Coordinated Development of Yeast Colonies: Quantitative Modeling of Diffusion‐Limited Growth – Part 2

A mathematical model was developed which described the growth of yeast colonies based on the assumptions that (i) these populations were built up of single cells whose proliferation was (ii) exclusively controlled by nutrient availability in the environment. The model was of a hybrid cellular automaton type and described discrete cells residing on a one‐dimensional lattice as well as on continuously distributed nutrients. Experimental results and numerical calculations were compared to elucidate under which cultivation conditions the diffusion‐limited growth (DLG) was the major construction principle in yeast colonies. Simulations were scaled to the growth of Yarrowia lipolytica and Candida boidinii colonies under carbon and nitrogen limitation. They showed that nutrient‐controlled growth of the individual cells resulted in DLG of the population. Quantitative predictions for the spatio‐temporal development of the cell‐density profile inside a growing yeast mycelium were compared to the growth characteristics of the model yeast mycelia. Only for the carbon‐limited growth of C. boidinii colonies on glucose as the limiting nutrient resource did the DLG model reproduce the cell‐density profile estimated at the end of the cultivation. Under all other cultivation conditions, strong discrepancies between calculations and experimental results were evident precluding DLG as the ruling regulatory mechanism. Thus, whether or not the development of a yeast population could be described by a DLG scenario, was strongly dependent on the particular cultivation conditions and the applied yeast species. In those cases for which the DLG hypothesis failed to explain the observed growth patterns, the underlying assumptions, i.e., the complete absence of nutrient translocation between the individual cells inside the yeast mycelia as well as the exclusively nutrient‐controlled proliferation of the cells, have to be reevaluated. The presented study demonstrated how the mathematical analysis of growth processes in yeast populations could assist the experimental identification of potential regulatory mechanisms.     

Biosorption Processes for Natural and Waste Water Treatment – Part II: Experimental Studies and Theoretical Model of a Biosorption Fixed Bed

A model has been developed for fixed‐bed biosorption performance, i.e. combined action of adsorption of organic water contaminants and their biological destruction in a column. The model contains an adsorption isotherm of the Freundlich type, adsorption kinetics by an overall film mass transfer (Glueckauf equation), maximum bacterial growth,and biological aerobic destruction (Monod model) of the organics by exoenzymes. Bacteria can not penetrate into the pores of the adsorbent. The model was tested using the system aqueous solution of aniline/Pseudomonas putida/Polysorb 40/100. Breakthrough curves in shorter columns have been measured and a velocity‐dependent steady‐state exit concentration was achieved. These curves could be simulated with sufficient accuracy on the basis of isotherm data, mass transfer coefficients and values of biological growth and destruction activity estimated from independent measurements.     

Hermansky–Pudlak Syndrome: Vesicle Formation from Yeast to Man

The disorders known as Hermansky–Pudlak syndrome (HPS) are a group of genetic diseases resulting from abnormal formation of intracellular vesicles. In HPS, dysfunction of melanosomes results in oculocutaneous albinism, and absence of platelet dense bodies causes a bleeding diathesis. In addition, some HPS patients suffer granulomatous colitis or fatal pulmonary fibrosis, perhaps due to mistrafficking of a subset of lysosomes. The impaired function of specific organelles indicates that the causative genes encode proteins operative in the formation of certain vesicles. Four such genes, HPS1, ADTB3A, HPS3, and HPS4, are associated with the four known subtypes of HPS, i.e. HPS‐1, HPS‐2, HPS‐3, and HPS‐4. ADTB3A codes for the β3A subunit of adaptor complex‐3, known to assist in vesicle formation from the trans‐Golgi network or late endosome. However, the functions of the HPS1, HPS3, and HPS4 gene products remain unknown. These three genes arose with the evolution of mammals and have no homologs in yeast, reflecting their specialized function. In contrast, all four known HPS‐causing genes have homologs in mice, a species with 14 different models of HPS, i.e. hypopigmentation and a platelet storage pool deficiency. Pursuit of the mechanism of mammalian vesicle formation and trafficking, impaired in HPS, relies upon investigation of these mouse models as well as studies of protein complexes involved in yeast vacuole formation.     

Converting the Yeast Arginine Can1 Permease to a Lysine Permease

Amino acid uptake in yeast cells is mediated by about 16 plasma membrane permeases, most of which belong to the amino acid-polyamine-organocation (APC) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Although Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H⁺-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a three-dimensional Can1 structural model based on the crystal structure of the bacterial APC family arginine/agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q and E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H⁺ coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport.     

Analysis of the budding yeast pH 4–7 proteome in meiosis

Meiosis, the developmental programme generating haploid gametes from diploid precursors, requires two cell divisions and many innovations. In budding yeast, a large number of genes are expressed exclusively during meiosis while others are repressed compared to vegetative growth. Microarray analysis has shown that gene expression during meiosis is highly regulated, and has been used to classify yeast genes according to meiotic temporal expression pattern. In this study, we have begun to investigate the kinetics of meiotic protein expression using a proteomics approach. 2‐D DIGE was used to characterise the temporal protein expression patterns of the budding yeast pH 4–7 proteome in meiosis. More than 1400 meiotic protein spots were visualised and at least 63 spots were temporally regulated during meiosis in a statistically significant manner. Gel spots with significant expression changes were excised and 26 unique proteins were identified using LC‐MS/MS. The identified proteins could be classified into functional categories and the genes encoding a number of these were previously shown to be involved in yeast sporulation and meiosis. This data set was used to assemble the first differential 2‐D PAGE map of budding yeast meiosis, which can be accessed through a web server. This work represents one of the first quantitative proteomic analyses of meiosis in yeast and will provide a valuable resource for future investigations.     

From Enzyme Kinetics to Metabolic Network Modeling – Visualization Tool for Enhanced Kinetic Analysis of Biochemical Network Models

Model‐based analysis of enzyme kinetics allows the determination of optimal conditions for their use in biocatalysis. For biotransformations or fermentative approaches the modeling of metabolic pathways or complex metabolic networks is necessary to obtain model‐based predictions of steps which limit product formation within the network. To set up adequate kinetic models, relevant mechanistic information about enzyme properties is required and can be taken from in vitro studies with isolated enzymes or from in vivo investigations using stimulus‐response experiments which provide a lot of kinetic information about the metabolic network. But with increasing number of reaction steps and regulatory interdependencies in the network structure the amount of simulation data dramatically increases and the simulation results from the dynamic models become difficult to analyze and interpret. Demonstrated for an Escherichia coli model of the central carbon metabolism, methods for visualization and animation of simulation data were applied and extended to facilitate model analysis and biological interpretation. The dynamic metabolite pool and metabolic flux changes were visualized simultaneously by a software tool. In addition, a new quantification method for enzyme activation/inhibition was proposed, and this information was implemented in the metabolic visualization.     

Nutrient Fluxes from Land to Sea: Consequences of Future Scenarios on the Oder River Basin – Lagoon – Coastal Sea System

Eutrophication management is still one of the major challenges in the Baltic Sea region. Intense transformation processes in several Baltic Sea states have led to drastic changes in e.g., landuse and thereby nutrient emissions and water quality. Several future development directions are possible. The Oder catchment – lagoon – coastal water system serves as a pilot study area, since it has a major influence on the nutrient loads into the Baltic Sea and about 90% of the catchment is located in Poland, a state with transitional economy. Different scenarios for landuse changes in the Oder catchment are developed and their consequences on nutrient emissions simulated. Next to politically induced changes of agricultural landuse in general, specific aspects such as cultivation of energy maize and increased animal stocks are considered. Nitrogen emissions are likely to increase due to agricultural landuse changes whereas phosphorus emissions will not change or even decrease according to the application of the EC‐Urban Waste Water Treatment Directive. Resulting nitrogen loads to the Oder Lagoon could increase up to 23%, phosphorus loads could decrease by 11% compared to 2005. These trends may lead to higher nitrogen availability compared to phosphorus at least in the Oder lagoon. Interannual differences in discharge also have profound effects on nutrient emissions. A good status of the Oder river basin – lagoon – coastal sea system according to EC‐Directives is not very likely to be achieved under the investigated circumstances. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Adaptation to β‐myrcene catabolism in Pseudomonas sp. M1: An expression proteomics analysis

β‐Myrcene, a monoterpene widely used as a fragrance and flavoring additive, also possesses analgesic, anti‐mutagenic, and tyrosinase inhibitory properties. In order to get insights into the molecular mechanisms underlying the ability of Pseudomonas sp. M1 to catabolize β‐myrcene, an expression proteomics approach was used in this study. Results indicate that the catabolic enzyme machinery for β‐myrcene utilization (MyrB, MyrC, and MyrD and other uncharacterized proteins) is strongly induced when β‐myrcene is present in the growth medium. Since an M1 mutant, lacking a functional 2‐methylisocitrate dehydratase, is not able to grow in mineral medium with β‐myrcene or propionic acid as the sole C‐source, and also based on the expression proteomic analysis carried out in this study, it is suggested that the β‐myrcene catabolic intermediate propionyl‐CoA is channeled into the central metabolism via the 2‐methylcitrate cycle. Results also suggest that the major alteration occurring in the central carbon metabolism of cells growing in β‐myrcene‐containing media is related with the redistribution of the metabolic fluxes leading to increased oxaloacetate production. Other up‐regulated proteins are believed to prevent protein misfolding and aggregation or to play important structural roles, contributing to the adaptive alteration of cell wall and membrane organization and integrity, which are essential features to allow the bacterium to cope with the highly lipophilic β‐myrcene as C‐source.     

Yeast telomere length varies in response to changes in the amount of polyC1 – 3A in the cell

Summary Yeast chromosomes terminate in a GC-rich tail of DNA. Previous investigations have shown that the length of this tail can change in response to genetic variation. Here we present data that show that the length can also alter in response to changes in the amount of the GC-rich DNA found elsewhere in the nucleus.     

Biosorption Processes for Natural and Wastewater Treatment – Part 1: Literature Review

An analysis is made of the literature on the mechanism of biosorption‐bioregeneration of organic substances mainly on activated carbon (AC). There have advanced two hypotheses on the biosorption mechanism: the Rodman exoenzymatic hypothesis and the bioregeneration approach of Andrews and Chi Tien. In addition, it was shown that the principal mechanism responsible for the removal of both biodegradable and bioresistant compounds is biological oxidation, but only in combination with adsorption on activated carbon. The authors examine the role of the filter medium in biosorption, factors affecting efficiency of biosorption and bioregeneration of AC and technological aspects of the application of biosorptive processes.     

Studies on cytochrome c. Part I. Synthesis of the protected hexadecapeptide (sequence 1–16) of Baker's Yeast iso-1-cytochrome c

The general strategy for the synthesis by conventional procedure of N- and C-terminal fragments and of the entire sequence of baker's yeast iso-1-cytochrome c is discussed. The synthesis of the N-benzyloxycarbonylhexadecapeptide tert-butoxycarbonylhydrazide corresponding to the sequence 1–16 of the apoprotein is described in detail.     

Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.     

Nodding Capitula in Superpáramo Asteraceae: An Adaptation to Unpredictable Environment1

Nodding capitula are a striking feature in several Asteraceae species of the Andean superpáramo. In this study, the thermal environment of the nodding inflorescence in Culcitium canescens Humb. & Bonpl. was surveyed by a short-term microclimatic measurement. Temperatures of the nodding inflorescences were higher than temperatures of the surrounding air during the day, while the respective temperatures were comparable during the night. Also, compared to other parts of the plant, the nodding inflorescence provided favorable temperature conditions. An experiment showed that the radiation reflected from the substratum explained most of the variability in the inflorescence temperature. It is suggested that the nodding capitula have evolved to protect the flowers from snow and rain while at the same time ensuring sufficient temperatures for floral development.     

Forgotten Perfumery Plants – Part I: Balm of Judea

Perfumes have always been products of great importance, mainly composed of natural, valuable and vegetal raw materials. Today, some of them have completely disappeared in perfumery, even though they are part of our cultural heritage and were commonly used in the past. Balm of Judea is one of the most noble, rare and fascinating ingredient long used in perfumery and medicine, that is missing today. After years of research, we collected a resin and an essential oil (steam distillation of fresh aerial parts) from Commiphora gileadensis (L.) C.Chr . native from Saudi Arabia and cultivated in Israel. The aims of this study were to i) identify the main reasons of the loss of the balm of Judea, ii) characterize the volatile composition of the resin and the essential oil and iii) evaluate their olfactory profile and assess their biological activity. Eighty‐three compounds were identified in the resin, by a combination of GC‐MS and GC/FID techniques, using direct injection and HS‐SPME. α‐Pinene (24.0 %), sabinene (43.8 %), β‐pinene (6.3 %) and cymene (3.6 %) were the main identified compounds, giving an intense, terpenic and lemony smell to the resin. Anti‐inflammatory, wound‐healing and whitening activities were highlighted. Sabinene (22.7 %), terpinen‐4‐ol (18.7 %), α‐pinene (14.4 %) and cymene (13.6 %) were identified as the main components of the essential oil, giving a spicy, woody and lemony fragrance. Anti‐inflammatory and whitening activities were emphasized.     

Differential Role of HAMP-like Linkers in Regulating the Functionality of the Group III Histidine Kinase DhNik1p

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1–5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1–5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1–5 protein inside experimental data-based models showed how two chains of HAMP1–5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1–5 structure mediate its function.     

酵母豆蔻酰辅酶A：蛋白质N端转酰基酶基因的克隆与表达

利用ＰＣＲ技术,从酵母染色体中扩增得到酵母豆蔻酰－ＣｏＡ：蛋白质Ｎ端转酰基酶（ＹＳＣＮＭＴ）基因,并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋载体中。由ＤＮＡ全序测定表明,获得了ＹＳＣＮＭＴ编码基因。进一步构建了Ｔ７Ｐｒｏｍｏｔｅｒ控制下的含上述完整ＹＳＣＮＭＴ编码基因的表达质粒ｐＭＦＴ７－５－ＮＭＴ,转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。通过ＳＤＳ－ＰＡＧＥ分析,观察到一与理论分子量一致的诱导条带（约５３ｋＤ）,占全菌蛋白的３９％左右,且可溶性部分约占上清液中全部蛋白的３４％。经一步Ｐ１１磷酸纤维素阳离子交换柱层析,将其纯化到纯度达９７％以上．纯化的表达产物经Ｎ端氨基酸序列分析,所测定的Ｎ端５个氨基酸的序列,与从克隆的ＹＳＣＮＭＴ基因推出的氨基酸序列完全一致（不含Ｎ端Ｍｅｔ）。对所得的ＹＳＣＮＭＴ进行酶活力鉴定,观察到了明显的活力。     

Some highlights in the history of fungi in medicine – A personal journey

The history of the role of fungi in the development of medicine has largely been neglected. Here the early recognition of the germ theory will be discussed in relation to surface infections, the “cholera fungus theory” and recognition of the phenomenon of microbial antagonism. This leads to a short review of the history of those antibiotics produced by fungi, notably penicillin. Finally, the history of the recognition of the potential role fungi play as casual agents of cancer will be discussed; although currently neglected, this work may prove to be of future significance. This history of medical mycology is discussed from the standpoint of my own interest and research into this fascinating, and often overlooked area of medical history.