic, monoaminergic, and glutamatergic changes, still observed 3 months after the asphyxiant insult, may reflect their involvement in the pathomechanisms of PA and indicate mechanisms leading to long-term complications of PA. The variable consequences on the individual markers in several brain regions may be explained by specific susceptibility of cholinergic, monoaminergic, and glutamatergic neurons to the asphyxiant insult.

Histological changes and neurotransmitter levels three months following perinatal asphyxia in the rat.

The involvement of excitatory amino acids (EAA) in the pathogenesis of hypoxic-ischemic states is well-documented. Information on the role of overexcitation by EAA in perinatalasphyxia (PA), however, is limited and data from adult models cannot be directly extrapolated to immature systems. Moreover, most adult models of ischemia are representing stroke rather than PA. We decided to study long term effects in a non-invasive rat model of PA resembling the clinical situation three months following the asphyctic insult. Morphometry on Nissl - stained sections was used to determine neuronal death in frontal cortex, striatum, hippocampus CA1, hypothalamus and cerebellum L1, and the amino acids glutamate, glutamine, aspartate, GABA, taurine, arginine as well as histamine, serotonin and 5-hydroxy-indoleacetic acid were determined in several brain regions and areas. Morphometry revealed that neuronal loss was present in the hippocampal area CA1 in all groups with PA and that morphological alterations were significantly higher in the cerebellar granular layer. The prominent light microscopical finding in all areas of asphyctic rats studied was decreased Nissl-staining, suggesting decreased cellular RNA levels. Glutamate, aspartate and glutamine were significantly elevated in the hypothalamus of asphyctic rats probably indicating overstimulation by EAA. Excitotoxicity in this area would be compatible with findings of emotional / behavioral deficits observed in a parallel study in our model of PA. Our observations point to and may help to explain behavioral and emotional deficits in Man with a history of perinatal asphyxia.

Sustained hyperglycemia in vitro down-regulates the GLUT1 glucose transport system of cultured human term placental trophoblast: a mechanism to protect fetal development?

The trophoblast of human placenta is directly exposed to the maternal circulation. It forms the main barrier to maternal-fetal glucose transport. The present study investigated the effect of sustained hyperglycemia in vitro on the glucose transport system of these cells. Trophoblasts isolated from term placentas and immunopurified were cultured for 24, 48, and 96 h in DMEM containing either 5.5 (normoglycemia) or 25 mmol/l D-glucose (hyperglycemia), respectively. Initial uptake of glucose was measured using 3-O-[14C]methyl-D-glucose. Kinetic parameters were calculated as K(M) = 73 mmol/l and Vmax = 29 fmol s(-1) per trophoblast cell. Uptake rates of cells cultured under hyperglycemic conditions did not differ at exogenous D-glucose concentrations in the physiological range (1, 5.5, 10, and 15 mmol/l), but were significantly decreased by 25% (P<0.05) at diabetes-like concentrations (20 and 25 mmol/l) as compared to normoglycemic conditions. This effect was due to a decrease in Vmax (-50%), whereas K(M) remained virtually unaffected. GLUT1 mRNA levels were lower by 50% (P<0.05; Northern blotting) and GLUT1 protein was reduced by 16% (P<0.05; Western blotting) in trophoblast cells cultured under hyperglycemic vs. normoglycemic conditions. We conclude that prolonged hyperglycemia in vitro reduces trophoblast glucose uptake at substrate concentrations corresponding to blood levels of poorly controlled diabetic gravidas. This effect is due to diminished GLUT1 mRNA and protein expression in the trophoblast.

A novel monospecific IgG2/lambda-autoantibody with specificity for a mitochondrial antigen: evidence for an antigen-driven pathogenetic B-cell response in rheumatoid synovial tissue, induced by tissue alteration.

To elucidate the pathogenic role of synovial B cells in rheumatoid arthritis (RA), nine human IgG/lambda-secreting B-cell hybridomas from rheumatoid synovial tissue of a patient with definite RA were screened by enzyme-linked immunosorbent assay and indirect immunofluorescence on tissue cryosections for detection of antibodies against autoantigens. One IgG2/lambda monoclonal antibody (mAb) from the B-cell hybridoma ELG211/15/63 (= hybr63) exhibited intense immunofluorescence reactivity in the cytoplasm of chondrocytes and epithelial cells of the gastrointestinal tract, especially in parietal cells of gastric mucosa (human and mouse tissue), representing a mitochondrial pattern. This result was confirmed by morphometric analysis of immunoelectron microscopy data, exhibiting a significantly higher labeling density in mitochondria (p < or = 0.001) than in the cytoplasmic background, with predominant staining in the inner mitochondrial membrane and mitochondrial matrix (p < or = 0.05). Immunoblotting experiments carried out with gastric mucosa, and a mitochondrial protein preparation revealed two major proteins of 38 and 50 kd under reducing conditions. The analysis of the IgV(H) genes from this B-cell hybridoma showed highest homology to the human germline gene DP53 (96%). The IgV(L) region gave highest homology to the human germline gene DP5 (93%). In the complementarity-determining regions, residues of the H- and L-chain variable regions replacement mutations only indicated that this B-cell clone had been antigen-selected for its affinity (ratio of replacement to silent mutations: > or = 7). To analyze the in vivo expansion of the B-cell clone, primers specific for the V(H) to D to J(H) rearrangement of this B-cell hybridoma were used. Specific amplifications could be detected within part of the synovial tissue but not within the cells of the synovial fluid and peripheral blood of the patient. The ability of the IgG2/lambda mAb to induce an inflammatory reaction was tested by intraperitoneal application in severe combined immunodeficiency (SCID) mice, which resulted in an inflammatory, predominantly granulocytic infiltration of the peritoneum. Consequently, intrasynovial cell death or cartilage destruction seems to be a possible source of liberation of mitochondrial antigens, inducing a local, antigen-driven IgG2/lambda B-cell response with the ability to induce an inflammatory reaction. These data suggest that tissue destruction may serve as a source of arthritogenic antigens that perpetuate and amplify the local pernicious inflammatory process in RA synovialitis.

Ribosomal gene transcription is organized in foci within nucleolar components.

We investigated how only three morphologically distinguished nucleolar components can integrate the many necessary tasks in ribosome biogenesis. For the mapping of ribosomal (r)DNA transcription loci, we combined non-autoradiographic in situ transcription assays with the immunological analysis of the ultrastructural distribution of transcription-associated proteins, i.e., polymerase I, the human polymerase I-specific upstream binding factor, and topoisomerase I. Furthermore, we visualized the nascent transcripts simultaneously with the rDNA in the nucleoli. All tested transcription proteins were found in both the fibrillar center and the dense fibrillar component (DF) of nucleoli in human cells. In the DF the nascent transcripts, detected by bromouridine incorporation, were found colocalized with the transcription proteins only within circumscribed regions. We did not observe colocalization of rDNA with nascent transcripts within the fibrillar centers, which corroborates the view that transcription proteins in this component are rather inactive. Our results suggests that only a minor portion of the DF is involved in transcriptional activity. Transcription appears to be confined to small foci, which exist close to or associated with the DF. Our results are in favor of the view that the DF has different functions which are localized in subcompartments of the DF.

Deficient transcription of subunit RPA 40 of RNA polymerase I and III in heart of rats with neonatal asphyxia.

RNA polymerases transcribe nuclear genes for ribosomal RNA thus representing ribosomal biogenesis. RNA polymerase I transcribes class I genes, coding for large ribosomal RNA and is located in the nucleolus. RNA polymerase III transcribes class III genes, those that encode a number of small ribosomal RNA molecules. Both RNA polymerases form ribosomal biogenesis in a concerted action and have a common subunit, RPA40, essential for function and integrity. The aim of our study was to study the influence of hypoxia/asphyxia on transcription of this subunit as deterioration of ribosomal biogenesis may not be compatible with life. To test this hypothesis we used a nonsophisticated model of neonatal asphyxia. Rat pups were exposed to various asphyctic periods up to twenty minutes and heart tissue was taken for the evaluation of mRNA RPA40 levels, pH measurements and histological evaluation of the nucleolus by silver staining. mRNA RPA40 levels gradually decreased with the length of the asphyctic period paralleling the decrease of pH. Silver staining was remarkably decreased at the asphyctic period of 20 minutes. Our findings of decreased transcription of this essential RNA polymerase subunit indicate impairment of the ribosomal RNA synthetizing machinery and the histological findings suggest its structural relevance. This is the first in vivo observation of deteriorated RNA polymerase in asphyxia/hypoxia.

Decrease of heart protein kinase C and cyclin-dependent kinase precedes death in perinatal asphyxia of the rat.

Acidosis, energy depletion, overstimulation by excitatory amino acids, and free radical-mediated reactions are the major current concepts for the explanation of damage and death resulting from asphyxia. Impaired phosphorylation by protein kinase C (PKC) represents another mechanism incriminated for cell death. We used an unsophisticated perinatal asphyxia model to study heart protein kinases PKC and cyclin dependent kinase (CDK). Tissue pH, ATP, the antioxidant enzymes superoxide dismutase, catalase, and glutathion peroxidase, lipid peroxidation products, carbonyls, and aromatic hydroxylation were also tested. Electron spin resonance was applied to demonstrate the possible presence of radical adducts. An ELISA method was used to determine cell death. PKC activity and mRNA decreased with the length of the asphyctic periods and were paralleled by CDK and pH, whereas cell death gradually increased. No evidence was found for the involvement of active oxygen species or a radical adduct, and no energy depletion was observed. We conclude that impaired protein phosphorylation and/or acidosis may play a role in the pathobiochemistry of death from perinatal asphyxia in the rat.

Homocysteine increases cyclin-dependent kinase in aortic rat tissue.

BACKGROUND: Hyperhomocyst(e)inemia is strongly associated with occlusive arterial disease. A direct effect of homocysteine on the proliferation of smooth muscle cells was proposed recently. This observation led us to examine the effect of homocysteine on cyclin-dependent kinase, the starter of mitosis and reflecting proliferation. METHODS AND RESULTS: Seventy Him:OFA rats were divided into seven groups. For 12 weeks, 10 rats were fed homocysteine 25 mg/kg body weight per day, 10 were fed 50 mg/kg body wt per day, and 10 were fed 100 mg/kg body weight per day; 10 were given homocysteic acid 100 mg/kg body weight per day, 10 were administered cysteine 100 mg/kg body weight per day, and 10 were given ascorbic acid 270 mg/kg body weight per day. Ten remained untreated and served as controls. Aortic cyclin-dependent kinase was determined at the transcriptional (mRNA) and protein levels. Phosphokinase C and aortic homocyst(e)ine also were evaluated in aortic tissue. Aortic cyclin-dependent kinase protein was significantly (P = .0001) elevated in the three homocysteine-treated groups, and mRNA cyclin-dependent kinase levels were significantly elevated in the rats given the 50 and 100 mg/kg body weight per day protocol. Endothelial damage was shown at higher homocysteine doses as reflected by circulating ACE and von Willebrand factor changes. Proliferation of cells of the aortic wall by bromodeoxyuridine incorporation could be shown in the high-dose homocysteine group only. CONCLUSIONS: Our findings indicate that homocysteine specifically stimulates aortic cyclin-dependent kinase at the transcriptional level, with the possible consequence of proliferation of aortic cells as revealed by incorporation of bromodeoxyuridine in the aortic wall.

Nuclear architecture and ultrastructural distribution of poly(ADP-ribosyl)transferase, a multifunctional enzyme.

A monospecific autoimmune serum for poly(ADP-ribosyl)transferase (pADPRT) was used to localise the enzyme in ultrastructural cellular compartments. We detected enzyme in mitochondria of HeLa and Sertoli cells. Within the nucleoplasm the enzyme concentration was positively correlated with the degree of chromatin condensation, with interchromatin spaces being virtually free of pADPRT. During spermatogenesis we observed a gradual increase of the chromatin associated pADPRT that parallelled chromatin condensation. The highest concentration was seen in the late stages of sperm differentiation, indicating the existence of a storage form in transcriptionally inactive nuclei. In nucleoli pADPRT is accumulated in foci within the dense fibrillar component. Such foci are seen in close spatial relationship to sites of nucleolar transcription as revealed by high resolution immunodetection of bromouridine uptake sites. It is suggested that nucleolar pADPRT plays a role in preribosome processing via the modification of nucleolus specific proteins that bind to nascent transcripts and hence indirectly regulates polymerase I activity. The persisting binding of pADPRT to ribonucleoproteins may explain the observed disperse enzyme distribution at lower concentrations in the granular component. The fibrillar centres seem to contain no pADPRT. We conclude that known compounds of fibrillar centres like polymerase I are unlikely candidates for modification via direct covalent ADP-ribosylation.