Drosophila KASH-domain protein Klarsicht regulates microtubule stability and integrin receptor localization during collective cell migration.

During collective migration of the Drosophila embryonic salivary gland, cells rearrange to form a tube of a distinct shape and size. Here, we report a novel role for the Drosophila Klarsicht-Anc-Syne Homology (KASH) domain protein Klarsicht (Klar) in the regulation of microtubule (MT) stability and integrin receptor localization during salivary gland migration. In wild-type salivary glands, MTs became progressively stabilized as gland migration progressed. In embryos specifically lacking the KASH domain containing isoforms of Klar, salivary gland cells failed to rearrange and migrate, and these defects were accompanied by decreased MT stability and altered integrin receptor localization. In muscles and photoreceptors, KASH isoforms of Klar work together with Klaroid (Koi), a SUN domain protein, to position nuclei; however, loss of Koi had no effect on salivary gland migration, suggesting that Klar controls gland migration through novel interactors. The disrupted cell rearrangement and integrin localization observed in klar mutants could be mimicked by overexpressing Spastin (Spas), a MT severing protein, in otherwise wild-type salivary glands. In turn, promoting MT stability by reducing spas gene dosage in klar mutant embryos rescued the integrin localization, cell rearrangement and gland migration defects. Klar genetically interacts with the Rho1 small GTPase in salivary gland migration and is required for the subcellular localization of Rho1. We also show that Klar binds tubulin directly in vitro. Our studies provide the first evidence that a KASH-domain protein regulates the MT cytoskeleton and integrin localization during collective cell migration.

A cardiovascular simulator tailored for training and clinical uses.

OBJECTIVE: In the present work a cardiovascular simulator designed both for clinical and training use is presented. METHOD: The core of the simulator is a lumped parameter model of the cardiovascular system provided with several modules for the representation of baroreflex control, blood transfusion, ventricular assist device (VAD) therapy and drug infusion. For the training use, a Pre-Set Disease module permits to select one or more cardiovascular diseases with a different level of severity. For the clinical use a Self-Tuning module was implemented. In this case, the user can insert patient's specific data and the simulator will automatically tune its parameters to the desired hemodynamic condition. The simulator can be also interfaced with external systems such as the Specialist Decision Support System (SDSS) devoted to address the choice of the appropriate level of VAD support based on the clinical characteristics of each patient. RESULTS: The Pre-Set Disease module permits to reproduce a wide range of pre-set cardiovascular diseases involving heart, systemic and pulmonary circulation. In addition, the user can test different therapies as drug infusion, VAD therapy and volume transfusion. The Self-Tuning module was tested on six different hemodynamic conditions, including a VAD patient condition. In all cases the simulator permitted to reproduce the desired hemodynamic condition with an error<10%. CONCLUSIONS: The cardiovascular simulator could be of value in clinical arena. Clinicians and students can utilize the Pre-Set Diseases module for training and to get an overall knowledge of the pathophysiology of common cardiovascular diseases. The Self-Tuning module is prospected as a useful tool to visualize patient's status, test different therapies and get more information about specific hemodynamic conditions. In this sense, the simulator, in conjunction with SDSS, constitutes a support to clinical decision - making.

Application of a user-friendly comprehensive circulatory model for estimation of hemodynamic and ventricular variables.

PURPOSE: Application of a comprehensive, user-friendly, digital computer circulatory model to estimate hemodynamic and ventricular variables. METHODS: The closed-loop lumped parameter circulatory model represents the circulation at the level of large vessels. A variable elastance model reproduces ventricular ejection. The circulatory model has been modified embedding an algorithm able to adjust the model parameters reproducing specific circulatory conditions. The algorithm reads input variables: heart rate, aortic pressure, cardiac output, and left atrial pressure. After a preliminary estimate of circulatory parameters and ventricular elastance, it adjusts the amount of circulating blood, the value of the systemic peripheral resistance, left ventricular elastance, and ventricular rest volume. Input variables and the corresponding calculated variables are recursively compared: the procedure is stopped if the difference between input and calculated variables is within the set tolerance. At the procedure end, the model produces an estimate of ventricular volumes and Emaxl along with systemic and pulmonary pressures (output variables). The procedure has been tested using 4 sets of experimental data including left ventricular assist device assistance. RESULTS: The algorithm allows the reproduction of the circulatory conditions defined by all input variable sets, giving as well an estimate of output variables. CONCLUSIONS: The algorithm permits application of the model in environments where the simplicity of use and velocity of execution are of primary importance. Due to its modular structure, the model can be modified adding new circulatory districts or changing the existing ones. The model could also be applied in educational applications.

Defensive conditioning-related functional heterogeneity among nuclei of the rat amygdala revealed by c-Fos mapping.

The amygdala is a complex forebrain structure proposed to play a pivotal role in fear conditioning circuitry. In this study, c-Fos immunomapping was applied to investigate the functional activation of particular amygdalar nuclei following a 50-trial training session of two-way active avoidance reaction. To dissect distinctive responses displayed by the animals and to cluster them into groups of correlated behaviors, factor analysis was employed. The training procedure resulted in an increase of c-Fos expression within the cortical, medial, lateral and basolateral, but not central, nuclei. The expression in the cortical nucleus correlated negatively with grooming behavior, whereas c-Fos immunolabeling of the other three subdivisions of the amygdala could be associated with the number of intertrial responses. No correlation was observed between c-Fos expression and avoidance reactions performed or the amount of shock received by the animal. The results obtained with c-Fos mapping of various regions of rat amygdala, combined with a fine dissection of behavioral repertoire, imply that there are specific functional links between particular parts of the structure and distinctive behaviors that reflect various emotional states of the animal.

Inhibitory effects of fumagillin and its analogue TNP-470 on the function, morphology and angiogenesis of an oestrogen-induced prolactinoma in Fischer 344 rats.

The process of angiogenesis occurs in many physiological states, but it is also essential for the growth of solid tumours and metastasis formation. An abnormal arterial vascularization has been shown in prolactin-secreting pituitary adenomas induced by prolonged treatment with oestrogens in Fischer 344 (F344) rats. It is thought that anti-angiogenic agents might be useful in therapy for these tumours. Fumagillin and its analogue TNP-470 are known to inhibit endothelial cell proliferation selectively, but their effect on lactotroph cell secretory function and prolactinoma formation has not yet been described. The aim of the present study was to examine the effects of fumagillin and TNP-470 on prolactin secretion, and morphological and vascular changes within the anterior pituitary in long-term oestrogen-treated male F344 rats in vivo and in vitro. As expected, 7 weeks after s.c. implantation of Silastic tubes containing 10 mg diethyl-stilboestrol (DES), a very high rise in serum prolactin levels was found. Both angiogenesis inhibitors injected s.c. at doses of 10 mg/kg body weight for 24 days attenuated the stimulatory effect of DES on prolactin production and release. They also diminished prolactin cell density and inhibited cell proliferation expressed as the number of anterior pituitary cells labelled with bromodeoxyuridine (BrdU), but the effect of TNP-470 was minor compared with fumagillin. Both angioinhibitors suppressed neo-vascularization within the anterior pituitary with similar potency but, on the other hand, they did not affect DES-induced increases in prolactin secretion from cultured rat pituitary cells and cell proliferation in vitro. In conclusion, our results provide strong evidence for the anti-tumour and anti-prolactin activity of angiogenesis inhibitors in the experimentally oestrogen-induced pituitary adenoma; this might be mediated indirectly through the inhibition of angiogenesis.

The effect of somatostatin analog octreotide on diethylstilbestrol-induced prolactin secretion, cell proliferation and vascular changes in the rat anterior pituitary gland.

The effects of diethylstilbestrol (DES) and of long-acting somatostatin analog, octreotide (SMS) on the rat anterior pituitary microvasculature have been studied by means of computer-assisted image analysis. Additionally, the effects of DES and SMS on prolactin secretion and anterior pituitary cell proliferation have been studied, as well. The vascularization was visualized using Selye's method modified by Poely et al. (1964). The prolactin serum levels were estimated by radio-immunoassay. The proliferation indices were assessed using bromodeoxyuridine incorporation assay. As expected, it was found that DES sharply increased serum prolactin levels and enhanced cell proliferation in the anterior pituitary gland. DES also induced changes in parameters of vascularization. Simultaneous treatment of rats with SMS inhibited the DES-induced elevation of prolactin levels and pituitary cell proliferation. It also suppressed some but not all DES-induced changes in the anterior pituitary vascularization. These data suggest that the angio-inhibitory activity of SMS might be involved in its anti-tumor action on pituitary adenomas, but not as a sole or principal mechanism.

Effects of octreotide on propylotiouracil-induced goiter in rats: a quantitative evaluation.

To evaluate the possible antigoitrogenic effect of somatostatin, the influence of long-acting somatostatin analog--octreotide--on experimental goiter developed in rats treated with propylthiouracil was examined. Goiter formation was assessed by measurement of the main histological compartments of the thyroid as well as by morphometric analysis of the vascularization and blood supply of the gland. Although treatment with octreotide did not prevent the goiter formation, it clearly reduced blood supply and vascularization of the thyroid and counteracted propylthiouracil-induced increase in the relative volume of follicular epithelium. To conclude, the somatostatin analog--octreotide--is effective in reduction of goiter vascularisation. This finding provides a rationale for the clinical trials of the treatment of hypervascular goiter by somatostatin analogs.

The effect of angiotensin II receptor antagonists on diethylstilbestrol-induced vascular changes in the rat anterior pituitary gland: a quantitative evaluation.

The effects if diethylstilbestrol (DES) and of angiotensin II (Ang II) receptor antagonists, such as losartan (selective AT1 receptor antagonist) or PD 123319 (selective AT2 receptor antagonist) on the anterior pituitary microvasculature were studied by means of computer-assisted image analysis. The vascularization was visualized using Selye's method modified by Poely et al. (1964). It was found that DES induced a sharp increase in vessel area, mean vessel diameter and perimeter, whereas mean vessel number was reduced. These DES-induced changes were inhibited by simultaneous administration of losartan. On the other hand, PD 123319 was less effective. These findings suggest an involvement of Ang II, acting mainly via AT1 receptors, in the mechanism of estrogen-induced vascular changes in the rat anterior pituitary gland.

CS modality transfer of two-way avoidance in rats with central and basolateral amygdala lesions.

Post-lesion acquisition of two-way avoidance and subsequent transfer to two warning signals (conditioned stimulus, CS) of different modality were investigated in 60 rats. In Experiment I the animals were originally trained with less salient (darkness) CS, then transferred to more salient compound (darkness and white noise), and finally to white noise CS. The opposite arrangement of the conditioned stimuli (CSi) during the subsequent stages was employed in Experiment II. In control animals, avoidance acquisition was faster and the intertrial responding (ITR) rate lower with the auditory than with the visual CS. Lesioned rats learned avoidance responses more slowly, independently of CS modality. The transfer to other CSi revealed dramatic between-group difference in the level and consistency of avoidance response, shuttle-box latencies and ITR rate. In control animals, transfer to more salient CSi enhanced avoidance performance, whereas change to less salient CS decreased it. Rather small changes in shuttle-box performance and consistency of avoidance response due to CS modality were seen in rats with the basolateral lesions. In contrast, central nucleus injury caused a strong deterioration in the avoidance transfer, especially when the visual CS followed the acoustic one. The results indicate differential involvement of the basolateral and central amygdala nuclei in stimulus-processing mechanisms of instrumental defensive behavior.

The role of conditioned stimulus termination in short-latency avoidance responding in cats.

The behavioral effects of two procedures for bar-pressing avoidance training in cats were studied. In one procedure conditioned stimulus (CS) termination was response-contingent on both shock and non-shock trials; in the other the minimal duration of the CS was equal to the CS-US (unconditioned stimulus) interval. When avoidance responses did not terminate the CS short-latency avoidance responses were not acquired, the cats made more intertrial responses, and removal of the proreal and orbital gyri interfered more with avoidance responding than was observed in the other group. Abolition of shock application and introduction of a fixed duration of the CS resulted in extinction of the avoidance responses, which was more rapid in cats trained under the response contingent CS termination procedure. The data suggest that responses performed during the CS-US interval should be divided into two subclasses: short-latency responses which not only avoid pain but also avoid fear conditioned to the CS, and long-latency responses which avoid pain and escape from the fear state.

Effects of partial lesion of dorsal hippocampal afferent and GM1 ganglioside treatment on conditioned emotional response and hippocampal afferent markers in rats.

Acquisition of the conditioned emotional response (CER) in 32 male hooded rats previously learned to press a bar for food and divided into four groups was studied. Two groups received electrolytic lesions of the dorsal hippocampal afferent and were thereafter injected either with GM1 ganglioside (30 mg/kg daily) or with buffer. Two remaining groups were sham operated and similarly injected. The partial hippocampal deafferentation evoked immediate enhancement of bar presses rate which persisted during the 2-week period of testing. CER training undertaken 2 days after surgical procedures appeared unsuccessful, whereas similar training with a cue of different modality initiated a week later resulted in acquisition of conditioned suppression of bar presses in all groups. Toward the end of training the conditioned suppression was more pronounced in lesioned than in control rats. The GM1 injections attenuated the conditioned suppression in control rats, presumably due to an antinociceptive role of ganglioside treatment. Behavioural training did not change the normal distribution pattern in cholinergic and serotonergic hippocampal afferent markers showing dorso-ventral gradient along longitudinal axis. The lesion-induced decrease pattern was also not affected. However, in contrast to previous findings in non-trained animals, the GM1 treatment was not effective in protecting against degenerative changes in the hippocampus of trained rats.

Morphology of the microvascular bed in primary human carcinomas of lung. Part I: Three--dimensional pattern of microvascular network.

Three-dimensional patterns of the microvascular network of four main types of human lung carcinomas (squamous cell carcinoma, oat cell carcinoma, adenocarcinoma and large cell carcinoma) have been described. Investigation has been conducted in 18 autopsy cases by x-ray microangiography, and in 52 autopsy and surgical cases by modified benzidine-nitroprusside method for colouring the erythrocyte column in blood vessel. Specimens for the investigation were collected from standardized places of the tumor margin. Eight essential spatial structures of which the carcinomas' microvascular bed is built have been distinguished. Some characteristic features of microvascular network of highly differentiated squamous cell carcinomas, adenocarcinomas and oat cell carcinoma have been described. It has been concluded also that large cell carcinomas, poorly differentiated squamous cell carcinomas as well as adenocarcinomas do not produce any characteristic and repeated pattern of microvascular bed. One of the main elements of the neoplasm structure influencing the growth rate of the tumor is its peripherial vascular bed. It determines the access of chemotherapeutics, oxygen and components participating in the immunological response also. All of those factors are significant elements while considering the curability.

Morphology of the microvascular bed in primary human carcinomas of lung. Part II. Morphometric investigations of microvascular bed of lung tumors.

A comparative investigations of peripheral microvascular bed in various histological types of human primary lung carcinomas (squamous cell carcinomas, oat-cell carcinomas, adenocarcinomas and large cell carcinomas) have been performed. The study was performed on 52 cases of lung carcinoma (there were 49 cases from autopsy and 3 cases from surgery) and on 8 normal comparative cases for evaluation of unchanged bronchial mucosa. Tissue blocks were obtained from standardized parts of bronchus and contained the intra- and extrabronchial parts of tumour as well as the bordering bronchial mucosa. The relative capacities of: 1. anaplastic cells, 2. connective tissue in stroma, 3. intraluminal space of blood vessels, 4. degenerative anaplastic cells, 5. necrotic detritus, and 6. haemorrhages and erytrorrhages were estimated in carcinomatous tissue by the hit points method. In bronchial mucosa the capacities of: 1. intraluminal space of blood vessels, and 2. haemorrhages and erythrorrhages were recorded. Furthermore, the structure of peripheral microvascular bed was evaluated by constructing the distributive series of diameters of blood vessels. These distributive series were approximated with the analitical function. It was found that the largest capacity of blood vessels (with an extremely developed capillary segment of vascular bed) is in the intrabronchial part of squamous cell carcinomas. In the extrabronchial part of this type of carcinoma, the capacity of blood vessel is about three-times smaller than in the intrabronchial part (3.36% and 9.26%, respectively). The finest blood vessels of extrabronchial part undergo the most considerable reduction, in comparison with the ones in the intrabronchial part of squamous cell carcinoma. Oat-cell carcinomas show the smallest difference in vascularization of intra- and extrabronchial parts of tumor (4.38% and 3.33%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Cueing properties of the decrease of white noise intensity for avoidance conditioning in cats.

In the main experiment two groups of 6 cats each were trained in active bar-pressing avoidance to a CS consisting of either a 10 dB or 20 dB decrease of the background white noise of 70 dB intensity. The two groups did not differ in rapidity of learning, however cats trained to the greater change .in background noise performed avoidance responses with shorter latencies than did cats trained to smaller change. Within-groups comparisons of cumulative distributions of response latencies for consecutive Vincentized fifths of avoidance acquisition showed the greatest changes in the region of latencies longer than the median latency of instrumental responses. On the other hand, the effects of CS intensity found in between-groups comparisons were located in the region of latencies shorter than the median latency of either group. Comparisons with data obtained in a complementary experiment employing additional 17 cats showed that subjects trained to stimuli less intense than the background noise level were marked by an exceptionally low level of avoidance responding with latencies shorter than 1.1 s, which was lower than expected from the probability of intertrial responses for this period of time. Due to this property of stimuli less intense than the background, the distributions of response latencies were moved to the right, in effect, prefrontal lesions influenced a greater part of latency distributions than in cats trained to stimuli more intense than the background.

Changes of behavioral strategy as a mechanism of relearning instrumental responses after cerebral lesions.

Several examples of functional recovery of instrumental responding after cerebral lesions are reviewed. Small incision on the boundary between the sensory and the motor cortex of a dog changed the saliency not only of the tactile but also of the auditory conditioned stimuli, eliciting the preoperatively acquired alimentary instrumental response. Prefrontal lesions in cats affected differently each of the several learning processes involved in the performance of the avoidance response: they enhanced the classically conditioned component, attenuated the motivational role of fear but left the motor component intact. Preoperatively acquired interrelations of these components underwent substantial changes, leading to the restoration of the previous level of avoidance of pain. However, a highly adaptive mode of behavior, namely avoidance of the state of fear, was permanently impaired. After small electrolytic lesions of amygdalar nucleus centralis cats were handicapped in the acquisition of escape from pain responses. The responses to low shock intensities were especially diminished. Improvement of the escape from pain responses was observed after a prolonged increase of the intertrial response frequency. The effectiveness of both escape and avoidance learning depended on the duration of the postoperative recovery period prior to instrumental training. All these data indicated that cerebral lesions changed the interactions between different parts of the neuronal circuitry participating in the learning and performance of a given instrumental response.

Intertrial responses in defensive instrumental learning.

Data by the author, as well as from other laboratories, show residual fear as the main source of intertrial responses (ITR) during defensive instrumental training in rats, cats and dogs. Between-subject differences in ITR rate arise in the course of training. The differences depend on training conditions and on subject's emotionality. Under the same conditions higher ITR rates correspond to more rapid avoidance learning. The increase of ITR rate is regularly observed in the following circumstances: at early stages of training, after transfer to a less salient discriminative stimulus, and after a change of the task requirements. The decrease of ITR rate corresponds to the plateau level of avoidance or escape performance. Both a lower ITR rate and reduced flexibility of behavior are found after lesions of the amygdala nuclei.

Changes in avoidance response latencies after prefrontal lesions in cats: group versus individual data.

Clear differences in proportion of short-latency bar-pressing avoidance responses were observed in two groups of cats trained to the same acoustic CS but against backgrounds of different intensities. Removal of the proreal and orbital gyri resulted in transient impartment of avoidance performance and in permanent decrease of short-latency avoidance responses. In the course of post-operative retraining the probability of avoidance responses increase, but mostly performed with latencies longer than those affected by prefrontal lesion. Similar effects of lesions and post-operative retraining were observed both on group averages and on individual data. Analysis of individual data indicates that the higher proportion of short-latency responses before operation, the more pronounced post-operative changes in response latencies.

Effects of prefrontal lesions on avoidance and escape reflexes.

Retention of the avoidance and escape reflexes after removal of proreal and orbital gyri has been studied in adult cats. The ability to perform short-latency avoidance responses was severely impaired after prefrontal lesions, whereas no effect was found either on avoidance responses executed with longer latencies or on escape responses. Severity of impairment of the avoidance reflex was related to the pre-operative level of performance of the short-latency avoidance responses, which depend on the CS intensity, duration of the CS-US interval, and the type of inhibitory training. The post-operative retraining of the avoidance reflex performance was exclusively due to the increase in the proportion of avoidance responses executed with longer latencies. On the basis of neurophysiological and behavioral data it is postulated that different physiological mechanisms are responsible for execution of short-latency avoidance responses and avoidance responses with longer latencies. Only the first mechanism is destroyed after prefrontal lesions in cats.

Involvement of the partial reinforcement procedure in reward training: open address.

The analysis of the course of reward training presented in early Konorski and Miller’s papers (1933, 1936) indicates that the appearance of active instrumental responses is related with the introduction of the partially reinforced, excitatory classically conditioned procedure. It is postulated that the mechanism underlying the effects of this procedure on the shaping of instrumental responses is similar to that proposed by Konorski (1967) for the effects of inhibitory classically conditioned stimuli on the performance of the instrumental response.

The amount of decrease of the background white noise intensity as a cue for differentiation training.

The course of differentiation learning, using the conditioned emotional response (CER) method, was investigated in two groups of 16 rats. The discriminative stimuli consisted of decreases in the 80 dB background white noise to either 70 dB or 60 dB. Differentiation learning was more efficient with the larger decrease of background noise intensity as the CS(+) and the smaller decrease as the CS(-) than vice versa.