Hydrophilic peptides derived from the transframe region of Gag-Pol inhibit the HIV-1 protease

The HIV-1 transframe region (TFR) is between the structural and functional domains of the Gag-Pol polyprotein, flanked by the nucleocapsid and the protease domains at its N and C termini, respectively. Transframe octapeptide (TFP) Phe-Leu-Arg-Glu-Asp-Leu-Ala-Phe, the N terminus of TFR, and its analogues are competitive inhibitors of the action of the mature HIV-1 protease. The smallest, most potent analogues are tripeptides: Glu-Asp-Leu and Glu-Asp-Phe with Ki values of approximately 50 and approximately 20 microM, respectively. Substitution of the acidic amino acids in the TFP by neutral amino acids and d or retro-d configurations of Glu-Asp-Leu results in an >40-fold increase in Ki. Protease inhibition by Glu-Asp-Leu is dependent on a protonated form of a group with a pKa of 3.8; unlike other inhibitors of HIV-1 protease which are highly hydrophobic, Glu-Asp-Leu is extremely soluble in water, and its binding affinity decreases with increasing NaCl concentration. However, Glu-Asp-Leu is a poor inhibitor (Ki approximately 7.5 mM) of the mammalian aspartic acid protease pepsin. X-ray crystallographic studies at pH 4.2 show that the interactions of Glu at P2 and Leu at P1 of Glu-Asp-Leu with residues of the active site of HIV-1 protease are similar to those of other product-enzyme complexes. It was not feasible to understand the interaction of intact TFP with HIV-1 protease under conditions of crystal growth due to its hydrolysis giving rise to two products. The sequence-specific, selective inhibition of the HIV-1 protease by the viral TFP suggests a role for TFP in regulating protease function during HIV-1 replication.     

Mast cell activation and migration to lymph nodes during induction of an immune response in mice

The mast cell response in skin and lymph nodes was examined during the sensitization phase of dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in mice. Degranulation of 62% of mast cells in DNFB-exposed skin was evident within 30 min of a dual application of DNFB, reaching a peak of 77% at 24 h, and persisting in 42% after 5 d. Abundant expression of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNAs and proteins was observed in keratinocytes, and mast cell degranulation was significantly inhibited after administration of neutralizing antibodies to MIP-1alpha, but not MIP-1beta. During DNFB sensitization, the mast cell density in the skin decreased by half, concurrent with a fivefold expansion of mast cell numbers in draining lymph nodes. Fluorescent-labeled mast cells injected into the skin appeared in draining lymph nodes after application of DNFB, followed by subsequent migration to the spleen. In lymph nodes, mast cells were an abundant and predominant source of MIP-1beta, neutralization of which partially inhibited T lymphocyte recruitment. These results indicate that mast cells contribute to the induction of this primary immune response by activation at and migration from the site of antigen encounter to draining lymph nodes, wherein they mediate T lymphocyte recruitment by production of MIP-1beta.     

Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitoylphosphatidylcholine bilayers to phospholipase A2

Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperature near the main bilayer phase transition and calcium concentration modulate the effectiveness of the reaction products. The purpose of this study was to examine the individual contributions of lysolecithin and palmitic acid to the susceptibility of dipalmitoylphosphatidylcholine vesicles and to rationalize the effects of temperature and calcium. Various fluorescent probes (Prodan, Laurdan, pyrene-labeled fatty acid, and dansyl-labeled phospholipid) were used to assess changes in the ability of the reaction products to perturb the bilayer and to affect the interactions with the enzyme. Un-ionized palmitic acid decreased bilayer polarity and perturbed the membrane surface exposing some of the Prodan to bulk water. Lysolecithin increased bilayer polarity and the rate of dipolar relaxation in response to the excited states of Laurdan and Prodan. A combination of the individual contributions of each product was observed when palmitic acid and lysolecithin were present together at low calcium, and the effects of lysolecithin dominated at high calcium. Palmitic acid, but not lysolecithin, promoted the binding of phospholipase A2 to the bilayer surface in the absence of calcium. Lysolecithin reduced the ability of fatty acid to enhance binding apparently by altering the structure of fatty acid domains in the membrane. Furthermore, increased temperature and ionization of the fatty acid tended to cause segregation of bound phospholipase A2 into domains poor in phospholipid content which presumably impeded bilayer hydrolysis. In contrast, un-ionized palmitic acid and lysolecithin promoted hydrolysis by augmenting a step distal to the adsorption of enzyme to the bilayer. This kinetic response to lysolecithin was calcium-dependent. A model accounting for these varied influences of the reaction products is presented.     

Influence of clinical mastitis during early lactation on reproductive performance of Jersey cows

In the common goby, Pomatoschistus microps (Pisces, Gobiidae), males build nests under mussel shells where they care for the eggs until hatching. To investigate why male common gobies cannibalize their own eggs (filial cannibalism), we conducted a feeding experiment. Males given little food ate from their eggs more often than males given food in excess. However, males given mussel meat in excess did not eat more of their eggs than males fed with both mussel meat in excess and goby eggs. This may suggest that male common gobies cannibalize their eggs to obtain energy rather than essential nutrients lacking in other diets. Moreover, males ate their whole clutch if it was exceptionally small regardless of food treatment, suggesting that males stop investing in their clutch if its reproductive value is less than the cost of guarding it. Thus, whole clutch cannibalism and partial clutch cannibalism seem to be governed by different factors. Furthermore, poorly built nests were associated with starved males, suggesting that nest concealing is costly. There was an association between how well the nest was built and partial clutch filial cannibalism, suggesting that the appearance of the nest may indicate the condition of the male, and thus the risk of filial cannibalism. Copyright 1998 The Association for the Study of Animal Behaviour.     

Mechanisms of insulin resistance in experimental hyperinsulinemic dogs

The aim of this work was to identify which proteins in horse dander extracts are allergens and to characterise them. Two-dimensional PAGE showed that horse dander preparations are composed of up to 50 proteins, all having acidic isoelectric points in the pH range 3-4.5. Immunoblots of two-dimensional PAGE were used to compare the reactivity of the proteins with IgE from 23 allergic patients. Patient sera were divided into two main groups recognising either allergens of 18.5 kDa or proteins of 27-29 and 31 kDa. The proteins of 27-29 kDa and 31 kDa were all N-glycosylated and their glycan chains seem to play a role in the binding of IgE from allergic patients. The sugar composition of their carbohydrate moiety was determined and lectin-binding experiments indicated presence of terminal sialic acid linked alpha-(2-->6) to galactose, galactose linked beta-(1-->4) to N-acetylglucosamine, and possibly presence of sialic acid linked alpha-(2-->3) to galactose. The 27-29-kDa glycoproteins had heterogeneous isoelectric points, most probably due to different degrees of sialylation in their oligosaccharide chains. The two 18.5-kDa allergens exhibited slightly different isoelectric points and their N-terminal sequences were identical, showing that they most likely were isoforms of the same protein. Sequence analyses revealed that their N-terminal sequences are similar to proteins belonging to the lipocalin family. We named the two 18.5-kDa proteins Equ c 2.0101 and Equ c 2.0102, according to International Allergen Nomenclature recommendations [King, T. P., Hoffman, D., Lowenstein, H., Marsh, D. G., Platts-Mills, T. A. E. & Thomas, W. (1995) J. Allergy Clin. Immunol. 96, 5-14]. The N-terminal of the allergens of 27-29 kDa were blocked and their sequences were not determined. Their amino acid compositions were determined and comparison with acidic mammalian proteins in the Swiss-Prot database revealed high scores with lipocalin proteins. This suggests that the glycosylated horse dander allergens belong to the lipocalin family, like Equ c 2.0101 and Equ c 2.0102.     

Assessment of sperm count in rural population of central India

26 clinician trainees' recollections of experiences in a diagnostic preschool program were analyzed in terms of strength and weaknesses of the program.     

Attenuated hematopoietic response to granulocyte-macrophage colony-stimulating factor in patients with acquired pulmonary alveolar proteinosis

The pathogenesis of acquired pulmonary alveolar proteinosis (PAP), a rare lung disease characterized by excessive surfactant accumulation within the alveolar space, remains obscure. Gene-targeted mice lacking the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) or the signal-transducing beta-common chain of the GM-CSF receptor have impaired surfactant clearance and pulmonary pathology resembling human PAP. We therefore investigated the hematopoietic effects of GM-CSF in patients with PAP. The hematologic response of 5 infants with congenital PAP to 5 microgram/kg/d was of normal magnitude. By contrast, despite normal expression of GM-CSF receptor alpha- and beta-common chains on peripheral blood myelomonocytic cells (n = 6) and normal binding affinity of bone marrow mononuclear cells for GM-CSF (n = 3), each of the 12 patients with acquired PAP treated displayed impaired responses to GM-CSF; 5 microgram/kg/d produced only minor eosinophilia, and doses of 7.5 to 20 microgram/kg were required to induce >/=1.5-fold neutrophil increments in the 3 patients who underwent dose-escalation. However, neutrophilic responses to 5 microgram/kg granulocyte colony-stimulating factor (G-CSF) were normal (n = 4). In vitro, the proportion of hematopoietic progenitors responsive to GM-CSF (16.1% +/- 8.9%; P = .042) or interleukin-3 (IL-3; 19.3% +/- 7.7%; P = .063), both of which utilize the beta-common chain of the GM-CSF receptor complex, were reduced among patients with acquired PAP (n = 4) compared with normal bone marrow donor controls (47.2% +/- 25.9% and 40.9% +/- 18.6%, respectively). In the one individual who had complete resolution of lung disease during the period of study, this was temporally associated with correction of this defective in vitro response to GM-CSF and IL-3 on serial assessment. These data establish that patients with acquired PAP have an associated impaired responsiveness to GM-CSF that is potentially pathogenic in the development of their lung disease. Based on these observations, we propose a model of the pathogenesis of acquired PAP that suggests the disease arises as a consequence of an acquired clonal disorder within the hematopoietic progenitor cell compartment.     

Large-scale functional connectivity in associative learning: interrelations of the rat auditory, visual, and limbic systems

Large-scale functional connectivity in associative learning: interrelations of the rat auditory, visual, and limbic systems. J. Neurophysiol. 80: 3148-3162, 1998. Functional relations between specialized parts of the brain may be important determinants of learned behaviors. To study this, we examined the interrelations of the auditory system with several extraauditory structures in two groups of rats having different behavioral histories. Both groups were trained to associate a tone conditional stimulus (CS) with an aversive unconditional stimulus (US). For one group, a light presented with the tone predicted the absence of the US (group TL-). In the other group, the light was a neutral stimulus (group TL0). Fluorodeoxyglucose (FDG) incorporation was measured in the presence of the tone-light compound. Because the tone-light compound was physically identical for both groups, neural differences between groups reflected differences in the learned associative properties of the stimuli. Covariances of FDG uptake in the auditory system and extraauditory structures were examined using partial least squares. Three strong covariance or functional connectivity patterns were identified. The first pattern mainly reflected similarities between groups, with strong interrelations between the subcortical auditory system and the thalamocortical visual system, cerebellum, deep cerebellar nuclei, and midline thalamus. This pattern of interactions may represent part of a common circuit for relaying the associative value of the tone CS to the cerebellum and the midline thalamus. The external nucleus of the inferior colliculus and medial division of the medial geniculate nucleus were associated more strongly with this pattern for group TL-, which was interpreted as representing the change of the associative value of the tone by the light, mediated through extraauditory influences on these two regions. A second pattern involved midbrain auditory regions, superior colliculus, zona incerta, and subiculum and was stronger for group TL0. The relations between midbrain structures may represent the excitatory conditioned response (CR) evoked by the tone in this group. The final pattern was strongest in group TL- and involved interrelations of the thalamocortical auditory system with hippocampus, basolateral amygdala, and hypothalamus. This pattern may represent the learned inhibition of the CR to the tone in the presence of the light. These findings are consistent with behavioral studies suggesting that at least two types of associations are formed during associative learning. One is the sensory relation of the stimuli and another is the relation between the CS and the affective components of the US. These behavioral associations are mapped to the patterns of functional connectivity between auditory and extraauditory regions.