Cumene hydroperoxide induced changes in calcium homeostasis in cultured neonatal rat heart cells.

The relationship between oxidative stress induced cell necrosis and perturbation of intracellular calcium homeostasis was investigated in cultured myocytes. Cultured neonatal rat heart cells were loaded with fura-2 AM to measure cytosolic free calcium ([Ca2+]i). Probenecid, an inhibitor of organic anion transport, was present during the experiment to reduce efflux of fura-2 from the cytoplasm. Cells were exposed to cumene hydroperoxide, a toxic organic hydroperoxide that is known to induce oxidative stress in myocytes. The efficacy of the protective agents Trolox C (a vitamin E analogue) and chlorpromazine (a phospholipase inhibitor) on cumene hydroperoxide induced cell injury was determined. [Ca2+]i in control cells was constant (60 nM) during an incubation time of 45 min. Probenecid did not affect [Ca2+]i levels or cell viability under the experimental conditions. Cumene hydroperoxide caused a sustained rise in [Ca2+]i starting after 5-10 min, to a level of 1300 nM at 45 min. After 20-25 min the viability of the heart cells started to decline and after 45 min 44% of the cells were irreversibly injured. The loss of cell viability was expressed as percentage decrease of the fluorescence at 360 nm (the calcium independent wavelength), since the percent release of cellular alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) activity equalled the percent decrease of the fluorescence at 360 nm. Trolox C and chlorpromazine almost completely prevented the cumene hydroperoxide induced alpha-HBDH release. The [Ca2+]i of myocytes incubated with cumene hydroperoxide in combination with Trolox C rose to 1000 nM without affecting cell viability. The cumene hydroperoxide induced rise in [Ca2+]i was markedly reduced by chlorpromazine (at t = 45 min, [Ca2+]i = 360 nM). Addition of Trolox C to untreated cells did not influence [Ca2+]i, whereas chlorpromazine alone induced a slight increase of [Ca2+]i up to 360 nM with complete preservation of cell viability. Trolox C and chlorpromazine are very effective inhibitors of cumene hydroperoxide induced perturbation of calcium homeostasis and subsequent cell death. A role for peroxidation of membrane phospholipids and activation of calcium dependent phospholipase in the cascade of events leading to irreversible injury is suggested.


Neural regulation of heart rate variability in endurance athletes and sedentary controls.

The aim was to examine the cardiac autonomic responses to orthostatic stress and recovery from steady state exercise in endurance trained athletes and sedentary subjects. The power spectrum of heart rate variability was measured before and after exercise in 10 male long distance runners and 14 male sedentary control subjects. Both groups were comparable in sex, age, and body mass index. Continuous ECG recordings were obtained during the following physiological manoeuvres: 45 min supine rest state; 10 min standing; 15 min steady state exercise at 50% maximum workload, and 15 min while supine during post-exercise recovery. The resting heart rate of athletes was lower than controls, at 52(SD 4.9) v 67(8.7) beats.min-1, p < 0.001. Power spectrum analysis was performed using autoregressive modelling. The resting high frequency (HF) vagal component was higher in athletes than controls, at 62 (10.7) v 44(22.4) beats.min-1.Hz-1, p < 0.05. The resting low frequency (LF) peak power was significantly reduced in athletes, at 54(9.9) v 70(19.5) in control, p < 0.05. Although no group differences were observed during upright posture or exercise, the LF:HF area ratio had already returned to pre-exercise levels within 5 min of recovery in athletes. Conversely, it required up to 15 min of recovery before a noticeable decrease in the LF:HF area ratio was seen in controls. These data support the hypothesis that endurance training modifies heart rate control in whole or in part through neurocardiac mechanisms.


Comparison of laser fluorescence and longitudinal microradiography for quantitative assessment of in vitro enamel caries.

A new quantitative, non-destructive method using laser-induced fluorescence (LAF) was compared with longitudinal microradiography (LMR) for assessment of mineral changes in enamel slices using an in vitro caries model. Ten enamel slices, cut longitudinally from sound natural smooth surfaces of human teeth, were exposed to de- and remineralization in a pH-cycling model. The enamel slices were subjected to LAF and LMR measurements before and at 2, 4, 7, and 9 days of demineralization. For LAF, the average fluorescence radiance decreased during the demineralization period with 11% by day 2 and 49% by day 9. For LMR, the corresponding average loss of mineral content changed with 0.01 and 0.10 kg.m-2 over the same time period. The mineral losses in each individual enamel slice measured with the two techniques were strongly correlated, r = 0.97. The Spearman rank correlation coefficient for all LAF and LMR demineralization results was 0.86. The precision (coefficient of variation) for LAF was 3.1%, corresponding to 0.005 kg.m-2, and the repeatability error for LMR was 0.02 kg.m-2, indicating a lower discrimination threshold for LAF compared to LMR. It was concluded that the new, sensitive, non-destructive LAF method provides possibilities for further improvement in the quantification of initial caries lesions in natural smooth enamel surfaces for use in in vitro studies. Furthermore, it offers potential in in situ caries studies as well as a tool in the diagnosis of early enamel caries in vivo.


Fluoride and calcium content of enamel organ, muscle, liver and plasma in rats.

Nine-day-old rats were given oral fluoride doses (0.5 micrograms F/g body weight). Plasma, enamel organ, muscle and liver samples were collected from nondosed pups (baseline) and 15, 30, 60 and 120 min after the dose. Samples were assayed for fluoride and calcium concentrations. Adult rats were given 20 ppm F water for 6 weeks, and the same tissues were sampled and assayed for fluoride concentrations only. In the first phase of the study, enamel organ had significantly higher fluoride and calcium content than liver and muscle. In adult rats the fluoride content of enamel organ was again significantly higher than for liver and muscle.


High resolution fluoride profiles of artificial in vitro lesions treated with fluoride dentifrices and mouthrinses during pH cycling conditions.

A pH cycling model which incorporated a severe demineralization component was used to evaluate fluoride uptake and lesion progression for each of a NaF-based dentifrice, NaF-based mouthrinse and a monofluorophosphate (MFP)-based dentifrice. Simultaneous transverse fluoride and calcium profiles across the resulting artificial lesions were obtained using a proton microprobe technique with a resolution of approximately 10 microns. Longitudinal microhardness testing and proton microprobe calcium profiles were used to determine the extent of lesion progression (delta Z) with respect to untreated controls. Under the pH cycling conditions of the present study, the NaF dentifrice and mouthwash were observed to have a considerably higher uptake of fluoride in the lesion than the MFP dentifrice. Although the mineral content profiles of the lesions differed for treatments with each of the fluoride products, the differences were not significantly different in this model.


Effect of slow-release chlorhexidine mouthguards on the levels of selected salivary bacteria.

This study evaluated the effect of an alternate delivery system for chlorhexidine on salivary levels of mutans streptococci (MS) and other selected oral bacteria. On the basis of salivary MS levels > or = 10(4) CFU/ml, 22 subjects were enrolled. All caries lesions were restored prior to treatment. Two pretreatment paraffin-stimulated saliva samples were obtained for culturing at least 1 week apart. Complete-arch vacuum-adapted mouthguards were individually fabricated and coated internally with a 3% w/v chlorhexidine varnish. Mouthguards were worn for an average of 7 h/night for 7 nights. Saliva samples were obtained immediately after treatment and 1 and 3 months later. There was no significant change in levels of any of the monitored bacteria between the two pretreatment samples. A significant and specific reduction in salivary MS levels was observed in the three after-treatment samples as compared with baseline values (p < 0.001, p < 0.001, p < 0.05, respectively). The levels of Actinomyces viscosus were also measured, and there was a significant reduction immediately after treatment (p < 0.05), a return to baseline values at 1 month, and a significant increase above baseline values at 3 months, (p < 0.001). There was no significant change in saliva volume, in total numbers of facultatively anaerobic bacteria, or in levels of lactobacilli or Streptococcus sanguis. This treatment system is capable of significant and specific suppression of MS levels for up to 3 months without retreatment.


Effects of chlorhexidine-fluoride gel treatments in mothers on the establishment of mutans streptococci in primary teeth and the development of dental caries in children.

In a longitudinal 3-year study, 151 children were followed for the colonization of the primary dentition by mutans streptococci (MS) and for the development of dental caries. At the age of 1 year, the child-mother pairs were divided into three groups on the basis of the levels of MS in maternal saliva. In the experimental group, the mothers had MS levels higher than 10(5) CFU/ml, and they were given chlorhexidine (1%)-sodium fluoride (0.2%) gel treatments twice a year for 3 years. Two control groups were formed. In control group 1, the mothers also had high levels of MS, but no gel was given. In control group 2, the mothers had low (< 10(5) CFU/ml) baseline levels of salivary MS, and no chlorhexidine-fluoride gel was used. In the total study population, 16, 42, and 54% of the children were colonized by MS by the age of 2, 3, and 4 years, respectively. Most children harbored only Streptococcus mutans, but 2 had both S. mutans and Streptococcus sobrinus, and 2 had only S. sobrinus. Twenty-eight percent of the MS-positive children developed caries by the age of 4 years, whereas 4 out of 27 children with dental caries did not have any detectable MS in their plaque samples. Both the colonization by MS and the caries incidence were highest in control group 1 and lower in the experimental group and in control group 2. These observations suggest that the reduction of maternal salivary MS at the time of tooth emergence may delay, or perhaps even prevent, the colonization of MS in the children's primary dentition with a concomitant decline in caries incidence, even in a population with an already low prevalence of dental caries.


Effect of delmopinol on the viscosity of extracellular glucans produced by Streptococcus mutans.

The surfactant delmopinol, which is a new antiplaque agent with a low anti-microbial profile, was tested for its effects on the viscosity of bacterial extracellular glucans. Glucans were isolated from Streptococcus mutans broth supernatants incubated with 0.15 M sucrose in 50 mM sodium phosphate buffer at pH 6. The viscosity was measured in a shear rate range from 15 to 230 reciprocal seconds. The viscosity of the water-soluble glucan was found to be independent of shear rate whereas the water-insoluble glucan showed a strong shear thinning. The addition of delmopinol to preformed glucans did not affect the viscosity nor the shear rate dependence of the glucans. However, when present during synthesis of the polysaccharides, delmopinol was found to reduce the viscosity of both water-soluble and water-insoluble glucans by approximately 50% at the shear rates investigated. The reduction in viscosity for the water-soluble glucans was obtained at a delmopinol concentration of 0.32 mM (0.01%) and for the water-insoluble glucans at 3.2 mM delmopinol. The observed reduction of viscosity of glucans indicates that the in vivo stability of plaque matrix after delmopinol treatment would be lowered, which may lead to a reduction of plaque cohesion and thus facilitate mechanical plaque removal.


Effect of sugarcane chewing on plaque pH in rural Kenyan children.

In 5 rural Kenyan children, the effect of sugarcane chewing on plaque pH was compared with the effect of a mouthrinse with 10% sucrose at various intraoral sites. They all had poor oral hygiene and at least two carious cavities in occlusal surfaces of molars. pH measurements were conducted under field conditions using paladium touch microelectrodes connected to a battery-operated pH meter. There was a marked difference in pH response of non-carious approximal sites between maxilla and mandible, with the lowest values in the maxilla. However, the pH recovery following the instantaneous drop occurred in parallel even if most pH values had not returned to baseline values 30 min after the sucrose rinse. Following the sugarcane chewing, the pH fall was less pronounced on all sites, and within 5-10 min the values had returned to resting pH and even exceeded this. In carious cavities, a similar pattern was observed, although the acidity in these sites was more pronounced, also reflected in a lower resting mean pH. The main conclusion from this study is that sugarcane chewing yields a less pronounced pH drop and a quicker pH recovery in dental plaque than is seen following a mouthrinse with 10% sucrose. This difference probably results from stimulation of salivary flow associated with the chewing.


Effect of hard cheese exposure, with and without fluoride prerinse, on the rehardening of softened human enamel.

The in situ rehardening effect was compared between Cheddar cheese consumption and saliva secretion with and without a fluoride pretreatment on softened human enamel. Ten volunteers wearing prostheses which held slices of human enamel participated in this study. Average microhardness of enamel was determined on the surface at baseline, after exposing to an acidic beverage, after exposing to saliva and mastication of cheese, with and without a mouth F prerinse (10 ml Meridol containing 0.025% F). The rehardening was increased in the groups consuming cheese compared to the saliva controls. The effect was increased by an F prerinse; the initial hardness of the intact enamel surface, however, was not reached.


Effect of glutardialdehyde on secondary caries in situ.

In this paper the results are presented on the effect glutardialdehyde (GDA) on the in situ demineralization of human enamel and dentine around an artificial gap of 200 microns width. In this model secondary enamel and dentine caries is simulated in situ; the mineral loss was quantified. The results show that a 2-min application of a 2% acidic GDA solution reduces dentine demineralization substantially, but does not influence enamel demineralization. Microradiography showed a reduction of mineral loss values around the gap in dentine by about 30%. The beneficial effect of GDA on dentine is presumably a combined result of collagen fixation, the reduced diffusion of calcium and phosphate ions out of the dentine lesion, and antibacterial action. The results indicate that GDA might play a role in the future reduction or prevention of secondary dentine caries.


Effect of oral care habits on caries in adolescents.

Data on toothbrushing habits were collected during a 3-year caries clinical trial of sodium monofluorophosphate toothpastes in Lanarkshire, Scotland, involving 3,005 schoolchildren of mean age 12.5 years at baseline. Stated normal brushing frequency and oral rinsing method after brushing were recorded. Half the panel indicated they rinsed their mouths after toothbrushing using a beaker. The proportion of the panel brushing once per day or more increased during the trial. Differences in oral habits were observed between the sexes, with 42% of girls and 52% of boys being non-beaker rinsers and 73% of girls, but only 44% of boys, brushing their teeth at least twice per day. Twice-a-day brushers had a consistently lower caries increment than less frequent brushers. This was also seen in the baseline prevalence data, but did not account for all incremental differences noted. Subjects using beakers had consistently higher increments than non-beaker rinsers. Again, this difference could not be explained by variations in baseline prevalence. Differences in the caries increment were also observed between boys and girls, these appearing to be linked both to the cumulative effect of male/female habit variations plus a difference in the baseline caries prevalence. A dose response to the three fluoride levels, i.e 1,000, 1,500, and 2,500 ppm F, was seen for the different habit combinations which again could not be explained by differences in the baseline caries prevalence.


Clinically undetected occlusal dentine caries: a radiographic comparison.

The occlusal bite-wing radiograph evaluations of 131 14-year-old and of 123 17- and 20-year-old Dutch persons were compared with the clinically collected observations. The clinical observations and the bite-wing radiographs were collected as part of a longitudinal epidemiological survey conducted in 1987 and 1990. Comparison of the evaluations of the first and second molar teeth showed significantly more radiolucencies in all three age groups than expected clinically. Of the clinically judged 'sound' surfaces, 26% in the 14-, 37.5% in the 17- and 50% in the 20-year-old group showed a radiolucency on the bite-wing radiograph. In the 14-, 17- and 20-year-old age group a radiolucency was noticed on the bite-wing radiograph in 32, 44 and 58% of the sealed teeth, respectively. The radiolucencies below the occlusal enamel of sealed teeth were most likely due to the problems in diagnosing occlusal dentine lesions correctly with the clinical diagnostic methods. Therefore not only approximal but also occlusal bite-wing radiograph judgement before sealant application seems to be advisable now. Because of the numbers of clinically undetected lesions it may be advisable to reconsider the criteria for occlusal diagnosis in epidemiological surveys and to judge not only the approximal but also the occlusal surfaces in epidemiological surveys when bite-wing radiographs are available.


Class II amalgam restorations, glass-ionomer tunnel restorations, and caries development on adjacent tooth surfaces: a 3-year clinical study.

Eighteen caries-active adolescents, each having both a class II conventional amalgam (Dispersalloy) and a glass-ionomer (Ketac Silver) tunnel restoration of the same age placed on a contralateral tooth, were part of a 3-year clinical study. At the 1- and 2-year examinations all test restorations were assessed as acceptable in all patients. At the 3-year assessment three amalgam restorations failed due to recurrent caries, and one glass-ionomer restoration failed due to marginal ridge fracture. During the entire study period, the requirement of restorative therapy because of primary proximal caries was significantly reduced (p < 0.05) on tooth surfaces adjacent to the glass-ionomer restorations as compared with that on tooth surfaces adjacent to the amalgam restorations.


Morphological characteristics of different types of neurons in the ventrooralis anterior, ventrooralis internus, and ventrooralis posterior nuclei in the human thalamus.

1. Golgi-Kopsch preparations of the oral ventral nuclei of human thalamus were analyzed in an attempt to classify the neuronal types. 2. Three types of neurons are described for the first time in humans. Type I neurons are large or medium in size and bear dendrites with protrusions, spines, and short hair-like appendages. Some have a radiate dendritic arbor and others have dendrites grouped in tufts. The dendritic trees of these neurons are dense. 3. Type II neurons are medium or small in size with less dense dendritic trees. These cells have somatic as well as dendritic appendages of different forms. 4. Relatively rare is a type of very small neurons, type III, with few and sparsely branching dendrites.


[Progression of chronic glomerulonephritis. Myths and facts].

Chronic glomerulonephritis is still the most frequent cause of irreversible renal failure. The aetiology of the majority of glomerulonephritis is unknown but it seems that they are genetically determined. In their genesis immunological factors participate but in their development and progression non-immunological factors are also involved. The author presents a review of contemporary therapeutic possibilities.


[Evaluation of glomerular filtration in patients after renal transplantation and treatment with cyclosporin A].

According to some findings (Ross et al. 1987) the creatinine plasma concentration (Pcr) is an inaccurate indicator of glomerular filtration (GFR) in patients with a transplanted kidney, treated with cyclosporin A (CyA), who are in a stabilized state of renal function. In the submitted work the authors investigated whether the inaccuracy of the assessed GFR based on Pcr or creatinine clearance (Ccr) is greater than in patients with various chronic renal diseases who at the time of examination did not take any drugs. The investigation was made in 30 patients with a transplanted kidney treated with CyA as well as azathioprine and prednisone. The authors examined also 51 patients with various chronic renal diseases, mostly chronic glomerulonephritis or tubulo-interstitial nephritis. The GFR value was assessed on the basis of polyfructosan clearance (CPF). In subjects with a transplanted kidney a significant linear relationship was found between Ccr and CPF (r = 0.829, p < 0.001). A similar relationship was found in patients with chronic renal disease (r = 0.935, p < 0.001). The regression lines characterize this relationship in both groups and do not differ significantly. Between values of Pcr and CPF a significant relationship of a hyperbolic character was found in both groups (r = 0.693, p < 0.001 and r = 0.741, p < 0.001 resp.). The hyperbolic relations found in the examined groups did not differ significantly. The findings confirm that a normal or only slightly elevated Pcr value can be associated with a markedly reduced GFR, in some instances to as much as one third of the normal values.(ABSTRACT TRUNCATED AT 250 WORDS)


[Clinical study of biocompatibility of dialysis membranes made from non-modified and modified cellulose].

Basic biocompatibility parameters of dialysis membranes made of non-substituted regenerated cellulose (NRC) and cellulose membranes with hydroxyl groups substituted, to a higher (H) or lower (L) degree, by dl-ethyl-amino-ethyl groups (DEAE), or by acetate (CA) were investigated in a 16-week clinical study, involving 10 long-term haemodialysis patients. In the 15th minute of dialysis, the decrease in blood leukocyte count, while using NRC (0.24 +/- 0.03 of baseline value, arithmetic mean +/- SEM) was deeper compared with that seen in DEAE-L (0.88 +/- 0.10, p < 0.001), in DEAE-H (0.79 +/- 0.10, p < 0.01), and in CA (0.73 +/- 0.05. p < 0.05). In the 15th minute of the procedure, C5a concentrations, reflecting complement activation, were higher in NRC (4.4 +/- 0.51 micrograms/L) than in DEAE-L (1.41 +/- 0.22, p < 0.001), in DEAE-H (1.68 +/- 0.47, p < 0.01), and in CA (1.68 +/- 0.22, p < 0.01). Activated clotting times were, in the 10th minute of the procedure, significantly longer in NRC (2.94 +/- 0.37 of baseline value) than in DEAE-H (1.74 +/- 0.10, p < 0.05) and, by the end of dialysis, the difference between these membranes (NRC: 1.47 +/- 0.21, DEAE-H: 0.85 +/- 0.08, p = 0.07) was close to the level of statistical significance. The authors conclude: 1. Substitution of the hydroxyl groups of regenerated cellulose reduces the decrease in leukocyte count and complement activation in the initial phase of haemodialysis. 2. At the same time, substitution by DEAE groups may raise thrombogenicity, as indicated by the shorter activated clotting times.(ABSTRACT TRUNCATED AT 250 WORDS)


[Rapidly progressing glomerulonephritis with antibodies against cytoplasm of neutrophilic leukocytes].

The authors describe the clinical, laboratory and morphological findings, the clinical course and response to treatment in three patients with rapidly progressing glomerulonephritis where positive antibodies against the cytoplasm of neutrophil granulocytes (ANCA) were found. The authors confront their own clinical experience with data in the literature. Examination of ANCA is not only a significant contribution towards a more accurate diagnosis of renal vasculitis, but also an indicator of the activity of the disease and thus of the effectiveness of immunosuppressive treatment.


[14 years of the peritoneal dialysis program at the internal medicine department in Strahov (1978-1991)].

The authors present an account of 14 years experience of the department as regards treatment of chronic renal failure by peritoneal dialysis. Initial experience revealed that this method is feasible as an alternative of haemodialyzation treatment even in case of limited technical possibilities (using the so-called bottle system) but treatment was associated with a high incidence of peritonitis. During the subsequent period the incidence of peritonitis was substantially reduced in conjunction with the elaboration of hygienic provisions, enlightment of patients and a change from the regime of continual exchanges in a home environment to a regime of intermittent peritoneal dialysis performed mostly in hospital. Experience assembled in the department was also important. The longest period of treatment is 60 months. In another 18 patients it is more than 20 months. The most frequent cause of termination is loss of the ultrafiltration capacity of the peritoneum. Several patients had successful transplantations. The prolonged experience can be used at present when the so-called bag system is introduced.


[Interesting urinary findings in acute intermittent porphyria].

The authors describe an uncommon case of a patient with acute intermittent porphyria whose urine on standing, when exposed to light and air, turned dark and eventually black. The reason was increased excretion of porphobilin. The urine continued moreover increased amounts of Thormählen-positive substances, typical melanogens were not detected, however. The authors wish to draw attention to possible diagnostic errors which arise due to erroneous interpretation of laboratory results.


[Present views on classification of cerebrovascular diseases].

In conjunction with the declaration of the "brain decade" which was called for by the unfavourable position as regards early diagnosis and by inadequate treatment of cerebral diseases, the author reflects on the importance of classification of cerebrovascular diseases. He submits a historical review of different classifications of cerebrovascular diseases starting with the international classification of diseases introduced into practice as early in 1893, the clinical and research classification elaborated by Millikan in 1975--both are still used by WHO--to the most recent suggestions of Hachinski of 1990 and the Classification of spontaneous intracerebral haematoma by Mizukami elaborated in 1985. The paper contains also the author's own modification of the clinical and research classification.


[The effect of isometric loading on diastolic left ventricular filling in healthy individuals studied by the Doppler technic].

The authors examined by Doppler echocardiography 30 subjects aged 21-30 years, exposed during examination to an isometric load by using a manual balloon dynamometer. The authors investigated different indicators of left ventricular filling during diastole and their indexes which evaluate indirectly the left ventricular diastolic function. The assessed correlations of some indicators with the quantitative expression of the isometric load (double product) were as follows: peak speed E - r = 0.3671 (p < 0.05), peak speed A - r = 0.6098 (p < 0.001), index of rations of peak speeds E/A - r = 0.6098 (p < 0.0001, sum of speed--time integrals TVIE + TVIA - r = 0.6469 (p < 0.001) and the time of retardation of the early filling phase of the left ventricle related to the heart rate DCT/TF - r = 0.7249 (p < 0.001). A statistically significant increase during the load was recorded in the peak speed A and a reduction of the E/A ratio (p < 0.01). The time speed integral TVIE increased (p < 0.01), both indexes TVI E + A and TVI E/A declined (p < 0.05). The time of retardation of the early filling stage to the early filling time DCT/DFT increased (p < 0.05) and DCT in relation to the heart rate DCT/TF declined significantly (p < 0.01). There was an insignificant decline of the peak speed of early filling E and its time-speed integral, TVI E. The sum of time-speed integrals TVIE + A did not change significantly.(ABSTRACT TRUNCATED AT 250 WORDS)


[The character of left ventricular filling measured by pulsed Doppler echocardiography in patients with various degrees of arterial hypertension].

The authors assessed, using Doppler echocardiography under standard conditions, indicators of left ventricular filling in 95 patients, mean age 52 years, with arterial hypertension of varying severity. The patients were divided into four groups: Group I--Patients with severe hypertension and concentric hypertrophy of the left ventricle (LV). Group II--Patients with severe hypertension without organ changes. Group III--Patients with mild hypertension without organ changes. Group IV--Patients with severe hypertension, hypertrophy of the LV and signs of its failure grade II according to Killip's classification. The results were compared with findings in a group of 30 normal subjects of similar age. In all groups of patients statistically significant differences were found in the type of left ventricular filling, as compared with normal subjects. In less severe affection of left ventricular function, where impaired active relaxation of musculature predominates (groups I, II and III), the left ventricular filling during early diastole was reduced, slower and its main part was shifted to the late stage of diastole. The ratio of the late and early flow rate through the mitral orifice Av/Ev and the ration of time/rate integrals of these rates, TVIA/TVIE, were elevated, as compared with normal subjects, similarly as the time value of retardation of early diastolic flow through the mitral orifice corrected with regard to the heart rate, DT/TF. The value of maximal flow through the mitral orifice during early diastole, PFRB was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)


[Functional determination of C1-inhibitors in human blood].

The author describes the method of assessment of the C1-inhibitor, the principle being activation of plasma prekallikrein by Hageman factor fragment (HFf) to the active enzyme kallikrein which splits the specific chromogenic substrate NO-Pro-Phe-Arg-pNA. In the presence of acetone the influence of C1-INH is eliminated and the assessed amount of kallikrein corresponds to the prekallikrein plasma level. In a parallel estimation without acetone after formation of the C1-INH complex the residual amount of kallikrein is assessed. The difference between the two levels is proportional to the formed enzyme-inhibitor complex. The method was tested on plasma of healthy donors and the results were compared with assessment of C1-INH by a modification of Schapira's method (6) (correlation coefficient r = 0.88) and with Cullmann's method (8) (r = 0.61). The advantage of the proposed method is that it does not require commercially unavailable enzyme preparations, that it uses as activating agent HFf concentrate, prepared in the laboratory and that along with the C1-INH level also the plasma level of prekallikrein is assessed.


[Present possibilities of use of intravascular ultrasound examinations].

Catheter intravascular imaging is currently used as an adjunct to contrast angiography and angioscopy in peripheral and coronary arteries. At present the smallest catheters are 3.0 F (1.0 mm). Frequencies used are from 10 to 40 Mz, which allow high resolution imaging of structures close to the transducer. Mechanical and phased array systems are used, introduced either on the wire or through fine plastic catheter. More recent is the monorail system improving the flexibility and tracking of the mechanical systems. The review presents current status of catheters and diagnostic possibilities. Normal arteries have typical three layered appearance (intima, media, adventitia). Ultrasound can be reliably used to distinguish abnormal vascular segments. Atherosclerotic plaques can be subclassified on the basis of plaque composition. Thrombus is characterized by granular appearance with varying sonographic gray levels. Dissection and intimal flaps are also visualized by intravascular ultrasound. Potential utility of intravascular ultrasound in the assessment of endovascular interventions and its limitations are discussed.


[Energy expenditures in patients with acute and chronic catabolic conditions].

The authors investigated the energy expenditure and utilization of different nutrients by indirect calorimetry. They examined three groups of subjects: patients with acute febrile diarrhoeal diseases (n = 36), patients with subacute to chronic severe febrile diseases (n = 10) and healthy subjects (n = 21). The measurements were made three times a day. In all groups the energy requirements of the organism were met in the morning above all by fats, in the course of the day the ration of utilized carbohydrates increased markedly. The authors discuss the problem of optimal nutrition of patients in a critical condition from the quantitative and qualitative aspect.


[Use of walking in the evaluation of aerobic fitness].

The reliability and validity of assessment of the maximal oxygen consumption as a criterion of aerobic fitness by the 2000 m walking test was established in 38 students of the military secondary school (mean age 17.8 +/- 0.4 years). The reliability of the 2000 m walking test is very high, during repeated estimations in the course of one week we find a correlation coefficient of r = 0.82 (p < 0.001). There is also a close correlation of 2000 m walking time and 2000 m running time (r = 0.67, p < 0.001). A group of 18 students selected at random from the above group was examined in the laboratory on a treadmill up to vita maxima. A close correlation was found between the results during walking and the maximal oxygen consumption (VO2max.kg-1) (r = -0.59 and -0.60) and also the 2000 m running time and VO2max.kg-1 (r = 0.59), p < 0.01 in all instances. The standard error of the mean of the maximal oxygen consumption estimate from the 2000 m walk is 3.44 ml.kg-1 x min-1 (5.7%) and from the running time of the same distance 3.49 ml.kg-1 x min-1 (5.8%). These results justify the conclusion that the 2000 m rapid walking test (normal style) is a simple alternative way for estimating the maximal oxygen consumption and thus also for evaluating the level of aerobic fitness with an accuracy which matches that, when running is used for the test.


[Initial experience with the treatment of endobronchial lesions using the Nd:YAG laser].

Endobronchial treatment by means of a laser acquired during the past ten years a firm place in bronchology. In the submitted paper the authors present their initial experience with this treatment in patients of the Pneumological Clinic in Prague-Veleslavín. In the course of 12 months since when there is a Nd:YAG laser, Sharplan Co. at the clinic, 52 patients were treated by this method. Forty of them suffered from lung cancer. Three had other types of tumours. Eight patients suffered from benign cicatricial stenoses and one female patient aspirated a foreign body. A total of 124 bronchoscopies, using a laser, were performed, on average 2.4 per patient. In 75% of patients with lung cancer recanalization was achieved, the trachea was dilated in all patients with cicatricial stenoses, however, only in one quarter of them complete restitution of the lumen was achieved. Hitherto assembled experience confirms the value of laser treatment for improving the life and its prolongation in patients with lung cancer and its importance for treatment of other endotracheobronchial processes. Three serious haemorrhages during operation were recorded, one female patient died. The use of the laser is simple, its operation smooth.


[One-stage surgery in concomitant heart and lung disease].

The authors evaluate the advantages and disadvantages of one-stage operations in concomitant cardiac and pulmonary diseases and define the optimal indications for this procedure. The justification of one-stage operations in selected patients is confirmed by the authors' own experience assembled in two patients. One suffered from lung cancer and ischaemic heart disease and the other one from a tuberculoma and ischaemic heart disease. The one-stage operation in both patients was without complications.


Morphological analysis of the neurons in the area of the hypothalamic magnocellular dorsal nucleus of the guinea pig.

In the guinea-pig hypothalamus, a group of enkephalinergic cells forms a well-circumscribed nuclear area called the magnocellular dorsal nucleus (MDN). This nucleus gives rise to a prominent projection to the lateral septum: the hypothalamo-septal enkephalinergic pathway. In the present study, MDN neurons visualized by Golgi impregnation were subjected to morphological analysis in order to define the potential segregation of cellular types within the MDN. This study was complemented by additional observations of MDN neurons intracellularly injected by Lucifer yellow (LY) or horseradish peroxidase (HRP) during the in vitro incubation of hypothalamic slices. The following results were obtained from the analysis of 200 neurons: 163 Golgi-impregnated cells plus 37 injected cells (LY = 14; HRP = 23). Thirteen HRP-injected cells were precisely located in the MDN and 10 were located in the perifornical area surrounding the MDN. Four different cellular types were identified. Type-I neurons (41%) displayed a globular perikaryon, a variable number of primary dendrites that were poorly ramified, no preferential orientation, and an axon emerging from the perikaryon. Type-II neurons (30.5%) had a triangular perikaryon, three well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from the perikaryon. Type-III neurons (22%) exhibited a spindle-shaped perikaryon, two opposed well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from a primary dendrite. Type-IV neurons (6.5%), showed a globular perikaryon, a variable number of primary dendrites, poorly ramified dendrites, an orientation parallel to the third ventricle, and an axon whose orientation could not be identified. Neurons labeled after intracellular injection belonged to the first three cellular types.


Distribution of enteric nerve cells that project to the coeliac ganglion of the guinea-pig.

The digestive tract of the guinea-pig, from the esophagus to the rectum, was examined in detail to determine the distribution and relative abundances of neurons in these organs that project to the coeliac ganglion and the routes by which their axons reach the ganglion. A retrogradely transported neuronal marker, Fast Blue, was injected into the coeliac ganglion. The esophagus, stomach, gallbladder, pancreas, duodenum, small intestine, caecum, proximal colon, distal colon and rectum were analysed for labelled neurons. Retrogradely labelled neurons were found only in the myenteric plexus of these organs, and in the pancreas. No labelled neurons were found in the gallbladder or the fundus of the stomach, or in the submucous plexus of any region. A small number of labelled neurons was found in the gastric antrum. An increasing density of labelled neurons was found along the duodenum. Similarly, an increasing density of labelled neurons was found from proximal to distal along the jejuno-ileum. However, the greatest densities of labelled neurons were in the large intestine. Many labelled neurons were found in the caecum, including a high density underneath its taeniae. An increasing density of labelled neurons was found along the length of the proximal colon, and labelled neurons were found in the distal colon and rectum. In total, more labelled cell bodies occurred in the large intestine than in the small intestine. The routes taken by the axons of viscerofugal neurons were ascertained by lesioning the nerve bundles which accompany vessels supplying regions of the digestive tract. Viscerofugal neurons of the caecum project to the coeliac ganglion via the ileocaeco-colic nerves; neurons in the proximal colon project to the ganglion via the right colic nerves, and neurons in the distal colon project to the ganglion via the mid colic and intermesenteric nerves. Neurons in the rectum project to the coeliac ganglion via the intermesenteric nerves. These nerves (except for the intermesenterics) all join nerve bundles from the small intestine that follow the superior mesenteric artery. All viscerofugal neurons of the caecum were calbindin-immunoreactive (calb-IR) and 94% were immunoreactive for vasoactive intestinal peptide (VIP-IR). In the proximal colon, 49% of labelled neurons were calb-IR and 85% were VIP-IR. In the distal colon, 80% of labelled neurons were calb-IR and 71% were VIP-IR.


Neuronal influence on B and H human blood-group antigen expression in rat cochlear cultures.

The presence of B and H human blood-group antigens was analyzed by immunocytochemistry in rat cochleas developing either in vivo or in vitro. Developing animals, on embryonic day (E) 18 and postnatal day (P) 3, were used for in vivo studies. For in vitro studies, cochleas were removed at E18 and placed for 3 or 8 days in organotypic culture either directly or after partial spiral ganglion removal. Results from epithelial regions from cochleas developing in vivo were similar to those observed in corresponding areas of direct organotypic cultures where the innervation from spiral ganglion neurons was present. Antibodies to human blood group antigens, anti B and anti AB, selectively labeled hair cells. The intensity of labeling was weak at E18, but increased at P3 in vivo or after 3-8 days in organotypic culture. Anti H antibodies showed weak labeling of the apical surface of hair cells and other epithelial cells at E18; this labeling also increased at P3 or after 3-8 days in culture. In contrast, the non-innervated regions from organotypic cultures, where ganglia were partially removed, exhibited an epithelial disorganization and no hair cell labeling with any of the antibodies studied. The present findings suggest that human blood-group antigen expression on developing cochlear hair cells of rats may be related to afferent nerve fiber influence.


Immunocytochemical localization of Bombyx-PTTH-like molecules in neurosecretory cells of the brain of the migratory locust, Locusta migratoria. A comparison with neuroparsin and insulin-related peptide.

Using a monoclonal antibody directed against a synthetic pentadecapeptide corresponding to the N-terminus of the prothoracicotropic hormone (PTTH) of Bombyx mori, we report the presence of immunoreactive molecules in a large number of median neurosecretory cells of the pars intercerebralis of the migratory locust, Locusta migratoria. These cells correspond to the A1 cell type which we show to contain also neuroparsins, a family of predominant neurohormones of the migratory locust. In contrast, PTTH-like molecules are absent from A2 cells of the pars intercerebralis which contain Locusta insulin-related peptide (LIRP). Developmental studies show the presence of PTTH-related substances in neurosecretory cells of Locusta migratoria from late embryogenesis to adult development, including ageing vitellogenic female adults.


Organization of cells and extracellular matrix in mesenteric arteries of spontaneously hypertensive rats.

Biochemical studies have been used to assess the quantitative changes in elastin and collagen in hypertensive vs. normotensive arteries. However, the relative distribution and organization of these fibrous proteins is likely to be equal in importance to their absolute amounts. In this study we have used scanning electron microscopy in association with selective digestion techniques to assess the organization of cellular and extracellular components of the tunica media of mesenteric arteries of spontaneously hypertensive rats. Superior and small mesenteric arteries were digested with acid, alkali, or bleach to exposure cells, collagen, or collagen and elastin, respectively. We observed that hypertension does not cause a qualitative change in the 3-dimensional arrangement of cells, collagen, or elastin in spontaneously hypertensive arteries when compared to normotensive arteries. However, cells in the superior artery are significantly different in overall shape and surface features when compared to cells of small arteries. These differences in surface morphology of cells are present in hypertensive and normotensive vessels and suggest that superior and small mesenteric artery cells transmit load to the isotropic matrix in different ways. In the elasto-muscular superior artery, force is transmitted across digitations throughout the cell surface. In the muscular small artery, force is transmitted across the tapered, smooth cell surface.


Immunohistochemical localisation of natriuretic peptides in the heart and brain of the gulf toadfish Opsanus beta.

The distribution of natriuretic peptide immunoreactivity was determined in the heart and brain of the gulf toadfish Opsanus beta using the avidin-biotin peroxidase technique. Four antisera were used: the first raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); the third raised against rat atrial natriuretic peptide; and the fourth raised against eel atrial natriuretic peptide. Natriuretic peptide- and porcine brain natriuretic peptide-like immunoreactivity was observed in all cardiac muscle cells of the atrium. In the ventricle, natriuretic peptide-like immunoreactivity was found in all cardiac muscle cells, however, porcine brain natriuretic peptide-like immunoreactivity was confined to muscle cells adjacent to the epicardium. There was no discernible difference in the distribution of natriuretic peptide-like immunoreactivity and porcine brain natriuretic peptide-like immunoreactivity in the brain. Immunoreactive perikarya were observed only in the preoptic region of the diencephalon, and many immunoreactive fibres were found in the telencephalon, preoptic area, and rostral hypothalamus, lateral to the thalamic region. There was no immunoreactivity in any region of the hypophysis. A pair of distinct immunoreactive fibre tracts ran caudally from the preoptic area to the thalamic region, from which fibres extended to the posterior commissure, area praetectalis, dorsolateral regions of the midbrain tegmentum, and tectum.(ABSTRACT TRUNCATED AT 250 WORDS)


Cellular differences in the regeneration of murine skeletal muscle: a quantitative histological study in SJL/J and BALB/c mice.

Skeletal muscle regeneration in SJL/J and BALB/c mice subjected to identical crush injuries is markedly different: in SJL/J mice myotubes almost completely replace damaged myofibres, whereas BALB/c mice develop fibrotic scar tissue and few myotubes. To determine the cellular changes which contribute to these differential responses to injury, samples of crushed tibialis anterior muscles taken from SJL/J and BALB/c mice between 1 and 10 days after injury were analysed by light and electron microscopy, and by autoradiography. Longitudinal muscle sections revealed about a 2-fold greater total mononuclear cell density in SJL/J than BALB/c mice at 2 to 3 days after injury. Electron micrographs identified a similar proportion of cell types at 3 days after injury. Autoradiographic studies showed that the proportions of replicating mononuclear cells in both strains were similar: therefore greater absolute numbers of cells (including muscle precursors and macrophages) were proliferating in SJL/J muscle. Removal of necrotic muscle debris in SJL/J mice was rapid and extensive, and by 6 to 8 days multinucleated myotubes occupied a large part of the lesion. By contrast, phagocytosis was less effective in BALB/c mice, myotube formation was minimal, and fibrotic tissue conspicuous. These data indicate that the increased mononuclear cell density, more efficient removal of necrotic muscle, together with a greater capacity for myotube formation in SJL/J mice, contribute to the more successful muscle regeneration seen after injury.


Cellular immunity in an annelid (Nereis diversicolor, Polychaeta): production of melanin by a subpopulation of granulocytes.

We attempted to identify the nature and origin of the pigment produced by the marine worm Nereis diversicolor in order to isolate, in inert brown capsules, foreign objects introduced into its body cavity. This brown pigment, characterized by cytochemical techniques, could be a melanin. The activity of the enzyme phenoloxidase responsible for melanin biosynthesis was detected by enzyme cytochemistry techniques in vacuoles and the Golgi apparatus of coelomocytes activated by the presence of foreign bodies. Morphological techniques combined with a monoclonal immunological probe enabled us to establish that the "G2" granulocytes contain both the precursor of the pigment in dense bodies and the capacity for phenoloxidase synthesis when activated to encapsulate foreign bodies. The "G2" granulocyte may therefore be compared to a melanocyte in which melanin is not stored as in mammals, but immediately extruded following synthesis in the form of a thick fluid.


Differential expression of four genes encoding molluscan insulin-related peptides in the central nervous system of the pond snail Lymnaea stagnalis.

In the pond snail Lymnaea stagnalis, the growth regulating system consists of (1) about 200 neuroendocrine light green cells, located in four clusters in the cerebral ganglia, and (2) the paired canopy cells, located in the lateral lobes. These cells express genes encoding the molluscan insulin-related peptides (MIPs). Six MIP genes have previously been identified. Four of these (I, II, III and V) are expressed in the light green cells and the canopy cells. The MIP-VI gene is a pseudogene. In the present in situ hybridization study, using oligonucleotide probes specific to the transcripts of MIP-I, -II, -III, -IV and -V, no signal was obtained with the MIP-IV probe, indicating that gene IV is also a pseudogene. With the other four probes, two types of light green cells were distinguished. Type-A cells express all four MIP genes, whereas type-B cells do not (or only faintly) express the MIP-I gene. Gene III is relatively strongly expressed in type-B cells. Genes II and V are moderately expressed in both cell types. Type-A cells are mainly located in the periphery of the clusters, whereas type-B cells are present in the center. The canopy cell resembles type-A light green cells. The expression levels of the MIP-II and MIP-V genes are low in the canopy cell. The expression pattern of the MIP genes correlates with the staining pattern of the anti-MIP-C antibody, which has been raised to a synthetic C-fragment shared by MIP-I, -II and -V. Type-A cells stain more intensely with the antibody than type-B cells.


The origin of the luteinizing hormone-releasing hormone (LHRH) neurons in newts (Cynops pyrrhogaster): the effect of olfactory placode ablation.

Neurons containing luteinizing hormone-releasing hormone (LHRH) are first detected in newt embryos (Cynops pyrrhogaster) in the olfactory epithelium and ventromedial portion of the olfactory nerve, after which they sequentially appear in the intracerebral course of the terminal nerve at prometamorphosis, and in the septo-preoptic area at postmetamorphosis. In adults, however, LHRH-immunoreactive cells are rarely seen in the nasal region, and their distribution shifts into the brain, suggesting their migration. In order to ascertain the origin and possible migration route of these neurons in newt larvae, the effect of unilateral or bilateral olfactory placodectomy on the LHRH neuronal system has been studied. Removal of the olfactory placode results in the absence of LHRH-immunoreactive cells in the nasal and brain regions of the operated side, whereas the subsequent growth and the LHRH-immunoreactive cellular distribution in the contralateral side are identical to those of normal larvae. Following bilateral placodectomy, no LHRH immunoreactivity is detected on either side of the olfactory-brain axis. These results suggest that LHRH neurons of the newt, Cynops pyrrhogaster, originate in the olfactory placode and then migrate into the brain during embryonic development.


Widespread tissue distribution of rabbit calreticulin, a non-muscle functional analogue of calsequestrin.

Calreticulin was identified in a variety of rabbit tissues by Western blot analysis. Indirect immunofluorescence studies on cultured cells or frozen sections from the corresponding tissues revealed that the protein was distributed to the endoplasmic reticulum or sarcoplasmic reticulum. Calreticulin was found to be an abundant calcium-binding protein in non-muscle and smooth muscle cells and a constituent calcium-binding protein in cardiac and skeletal muscle. From the immunoblot data, calreticulin may exist as an isoform in rabbit neural retina. The present study establishes the ubiquity of calreticulin in intracellular calcium binding.


Morphogenesis of the photoreceptor outer segment during postnatal development in the mouse (BALB/c) retina.

Disc formation of rod photoreceptor cells in developing BALB/c mice retinas was studied by rapid freeze, freeze-substitution, freeze-etching, immunocytochemistry, and myosin S-1 decoration methods. Freeze-substituted photoreceptor cells contained variously shaped vesicles in the apical swelling of the connecting cilium or the base of the outer segment during postnatal development. Rapid freezing successfully arrested pinocytosis; the fusion of small vesicles to give large ones, and the compression of certain vesicles (0.3-0.6 micron) appears to lead gradually to the formation of the so-called discs. We therefore propose that membranous discs are formed by the fusion of small pinocytotic vesicles and their subsequent compression. Discs formed in this way were partially stacked, but were ordered at random during the early developmental stages. During development, a partial stack of discs was progressively rearranged to a regular form as seen in mature outer segments. Cytoskeletal actin was expected to be involved in the disc formation; it was demonstrated in the distal axoneme of the connecting cilium during development and showed no change in its distribution. However, the polarity of the actin filaments, as revealed by myosin S-1 decoration in early developmental stages, was much more variable than in the adult. Barbed ends of actin filaments were associated with the plasma membrane or the membrane of vesicles. We also found actin filaments coiled up helically on ciliary microtubules.


Smooth muscle cells in the walls of ovarian follicles in the Japanese quail.

The walls of pre-ovulatory follicles of the Japanese quail were examined at the ultrastructural level for the presence of cells displaying the typical morphological features of smooth muscle cells. These characteristics were found in the cells of the chordae, the tunica albuginea, and the theca externa. Small, elongated cells, containing microfilaments, were observed in the theca of prelampbrush follicles localized in the ovarian cortex. These thecal cells were considered as the putative precursors of the thecal smooth muscle cells of the pre-ovulatory follicle. The difference between the smooth muscle cells of the pre-ovulatory follicle and those in the wall of the most recent post-ovulatory follicle is the contracted state of the latter, which is most evident in the cells of the theca externa. It can be concluded that the cells of the theca externa are smooth muscle cells which are mainly contracted during the ovulatory process. A comparison was made with other vertebrate species.


Dye coupling in the corneal endothelium: effects of ouabain and extracellular calcium removal.

The effects of ouabain and extracellular calcium removal on gap junctional coupling of isolated rabbit corneal endothelium was examined using a modified dye-spread technique. This technique is a modification of a microelectrode procedure that now utilizes patch electrodes connected to a current-clamp circuit for dye iontophoresis and a shuttering system in the excitation light path to reduce phototoxic effects in the monolayer. It was found that a significant degree of junctional uncoupling occurred after 45 min of exposure to ouabain, quantified as a reduction in the effective diffusion coefficient of injected Lucifer yellow CH: 1.74 x 10(-7) cm2/s (control) versus 0.43 x 10(-7) cm2/s (ouabain-treated). It was also determined that no gap junctional uncoupling occurs after extended exposure (up to 3.5 h) to a calcium-free extracellular environment.


Postembryonic development of leucokinin I-immunoreactive neurons innervating a neurohemal organ in the turnip moth Agrotis segetum.

In the abdominal ganglia of the turnip moth Agrotis segetum, an antibody against the cockroach neuropeptide leucokinin I recognizes neurons with varicose fibers and terminals innervating the perisympathetic neurohemal organs. In the larva, the abdominal perisympathetic organs consist of a segmental series of discrete neurohemal swellings on the dorsal unpaired nerve and the transverse nerves originating at its bifurcation. These neurohemal structures are innervated by varicose terminals of leucokinin I-immunoreactive (LKIR) fibers originating from neuronal cell bodies located in the preceding segment. In the adult, the abdominal segmental neurohemal units are more or less fused into a plexus that extends over almost the whole abdominal nerve cord. The adult plexus consists of peripheral nerve branches and superficial nerve fibers beneath the basal lamina of the neural sheath of the nerve cord. During metamorphosis, the LKIR fibers closely follow the restructuration of the perisympathetic organs. In both larvae and adults the LKIR fibers in the neurohemal structures originate from the same cell bodies, which are distributed as ventrolateral bilateral pairs in all abdominal ganglia. The transformation of the series of separated and relatively simple larval neurohemal organs into the larger, continuous and more complex adult neurohemal areas occurs during the first of the two weeks of pupal life. The efferent abdominal LKIR neurons of the moth Agrotis segetum thus belong to the class of larval neurons which persist into adult life with substantial peripheral reorganization occurring during metamorphosis.


Postnatal development and testosterone-dependence of GP-83 and GP-49, two sperm maturation-related glycoproteins in BALB/c mouse epididymis.

The secretion of sperm maturation-related molecules by the epididymis is subjected to developmental and hormonal regulation. In the BALB/c mouse, we found that GP-83 and GP-49, two sperm maturation-related glycoproteins, were secreted by the epididymis. The present study investigated the postnatal development and testosterone-dependence of these two molecules. Histochemical localization in paraffin sections revealed that wheat-germ agglutinin (WGA)-binding sites were first present in the epididymis of 4-week-old mice. The distribution of WGA-binding sites was the same as that of more mature mice, i.e., it was first found in the principal cells of the corpus epididymidis, and gradually appeared in the contents of epididymal tubules. On WGA blots, GP-83 and GP-49 were identified in the corpus, and GP-83 was identified in the cauda of the epididymis. In mice that had received unilateral orchiectomy at 4 weeks of age, GP-83 and GP-49 were present in both intact and orchiectomized epididymides 4 weeks after the operation. In the epididymides of mice that had received bilateral orchiectomy, GP-83 and GP-49 were barely identifiable. However, the presence of these two molecules was restored if testosterone was supplemented immediately after orchiectomy. These results indicate that GP-83 and GP-49 are secreted de novo in the epididymis, and that the secretion of these two molecules is developmentally regulated and androgen-dependent.


Quantitative morphology of stimulation-induced damage in rabbit fast-twitch skeletal muscles.

The purpose of this study was to examine the contention that stimulation-induced damage, resulting in degeneration with subsequent regeneration, plays a major role in the transformation of fibre type brought about by chronic electrical stimulation. Data from histological and histochemical sections of 9-day-stimulated rabbit fast-twitch muscles were analysed with multivariate statistical techniques. Fibre degeneration and regeneration varied non-systematically between sample areas at any given cross-sectional level. In the extensor digitorum longus muscle, but not in the tibialis anterior, there was more degeneration in proximal than in distal portions of the muscle. The extensor digitorum longus muscle consistently showed more degeneration than the tibialis anterior muscle. Degeneration was less extensive for an intermittent pattern of stimulation that delivered half the aggregate number of impulses of continuous stimulation. Degeneration and regeneration varied markedly between individual rabbits in each of the groups. Sections that revealed the most degeneration and regeneration also had more fibres that reacted positively with an anti-neonatal antibody. Rigorous analysis of different sources of variation has helped to explain apparent conflicts in the literature. The incidence of muscle fibre damage in the stimulated tibialis anterior muscle is low, showing that the contribution of degenerative-regenerative phenomena to fibre type conversion in this muscle is insignificant.


Bromodeoxyuridine-immunohistochemistry on cellular differentiation and migration in the fundic gland of Xenopus laevis during development.

Cellular differentiation and migration in the fundic glands of adult and larval Xenopus laevis have been examined using bromodeoxyuridine-immunohistochemistry. In the adult fundic gland, cumulative labeling with bromodeoxyuridine revealed a proliferative cell zone between the surface mucous cells and mucous neck cells, in what is referred to as the neck portion of the gland. The labeling-index of mucous neck cells had rapidly increased by week-5. The labeling-index of oxynticopeptic cells showed a more delayed increase until week-7, coincident with the decrease in the labeling of mucous neck cells. In the immature fundic glands of larvae, the labeled proliferating cells were randomly distributed throughout the developing gastric mucosa. During metamorphosis, the labeling-index of immature epithelial cells was highest at stage 63. Following administration of bromodeoxyuridine at this stage, there was no significant loss of labeled epithelial cells during the metamorphosing period. Furthermore, there was no significant difference in the labeling-indices among the epithelial cells, such as surface mucous cells/generative cells, mucous neck cells, and oxynticopeptic cells, 7 days after administration. Cellular differentiation and migration pathways of epithelial cells in the fundic gland of adult X. laevis and its larvae are discussed.


Immunolocalization of inhibin alpha-subunit in the human testis. A light- and electron-microscopy study.

The localization of inhibin alpha-subunit in the human testis was studied at the light- and electron-microscope level with immunostaining techniques. Antibodies against specific fragments of porcine and human inhibin alpha-subunits were utilized. At light microscopy, inhibin alpha-subunit immunoreactivity was detected in Sertoli cells, spermatocytes and in some Leydig cells. At electron microscopy, gold labeling was found in the cisternae of the Golgi apparatus and in the endoplasmic reticulum of Sertoli and Leydig cells. Gold labeling for inhibin was also found in coated vesicles in the cytoplasm of Sertoli cells as well as in coated pits and coated vesicles in the cytoplasm of some spermatocytes. The results of the present study suggest that, in the human testis, inhibin is produced by Sertoli and Leydig cells and is taken up by spermatocytes, on which it might act in a paracrine manner.


In vivo effects of tunicamycin on chondrocytes of rat mandibular condyles as revealed by lectin cytochemistry.

The in vivo effects of tunicamycin on the glycosylation of proteoglycans and link protein in rat mandibular condylar chondrocytes were studied by ultrastructural lectin histochemistry. The binding of wheat-germ agglutinin was shown by using anti-lectin antibody followed by protein A-gold complex. In normal rats, wheat-germ agglutinin labeling was restricted to trans cisternae and vacuoles of the Golgi complex, whereas it was observed in neither the cis region of the Golgi complex nor in the rough endoplasmic reticulum. By 3 h after the drug administration, wheat-germ agglutinin binding sites on the disorganized Golgi vacuoles were dramatically reduced in number. At 6 h after the drug administration, the lectin binding sites on the Golgi vacuoles were restored. These results demonstrate that the in vivo use of tunicamycin in combination with histochemical analysis using lectin probes is of significant value for the study of protein glycosylation in chondrocytes of the rat mandibular condyle.


Neuropeptides regulate the cardiac activity of a prosobranch mollusc, Rapana thomasiana.

Involvement of neuropeptides in the regulation of cardiac activity in a prosobranch mollusc, Rapana thomasiana, was studied physiologically as well as immunohistochemically. A catch-relaxing peptide (CARP) showed strong inhibitory effects on the heart with a lower threshold than acetylcholine. The action of CARP was in contrast to that of another neuropeptide, FMRFamide, which has previously been shown to enhance the heart beat. Benzoquinonium blocked the effects of acetylcholine and stimulation of right cardiac nerves 1 and 3 b, but not those of CARP, suggesting that the effects of nerve stimulation are mainly due to the release of acetylcholine. Immunohistochemical examinations demonstrated that FMRFamide-like and CARP-like immunoreactive neurons are distributed in the visceral ganglia. Although a neuron appeared to show weak immunoreactivity to both antisera, evidence for the coexistence of peptides in a single neuron was not exhibited. Positive immunoreactivity to FMRFamide and CARP antisera also appeared in right cardiac nerves 1 and 3. In the heart, FMRFamide- and CARP-like immunoreactive fibers were restricted to the atrium and the aortic end of the ventricle, consistent with the morphological observation of innervation. The present results suggest that FMRFamide- and CARP-like peptides are involved in regulating the heart beat.


Fine structure of the neurohemal sinus gland of the shore crab, Carcinus maenas, and immuno-electron-microscopic identification of neurosecretory endings according to their neuropeptide contents.

The sinus gland of the shore crab, Carcinus maenas, is a compact assembly of interdigitating neurosecretory axon endings abutting upon the thin basal lamina of a central hemolymph lacuna. Four types of axon endings are distinguishable by the size distribution, shape, electron density and core structure of their neurosecretory granules. One additional type of axon ending is characterized by electron-lucent vacuoles and vesicles. The axon profiles are surrounded by astrocyte-like glial cells. Various fixations followed by epoxy- or Lowicryl-embedding were compared in order to optimize the preservation of the fine structure of the granule types and the antigenicity of their peptide hormone contents. By use of specific rabbit antisera, the crustacean hyperglycemic, molt-inhibiting, pigment-dispersing, and red-pigment-concentrating hormones were assigned to the four distinct granule types which showed no overlap of immunostaining. Epi-polarization microscopy and ultrathin section analysis of immunogold-stained Lowicryl-embedded specimens revealed that immunoreactivity to Leu-enkephalin and proctolin is co-localized with molt-inhibiting hormone immunoreactivity in the same type of granule. The size and core structure of the immunocytochemically identified granule types vary little with the different pretreatments but, in some cases, to a statistically significant extent. The present results are compared with those from earlier studies of sinus glands in different crustaceans. The methods of granule identification used in this study supplement the classical approach in granule typing; they are easier to perform and more reliable for the analysis of release phenomena in identified secretory neurons supplying the neurohemal sinus gland.


Pituitary adenylate cyclase-activating polypeptide (PACAP): occurrence in rodent pancreas and effects on insulin and glucagon secretion in the mouse.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that occurs in several tissues, e.g., in the gut. We have studied PACAP-like immunoreactivity in the pancreas of rat and mouse, and the effects of PACAP-38 on basal and stimulated insulin and glucagon secretion in the mouse. Immunofluorescence staining demonstrated the presence of PACAP-like immunoreactivity in nerve fibers in both the rat and mouse pancreas. The nerve fibers were seen in the exocrine pancreas and surrounding the islets. Occasionally, the nerve fibers occurred within the islets. Most PACAP-positive nerve fibers innervated the intrapancreatic ganglia, although no nerve cell bodies contained PACAP-like immunoreactivity. In-vivo experiments in mice revealed that basal plasma glucagon levels were increased by PACAP-38 injected intravenously at dose levels exceeding 1.8 nmol/kg. Furthermore, PACAP-38 (7 nmol/kg) potentiated the plasma glucagon response to the cholinergic agonist carbachol (0.16 mumol/kg). This potentiation was reduced to simple addition by pretreatment with a combined alpha- and beta-adrenergic blockade by phentolamine (35 mumol/kg) and propranolol (8.5 mumol/kg). Moreover, PACAP-38 inhibited a carbachol-induced increase in the level of plasma insulin in the absence but not in the presence of adrenergic blockade. PACAP-38 increased basal plasma insulin levels and increased basal plasma glucose levels 6 min and 10 min, respectively, after injection of the peptide. We conclude that PACAP-like immunoreactivity exists in nerve fibers innervating the mouse and rat pancreas, particularly the intrapancreatic ganglia, and that PACAP-38 augments both basal and carbachol-stimulated glucagon secretion in the mouse.


The role of cell proliferation and migration in the development of a neo-intimal layer in veins grafted into arteries, in rats.

The development of a thickened (hyperplastic) fibro-cellular neo-intima is a significant event in the adaptation of a vein grafted into an artery. The histogenesis of tissues in vein grafts was explored in a rat model where the source of endothelial and smooth muscle cells was from the adjacent artery. Cell proliferation was assessed by the incorporation of tritiated thymidine and autoradiography, up to 18 months after grafting. Cell migration was detected by prelabelling in the first 5 days after grafting and sampling at later times. The proliferation of cells in the arterial media adjacent to the graft was elevated above control levels as early as 2 days after grafting; it was maximal at 3 days and returned to low levels by day 21. During the first week, prelabelled smooth muscle cells in the tunica media of the adjacent artery migrated to the subendothelial space, where they continued to proliferate to produce arterial intimal hyperplasia. The migration of endothelial and smooth muscle cells proceeded across the anastomosis to populate the vein graft neo-intima, where smooth muscle cells continued to proliferate until 28 days after grafting. Cell migration and proliferation were significant factors in the histogenesis of vein graft neo-intimal hyperplasia in this model. These processes were controlled, perhaps by local regulatory factors, to form a vein graft, the wall of which was similar in thickness and structure to that of the host artery.


Calbindin D-28k-positive neurons in the rat olfactory bulb. An immunohistochemical study.

We have studied the distribution of calbindin D-28k immunoreactivity in the rat olfactory bulb using specific monoclonal antibodies and the avidin-biotin-immunoperoxidase method. The largest number of positive neurons was located in the periglomerular layer. These neurons were identified as periglomerular cells; they have been described also by other authors as calbindin-positive elements. Close to these neurons, a second population of nerve cells was identified as superficial short-axon neurons. The remaining layers showed a smaller number of stained elements. Other labeled neurons were located along the external border of the external plexiform layer; the scarce neurons marking its internal border were identified as van Gehuchten cells. No immunoreactive structures were found in the mitral cell layer, although we observed another population of immuno-stained short-axon cells at its internal border. Some reactive structures, identified by us as horizontal and vertical cells of Cajal, were located in the boundary zone between the internal plexiform layer and the granule layer. In the white matter, we found a neuronal type characterized by its large size and oriented arborization of varicose dendrites.


Glomerular bypass shunts and distribution of glomeruli in the kidney of the lesser spotted dogfish, Scyliorhinus caniculus.

Scanning electron microscopy of corroded resin casts of the renal vasculature of Scyliorhinus caniculus has revealed a novel vascular pathway arising from the afferent arteriole and bypassing the glomerulus. This glomerulus bypass shunt occurred in 36% of the glomerular casts examined. The shunt ran to join a peritubular network of capillaries and thereby offers the potential to vary the degree of glomerular perfusion and control the proportion of active glomeruli. In 29% of glomeruli two efferent arterioles drained the capillary knot. Glomeruli were located close to the dorsal margin of the posterior mass of the kidney, and towards the lateral edge of the anterior lobes of the kidney of female dogfish. In male dogfish, glomeruli were evenly distributed through the posterior mass of kidney, while in female dogfish 89% of glomeruli occurred in the posterior mass and 11% of glomeruli were located within the small anterior lobes.


Organization of the ciliary basal apparatus in embryonic cells of the sea urchin, Lytechinus pictus.

The basal apparatus of embryonic cells of the sea urchin Lytechinus pictus was examined by transmission electron microscopy and compared with the basal apparatus of other metazoan cells. The basal apparatus in these cells is associated with a specialized region of the apical cell surface that is encircled by a ring of microvilli. The basal apparatus includes several features that are common to all ciliated cells, including a basal body, basal foot, basal foot cap, and striated rootlet. However, a component not seen in the basal apparatus of other species has been observed in these cells. This structure is continuous with the striated rootlet, and its ultrastructure indicates that it is composed of the same components as the rootlet. This structure extends from the junction of the basal body and striated rootlet to the cortical region that surrounds the basal body. Based on its morphology and position, this structure is referred to as a striated side-arm. The striated side-arm is always aligned in the plane of the basal foot. Thus, both of these structures extend from the basal body in the plane of the effective stroke. It is suggested that the striated side-arm serves to stabilize the basal apparatus against force exerted by the cilium.


Localization of calcitonin gene-related peptide and islet amyloid polypeptide in the rat and mouse pancreas.

It was previously demonstrated that the two chemically related peptides calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) both occur in the pancreas. We have now examined the cellular localization of CGRP and IAPP in the rat and the mouse pancreas. We found, in both the rat and the mouse pancreas, CGRP-immunoreactive nerve fibers throughout the parenchyma, including the islets, with particular association with blood vessels. CGRP-immunoreactive nerve fibers were regularly seen within the islets. In contrast, no IAPP-immunoreactive nerve fibers were demonstrated in this location. Furthermore, in rat islets, CGRP immunoreactivity was demonstrated in peripherally located cells, constituting a major subpopulation of the somatostatin cells. Such cells were lacking in the mouse islets. IAPP-like immunoreactivity was demonstrated in rat and mouse islet insulin cells, and, in the rat, also in a few non-insulin cells in the islet periphery. These cells seemed to be identical with somatostatin/CGRP-immunoreactive elements. In summary, the study shows (1) that CGRP, but not IAPP, is a pancreatic neuropeptide both in the mouse and the rat; (2) that a subpopulation of rat somatostatin cells contain CGRP; (3) that mouse islet endocrine cells do not contain CGRP; (4) that insulin cells in both the rat and the mouse contain IAPP; and (5) that in the rat, a non-insulin cell population apparently composed of somatostatin cells stores immunoreactive IAPP. We conclude that CGRP is a pancreatic neuropeptide and IAPP is an islet endocrine peptide in both the rat and the mouse, whereas CGRP is an islet endocrine peptide in the rat.


Immunolocalization of synexin (annexin VII) in adrenal chromaffin granules and chromaffin cells: evidence for a dynamic role in the secretory process.

Synexin (annexin VII) is a Ca(2+)- and phospholipid-binding protein which has been proposed to play a role in Ca(2+)-dependent membrane fusion processes. Using a monoclonal antibody against synexin, Mab 10E7, and immunogold, we carried out a semiquantitative localization study of synexin in bovine adrenal medullary chromaffin granules, and in resting and nicotine-stimulated adrenal chromaffin cells. Isolated chromaffin granules contained very little synexin, whereas chromaffin granules aggregated with synexin (24 micrograms/mg) and Ca2+ (1 mM) clearly showed synexin-associated immunogold particles in the vicinity of the granule membrane (1.88 gold particles per granule profile). In isolated, cultured adrenal chromaffin cells, synexin was present in the nucleus (5.5 particles/microns 2) and in the cytosol (5.3 particles/microns 2), but mainly around the granule membrane in the granular cell area (11.7 particles/microns 2). During the active phase of cholinergically stimulated catecholamine secretion, the amount of synexin label was reduced by 33% in the nucleus, by 23% in the cytosol, and by 51% in the granule area. The plasma membrane contained a small amount of synexin, which did not significantly change upon stimulation of the cells. We conclude that synexin is involved in the secretory process in chromaffin cells.


Species variability in the expression of met- and leu-enkephalin-like immunoreactivity in mammalian Merkel cell dense-core granules. A light- and electron-microscopic immunohistochemical study.

Immunogold staining failed to show met-enkephalin immunoreactivity in the Merkel cell dense-core granules of rats when examined by electron microscopy, but showed gold particle staining in the Merkel cell dense-core granules of mice and nude mice. Merkel cells of hamster, guinea pig, rabbit, cat and dog were also examined using a similar method, and different antisera dilutions. Immunogold particles were consistently found in the dense-core granules of mice and nude mice at all antisera dilutions, but not in the other species, except in the dog, where a very low labelling response was encountered. Merkel cells from skin touch domes or sinus hair follicles, did not exhibit any difference in peptide expression as far as met-enkephalin immunoreactivity was concerned. In addition, all species studied, including mice and nude mice, did not show leu-enkephalin immunoreactivity in their Merkel cell dense-core granules. It is concluded that species variability in peptide expression occurs in the Merkel cell dense-core granules, and may be closely related to the different methodologies used.


Some peptide-like colocalizations in endocrine cells of the pyloric caeca and the intestine of Oncorhynchus mykiss (Teleostei).

The coexistence of immunoreactivities to cholecystokinin, glucagon, glucagon-like peptide 1, salmon pancreatic polypeptide, neuropeptide tyrosine, and peptide tyrosine tyrosine was studied immunocytochemically, revealing for the first time in fish intestine the existence in the same cell of immunoreactivities to cholecystokinin-glucagon/glucagon-like peptide 1, cholecystokinin-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-neuropeptide tyrosine, salmon pancreatic polypeptide tyrosine tyrosine, and glucagon/glucagon-like peptide 1-peptide tyrosine tyrosine. Colocalization of cholecystokinin-salmon pancreatic polypeptide was observed only in the pyloric caeca of the rainbow trout Oncorhynchus mykiss, while the other colocalizations also occurred in proximal and middle intestinal segments. In all cases, endocrine cells immunoreactive to only one of the paired antisera were detected except for anti-glucagon and anti-glucagon-like peptide 1, which always immunostained the same cells.


Dark and light zones of germinal centres of the human tonsil: an ultrastructural study with emphasis on heterogeneity of follicular dendritic cells.

The cellular composition of the dark and light zones of germinal centres in human tonsils was quantitatively determined by electron microscopy. In addition to the well known germinal-centre B-cells, we defined the cleaved blast as a new distinct cell type in the germinal centre. The dark and the light zones clearly differed in their content of lymphoid and non-lymphoid germinal-centre cells. The dark zone was characterized by higher frequencies of cleaved blasts and small centroblasts, a higher blast-centrocyte ratio and a higher incidence of mitotic figures. In contrast, the light zone had higher frequencies of centrocytes, centroplasmacytoid cells, lymphocytes and follicular dendritic cells (FDC) and an inverted blast-centrocyte ratio. Seven distinct appearances of FDC (FDC.1-FDC.7) could be recognized on the basis of their ultrastructure. The distribution pattern of these subtypes differed between the dark and light zone. The undifferentiated subtypes FDC.2 and FDC.3 predominated in the dark zone. In contrast, the highly differentiated subtypes FDC.4 and FDC.5 were present at a much higher density in the light zone. These findings suggest that the dark and light zones are different compartments with regard to proliferation and differentiation of germinal-centre B-cells and that both compartments have a specific microenvironment influenced by FDC.


A novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), in human intestine: evidence for reduced content in Hirschsprung's disease.

A novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), exhibits sequence homology with vasoactive intestinal polypeptide (VIP) and occurs in the mammalian brain, lung and gut. The distribution of PACAP in ganglionic and aganglionic portions of the large intestine of patients with Hirschsprung's disease was examined by immunohistochemistry and radioimmunoassay. PACAP-immunoreactive nerve fibers were distributed in all layers of the ganglionic and aganglionic segments of the intestine, although they were less numerous in the latter, and PACAP-immunoreactive nerve cell bodies were seen in the ganglionic portion of the intestine. The concentration of immunoreactive PACAP was lower in the aganglionic than in the ganglionic segment of the intestinal wall. PACAP and VIP were found to coexist in both ganglionic and aganglionic segments of the intestine. Apparently, PACAP participates in the regulation of gut motility. The scarcer PACAP innervation of the aganglionic segment may contribute to the defect in intestinal relaxation seen in patients with Hirschsprung's disease.


Distribution of the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues.

The hepatic alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP) binds and endocytoses alpha 2-macroglobulin-proteinase complexes in plasma. In addition, it binds lipoproteins, a novel 40 kDa protein, and complexes between plasminogen activators and plasminogen activator inhibitor type-1. This study shows, for the first time, the tissue distribution of alpha 2MR/LRP as determined by immunohistochemistry with specific monoclonal antibodies. The analysis revealed alpha 2MR/LRP-expression in a restricted spectrum of cell types, including neurons and astrocytes in the central nervous system, epithelial cells of the gastrointestinal tract, smooth muscle cells, fibroblasts, Leydig cells in testis, granulosa cells in ovary, and dendritic interstitial cells of kidney. Monocyte-derived cells displayed marked alpha 2MR/LRP expression in the phagocytes of liver, lung and lymphoid tissues, but no or low expression in antigen-presenting cells including Langerhans' cells of the skin. The high abundance of alpha 2MR/LRP in certain cell types of most organs suggests two main routes for alpha 2MR/LRP-mediated ligand clearance: (1) systemic removal in liver of circulating ligands, and (2) non-hepatic interstitial removal in different organs, including the brain.


Ultrastructural evidence of indirect and direct autonomic innervation of human Leydig cells: comparison of neonatal, childhood and pubertal ages.

Recent physiological studies have indicated an autonomic influence on the secretion of testosterone from Leydig cells in humans and laboratory animals. Furthermore, a few studies have shown enhanced autonomic control of Leydig cell function in immature, relative to mature, laboratory animals. In the current ultrastructural study of the human testicular interstitium the morphology of autonomic components is described from neonatal, childhood and pubertal ages. Autonomic nerve fibers and varicosities with neurotransmitter vesicles are described in proximity to Leydig cells. The observed autonomic terminals are classified by vesicle morphology into three general types: (1) Type I with predominantly small agranular vesicles (30-60 nm) and occasional larger granular vesicles (100 nm). This type is morphologically consistent with being cholinergic. (2) Type II with predominantly small granular vesicles (30-60 nm), as well as sporadic large granular vesicles. These are morphologically consistent with adrenergic terminals. (3) Type III which exhibit numerous large granular vesicles of mixed size. Evidence of autonomic terminals is encountered most frequently in childhood biopsies, age 3 to 10 years. The neonatal specimen (4 months) is noteworthy in that many of the Schwann cells appear immature and no adrenergic terminals are observed. In contrast, terminals morphologically consistent with being adrenergic are common in the childhood series of biopsies. Although the vast majority of the autonomic terminals are associated with Leydig cells indirectly as "boutons en passant", separated by approximately 150 nm to more than a micron, evidence of direct contact (20 nm) of autonomic terminals with Leydig cells is presented.(ABSTRACT TRUNCATED AT 250 WORDS)


Histological distribution and developmental changes of tropomyosin isoforms in three chicken digestive organs.

Histological localization of tropomyosin isoforms in three digestive organs from embryonic and adult chickens was performed by using rabbit antisera against chicken skeletal muscle tropomyosin and against low-Mr-type tropomyosin from chicken small intestine mucosa. The former antiserum (named TM-SH) reacted with alpha, beta, and high-Mr-type isoforms, and the latter (named TM-HL) reacted with alpha, beta, high-Mr-type and low-Mr-type isoforms, alpha and beta Isoforms were detected in muscle cells of the muscular layer and the muscularis mucosa. Low-Mr-type isoforms, however, were detected along the cell membrane and cytoplasm of almost all nonmuscle cells, especially in terminal webs of epithelial cells. Developmental changes of tropomyosin isoforms in digestive organs were studied by two-dimensional gel electrophoresis and image analysis. The relative amounts of alpha and beta isoforms increased in the course of development, but those of low-Mr-type and high-Mr-type isoforms decreased.


Correlation of extracellular matrix components with the cytoarchitecture of mouse Peyer's patches.

The distribution patterns of extracellular matrix elements were determined to ascertain whether they play a role in the localization of lymphocytes in discrete T-cell, B-cell and dome antigen-processing domains within Peyer's patches. Antibodies against collagen types I, III and IV, laminin and fibronectin were applied to cryosections of mouse Peyer's patches and localized by direct or indirect immunoperoxidase methods. T-cell domains were identified with a monoclonal antibody against Thy-1.2. Labeled reticular fibers in distinctive patterns were more numerous in parafollicular and dome areas than within follicles. Germinal centers contained few such fibers. In parafollicular areas, fibers were oriented predominantly toward follicle domes; their distribution corresponded to T-cell zones and lymphocyte traffic areas, with their orientation being parallel to the migration pathways of lymphocytes from high endothelial venules to the antigen-processing domes. Subepithelial and subendothelial basal laminae were immunopositive for type-IV collagen, laminin and fibronectin. The dome subepithelial basal lamina had pore-like discontinuities through which lymphocytes migrated to and from the epithelium. The correspondence of the distribution patterns of extracellular matrix to specific functional domains of Peyer's patches suggests that this matrix provides a structural framework for lymphocyte migration and localization.


Electron microscopy of a double helical tubular filament in keyhole limpet (Megathura crenulata) hemolymph.

A approximately 25 nm hollow double helical filament has been detected ultrastructurally in the cell-free supernatant from hemolymph of the keyhole limpet Megathura crenulata (Gastropoda: Prosobranchia: Fissurellidae). Subsequently, much higher concentrations of this material were found in the cell pellet from hemolymph. Both negative staining and thin sectioning have been performed in an attempt to obtain a preliminary structural characterization of this new filament. It is proposed that the filaments are released or secreted from blood hemocytes in response to bleeding, but it has not been possible to define absolutely an intracellular organelle containing this material. It is shown that the hollow filaments, which have fine fibrillar surface extensions, are readily distinguishable from collagen fibrils and from tubular (multi-decameric) keyhole limpet hemocyanin (KLH).


Immunohistochemical localization and radioenzymatic measurements of serotonin (5-hydroxytryptamine) in hearts of Aplysia and several bivalve mollusks.

Serotonin immunoreactivity was localized in hearts of the opisthobranch gastropod, Aplysia californica (sea hare) and several species of bivalve mollusks, the heterodonts, Mercenaria mercenaria (quahog or cherry stone clam), Protothaca staminea (little neck clam), and the pteriomorphs, Hinnites multirugosus (rock scallop), Crassostrea virginica (eastern oyster), Mytilus edulis (eastern mussel), and Geukensia demissa (ribbed mussel). In addition, serotonin was assayed in the ventricles, auricles and heart-associated tissues in A. californica, M. mercenaria, H. multirugosus, and G. demissa with a sensitive radioenzymatic assay. Serotonin concentrations and the density of innervation were significantly higher in members of the subclass Heterodonta compared to the subclass Pteriomorpha. Serotonin immunoreactivity was observed in all species surveyed except G. demissa, which also contained relatively low concentrations of serotonin. Varicose fibers presumably corresponding to release sites were localized in the ventricles, auricles, and the auricular-ventricular valves. We hypothesize that in the species where serotonin-immunoreactive fibers are present, serotonin serves to modulate cardiac myogenic activity. The significance of the observed distribution and concentration of serotonin to the physiological effects of serotonin on cardiac function in these species is discussed.


Species differences in the immunoreactive patterns of calcitonin gene-related peptide in the pancreas.

In the pancreas, calcitonin gene-related peptide (CGRP) immunoreactivity has been described in nerve fibers and in distinct types of islet cells. This unique, apparently species-specific cell-type expression prompted the present investigation to clarify further the pattern of CGRP immunoreactivity in different mammalian species (i.e., different strains of rats, mice, guinea pigs, rabbits, cats, dogs, pigs, and humans) commonly used for functional and anatomical studies of the pancreas by means of immunohistochemistry using three different CGRP antibodies. In each species, CGRP-immunoreactive neurites innervate the exocrine and endocrine compartments, the vasculature, and the intrapancreatic ganglia, where they form dense networks encircling unstained cell bodies. The only exception is the pig pancreas, where the islets appear to be devoid of immunoreactive fibers. The overall density of immunoreactive pancreatic axons in different species is as follows: rat, mouse, and rabbit greater than guinea pig greater than or equal to pig and cat much greater than dog and human. CGRP-immunoreactive endocrine cells appear to be restricted to the rat pancreas, where they form a subpopulation of somatostatin-containing D cells. In contrast, in mouse, guinea pig, cat, dog, and human pancreas, a homogeneous staining of the core of the islets, where insulin-producing B cells are located, was visualized in sections incubated with the rabbit CGRP antiserum at 4 degrees C, but not at 37 degrees C (an incubation temperature that does not affect the islet cell staining in the rat nor the fiber labeling in any species). Furthermore, the staining of islet B cells was not reproducible with all the CGRP antibodies used, all of which comparably stain nerve fibers in each species, and islet D cells in the rat. Immunoreactive islet cells were not visualized in pig and rabbit pancreas. These results are consistent with the hypothesis that the expression of CGRP in nerve fibers is a common feature of mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to the D cells of the rat pancreas.


Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I.

An antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electron-microscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hind-gut.