Distribution of NADPH Diaphorase-Exhibiting Primary Afferent Neurons in the Trigeminal Ganglion and Mesencephalic Trigeminal Nucleus of the Rabbit

1. Nitric oxide (NO) is highly reactive gaseous molecule to which many physiological and pathological functions have been attributed in the central (CNS) and peripheral (PNS) nervous system. The present investigation was undertaken to map the distribution pattern of the enzyme responsible for the synthesis of NO, nitric oxide synthase (NOS), and especially its neuronal isoform (nNOS) in the population of primary afferent neurons of the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN) of the rabbit.2. In order to identify neuronal structures expressing nNOS we applied histochemistry to its specific histochemical marker nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd).3. We found noticeable amount of NADPHd-exhibiting primary afferent neurons in TG of the rabbit under physiological conditions. The intensity of the histochemical reaction was highly variable reaching the maximum in the subpopulation of small-to-medium-sized neurons. The large-sized neurons were only weakly stained or actually did not posses any NADPHd-activity. In addition, NADPHd-positive nerve fibers were detected between clusters of the ganglionic cells and in the peripheral branches of the trigeminal nerve (TN). NADPHd-exhibiting MTN neurons were noticed in the whole rostrocaudal extent of the nucleus even though some differences were found concerning the ratio of NADPHd-positive versus NADPHd-negative cell bodies. Similarly, we observed striking diversity in the intensity of NADPHd histochemical reaction in the subpopulations of small-, medium-, and large-sized MTN neurons.4. The predominant localization of NADPHd in the subpopulation of small-to-medium-sized TG neurons which are generally considered to be nociceptive suggests that NO probably takes part in the modulation of nociceptive inputs from the head and face. Furthermore, we tentatively assume that NADPHd-exhibiting MTN neurons probably participate in transmission and modulation of the proprioceptive impulses from muscle spindles of the masticatory muscles and mechanoreceptors of the periodontal ligaments and thus provide sensory feedback of the masticatory reflex arc.     

Crystal structure of a Cbf5-Nop10-Gar1 complex and implications in RNA-guided pseudouridylation and dyskeratosis congenita

H/ACA RNA-protein complexes, comprised of four proteins and an H/ACA guide RNA, modify ribosomal and small nuclear RNAs. The H/ACA proteins are also essential components of telomerase in mammals. Cbf5 is the H/ACA protein that catalyzes isomerization of uridine to pseudouridine in target RNAs. Mutations in human Cbf5 (dyskerin) lead to dyskeratosis congenita. Here, we describe the 2.1 A crystal structure of a specific complex of three archaeal H/ACA proteins, Cbf5, Nop10, and Gar1. Cbf5 displays structural properties that are unique among known pseudouridine synthases and are consistent with its distinct function in RNA-guided pseudouridylation. We also describe the previously unknown structures of both Nop10 and Gar1 and the structural basis for their essential roles in pseudouridylation. By using information from related structures, we have modeled the entire ribonucleoprotein complex including both guide and substrate RNAs. We have also identified a dyskeratosis congenita mutation cluster site within a modeled dyskerin structure.     

Mannopeptimycin esters and carbonates, potent antibiotic agents against drug-resistant bacteria

A series of ester and carbonate derivatives of the glycopeptide mannopeptimycin alpha (1) with potent activity against G+ bacteria, including the methicillin-resistant staphylococci and vancomycin-resistant enterococci, was synthesized. The SAR data obtained from natural and semisynthetic compounds demonstrated the importance of a hydrophobic group in the terminal mannosyl moiety for antibacterial activity.     

The Effect of Cauda Equina Constriction on Nitric Oxide Synthase Activity

Nitric oxide synthase (NOS) activity was studied in the gray and white matter regions of the spinal cord 2 and 5 days after multiple cauda equina constrictions of the central processes of L7-Co5 dorsal root ganglia neurons. The results show considerable differences in enzyme activity in the thoracic, upper lumbar, lower lumbar, and sacral segments. Increased NOS activity was observed at 2 days after multiple cauda equina constrictions in the dorsal, lateral, and ventral columns of the lower lumbar segments and in the ventral column of the upper lumbar segments. The values returned to control levels within 5 postconstriction days. In the lateral columns of thoracic segments taken 2 and 5 days after surgery, NOS activity was enhanced by 54% and 55% and in the upper lumbar segments by 130% and 163%, respectively. Multiple cauda equina constrictions performed surgically for 2 and 5 days caused a significant increase in NOS activity predominantly in the gray matter regions of thoracic segments. A quite different response was found 5 days postconstriction in the upper lumbar segments, where the enzyme activity was significantly decreased in the dorsal horn, intermediate zone, and ventral horn. No such extreme differences could be seen in the lower lumbar segments, where NOS activity was significantly enhanced only in the ventral horn. The data correspond with a higher number of NOS immunoreactive somata, quantitatively evaluated in the ventral horn of the lower lumbar segments at 5 days after multiple cauda equina constrictions. While the great region-dependent heterogeneity in NOS activity seen 2 and 5 days after multiple cauda equina constrictions is quite apparent and suggestive of an active role played by nitric oxide in neuroprotective or neurotoxic processes occurring in the gray and white matter of the spinal cord, the extent of damage or the degree of neuroprotection caused by nitric oxide in compartmentalized gray and white matter in this experimental paradigm would be possible only using longer postconstriction periods.     

gAd重组质粒的构建及其在大肠杆菌中的表达

为了建立人脂联素球状结构域(gAd)原核高效表达体系,分离纯化gAd并检测其生物活性,从淋巴细胞中提取人基因组DNA,PCR扩增出含有脂联素编码序列的片段,通过T-A克隆的方法克隆入pMD18-T载体中,然后设计适当的引物引入起始密码、终止密码以及相应的酶切位点,把脂联素球状结构域(gAd)基因亚克隆到表达载体pBV220,将序列鉴定正确的重组质粒转化入大肠杆菌DH5α中,用42℃温控诱导目的蛋白的表达;超声破菌,分离纯化包涵体,8mol/L尿素溶解,采用梯度稀释法复性重组蛋白,动物实验测定其活性。结果在大肠杆菌中成功实现了gAd稳定的以包涵体形式的高效表达,表达量约占全菌蛋白的20%,经聚丙烯酰胺凝胶电泳鉴定分离得到的包涵体具有较高纯度,复性后具有降低家兔血糖和游离脂肪酸的作用。为进一步研究gAd的生物学活性、作用机制奠定了基础。     

Single amino acids in the lumenal loop domain influence the stability of the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex of photosystem II (LHCIIb) is one of the most abundant integral membrane proteins. It greatly enhances the efficiency of photosynthesis in green plants by binding a large number of accessory pigments that absorb light energy and conduct it toward the photosynthetic reaction centers. Most of these pigments are associated with the three transmembrane and one amphiphilic alpha helices of the protein. Less is known about the significance of the loop domains connecting the alpha helices for pigment binding. Therefore, we randomly exchanged single amino acids in the lumenal loop domain of the bacterially expressed apoprotein Lhcb1 and then reconstituted the mutant protein with pigments in vitro. The resulting collection of mutated recombinant LHCIIb versions was screened by using a 96-well-format plate-based procedure described previously [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093], enabling us to test several thousand mutants for their ability to form stable pigment-protein complexes in vitro. At least one-third of the positions in the loop domain turned out to be sensitive targets; i.e., their exchange abolished formation of LHCIIb in vitro. This confirms our earlier notion that the LHCIIb loop domains contribute more specifically to complex formation and/or stabilization than by merely connecting the alpha helices. Among the target sites, glycines and hydrophilic amino acids are more prominently represented than hydrophobic ones. Specifically, the exchange of any of the three acidic amino acids in the lumenal loop abolishes reconstitution of stable pigment-protein complexes, suggesting that ionic interactions with other protein domains are important for correct protein folding or complex stabilization. One hydrophobic amino acid, tryptophan in position 97, has been hit repeatedly in independent mutation experiments. From the LHCIIb structure and previous mutational analyses, we propose a stabilizing interaction between this amino acid and F195 near the C-proximal end of the third transmembrane helix.     

4Pi microscopy reveals an impaired three-dimensional mitochondrial network of pancreatic islet β-cells,an experimental model of type-2 diabetes

Insulin production in pancreatic β-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the β-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic β-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an ～ 7-fold improved axial resolution (～ 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary β-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redox-sensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that β-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of β-cell condition, before their transplantations.     

可视化动缘探测系统检测心肌细胞舒缩功能

目的:分离成年大鼠心室肌细胞,利用可视化动缘探测系统检测其收缩/舒张功能.方法:常规酶解法分离制备钙耐受心肌细胞;用Ca2 荧光探剂fura-2/AM(0.5μmol/L)负载心肌细胞30 min(25℃)后,以含氧台氏液灌流并予电场刺激(0.5 Hz,5 ms),采用动缘探测系统检测心肌细胞收缩/舒张功能及细胞内荧光信号变化.结果:可视化动缘探测系统可实时同步观察和记录心肌细胞机械功能和细胞内钙瞬变变化,并可计算得到细胞收缩/舒张和钙瞬变的变化幅度、时程及速率等一系列指标,从而直接反映细胞水平的心肌功能改变.结论:可视化动缘探测系统为人们提供了一个在单心肌细胞水平探讨心肌肌源性功能及其改变的新方法,是当今心血管研究的一项重要技术.     

The evaluation of photosynthetic parameters in maize inbred lines subjected to water deficiency: Can these parameters be used for the prediction of performance of hybrid progeny?

The response of selected photosynthetic and morphological parameters of plants to drought was examined in 5 inbred lines of maize (Zea mays L.) and their 10 F1 hybrids. The aim of the study was to establish whether the photosynthetic performance of parental genotypes under drought conditions correlates with the performance of their progeny and whether the net photosynthetic rate, the chlorophyll fluorescence parameters or the content of photosynthetic pigments could be used as reliable physiological markers for early breeding generations. The relative importance of the additive and the nonadditive (dominance, maternal) genetic effects in the inheritance of these parameters was also assessed by means of the quantitative genetics analysis. The results showed that the nonadditive genetic effects associated with a particular combination of genotypes or a particular direction of crossing are at least equally and often even more important as the additivity and that these genetic effects almost totally change with the exposure of plants to drought conditions. This was reflected in the inability to predict the response of F1 hybrids to drought on the basis of the photosynthetic performance of their parents, which indicates that the practical usability of such parameters in maize breeding programs is rather limited.     

Genetic diversity and its effect on fitness in an endangered plant species, <Emphasis Type="Italic">Dracocephalum austriacum</Emphasis> L.

The aim of this study was to estimate genetic diversity and assess its importance for plant fitness in a species belonging to the most endangered species in Europe, Dracocephalum austriacum L., and to select the most valuable populations for conservation of genetic diversity within the species in the studied regions. We analyzed allozyme variation of 12 populations in three distinct regions (Czech Karst, Moravia and Slovak Karst) in Central Europe. The results showed high genetic diversity within populations (80.14%) and relatively low differentiation among populations within regions (9.42%) and between regions (10.45%). Seed production was significantly higher in larger, genetically more diverse and less inbred populations. The results suggest that genetic diversity has important effect on seed production in this species and thus can be expected to have strong direct consequences for plant fitness and vitality of the whole populations. They also show large variation in genetic diversity between populations and indicate which populations should get a priority in attempts to conserve all the genetic diversity within the region.