SVD-clustering, a general image-analyzing method explained and demonstrated on model and Raman micro-spectroscopic maps.

An image analyzing method (SVD-clustering) is presented. Amplitude vectors of SVD factorization (V1…Vi) were introduced into the imaging of the distribution of the corresponding Ui basis-spectra. Since each Vi vector contains each point of the map, plotting them along the X, Y, Z dimensions of the map reconstructs the spatial distribution of the corresponding Ui basis-spectrum. This gives valuable information about the first, second, etc. higher-order deviations present in the map. We extended SVD with a clustering method, using the significant Vi vectors from the VT matrix as coordinates of image points in a ne-dimensional space (ne is the effective rank of the data matrix). This way every image point had a corresponding coordinate in the ne-dimensional space and formed a point set. Clustering was applied to this point set. SVD-clustering is universal; it is applicable to any measurement where data are recorded as a function of an external parameter (time, space, temperature, concentration, species, etc.). Consequently, our method is not restricted to spectral imaging, it can find application in many different 2D and 3D image analyses. Using SVD-clustering, we have shown on models the theoretical possibilities and limitations of the method, especially in the context of creating, meaning/interpreting of cluster spectra. Then for real-world samples, two examples are presented, where we were able to reveal minute alterations in the samples (changing cation ratios in minerals, differently structured cellulose domains in plant root) with spatial resolution.

Pituitary Adenylate Cyclase Activating Polypeptide, A Potential Therapeutic Agent for Diabetic Retinopathy in Rats: Focus on the Vertical Information Processing Pathway.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects in different neuronal injuries, such as retinal degenerations. Diabetic retinopathy (DR), the most common complication of diabetes, affects the microvasculature and neuronal architecture of the retina. We have proven earlier that PACAP is also protective in a rat model of DR. In this study, streptozotocin-induced DR was treated with intravitreal PACAP administration in order to further analyze the synaptic structure and proteins of PACAP-treated diabetic retinas, primarily in the vertical information processing pathway. Streptozotocin-treated Wistar rats received intravitreal PACAP injection three times into the right eye 2 weeks after the induction of diabetes. Morphological and molecular biological (qRT-PCR; Western blot) methods were used to analyze retinal synapses (ribbons, conventional) and related structures. Electron microscopic analysis revealed that retinal pigment epithelium, the ribbon synapses and other synaptic profiles suffered alterations in diabetes. However, in PACAP-treated diabetic retinas more bipolar ribbon synapses were found intact in the inner plexiform layer than in DR animals. The ribbon synapse was marked with C-terminal binding protein 2/Bassoon and formed horseshoe-shape ribbons, which were more retained in PACAP-treated diabetic retinas than in DR rats. These results are supported by molecular biological data. The selective degeneration of related structures such as bipolar and ganglion cells could be ameliorated by PACAP treatment. In summary, intravitreal administration of PACAP may have therapeutic potential in streptozotocin-induced DR through maintaining synapse integrity in the vertical pathway.

Examination of calcium-binding protein expression in the inner ear of wild-type, heterozygous and homozygous pituitary adenylate cyclase-activating polypeptide (PACAP)-knockout mice in kanamycin-induced ototoxicity.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with diverse biological effects. It also occurs and exerts protective effects in sensory organs; however, little is known about its effects in the auditory system. Recently, we have shown that PACAP protects cochlear cells against oxidative-stress-induced apoptosis and homozygous PACAP-deficient animals show stronger expression of Ca(2+)-binding proteins in the hair cells of the inner ear, but there are no data about the consequences of the lack of endogenous PACAP in different ototoxic insults such as aminoglycoside-induced toxicity. In this study, we examined the effect of kanamycin treatment on Ca(2+)-binding protein expression in hair cells of wild-type, heterozygous and homozygous PACAP-deficient mice. We treated 5-day-old mice with kanamycin, and 2 days later, we examined the Ca(2+)-binding protein expression of the hair cells with immunohistochemistry. We found stronger expression of Ca(2+)-binding proteins in the hair cells of control heterozygous and homozygous PACAP-deficient mice compared with wild-type animals. Kanamycin induced a significant increase in Ca(2+)-binding protein expression in wild-type and heterozygous PACAP-deficient mice, but the baseline higher expression in homozygous PACAP-deficient mice did not show further changes after the treatment. Elevated endolymphatic Ca(2+) is deleterious for the cochlear function, against which the high concentration of Ca(2+)-buffers in hair cells may protect. Meanwhile, the increased immunoreactivity of Ca(2+)-binding proteins in the absence of PACAP provide further evidence for the important protective role of PACAP in ototoxicity, but further investigations are necessary to examine the exact role of endogenous PACAP in ototoxic insults.

Differential expression of PRLIPs, a pathogenesis-related gene family encoding class 3 lipase-like proteins in Arabidopsis.

In plants plenty of inducible defense-related proteins classified into 17 pathogenesis-related (PR) families have been described. Expression of homologous PR genes from the same family can be induced by the different defense hormones, like salicylic acid (SA), jasmonic acid (JA) or ethylene (ET), and are also regulated in a organ- or tissue-specific manner. A recently identified pathogenesis-related gene family, the PRLIP (pathogenesis-related lipase) has 9 members in Arabidopsis and their organization and expression pattern - as it is summarized in this study - is similar to the one of genes coding for other PR proteins. PRLIP3, PRLIP8 and PRLIP9 showed a relatively high expression in all tissues tested with a maximum in root (PRLIP3), stem (PRLIP8) or siliques (PRLIP9). The activity of PRLIP3 gene was further induced by SA and JA treatment. Other members (PRLIP1, PRLIP2, PRLIP4 and PRLIP6), however, were detected only in some of the tested organs. High levels of PRLIP1 mRNA occurred in all green tissues and in siliques, while in the latter PRLIP2 also displayed high expression. PRLIP6 and PRLIP4 exhibited root specific transcription while no mRNAs of PRLIP5 and PRLIP7 were detected in any plant tissues examined. In leaves SA treatment enhanced PRLIP1 and PRLIP2 expression, JA treatment induced PRLIP6 and ET treatment upregulated both PRLIP1 and PRLIP6. This organization and expression diversity of the PRLIP gene family is typical to plant PR genes suggesting the encoded proteins might serve essential functions in plant defense or priming.

Secondary structure of proteins adsorbed onto or embedded in polyelectrolyte multilayers.

The structural changes of bovine serum albumin (BSA) and hen egg white lysozyme (HEL) upon their adsorption onto the surface or their embedding into the interior of poly(allylamine hydrochloride)-(poly(styrenesulfonate) (PAH-PSS) multilayer architectures were investigated by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The presence of the polyelectrolytes seems, as previously observed for fibrinogen (J. Phys. Chem. B 2001, 105, 11906-11916), to prevent intermolecular interactions and, thus, protein aggregation at ambient temperature. The secondary structure of the proteins was somewhat altered upon adsorption onto the polyelectrolyte multilayers. The structural changes were larger when the charges of the multilayer outer layer and the protein were opposing. The adsorption of further polyelectrolyte layers onto protein-terminated architectures (i.e., embedding the proteins into a polyelectrolyte multilayer) did not cause considerable further changes in their secondary structures. The capacity of the polyelectrolyte architectures to delay the formation of intermolecular beta-sheets upon increasing temperatures was not uniform for the studied proteins. PSS in contact with HEL could largely prevent the heat-induced aggregation of HEL. In contrast, PAH had hardly any effect on the aggregation of BSA. The differences are explained on the basis of protein-polyelectrolyte interactions, affected mostly by the nature and the strength of the ionic interactions between the polyelectrolyte-protein contact surfaces.

Membrane dynamics as seen by fourier transform infrared spectroscopy in a cyanobacterium, Synechocystis PCC 6803. The effects of lipid unsaturation and the protein-to-lipid ratio.

The roles of lipid unsaturation and lipid-protein interactions in maintaining the physiologically required membrane dynamics were investigated in a cyanobacterium strain, Synechocystis PCC 6803. The specific effects of lipid unsaturation on the membrane structure were addressed by the use of desaturase-deficient (desA(-)/desD(-)) mutant cells (which contain only oleic acid as unsaturated fatty acid species) of Synechocystis PCC 6803. The dynamic properties of the membranes were determined from the temperature dependence of the symmetric CH(2) stretching vibration frequency, which is indicative of the lipid fatty acyl chain disorder. It was found that a similar membrane dynamics is maintained at any growth temperature, in both the wild-type and the mutant cell membranes, with the exception of mutant cells grown at the lower physiological temperature limit. It seems that in the physiological temperature range the desaturase system of the cells can modulate the level of lipid desaturation sufficiently to maintain similar membrane dynamics. Below the range of normal growth temperatures, however, the extent of lipid disorder was always higher in the thylakoid than in the cytoplasmic membranes prepared from the same cells. This difference was attributed to the considerable difference in protein-to-lipid ratio in the two kinds of membranes, as determined from the ratio of the intensities of the protein amide I band and the lipid ester C&z.dbnd6;O vibration. The contributions to the membrane dynamics of an ab ovo present 'structural' lipid disorder due to the protein-lipid interactions and of a thermally induced 'dynamic' lipid disorder could be distinguished.

Separable contributions of ordered and disordered lipid fatty acyl chain segments to nuCH2 bands in model and biological membranes: a Fourier transform infrared spectroscopic study.

In this article, the assignment of the nu(C-H) stretching region of lipid molecules is revisited. This region is extensively used to follow lipid phase transitions, and especially the frequency shifts and bandwidth alterations in the nu(sym)CH2 band have been utilized in this respect. Here, we propose and prove that behind these phenomena there are pairs of component bands in the cases of both the nu(sym)CH2 and the nu(as)CH2 bands. The lower-frequency components of the pairs are assigned to the vibrations of CH2 groups on trans segments of the fatty acyl chains, while the higher-frequency components of the pairs are assigned to CH2 groups on gauche segments. To prove these assignments, we have shown that the nuCH2 frequencies are characteristic of the conformation of the lipid fatty acyl chain itself, and not the state of the whole lipid matrix. Curve fitting in fact revealed the conformer-specific components. With the use of singular value decomposition analysis we have demonstrated that the relative intensity changes in the components, and not the shifts in the whole bands, cause the observed shifts in the nuCH2 bands upon lipid phase transition. The results of this approach are presented for deuterium-saturated dioleoyl-phosphatidylcholine mixtures, for the gel --> liquid-crystalline phase transition of dipalmitoyl-phosphatidylcholine multilayers, and for a biological membrane, barley thylakoid. This refined assignment offers physically plausible reasoning for the observed phenomena and is able to explain frequency shifts and bandwidth changes observed previously upon lipid phase transitions, including their nonconcerted temperature dependences. In biological membranes, this interpretation allows the separation of protein- and membrane-dynamics-induced lipid conformational changes.

Structure and interactions of phycocyanobilin chromophores in phycocyanin and allophycocyanin from an analysis of their resonance raman spectra.

Raman spectra of phycocyanobilin, phycocyanin, and allophycocyanin were obtained at resonance with their visible and near-UV transitions. These spectra were empirically assigned with the help of 14N- and 15N-isotopic substitutions and comparisons with resonance Raman spectra of phycoerythrin. These results confirm the previously suggested assignment of a conformation-sensitive band around 1239-1246 cm-1 to a mode involving nu CmH and nu CN coordinates. Computer-assisted decomposition of the complex, conformation-sensitive 1580-1670-cm-1 region yielded five components that we labeled I-V. The previously described spectral changes observed upon monomerization and denaturation in resonance Raman spectra of phycocyanin and allophycocyanin essentially arise from changes in the relative intensities of these components. Component I (around 1649-1651 cm-1) and component III (1621-1624 cm-1) originate predominantly from nu C=C at C15 of the chromophore. Their relative intensity ratio reflects the relative amounts of C15-Z-anti and C15-Z-syn methine bridge conformations, respectively. Component II (1633-1638 cm-1) is ascribed to a nu C=C mode of pyrrole rings; it is not sensitive to the chromophore conformation. Component IV is also conformation-insensitive and originates from nu C=N and nu C=C coordinates, most likely from ring C. Component V (1591-1594 cm-1) involves a nu C=N coordinate in ring D, coupled to a nu C=C coordinate of the C15 methine bridge. The implications of the present assignments on those of resonance Raman active modes of phytochrome are discussed. A consistent set of correlations between chromophore conformations and resonance Raman data is obtained for both phycobiliproteins and phytochrome.

In situ modification of the phospholipid environment of native rabbit sarcoplasmic reticulum membranes.

A water soluble hydrogenation catalyst (palladium di(sodium alizarine monosulphonate)) in a deuterium-containing environment has been used for the in situ insertion of deuterium atoms into the fatty acyl chains of biological membranes. The thermotropic response of the stretching vibrations of the formed C-D bonds, as detected by Fourier transform IR spectroscopy, was used as a selective probe of biological membrane structure. Partial deuteration of unsaturated fatty acyl chains coupled with IR detection potentially provides a means for detecting specific biological roles of particular lipid classes. In the current study of sarcoplasmic reticulum membranes and purified phospholipid/CaATPase vesicles, it is also shown that vC-D monitors change at particular membrane locations which may remain undetected through the CH2 symmetric stretching frequency, a widely used IR spectral parameter. The latter reflects the average environment of the acyl chains. The approach described here may be suitable for wide applications to the study of biomembranes.

Homogeneous catalytic deuteration of fatty acyl chains as a tool to detect lipid phase transitions in specific membrane domains: a Fourier Transform Infrared spectroscopic study.

Synthetic phospholipid molecules have been deuterated by using a water soluble catalyst and deuterium gas. The physical state of both deuterated segments and unaffected bulk part of the lipid molecules can be monitored simultaneously by Fourier Transform Infrared Spectroscopy. It is shown on multilamellar phospholipid systems that the deuterated segments can be used as structural probes. Whereas the nu(C-H) frequencies represent an average conformational order along all the alkyl chains present, by following changes in nu(C-D) vibrations, mobility of those membrane domains deuterium labeled at specific depths in the hydrocarbon core can be estimated. The potential importance of this new approach in the study of biological membranes is discussed.

Chromophore conformational analysis in phycocyanin and in related chromopeptides by surface enhanced Raman spectroscopy.

Chromopeptides got from phycocyanin by proteolytic digestion do not preserve the extended chromophore conformations characteristic to the native protein. Chromophore conformations in the chromopeptides showed heterogenity varying between completely folded and semi-extended states. Indications were found that the silver sol-phycocyanin interaction involves the UV electronic transition of the biliprotein which may explain why the visible excited surface enhanced Raman spectra were similar not to the visible excited but to the UV-excited resonance Raman spectrum of phycocyanin.

Resonance Raman spectra of phycocyanin, allophycocyanin and phycobilisomes from blue-green alga Anacystis nidulans.

Resonance Raman spectra of native C-phycocyanin, allophycocyanin and whole, intact phycobilisomes from the blue-green alga Anacystis nidulans (Synechococcus 6301) are reported. A tentative assignment for the more prominent resonance Raman bands is suggested. The possibly sensitive regions for inter-chromophore interactions in the case of phycobilisomes are also discussed.

Mn and Co toxicity in chlorophyll biosynthesis.

The metal ion-induced inhibition of tetrapyrrole biosynthesis was studied in the cyanobacterium Anacystis nidulans. The accumulation of protoporphyrin and Mg protoporphyrin due to the effect of Co(2+) and Mn(2+) treatment, respectively, pointed to two different sites of inhibition.