p21-activated kinase has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I

Acanthamoeba class I myosins are unconventional, single-headed myosins that express actin-activated Mg2+-ATPase and in vitro motility activities only when a single serine or threonine in the heavy chain is phosphorylated by myosin I heavy chain kinase (MIHCK). Some other, but not most, class I myosins have the same consensus phosphorylation site sequence, and the two known class VI myosins have a phosphorylatable residue in the homologous position, where most myosins have an aspartate or glutamate residue. Recently, we found that the catalytic domain of Acanthamoeba MIHCK has extensive sequence similarity to the p21-activated kinase (PAK)/STE20 family of kinases from mammals and yeast, which are activated by small GTP-binding proteins. The physiological substrates of the PAK/STE20 kinases are not well characterized. In this paper we show that PAK1 has similar substrate specificity as MIHCK when assayed against synthetic substrates and that PAK1 phosphorylates the heavy chain (1 mol of P(i) per mol) and activates Acanthamoeba myosin I as MIHCK does. These results, together with the known involvement of Acanthamoeba myosin I, yeast myosin I, STE20, PAK, and small GTP-binding proteins in membrane- and cytoskeleton-associated morphogenetic transformations and activities, suggest that myosins may be physiological substrates for the PAK/STE20 family and thus mediators of these events.     

Intraesophageal balloon distension test in Chagas' disease patients with noncardiac chest pain

Patients with Chagas' disease often have chest pain as a prominent symptom. The objective of this study was to compare the results of intraesophageal balloon distension in chagasic and nonchagasic patients with chest pain not caused by coronary obstruction. We studied 40 patients with chest pain and angiographically normal coronary arteries, 25 with a positive serologic test for Chagas' disease (Chagas group, 16 women, mean age 53+/-10 years), and 15 with a negative serologic test (control group, 11 women, mean age 46+/-10 years). All patients had radiologic and endoscopic examinations of esophagus, stomach, and duodenum, esophageal manometry with the acid infusion test in the distal esophagus, and intraesophageal balloon distension. None of them had esophageal dilation or any signs of cardiovascular disease. A 25-mm-long latex balloon located 10 cm above the lower esophageal sphincter was inflated and deflated over a period of 10 sec at 1-ml increments of air until the subjects reported chest pain or to a maximum volume of 20 mi. The test caused chest pain in 14 subjects in the control group (93%) and in 12 in the Chagas' disease group (48%, P < 0.05). The mean volume of air that caused chest pain was 10+/-3 ml in the control group and 15+/-4 ml in the Chagas' disease group (mean+/-SD, P < 0.05). The maximum intraesophageal pressure during the examination was higher in Chagas' disease patients with chest pain during balloon distension (60 +/- 21 mm Hg) than in patients who did not have chest pain (37 +/-18 mm Hg, P < 0.05) and did not differ from the control group (48+/-16 mm Hg, P > 0.05). With the other examinations there was no difference between groups or between patients with or without chest pain during the balloon distension test. Although esophagitis was observed in 47% of patients in the control group and in 40% of the Chagas' disease group, the acid infusion test was positive in 27% of patients in the control group and in 4% of patients in the Chagas' disease group. We conclude that, as compared to a group of patients with similar chest pain, chagasic patients are less sensitive to esophageal distension. Thus, it is unlikely that their chest pain is related to esophageal mechanisms.     

A GTPase-independent mechanism of p21-activated kinase activation. Regulation by sphingosine and other biologically active lipids

p21-activated kinases (PAKs) are serine/threonine kinases that have been identified as targets for the small GTPases Rac and Cdc42. PAKs have been implicated in cytoskeletal regulation, stimulation of mitogen-activated protein kinase cascades, and in control of the phagocyte NADPH oxidase. Membrane targeting of PAK1 induced increased kinase activity in a GTPase-independent manner, suggesting that other mechanisms for PAK regulation exist. We observed concentration- and time-dependent activation of PAK1 by sphingosine and several related long chain sphingoid bases but not by ceramides or a variety of other lipids. Although phospholipids were generally ineffective, phosphatidic acid and phosphatidylinositol also had stimulatory effects on PAK1. Lipid stimulation induced a similar level of PAK1 activity as did stimulation by GTPases, and the patterns of PAK1 autophosphorylation determined after partial tryptic digestion and two-dimensional peptide analysis were similar with each class of activator. Lipid stimulation of PAK1 activity was dependent upon intact PAK kinase activity, as indicated by studies with a kinase-dead PAK1 mutant. Treatment of COS-7 cells expressing wild type PAK1 with sphingosine, fumonisin B, or sphingomyelinase, all of which are able to elevate the levels of free sphingosine, induced increased activity of PAK1 as determined using a p47(phox) peptide substrate. Studies using PAK1 mutants suggest that lipids act at a site overlapping or identical to the GTPase-binding domain on PAK. The inactive sphingosine derivative N,N-dimethylsphingosine was an effective inhibitor of PAK1 activation in response to either sphingosine or Cdc42. Our results demonstrate a novel GTPase-independent mechanism of PAK activation and, additionally, suggest that PAK(s) may be important mediators of the biological effects of sphingolipids.     

Paternal origin of trisomy 21 following intracytoplasmic sperm injection (ICSI)

One important aspect in the debate on the genetic risks associated with intracytoplasmic sperm injection (ICSI) is the possible increased rate of chromosomal abnormalities in resulting pregnancies. ICSI was performed in a 27 year old man with asthenoteratozoospermia and his 25 year old wife. There was a spontaneous miscarriage at 9 weeks of gestation. Cytogenetic investigation revealed trisomy 21. Analysis of two polymorphic microsatellite markers showed that the additional chromosome was paternal. This is in contrast to the fact that the vast majority of trisomic concepti are maternal in origin. Identifying the parent of origin in trisomic conceptions achieved by ICSI may reveal whether ICSI is associated with an increased risk of paternally derived aneuploidy.     

The smallest known eukaryotic genomes encode a protein gene: towards an understanding of nucleomorph functions

Cryptomonads are unicellular algae with plastids surrounded by four membranes. Between the two pairs of membranes lies a periplastidal compartment that harbours a DNA-containing organelle, termed the nucleomorph. The nucleomorph is the vestigial nucleus of a phototrophic, eukaryotic endosymbiont. Subcloning of parts of one nucleomorph chromosome revealed a gene coding for an Hsp70 protein. We demonstrate the expression of this nucleomorph protein-coding gene and present a model for protein transport from the host to the endosymbiont compartment.     

Acute renal failure complicating nonfulminant hepatitis A

PURPOSE: To compare the orbital blood flow velocities of patients with long-standing ocular hypertension and patients with primary open-angle glaucoma. METHODS: Twenty patients with ocular hypertension were recruited from our clinic and underwent color Doppler imaging evaluation of their retrobulbar vessels. The blood flow velocities and resistance index of their central retinal artery, temporal short posterior ciliary artery, and ophthalmic artery were compared with those of 20 glaucoma patients individually matched for age and level of the highest untreated intraocular pressure ever recorded. RESULTS: Glaucoma patients had significantly lower peak systolic velocity and end-diastolic velocity than did patients with ocular hypertension in their central retinal artery (p < 0.001). No significant difference between the groups was observed in the other vessels studied. CONCLUSIONS: Glaucoma patients had lower blood flow velocity in the central retinal artery compared with that of ocular hypertension patients of similar age and level of untreated intraocular pressure. This might be important in the development of glaucomatous damage in those patients.