Progenetic species in polychaetes (Annelida) and problems assessing their phylogenetic affiliation

Progenesis is defined as the retention of ancestral juvenilecharacters by adult stages of descendants due to an accelerationof the sexual maturation and thus is often regarded as a fastevolutionary process. Several small, meiofaunal polychaetes,such as Dinophilidae, some "Dorvilleidae" (for example, Parapodrilus),and Protodrilida, exhibit morphological simplicity in that theylack features typical of larger polychaetes, for example, parapodiaand/or head appendages. Due to the general resemblance of adultmeiofaunal polychaetes to juveniles of larger forms, progenesishas been invoked to explain evolutionary origins of many smallertaxa with increasing frequency over the past 4 decades. In thisreview, I summarize the interstitial species of polychaetesfor which progenetic origin has been suggested on the basisof morphology. However, critical examination of morphologicaldata that includes larval features reveals that autapomorphiccharacters uniting supposed progenetic taxa to specific annelidlineages are often missing. Typically larval and juvenile characters,which are argued to support hypotheses of progenetic origin,are often widely dispersed, homoplastic features. Because ofthis situation, molecular data seem to be the most reliablesource for phylogenetic inference. However, other biologicaldata, for example, from life history and morphology, are necessitiesto substantiate the progenetic evolution of these species.     

Hspa4l-deficient mice display increased incidence of male infertility and hydronephrosis development

The Hspa4l gene, also known as Apg1 or Osp94, belongs to the HSP110 heat shock gene family, which includes three genes encoding highly conserved proteins. This study shows that Hspa4l is expressed ubiquitously and predominantly in the testis. The protein is highly expressed in spermatogenic cells, from late pachytene spermatocytes to postmeiotic spermatids. In the kidney, the protein is restricted to cortical segments of distal tubules. To study the physiological role of this gene in vivo, we generated mice deficient in Hspa4l by gene targeting. Hspa4l-deficient mice were born at expected ratios and appeared healthy. However, approximately 42% of Hspa4l(-/-) male mice suffered from fertility defects. Whereas the seminiferous tubules of Hspa4l(-/-) testes contained all stages of germ cells, the number of mature sperm in the epididymis and sperm motility were drastically reduced. The reduction of the sperm count was due to the elimination of a significant number of developing germ cells via apoptosis. No defects in fertility were observed in female mutants. In addition, 12% of null mutant mice developed hydronephrosis. Concentrations of plasma and urine electrolytes in Hspa4l(-/-) mice were similar to wild-type values, suggesting that the renal function was not impaired. However, Hspa4l(-/-) animals were preferentially susceptible to osmotic stress. These results provide evidence that Hspa4l is required for normal spermatogenesis and suggest that Hspa4l plays a role in osmotolerance.     

T-cell activation and B-cell depletion in chimpanzees treated with a bispecific anti-CD19/anti-CD3 single-chain antibody construct

BscCD19xCD3 is a bispecific single-chain antibody construct with exceptional cytotoxic potency in vitro and in vivo. Here, we have investigated the biological activity of bscCD19xCD3 in chimpanzee, the only animal species identified in which bscCD19xCD3 showed bispecific binding, redirected B-cell lysis and cytokine production comparable to human cells. Pharmacokinetic analysis following 2-h intravenous infusion of 0.06, 0.1 or 0.12 μg/kg of bscCD19xCD3 as part of a dose escalation study in a single female chimpanzee revealed a half-life of approximately 2 h and elimination of the bispecific antibody from circulation within approximately 8 h after the end of infusion. This short exposure to bscCD19xCD3 elicited a transient increase in serum levels of IFNγ, IL-6, IL-2, soluble CD25, and transiently upregulated expression of CD69 and MHC class II on CD8-positive cells. Cytokine release and upregulation of T-cell activation markers were not observed with vehicle controls. A multiple-dose study using 5 weekly doses of 0.1 μg/kg in two animals also showed transient cytokine release and an activation of peripheral T cells with a first-dose effect, accompanied by a transient lymphopenia. While oscillations of T-cell counts were relatively even during repeated treatments, the amplitudes of peripheral B cells declined with every infusion, which was not observed in a vehicle control animal. Our data show that bscCD19xCD3 can be safely administered to chimpanzees at dose levels that cause fully reversible T-cell activation and, despite a very short exposure time, cumulative loss of peripheral B lymphocytes. A clinical trial testing prolonged administration of bscCD19xCD3 (MT103) for improving efficacy is currently ongoing.     

Neonatal sympathectomy reduces NADPH oxidase activity and vascular resistance in spontaneously hypertensive rat kidneys

Neonatal sympathectomy reduces arterial pressure in spontaneously hypertensive rats (SHR). In SHR transplanted with a kidney from sympathectomized SHR, arterial pressure was lower and less Na+ sensitive than in SHR transplanted with a kidney from hydralazine-treated SHR. This study was performed to identify underlying renal mechanisms. Tests for differential renal mRNA expression of nine a priori selected genes revealed robust differences for renal medullary expression of the NADPH oxidase subunit p47phox. Therefore, we investigated the effects of neonatal sympathectomy on renal mRNA expression of NADPH oxidase subunits, NADPH oxidase activity, and renal function. In 10-wk-old sympathectomized SHR fed a 0.6% NaCl diet, medullary p47phox and gp91phox expression was 40% less than in hydralazine-treated SHR. Also, after a 1.8% NaCl diet, medullary p47phox mRNA expression was lower in sympathectomized than in hydralazine-treated SHR. We found lower cortical (-30%, P<0.01) and medullary (-30%, P<0.05) NADPH oxidase activities in sympathectomized than in hydralazine-treated or untreated SHR. Glomerular filtration rate, renal blood flow, medullary blood flow, and fractional Na+ excretion in kidney grafts from sympathectomized and hydralazine-treated donors (n=8 per group) were similar at baseline and in response to a 20-mmHg rise in renal perfusion pressure. Renal vascular resistance was lower in kidneys from sympathectomized than hydralazine-treated donors (25+/-2 vs. 32+/-4 mmHg.min.ml-1, P<0.05). The results indicate that the sympathetic nervous system contributes to the level of renal NADPH oxidase activity and to perinatal programming of alterations in renal vascular function that lead to elevated renal vascular resistance in SHR.     

Calpains: a physiological regulator of the endothelial barrier?

The intracellular protease calpain, abundant in endothelial cells (EC), is assumed to be inactive under physiological conditions but may account for Ca2+ -linked pathophysiological events. However, nonstimulated EC contained autolyzed, activated calpain. Adding 12-48 microM calpain inhibitor I (CI) or 0.5-1 microM of the novel, membrane-permeable conjugate of calpastatin peptide-penetratin (CPP) caused rapid rounding and retraction of cultured EC (phase contrast, capacitance) and translocation of Syk, Rac, and Rho to the membrane, signifying activation upon inhibition of calpain. Isolated hearts (guinea pig) perfused with 12 microM CI or 0.5 muM CPP developed coronary leak. We conclude that calpain is constitutively active in EC and regulates vascular permeability by governing cell-cell attachment.     

Subtractive hybridization for differential gene expression in mechanically unloaded rat heart

The objective of this study was to identify differentially expressed genes in the mechanically unloaded rat heart by suppression subtractive hybridization. In male Wistar-Kyoto rats, mechanical unloading was achieved by infrarenal heterotopic heart transplantation. Differentially expressed genes were investigated systematically by suppression subtractive hybridization. Selected targets were validated by Northern blot analysis, real-time RT-PCR, and immunoblot analysis. Maximal ADP-stimulated oxygen consumption (state 3) was measured in isolated mitochondria. Transplantation caused atrophy (heart-to-body weight ratio: 1.6 +/- 0.1 vs. 2.4 +/- 0.1, P < 0.001). We selected 1,880 clones from the subtractive hybridization procedure (940 forward and 940 reverse runs assessing up- or downregulation). The first screen verified 465 forward and 140 reverse clones, and the second screen verified 67 forward and 30 reverse clones. On sequencing of 24 forward and 23 reverse clones, 9 forward and 14 reverse homologies to known genes were found. Specifically, we identified reduced mRNA expression of complex I (-49%, P < 0.05) and complex II (-61%, P < 0.001) of the respiratory chain. Significant reductions were also observed on the respiratory chain protein level: -42% for complex I (P < 0.01), -57% for complex II (P < 0.05), and -65% for complex IV (P < 0.05). Consistent with changes in gene and protein expression, state 3 respiration was significantly decreased in isolated mitochondria of atrophied hearts, with glutamate and succinate as substrates: 85 +/- 27 vs. 224 +/- 32 natoms O.min(-1).mg(-1) with glutamate (P < 0.01) and 59 +/- 18 vs. 154 +/- 30 natoms O.min(-1).mg(-1) with succinate (P < 0.05). Subtractive hybridization indicates major changes in overall gene expression by mechanical unloading and specifically identified downregulation of respiratory chain genes. This observation is functionally relevant and provides a mechanism for the regulation of respiratory capacity in response to chronic mechanical unloading.     

Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli

Selenoprotein synthesis in Escherichia coli strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the gram-positive anaerobe Eubacterium acidaminophilum in E. coli was studied. Incorporation of ⁷⁵Se into glycine reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an E. coli wild-type strain and was fully absent in an E. coli SelB mutant. Selenoprotein synthesis, however, was strongly increased if selB and selC (tRNA^Sec) from E. acidaminophilum were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the E. coli SECIS element. However, mutations in these structures strongly reduced the amount of ⁷⁵Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using gst-lacZ translational fusions. In the presence of selB and selC from E. acidaminophilum, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in Desulfomicrobium baculatum (hydV), Treponema denticola (selD), and Campylobacter jejuni (selW-like gene) was also considerably enhanced in the presence of E. acidaminophilum selB and selC. This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in E. coli.     

Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.     

Reduction of the iron-sulfur clusters in mitochondrial NADH:ubiquinone oxidoreductase (complex I) by EuII-DTPA, a very low potential reductant

NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the mitochondrial electron transport chain. It contains a flavin mononucleotide to oxidize NADH, and eight iron-sulfur clusters. Seven of them transfer electrons between the flavin and the quinone-binding site, and one is on the opposite side of the flavin. Although most information about their properties is from EPR, the spectra from only five clusters have been observed, and it is difficult to match them to the structurally defined clusters. Here, we analyze complex I from bovine mitochondria reacted with a very low potential reductant, to impose a potential approaching -1 V. We compare the spectra with those from higher potentials and from the 24 kDa subunit and flavoprotein subcomplex, and model the spectra by starting from those with fewer components and building the complexity gradually. Spectrum N1a, from the 24 kDa subunit [2Fe-2S] cluster, is not observed in bovine complex I at any potential. Spectrum N1b, from the 75 kDa subunit [2Fe-2S] cluster, exhibits a lower potential than the N3, N4 and N5 spectra of three [4Fe-4S] clusters. In the lowest potential spectra an N5-type spectrum is observed at unusually high temperature (indicating a significant change to the cluster, or that two clusters have very similar g values), the relaxation rate of N1b increases (indicating that a nearby cluster has become reduced) and a new feature with an apparent g value of 2.16 suggests an interaction between two reduced clusters. The consequences of these observations for electron transfer in complex I are discussed.