Control of Specialized Metabolism by Signaling and Transcriptional Regulation: Opportunities for New Platforms for Drug Discovery?

Specialized metabolites are bacterially produced small molecules that have an extraordinary diversity of important biological activities. They are useful as biochemical probes of living systems, and they have been adapted for use as drugs for human afflictions ranging from infectious diseases to cancer. The biosynthetic genes for these molecules are controlled by a dense network of regulatory mechanisms: Cell-cell signaling and nutrient sensing are conspicuous features of this network. While many components of these mechanisms have been identified, important questions about their biological roles remain shrouded in mystery. In addition to identifying new molecules and solving their mechanisms of action (a central preoccupation in this field), we suggest that addressing questions of quorum sensing versus diffusion sensing and identifying the dominant nutritional and environmental cues for specialized metabolism are important directions for research.

Antennapedia/HS1 chimeric phosphotyrosyl peptide: conformational properties, binding capability to c-Fgr SH2 domain and cell permeability.

With the aim of interfering with the signaling pathways mediated by the SH2 domains of Src-like tyrosine kinases, we synthesized a tyrosyl-phospho decapeptide, corresponding to the sequence 392-401 of HS1 protein, which inhibits the secondary phosphorylation of HS1 protein catalyzed by the Src-like kinases c-Fgr or Lyn. This phospho-peptide was modified to enter cells by coupling to the third helix of Antennapedia homeodomain, which is able to translocate across cell membranes. Here we present CD and fluorescence studies on the conformational behavior in membrane-mimicking environments and on lipid interactions of Antennapedia fragment and its chimeric phosphorylated and unphosphorylated derivatives. These studies evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption on lipids. Experiments performed with recombinant protein containing the SH2 domain of c-Fgr fused with GST and with isolated erythrocyte membranes demonstrated that the presence of the N-terminal Antennapedia fragment only slightly affects the binding of the phospho-HS1 peptide to the SH2 domain. In fact, it has been shown that in isolated erythrocyte membranes, both phospho-HS1 peptide and its chimeric derivative greatly affect either the SH2-mediated recruitment of the c-Fgr to the transmembrane protein band 3 and the following phosphorylation of the protein catalyzed by the Src-like kinase c-Fgr. The ability of the chimeric phospho-peptide to enter cells has been demonstrated by confocal microscopy analysis.

Is megalencephaly specific to autism?

Several recent reports have described the presence of increased head circumference (megalencephaly) in patients with autism. Although some studies have described reports of megalencephaly in other disorders such as schizophrenia in adults, few such studies have been performed in children and adolescents. In the present study, the authors compared 20 subjects with autism/ pervasive developmental disorder (DSM-IV; all males; mean age = 10.9 years) with 20 controls with attention deficit hyperactivity disorder (DSM-IV; all males; mean age = 11.1 years). Four subjects and five controls had evidence of megalencephaly. In addition to their core symptoms, the autistic subjects with megalencephaly were hyperactive and impulsive. These findings suggest that megalencephaly may not be specific to autism, and when present, it may index the presence of additional symptoms such as hyperactivity and impulsivity.

Vestibular reflex changes following aminoglycoside induced ototoxicity.

The effects of aminoglycoside-induced changes on the vestibular system were evaluated in three subjects who suffered significant damage over the course of treatment. Measurements of vestibulo-ocular reflex (VOR) function were made using sinusoidal and pseudorandom rotational stimuli between 0 to 5 days after initiation of drug therapy, during drug therapy, and up to 579 days following drug administration. All subjects tested at baseline evaluations had normal VOR function. During aminoglycoside therapy there was a precipitous drop in both the VOR time and gain constants. The decline in VOR function was larger at lower stimulus frequencies compared to high frequencies, resulting in a relative preservation of function in the high frequencies. Over a period of about a year, VOR high frequency gains recovered to within normal limits. However, the VOR time constants showed only a modest recovery and remained well below the normal range.

Clustering of primary afferent fibers in peripheral nerve fascicles by sensory modality.

The spatial organization of cutaneous afferent fibers in the cat saphenous nerve was studied by recording from functionally identified units in split filaments. It was found that within each fascicle, fibers tend to be clustered together with others of like modality; they are not randomly distributed. These results suggest that the sensory modality of primary afferent neurons is determined prior to their innervation of the skin.

Sympathetic activation of unmyelinated mechanoreceptors in cat skin.

In single unit recordings from the saphenous nerve in anesthetized cats, afferent units were functionally identified as unmyelinated (C) mechanoreceptors. All units responded to stimulation of the sympathetic trunk with a repetitive discharge which was maintained throughout the period of stimulation. This discharge was asynchronous with respect to efferent activity and was unaffected by brief occlusion of the arterial blood supply. The mechanism underlying the strong excitatory action of the sympathetic system on these afferents is not known although changes in blood flow and temperature can be ruled out. It is suggested that the primary function of these afferents may be to signal conditions within the skin for regulatory purposes rather than to encode external mechanical events.

Sympathetically induced changes in the responses of slowly adapting type I receptors in cat skin.

The effects of sympathetic efferent activity on slowly adapting Type I receptors in the hairy skin of cats were studied by recording from single afferent units in the saphenous nerve. Stimulation of the sympathetic trunk at 10 Hz had predominantly excitatory effects, which were seen in some units as the development of a background discharge in the absence of overt mechanical stimulation, or in most other units as a reduction in the threshold for mechanical activation. These effects generally persisted throughout the 3-min period of sympathetic stimulation (SS). The percentage of afferent units that began to discharge during SS was significantly greater in female cats than in males (53% vs. 19%). An increase in the force exerted by the skin on the stimulus probe was also observed during SS. Several tests were conducted to assess possible neurochemical and mechanical mechanisms of action. Administration of the alpha-adrenergic blocking agent phentolamine produced a marked reduction in the sympathetic effects. However, histochemical analysis of sections from the touch domes showed no catecholamine fluorescence near the sensory fibers. Cessation of local blood flow just prior to SS, produced by occlusion of the descending aorta, had no apparent effect on the sympathetically induced changes in afferent activity. It was concluded that sympathetic activity has an excitatory action on most Type I afferents in the cat. Because this sympathetic action is neither replicated nor altered by aortic occlusion, it appears not to be mediated by changes in blood flow. It also appears not to be mediated by direct neurotransmitter action on the sensory receptor, because no catecholamine fluorescence was observed, yet the action was blocked by an alpha-adrenergic blocker. It is likely, therefore, that this sympathetic action is mediated by some unidentified mechanical response within the skin.

Sympathetic activation of A-delta nociceptors.

Primary afferent units in the saphenous nerve of cats, functionally identified as A-delta myelinated nociceptors, were tested for their responses to stimulation of the sympathetic trunk. The units were subdivided functionally into A-mechano-heat receptors (AMHs), which respond to both noxious heat and pressure, and high-threshold mechanoreceptors (HTMs), which respond only to pressure. No units of either subdivision were activated by sympathetic stimulation (SS) prior to noxious heating of their receptive fields. However, six of the seven AMH units with the highest mechanical thresholds (greater than 5 g von Frey) were activated by SS alone (10 Hz) after they had been sensitized by noxious heating of their receptive fields. Sensitized AMH units with lower mechanical thresholds (less than 5 g) were generally not activated by SS alone (1 of 22 units), and their responses to warming of their receptive fields were not altered by SS. The excitatory sympathetic action on AMH units was abolished by alpha- but not beta-andrenergic blockade in the two units tested. HTMs were unresponsive to SS even after repeated noxious heating of their receptive fields (15 units tested). The results of this study indicate that relatively high rates of sympathetic efferent activity (10 Hz) can induce firing in a small population of AMH receptors in damaged skin, specifically those units with high mechanical thresholds. This sympathetically evoked activity might trigger or exacerbate pain associated with skin damage; however, functional conclusions are difficult to draw, because of the scarcity of such units and the fact that the responses in some were brief and of low firing rates.