Examination of the role of the urokinase receptor in human colon cancer mediated laminin degradation.

The relevance of urokinase receptors to urokinase-mediated laminin degradation was investigated in cultured colon cancer. Six colon cancer cell lines degraded laminin in a plasminogen-dependent manner. The ability of the individual cell lines to cleave the glycoprotein correlated well (r2 = 0.9242) with the amount of urokinase recovered from the cell surface by a mild acid treatment. A radioreceptor assay indicated that colon cancer cells most active in degrading the laminin, possessed the largest number of urokinase receptors. Moreover, acid treatment which depletes the receptors of endogenous plasminogen activator augmented the specific binding of radioactive urokinase to the colon cancer cells by 12-200%. A cell line (HCT 116) which displayed 1.1 x 10(5) receptors/cell the majority of which were occupied with endogenous urokinase was selected and the effects of a urokinase receptor antagonist on laminin degradation determined. The peptide antagonist reduced laminin turnover by 60-80%. Morphological observations were consistent with these findings. Plasminogen-treated HCT 116 cells retracted from the culture dish and many cells were observed in the culture medium. This effect could be largely reversed by simultaneous treatment with the peptide antagonist. A poor correlation was found between laminin degradation and soluble urokinase (r2 = 0.1074). These data strongly argue for a role of the urokinase receptor in facilitating the action of the plasminogen activator in colon cancer.

Modulation of the urokinase receptor in human colon cell lines by N,N-dimethylformamide.

The present study documents the effect of the planar, polar differentiation promoter N,N-dimethylformamide (DMF) on urokinase binding to colon carcinoma cells. Exposure of the colon carcinoma cell lines to the agent resulted in enhanced specific binding of radioactive urokinase to all cells tested. Insulin binding to the cells was, however, unaffected by DMF. A DMF exposure period of 45 h was required to observe maximum urokinase binding to two representative cell lines FET and RKO. Optimal stimulation of both cell lines occurred with 0.8% DMF. Scatchard analysis revealed the dissociation constants to be unchanged by the agent with the increased binding of radioactive plasminogen activator reflecting an up-regulation of binding sites. In this regard, the cell line RKO upon exposure to DMF, displayed approx. 700,000 receptors/cell, the highest value published, to date, for any cell line.

International Conference on Harmonization--critical discussion of the biotech 'Specifications' document.

Since 1990, the International Conference on Harmonization (ICH) has served as the primary medium for the development of consistent, harmonized and scientifically based international standards that keep abreast of the complexity of rapidly evolving technologies, and health, safety and commerce issues. Of the 45 guidance documents generated to date, influencing the conduct of drug development at various points during its continuum, six are dedicated to biotechnology. 'Specifications', the last in the series, was completed in 1999. It is fully complimentary to the other five guidance documents in the ICH biotechnology compendium. The process of establishing product specifications (principles and applications) is functionally couched within the multifaceted strategy of ensuring quality and consistency, and is proving to be a fundamental resource in crafting future harmonized documents such as the Common Technical Document.

International collaborative study on the assay of commerically available high and low molecular weight urokinases.

Urokinases of different molecular weights are now commerically available. An international collaborative study (eight laboratories) has been conducted to investigate the effect of type and concentration of plasminogen on the assay of two different urokinase preparations against the International Reference Preparation (IRP). Considerable inter-laboratory variation in relative potency estimation was found, and a small effect of plasminogen concentration, independent of type, was apparent.

Rabbit fibrin: characterization of the separated chains.

In preparation for studies of the time course of production in vivo of the constituent chains of rabbit fibrin, we characterized the structural features of the fibrin molecule. Fibrin was isolated from plasma and reduced and alkylated. The alpha, beta, and gamma chains were separated by CM-cellulose chromatography and their molecular weights and amino acid compositions were determined. The gamma chain was sequenced 36 steps with 32 positive identifications and the beta chain, 12 steps with 12 identifications. No major differences between the sequences of these chains and those of man, chicken, and dog were noted.

Lysis of intracranial hematomas with urokinase in a rabbit model.

Urokinase (UK), a potent thrombolytic agent, was tested in a rabbit model for safety and efficacy in lysing intracranial hematomas. Intracerebral-intraventricular (IC-IV) hematomas were created by stereotaxically injecting 0.2 ml of clotted human blood into the frontal lobe and lateral ventricle of a total of 57 anesthetized adult New Zealand White rabbits (weighing 1.6 to 2.5 kg). Control animals received 0.2 ml of normal saline injected into the clot, and the experimental group received an equal volume of UK solution (50,000 units/ml) immediately after the clot injection. Some animals were sacrificed at 3 hours and others at 24 hours postinjection. At 3 hours, clot lysis had been achieved in nine (90%) of 10 UK-treated animals as compared to one (14%) of seven controls. By 24 hours, clots had been lysed in 10 (83%) of 12 UK-treated animals and in two (33%) of six controls. Overall, clot lysis was demonstrated in 19 (86%) of the 22 UK-treated animals and in only three (23%) of the 13 controls (p less than 0.001). There was no significant difference in results between these animals and a further set of 22 rabbits that were treated with UK or saline 24 hours after clot injection. There was no histological evidence of damage or inflammation noted on careful light microscopic examination of three to five sections from each brain, although findings consistent with encephalitozoonosis, an incidental protozoan infestation, were encountered in four animals. These studies suggest that UK may be safely and effectively employed for the lysis of intracranial hematomas in this animal model, and that a delay in therapy of up to 24 hours does not significantly compromise its efficacy.

Physiology of hemostasis.

The consequences of acute insults to the hemostatic system, whether congenital or acquired, frequently present a considerable challenge in diagnosis and therapy. Logical and effective management depends upon the proper identification of the hemostatic compartments involved; an appreciation for the considerably complex, delicately modulated interplays of various enzyme/inhibitor systems; and knowledge of the mechanism by which a variety of apparently unrelated disease processes precipitate sometimes catastrophic events--thrombosis/embolism/hemorrhage. We have attempted a logical review of basic mechanisms of hemostasis. The text is obligatorily brief, focusing on key elements of the biochemistry and physiology of the vessel wall, the platelet, and pertinent plasma factors. The section on plasma proteins pays particular attention to biocybernetic principles (positive/negative feedback loops) and to the interrelationship of enzyme systems involved in coagulation, fibrinolysis, kinin generation, and complement activation. No attempt was made to be encyclopedic. In the interest of brevity and clarity, the text has been limited to current concepts, with the reference material selected, whenever possible, in the form of review articles, volumes and monographs. We apologize for omissions. It is our belief that a working knowledge of basic mechanisms provides not only advantages in diagnostic/therapeutic management but also serves as a firm foundation for the development of novel diagnostic and therapeutic modalities.

Temporal correlation of some endocrine circadian rhythms in elderly subjects.

The aim of this chronobiological study was to investigate temporal correlations in the circadian patterns of 6 hormones, namely somatotrophic hormone (STH), prolactin (PRL), cortisol (F), aldosterone (ALD), insulin (IRI) and C-peptide (CP), assayed in systemic blood serum drawn at 07:00, 10:00, 13:00, 16:00, 19:00 and 22:00 h from an antecubital vein in 19 young subjects (aged 20-29 yr, comprising 10 males and 9 females; and 20 elderly subjects (aged 70-81 yr, comprising 10 males and 10 females). All subjects were sampled on a normal dietary sodium intake (120-140 mEq/24h) while following a social routine of diurnal activity (07:00-23:00) and nocturnal rest (23:00-07:00). Time-qualified data were analyzed by lead-lag correlation and by cosinor analysis. According to the lead-lag correlation findings, it would appear that the correlation which exists between several time-qualified series in young subjects is no longer present in elderly subjects. The circadian rhythms which were found to have lost their temporal correlations with advancing age were those between STH and IRI, STH and ALD, PRL and IRI, PRL and CP, and ALD and CP. It should be noted that the correlation between hormonal rhythms breaks down mainly on account of a peculiar age-related change in the magnitude of the circadian fluctuation. This chronological decline in amplitude led to the conclusion that the senescence of endocrine rhythmic functions is a biological phenomenon characterized by altered circadian variability.

Circadian rhythms of plasma renin activity and aldosterone: changes related to age, sex, recumbency and sodium restriction. Chronobiologic specification for reference values.

Plasma renin activity (PRA) and aldosterone (PA) levels are characterized by a circadian rhythmicity (CR). The present study revealed that this rhythmicity is influenced by several factors including posture, sodium intake and age. Time-qualified PRA and PA reference intervals can reduce the incidence of false positives and false negatives in a diagnostic work-up. The circadian rhythmicity of PRA and PA have been quantified in relation to posture, sodium intake and age. The cosinor procedure has been applied to quantify the properties of the circadian rhythmicity under these conditions. Chronograms and circadian parameters can be used to optimize the use of PRA and PA measurements in clinical practice. The chronobiological specification of reference values for PRA and PA is of valuable importance since the assessment of PRA and PA circadian rhythmicity has a diagnostic interest for a certain type of clinical disorder. It should be noted that several studies have described circannual variations for renin and aldosterone. The next step in the optimation of laboratory time-qualified reference values is the assessment of changes induced by the deterministic factors on a circannual domain.

Effects of a mild and prolonged restriction in sodium or food intake on the circadian rhythm of aldosterone and related variables.

The effect of a mild reduction in dietary sodium intake (-30 mEq/24 hr) and body weight (-2 kg/2 months) on circadian rhythms of urinary aldosterone (UA), sodium (UNa), potassium (UK), creatinine (UC) and volume (UV) have been investigated in nine clinically healthy subjects. The mild reduction in dietary sodium is associated with: (1) a decrease in the 24-hr excretion rate of UNa, UK and UV, and an increased mesor of UA and UC; (2) a lowered extent of the circadian variation for UNa, UK, UV and a greater amplitude for UA and UC (3) a later crest in the temporal phase for UK, UA, UC, an earlier phasic wave for UNa. The mild reduction in calorie intake resulting in a body weight loss is associated with a more pronounced decrease in the 24-hr excretion rate of UNa and UK, and in the extent of circadian fluctuation for UNa. Peculiar events are: (1) the decreased 24-hr excretion rate for UA, and the increased mesor for UV; (2) the extent variability increased for UV, decreased for UC. Such effect may have a practical resonance for heuristic physiology since the role of dietary sodium and food intake has been better clarified. Dietary sodium and food can be regarded as 'chronomodulatory agents' for the adrenal cortex since their adrenotropic influence is extended to the tonic as well as phasic secretion of aldosterone.

Migration of dog polymorphonuclear neutrophilic leukocytes to formylated peptides.

Formylated peptides are potent stimulants of polymorphonuclear neutrophilic leukocyte (PMN) migration from species such as humans and rabbits. Interestingly, PMNs from dogs, cats, pigs and cows have been reported as refractory to N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and generally are believed not to express formylpeptide receptors. Formylpeptides are a major component of conditioned media from E. coli cultures and believed to be a significant element in inflammatory responses elicited by E. coli. Our studies have found that E. coli filtrate was a potent stimulant of dog PMN migration. Inhibition of migration to E. coli filtrates by the antagonist t-botyloxycarbonyl-l-methionyl-l-leucyl-l-phenylalanine (t-boc-MLP) demonstrated that the migration was mediated through the formylated peptide receptor. Migration in response to peptides with higher affinity for the formylpeptide receptor than FMLP was further evidence for these receptors on the dog PMN. PMNs from dogs migrated in response to FMLP at high concentrations (100 microM); however, pretreatment with phorbol myristate acetate resulted in increased migration of dog PMNs in response to concentrations of FMLP as low as 1 pM. These results demonstrate that dog PMNs are responsive to formylpeptides and that these responses can be up-regulated by PMA. Thus PMNs from a species previously thought incapable of responding to formylpeptides can respond to formylpeptide analogs with high affinity for the receptor as well as be primed for enhanced migration to FMLP by PMA.

May a lymphocytic infiltration have a pathogenic role in an aldosterone-producing adrenal tumor?

A 38-year-old woman is described to have a primary hyperaldosteronism due to an aldosteronoma with foci of lymphocytic infiltration. The finding suggests: a concomitant lymphoid adrenalitis; or, an immunological attack to neoplastic cells. The hypothesis is that there may be a relationship in the association. The lymphocytic infiltrates could have a pathogenic role in the development of the aldosterone-producing adrenocortical neoplasm by interrupting some inhibitory mechanism(s) of the cells that secrete aldosterone.

[Clinical significance of anomalies of renin secretion in arterial hypertension]. / Significato clinico delle anomalie di secrezione reninica nella ipertensione arteriosa.

Aim of the present investigation is to define the clinical meaning of disorders in renin secretion in the field of vascular hypertensive diseases. In view of this scope, we report the results of a retrospective study on the levels of plasma renin activity assayed in renal veins, aorta and peripheral vein of 124 hypertensive, angiographically studied for a diagnostic work-up. The behavior of renal vein renin has been related to epidemiologic, clinical and etiopathogenetic factors. Results indicate that disorders in renin secretion occur in every type of arterial hypertension. However, some disorders are prevalent in a determined type of hypertensive disease. The incidence of disorders in renin secretion is different in relation to sex, age and duration of hypertension. Hypersecretion is prevalent in men and young hypertensives, while hyposecretion of renin is more frequent in the oldest hypertensive patients. Vascular damage is prevalent in men and young hypertensives, while hyposecretion of renin is more frequent in the oldest hypertensive patients. Vascular damage is prevalent in renin hyposecretive hypertensives. Hypersecretion and lateralization in renin release can be mainly encountered in the renovascular type of hypertension and, less frequently, in unilateral nephroparenchymal hypertension. However, totally lateralized hypersecretion can be detected even in essential hypertensive patients suggesting the possibility of false positives for the diagnosis of renovascular hypertension.