ATGL and HSL are not coordinately regulated in response to fuel partitioning in fasted rats

Prolonged fasting is characterized by lipid mobilization (Phase 2), followed by protein breakdown (Phase 3). Knowing that body lipids are not exhausted in Phase 3, we investigated whether changes in the metabolic status of prolonged fasted rats are associated with differences in the expression of epididymal adipose tissue proteins involved in lipid mobilization. The final body mass, body lipid content, locomotor activity and metabolite and hormone plasma levels differed between groups. Compared with fed rats, adiposity and epididymal fat mass decreased in Phase 2 (approximately two- to threefold) and Phase 3 (∼4.5-14-fold). Plasma nonesterified fatty acids (NEFA) concentrations were increased in Phase 2 (approximately twofold) and decreased in Phase 3 (approximately twofold). Daily locomotor activity was markedly increased in Phase 3 (∼11-fold). Compared with the fed state, expressions of adipose triglyceride lipase (ATGL; mRNA and protein), hormone-sensitive lipase (HSL; mRNA) and phosphorylated HSL at residue Ser660 (HSL Ser⁶⁶⁰) were increased during Phase 2 (∼1.5-2-fold). HSL (mRNA and protein) and HSL Ser⁶⁶⁰ levels were lowered during Phase 3 (∼3-12-fold). Unlike HSL and HSL Ser⁶⁶⁰, ATGL expression did not correlate with circulating NEFA, mostly due to data from animals in Phase 3. At this stage, ATGL could play an essential role for maintaining a low mobilization rate of NEFA, possibly to sustain muscle performance and hence increased locomotor activity. We conclude that ATGL and HSL are not coordinately regulated in response to changes in fuel partitioning during prolonged food deprivation, ATGL appearing as the major lipase in late fasting.     

Complete structure determination of the A chain of mistletoe lectin III from Viscum album L. ssp. album.

The complete primary structure of the A chain of mistletoe lectin III (ML3A), a type II ribosome-inactivating protein, was determined using proteolytic digests of ML3A, HPLC separation of the peptides, Edman degration and MALDI-MS. Based on our results, ML3A consists of 254 amino acid residues, showing a high homology to the A chain of isolectin ML1 with only 24 amino acid residue exchanges. A striking important structural difference compared with ML1A is the lack of the single N-glycosylation site in ML3A due to an amino acid exchange at position 112 (ML1A: NL112GS ==> ML3A: T112GS). The alignment of ML3A with the A chains of ML1, isoabrins, ricin D, Ricinus communis agglutinin and three lectins, identified from the Korean mistletoe Viscum album ssp. coloratum, demonstrates the rigid conservation of all amino acid residues, responsible for the RNA-N-glycosidase activity as reported for ricin D. In addition, the fully determined primary structure of ML3A will give further information about the biological mechanism of mistletoe lectin therapy.     

Aquaporin 1 - a novel player in spinal cord injury

The role of water channel aquaporin 1 (AQP-1) in uninjured or injured spinal cords is unknown. AQP-1 is weakly expressed in neurons and gray matter astrocytes, and more so in white matter astrocytes in uninjured spinal cords, a novel finding. As reported before, AQP-1 is also present in ependymal cells, but most abundantly in small diameter sensory fibers of the dorsal horn. Rat contusion spinal cord injury (SCI) induced persistent and significant four- to eightfold increases in AQP-1 levels at the site of injury (T10) persisting up to 11 months post-contusion, a novel finding. Delayed AQP-1 increases were also found in cervical and lumbar segments, suggesting the spreading of AQP-1 changes over time after SCI. Given that the antioxidant melatonin significantly decreased SCI-induced AQP-1 increases and that hypoxia inducible factor-1α was increased in acutely and chronically injured spinal cords, we propose that chronic hypoxia contributes to persistent AQP-1 increases after SCI. Interestingly; AQP-1 levels were not affected by long-lasting hypertonicity that significantly increased astrocytic AQP-4, suggesting that the primary role of AQP-1 is not regulating isotonicity in spinal cords. Based on our results we propose possible novel roles for AQP-1 in the injured spinal cords: (i) in neuronal and astrocytic swelling, as AQP-1 was increased in all surviving neurons and reactive astrocytes after SCI and (ii) in the development of the neuropathic pain after SCI. We have shown that decreased AQP-1 in melatonin-treated SCI rats correlated with decreased AQP-1 immunolabeling in the dorsal horns sensory afferents, and with significantly decreased mechanical allodynia, suggesting a possible link between AQP-1 and chronic neuropathic pain after SCI.     

Alterations in protein kinase activity following exposure of cultured human lymphocytes to modulated microwave fields

Cultures of human tonsil lymphocytes were exposed in a Crawford cell to a 450-MHz field (peak envelope intensity 1.0 mW/cm2), sinusoidally amplitude modulated (depth 80%) at frequencies between 3 and 100 Hz for periods up to 60 min. The Crawford cell was housed in a temperature-controlled chamber (35 degrees C) and control cultures were placed in the same chamber. Activity of cAMP-dependent protein kinase relative to controls remained unaltered by fields modulated at 16 or 60 Hz with exposures of 15, 30, and 60 min. By contrast, total non-cAMP-dependent kinase activity fell to less than 50% of unexposed control levels after 15 and 30 min exposures, but, despite continuing field exposure, returned to control or preexposure levels by 45 and 60 min. A smaller reduction (20-25%) also occurred with 60-Hz modulation and was also restricted to exposure durations of 15 and 30 min. CW 450-MHz fields were without effect. Reduced enzyme activity occurred with 16-, 40-, and 60-Hz modulation frequencies, but not with 3-, 6-, 80-, or 100-Hz modulation. The specific identity of this kinase is unknown. This rapid but transient reduction in lymphocyte protein kinase activity restricted to modulation frequencies between 16 and 60 Hz and to less than 30 min exposure is consistent with "windowing" with respect to modulation frequency and exposure duration.     

The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation

Rho-family GTPases are activated by the exchange of bound GDP for GTP, a process that is catalyzed by Dbl-family guanine nucleotide exchange factors (GEFs). The catalytic unit of Dbl-family GEFs consists of a Dbl homology (DH) domain followed almost invariantly by a pleckstrin-homology (PH) domain. The majority of the catalytic interface forms between the switch regions of the GTPase and the DH domain, but full catalytic activity often requires the associated PH domain. Although PH domains are usually characterized as lipid-binding regions, they also participate in protein-protein interactions. For example, the DH-associated PH domain of Dbs must contact its cognate GTPases for efficient exchange. Similarly, the N-terminal DH/PH fragment of Trio, which catalyzes exchange on both Rac1 and RhoG, is fourfold more active in vitro than the isolated DH domain. Given continued uncertainty regarding functional roles of DH-associated PH domains, we have undertaken structural and functional analyses of the N-terminal DH/PH cassette of Trio. The crystal structure of this fragment of Trio bound to nucleotide-depleted Rac1 highlights the engagement of the PH domain with Rac1 and substitution of residues involved in this interface substantially diminishes activation of Rac1 and RhoG. Also, these mutations significantly reduce the ability of full-length Trio to induce neurite outgrowth dependent on RhoG activation in PC-12 cells. Overall, these studies substantiate a general role for DH-associated PH domains in engaging Rho GTPases directly for efficient guanine nucleotide exchange and support a parsimonious explanation for the essentially invariant linkage between DH and PH domains.     

Rin-like, a novel regulator of endocytosis, acts as guanine nucleotide exchange factor for Rab5a and Rab22

RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis.