Photodynamic therapy: a new concept in medical treatment

A new concept in the therapy of both neoplastic and non-neoplastic diseases is discussed in this article. Photodynamic therapy (PDT) involves light activation, in the presence of molecular oxygen, of certain dyes that are taken up by the target tissue. These dyes are termed photosensitizers. The mechanism of interaction of the photosensitizers and light is discussed, along with the effects produced in the target tissue. The present status of clinical PDT is discussed along with the newer photosensitizers being used and their clinical roles. Despite the promising results from earlier clinical trials of PDT, considerable additional work is needed to bring this new modality of treatment into modern clinical practice. Improvements in the area of light source delivery, light dosimetry and the computation of models of treatment are necessary to standardize treatments and ensure proper treatment delivery. Finally, quality assurance issues in the treatment process should be introduced.

Biology and clinical utilization of mesenchymal progenitor cells

Within the complex cellular arrangement found in the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal progenitor cells (MPC). These cells can be expanded ex vivo and induced, either in vitro or in vivo, to terminally differentiate into at least seven types of cells: osteocytes, chondrocytes, adipocytes, tenocytes, myotubes, astrocytes and hematopoietic-supporting stroma. This broad multipotentiality, the feasibility to obtain MPC from bone marrow, cord and peripheral blood and their transplantability support the impact that the use of MPC will have in clinical settings. However, a number of fundamental questions about the cellular and molecular biology of MPC still need to be resolved before these cells can be used for safe and effective cell and gene therapies intended to replace, repair or enhance the physiological function of the mesenchymal and/or hematopoietic systems.

Alterations in proteins of bone marrow extracellular matrix in undernourished mice

The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM). Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5%) and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase) and laminin (4.8-fold increase) when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

Characterization of a methionine-rich protein from the seeds of Cereus jamacaru Mill. (Cactaceae)

We describe here the isolation and characterization of a major albumin from the seeds of Cereus jamacaru (Cactaceae), to which we gave the trivial name of cactin. This protein has a molecular mass of 11.3 kDa and is formed by a light chain (3.67 kDa) and a heavy chain (7.63 kDa). This protein was isolated using a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of cactin was determined and found to resemble that of the 2S seed reserve protein from the Brazil nut, a protein remarkable for its high methionine content. The usefulness of cactin as a molecular marker in the taxonomy of the Cactaceae is discussed.

Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa

A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30ºC. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the Km and Hill coefficient values were 0.35 mM and 1.01, respectively), alpha-naphthyl phosphate (the Km and Hill coefficient values were 0.44 mM and 0.97, respectively), ß-glycerol phosphate (the Km and Hill coefficient values were 2.46 mM and 1.01, respectively) and L-histidinol phosphate (the Km and Hill coefficient values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg2+, Zn2+ and Tris-HCl buffer, and is inhibited by Be2+, histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70ºC(half-life of 19.0 min, k = 0.036 min-1) as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min-1) in the same experiment.

Catecholamine response to exercise in individuals with different levels of paraplegia

The purpose of this study was to investigate the effect of the level of injury on the serum level of norepinephrine (Nor) and epinephrine (Epi) at rest and after maximal exercise in individuals with paraplegia. Twenty-six male spinal cord-injured subjects with complete paraplegia for at least 9 months were divided into two groups of 13 subjects each according to the level of injury, i.e., T1-T6 and T7-T12. Serum Nor and Epi concentrations were measured by HPLC-ECD, at rest (PRE) and immediately after a maximal ergospirometric test (POST). Statistical analysis was performed using parametric and non-parametric tests. Maximal heart rate, peak oxygen uptake, and PRE and POST Nor were lower in the T1-T6 than in the T7-T12 group (166 ± 28 vs 188 ± 10 bpm; 18.0 ± 6.0 vs 25.8 ± 4.1 ml kg-1 min-1; 0.54 ± 0.26 vs 0.99 ± 0.47 nM; 1.48 ± 1.65 vs 3.07 ± 1.44 nM). Both groups presented a significant increase in Nor level after exercise, while only the T7-T12 group showed a significant increase in Epi after exercise (T1-T6: 0.98 ± 0.72 vs 1.11 ± 1.19 nM; T7-T12: 1.24 ± 1.02 vs 1.89 ± 1.57 nM). These data show that individuals with paraplegia above T6 have an attentuated catecholamine release at rest and response to exercise as compared to subjects with injuries below T6, which might prevent a better exercise performance in the former group.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 ± 1.9 mV) compared to NWR (-69 ± 1.4 mV), NWR 1K-1C (-60.8 ± 1.6 mV), WKY (-67.1 ± 3.2 mV) and WKY 1K-1C (-56.9 ± 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 ± 0.86) when compared to arteries of all the other groups (NWR 0.93 ± 0.65, NWR 1K-1C 1.24 ± 0.45, WKY 1.22 ± 0.32, WKY 1K-1C 1.15 ± 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.

alpha-Tocopherol enhances tumour growth inhibition by cis-dichlorodiammine platinum (II)

Present studies indicate that alpha-tocopherol enhances the efficacy of cisplatin as demonstrated by inoculation of Dalton's lymphoma cells incubated with either cisplatin (5 or 10 µg/ml) alone or cisplatin + alpha-tocopherol (25 or 50 µg/ml) into C3H/He mice. Tumour cells (3 x 10(6) cells/mouse) incubated with cisplatin grow slowly in syngeneic mice as indicated by the late appearance of tumour. However, mice failed to develop tumour when inoculated with tumour cells incubated with cisplatin + alpha-tocopherol. When the animals were challenged with tumour cells (3 x 10(6) cells/mouse) on the 15th day after the initial inoculation, 30-50% survived more than 60 days, with 10% tumour-free survivors being observed in some groups. Antitumour activity was higher in mice receiving lymphoma cells (3 x 10(6) cells/mouse) preincubated with cisplatin + alpha-tocopherol compared to cisplatin alone. Tumour-bearing mice receiving cisplatin in combination with different concentrations of alpha-tocopherol exhibited significantly higher (P<0.001) intratumour platinum content (123-306%) but without any change in the kidney platinum content as compared to those receiving cisplatin (5 or 10 µg/ml) alone. Enhancement of cisplatin-induced tumour growth inhibition is probably due to the modulation of tumour cell membrane permeability by alpha-tocopherol. alpha-Tocopherol might increase the influx of cisplatin into tumour cells, causing the DNA repair machinery to be less efficient due to increased efficiency of adduct formation in the DNA molecule. This effect of alpha-tocopherol can render cisplatin more effective as an antitumour agent.

A priori estimation of accuracy and of the number of wells to be employed in limiting dilution assays

The use of limiting dilution assay (LDA) for assessing the frequency of responders in a cell population is a method extensively used by immunologists. A series of studies addressing the statistical method of choice in an LDA have been published. However, none of these studies has addressed the point of how many wells should be employed in a given assay. The objective of this study was to demonstrate how a researcher can predict the number of wells that should be employed in order to obtain results with a given accuracy, and, therefore, to help in choosing a better experimental design to fulfill one's expectations. We present the rationale underlying the expected relative error computation based on simple binomial distributions. A series of simulated in machina experiments were performed to test the validity of the a priori computation of expected errors, thus confirming the predictions. The step-by-step procedure of the relative error estimation is given. We also discuss the constraints under which an LDA must be performed.

Chronic intrathecal cannulation enhances nociceptive responses in rats

The influence of a chronically implanted spinal cannula on the nociceptive response induced by mechanical, chemical or thermal stimuli was evaluated. The hyperalgesia in response to mechanical stimulation induced by carrageenin or prostaglandin E2 (PGE2) was significantly increased in cannulated (Cn) rats, compared with naive (Nv) or sham-operated (Sh) rats. Only Cn animals presented an enhanced nociceptive response in the first phase of the formalin test when low doses were used (0.3 and 1%). The withdrawal latency to thermal stimulation of a paw inflamed by carrageenin was significantly reduced in Cn rats but not in Nv or Sh rats. In contrast to Nv and Sh rats, injection in Cn animals of a standard non-steroid anti-inflammatory drug, indomethacin, either intraperitoneally or into the spinal cord via an implanted cannula or by direct puncture of the intrathecal space significantly blocked the intensity of the hyperalgesia induced by PGE2. Cannulated animals treated with indomethacin also showed a significant inhibition of second phase formalin-induced paw flinches. Histopathological analysis of the spinal cord showed an increased frequency of mononuclear inflammatory cells in the Cn groups. Thus, the presence of a chronically implanted cannula seems to cause nociceptive spinal sensitization to mechanical, chemical and thermal stimulation, which can be blocked by indomethacin, thus suggesting that it may result from the spinal release of prostaglandins due to an ongoing mild inflammation.

Anti-hyperalgesic effect of electroacupuncture in a model of post-incisional pain in rats

Electroacupuncture has been proposed to be a low cost and practical method that allows effective pain management with minimal collateral effects. In this study we have examined the effect of electroacupuncture against the hyperalgesia developed in a model of post-incisional pain in rats. A 1-cm longitudinal incision was made through the skin and fascia of the plantar region of the animal hind paw. Mechanical hyperalgesia in the incision was evaluated 135 min after the surgery with von Frey filaments. The tension threshold was reduced from 75 g (upper limit of the test) to 1.36 ± 0.36 g (mean ± SEM) in control rats. It is shown that a 15-min period of electroacupuncture applied 120 min after surgery to the Zusanli (ST36) and Sanyinjiao (SP6) points, but not to non-acupoints, produces a significant and long-lasting reduction of the mechanical hyperalgesia induced by the surgical incision of the plantar surface of the ipsilateral hind paw. The tension threshold was reduced from 75 to 27.6 ± 4.2 g in animals soon after the end of electroacupuncture. The mechanical threshold in this group was about 64% less than in control. Electroacupuncture was ineffective in rats treated 10 min earlier with naloxone (1 mg/kg, ip), thus confirming the involvement of opioid mechanisms in the antinociceptive effects of such procedure. The results indicate that post-incisional pain is a useful model for studying the anti-hyperalgesic properties of electroacupuncture in laboratory animals.

Vasorelaxant effects of the potassium channel opener SR 47063 on the isolated human saphenous vein and rat aorta

The vasorelaxant effects of SR 47063 (4-(2-cyanimino-1,2-dihydropyrid-1-yl)-2,2-dimethyl-6-nitrochromene), a new K+-channel opener structurally related to levcromakalim, were examined in isolated human saphenous vein (HSV) and rat aorta (RA). HSV or RA rings were precontracted with either KCl or noradrenaline and cumulative relaxant concentration-response curves were obtained for SR 47063 (0.1 nM to 1 µM) in the presence or absence of 3 µM glibenclamide. SR 47063 potently relaxed HSV and RA precontracted with 20 mM (but not 60 mM) KCl or 10 µM noradrenaline in a concentration-dependent manner, showing slightly greater activity in the aorta. The potency of the effect of SR 47063 on HSV and RA was 12- and 58-fold greater, respectively, than that reported for the structurally related K+-channel opener levcromakalim. The vasorelaxant action of SR 47063 in both blood vessels was strongly inhibited by 3 µM glibenclamide, consistent with a mechanism of action involving ATP-dependent K+-channels.

Further analysis of open-respirometry systems: an a-compartmental mechanistic approach

A system is said to be "instantaneous" when for a given constant input an equilibrium output is obtained after a while. In the meantime, the output is changing from its initial value towards the equilibrium one. This is the transient period of the system and transients are important features of open-respirometry systems. During transients, one cannot compute the input amplitude directly from the output. The existing models (e.g., first or second order dynamics) cannot account for many of the features observed in real open-respirometry systems, such as time lag. Also, these models do not explain what should be expected when a system is speeded up or slowed down. The purpose of the present study was to develop a mechanistic approach to the dynamics of open-respirometry systems, employing basic thermodynamic concepts. It is demonstrated that all the main relevant features of the output dynamics are due to and can be adequately explained by a distribution of apparent velocities within the set of molecules travelling along the system. The importance of the rate at which the molecules leave the sensor is explored for the first time. The study approaches the difference in calibrating a system with a continuous input and with a "unit impulse": the former truly reveals the dynamics of the system while the latter represents the first derivative (in time) of the former and, thus, cannot adequately be employed in the apparent time-constant determination. Also, we demonstrate why the apparent order of the output changes with volume or flow.

Meal-feeding scheme: twenty years of research in Brazil

Naomi Shinomiya Hell was the first researcher to investigate the physiological adaptations to a meal-feeding scheme (MFS) in Brazil. Over a period of 20 years, from 1979 to 1999, Naomi's group determined the physiological and metabolic adaptations induced by this feeding scheme in rats. The group showed the persistence of such adaptations even when MFS is associated with moderate exercise training and the performance to a session of intense physical effort. The metabolic changes induced by the feeding training were discriminated from those caused by the effective fasting period. Naomi made an important contribution to the understanding of the MFS but a lot still has to be done. One crucial question still remains to be satisfactorily answered: what is the ideal control for the MFS?

The brain decade in debate: I. Neurobiology of learning and memory

This article is a transcription of an electronic symposium in which some active researchers were invited by the Brazilian Society for Neuroscience and Behavior (SBNeC) to discuss the last decade's advances in neurobiology of learning and memory. The way different parts of the brain are recruited during the storage of different kinds of memory (e.g., short-term vs long-term memory, declarative vs procedural memory) and even the property of these divisions were discussed. It was pointed out that the brain does not really store memories, but stores traces of information that are later used to create memories, not always expressing a completely veridical picture of the past experienced reality. To perform this process different parts of the brain act as important nodes of the neural network that encode, store and retrieve the information that will be used to create memories. Some of the brain regions are recognizably active during the activation of short-term working memory (e.g., prefrontal cortex), or the storage of information retrieved as long-term explicit memories (e.g., hippocampus and related cortical areas) or the modulation of the storage of memories related to emotional events (e.g., amygdala). This does not mean that there is a separate neural structure completely supporting the storage of each kind of memory but means that these memories critically depend on the functioning of these neural structures. The current view is that there is no sense in talking about hippocampus-based or amygdala-based memory since this implies that there is a one-to-one correspondence. The present question to be solved is how systems interact in memory. The pertinence of attributing a critical role to cellular processes like synaptic tagging and protein kinase A activation to explain the memory storage processes at the cellular level was also discussed.