Kinetics of protein release from yeast using enzymatic lysis for selective product recovery

The kinetics of release of four intracellular enzymes from different yeast cell locations using the Differential Product Release (DPR) method has been investigated. The method uses a combination of physical, chemical and biological agents such as lytic enzymes, an osmotic support and a spheroplast stabilizer. Using the DPR technique a wall enzyme, invertase, was released with a very high specific activity in the first step from a breadmaking strain ofS. cerevisiae. Maximum release could be obtained in this step when the incubation time was extended from 60 min to 100 min. Two cytosol enzymes, α-D-glucosidase and alcohol dehydrogenase were released in the second step. Fumarase was released in the third step almost instantaneously after disruption of the mitochondria which reduces considerably, by ca. 1 hour, the total incubation time of DPR. This paper investigates the kinetics of enzyme release during the 3 steps of DPR.     

The effects of naloxone on canine splanchnic arterial smooth muscle

The pharmacological properties of naloxone on vascular smooth muscle in vitro were examined using canine mesenteric arterial segments. Naloxone exerted two different effects on the artery: (A) naloxone at a high concentration (3 X 10(-4) M) produced a nonspecific vasodilation; and (B) naloxone at lower concentrations (3 X 10(-7), 3 X 10(-6), and 3 X 10(-5) M) augmented the vasoconstrictor effects of epinephrine and norepinephrine without altering KCl- or serotonin-induced constriction. Naloxone's augmenting effect on epinephrine-induced constriction was dose dependent. Even when the arterial strips were incubated in low calcium (0.8 mM) or calcium free Kreb's solution, naloxone (3 X 10(-5) M) still augmented epinephrine-induced constriction. With respect to naloxone's effect on another alpha-adrenoreceptor agonist, naloxone (3 X 10(-5) M) failed to alter phenylephrine-induced constriction. Naloxone's augmenting effect on norepinephrine-induced constriction was abolished when the specimens were incubated with 10(-5) M normetanephrine, while naloxone (3 X 10(-5) M) still augmented the constriction even when the specimens were incubated with 10(-5) M cocaine. These results suggest that naloxone at lower concentrations may augment the constrictor responses to catecholamines, at least in part, by inhibiting the extraneuronal uptake of those catecholamines.     

Prolactin administration during early postnatal life decreases hippocampal and olfactory bulb neurogenesis and results in depressive-like behavior in adulthood

Tight regulation of hormone and neurochemical milieu during developmental periods is critical for adequate physiological functions. For instance, activation of peptide systems during early life stress induces morphological changes in the brain resulting in depression and anxiety disorders. Prolactin (PRL) exerts different actions within the brain; it regulates neurogenesis and modulates neuroendocrine functions in the adult. However, PRL effects during early postnatal life are hardly known. Therefore, we examined whether neonatal administration of PRL influences cell survival in the hippocampal dentate gyrus (DG) and in the olfactory bulb (OB) and whether such influence results in behavioral consequences in adulthood. PRL-treated rat pups (13 mg/kg; PND1 to PND14), injected with BrdU at postnatal day 5 (PND5), showed a decrease in the density of DG BrdU/DCX and BrdU/NeuN-positive cells that survive at PND15. Similarly, PRL treatment decreased the density of BrdU + cells in the OB compared with VEH. Fluorojade B analysis showed no significant changes in the amount of cell death in the DG between the groups. Postnatal PRL administration induced a passive coping strategy in the forced swimming test in male and female adult rats when compared with control and vehicle groups. Corticosterone endogenous levels at PND12 were not affected by PRL or VEH treatment. Altogether, these results suggest that opposed to its effects in the adult, postnatal PRL treatment affects neurogenesis and results in psychopathology later in life. High PRL levels, as observed in neonates under several pathological states, might contribute to detrimental effects on the developing brain.     

Diurnal Variations of Striatal D2 Dopaminergic Receptors and its Relation with Haloperidol-induced Catalepsy

In spite of the clear evidences for the blockade of dopaminergic D2 receptors as the mechanism of action for haloperidol-induced catalepsy, the contribution of pharmacokinetic and pharmacodynamic aspects on the diurnal modulation of haloperidol-induced catalepsy is controversial. We studied the diurnal variations of striatal dopamine receptors and its relation with catalepsy diurnal variations. The [3 H]-spiperone binding to dopamine receptors had a clear rhythm with a peak at 00:00 to 03:00 h, and a trough at 12:00 to 18:00 h. Haloperidol-produced catalepsy measured with the four-cork test, also showed a clear rhythm, with a peak at 00:00 h and trough at 9:00 h. The dose-response curves at peak and trough of catalepsy had the same ED 50 (0.12 mg), with time-related changes in the maximal effect. Similar diurnal variations between catalepsy and dopamine receptor binding, indicate a relevant role of temporal pharmacodynamics of haloperidol on the modulation of its behavioral effects.     

Autophagy regulation depends on ER homeostasis controlled by lipid droplets

Macroautophagy (hereafter autophagy) is a highly conserved homeostasis and quality control process critically linked to neurodegeneration, metabolic diseases, cancer, and aging. A key feature of autophagy is the de novo formation of autophagosomes, double-membrane vesicular structures encapsulating cytoplasmic cargo for vacuolar turnover and recycling. The membrane rearrangements underlying nucleation, expansion, closure, and vacuolar fusion of autophagosomes are driven by multicomponent core autophagy machinery in cooperation with numerous factors involved in a variety of cellular processes. Our current understanding of the origin and contribution of diverse membrane sources to autophagosome biogenesis and of cellular functions enabling stress-appropriate autophagy responses critical for cell health and survival remains limited. Here, we summarize and discuss our recent findings analyzing the role of lipid droplets (LDs), conserved intracellular storage compartments for neutral lipids, for autophagy regulation. Our data indicate that LDs are dispensable as membrane sources, but fulfill critical functions for maintaining endoplasmic reticulum (ER) homeostasis, including buffering of newly synthesized fatty acids and maintenance of phospholipid composition, required for intact autophagy regulation and cell survival during nutrient stress.     

Land use,fallow period and the recovery of a Caatinga forest

Caatinga vegetation continues to be converted into mosaics of secondary forest stands, but the affect of this process on biodiversity has not yet been examined. We used 35 regenerating and old‐growth stands of Caatinga to examine the recovery of plant assemblages subsequent to slash‐and‐burn agriculture and cattle ranching/pasture in northeastern Brazil. Plant assemblages were contrasted in terms of community structure (stem density/basal area/species richness/diversity), functional (leaf habit/reproductive traits) and taxonomic composition. Soil attributes were also examined to infer potential drivers for cross‐habitat differences. As expected, plant assemblages clearly differed across a large set of community‐level attributes, including all trait categories relative to leaf habit and reproduction (pollination syndrome/floral color, size, type). Overall, old‐growth forest stands supported distinct and more diverse assemblages at the plot and habitat level; e.g., long‐lived tree species were almost exclusively found in old‐growth forest stands. For most attributes, plant assemblages subsequent to pasture exhibited intermediate values between those exhibited by old‐growth forest and those of agriculture‐related stands. Surprisingly, soils exhibited similar fertility‐related scores across habitats. Our results indicate that: (1) sprouting/resprouting represents an important mechanism of forest regeneration; (2) assemblage‐level attributes suggest recovery at distinct rates; (3) forest regeneration implies community‐level changes in both vegetative and reproductive functional attributes, including directional changes; (4) Caatinga is not able to completely recover in a period of 15‐yr following land abandonment; and (5) historical land use affects recovery rates and successional pathways/taxonomic trajectories. Seasonally dry tropical forests may intrinsically cover a wide range of patterns relative to successional model, recovery rates and successional pathways.     

The Influence of Spatial Attributes on Fragment Occupancy and Population Structure in the Mexican Mantled Howler (<Emphasis Type="Italic">Alouatta palliata mexicana</Emphasis>)

It is essential to document habitat occupancy patterns and population structure to facilitate the survival of primates in areas of anthropogenic disturbance. The overlapping of the Nearctic and Neotropical regions in the Olmec region of Mexico make this area particularly important as part of a natural biological corridor that harbors a high number of endemic species and connects the Atlantic and Pacific coastal plains. We surveyed Alouatta palliata mexicana (Mexican mantled howlers) in a 300-km² area to determine if fragment occupation and subpopulation structure were related to the spatial attributes of the fragments. We measured the fragment size and shape as well as the distances to the nearest road, human settlement, agricultural field, and nearest neighboring fragment. During 1 year (ca. 4500 fieldwork hours) we surveyed 48 fragments, 17 of which were occupied, and counted 198 howlers. Larger fragments that were farther from agricultural activities were more likely to be occupied. Subpopulation size and number of individuals in all age–sex classes increased in larger fragments that were closer to other fragments. We found more females and juveniles, as well as more females per male in fragments that were farther from roads and we found fewer immatures per female, females per male, and individuals per area in more irregular fragments. In addition, more males and immatures per female occurred in fragments that were farther from agricultural fields. The Olmec Region is located at the center of the geographic distribution of mantled howlers in Mexico, and could therefore play a fundamental role in maintaining the contact between different populations. However, our study suggests that mantled howlers are highly threatened by anthropogenic habitat disturbance in this area, mainly through the loss of their habitat and contact with humans.