Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201.

Whole-cell protein patterns of a psychrotrophic Bacillus cereus strain from cultures grown at 7 and 30 degrees C were compared. This analysis revealed that at least three major proteins are expressed at a significantly higher rate at 7 degrees C than at 30 degrees C. The most abundant of these cold-induced proteins was a small polypeptide of 7.5 kDa, designated CspA, of B. cereus. In addition, four small proteins very similar in size to CspA were seen on both 7 degrees C and 30 degrees C two-dimensional protein gels. Immunoblot analysis using B. cereus anti-CspA antibodies indicated that the five proteins described above plus an additional sixth protein not visible on silver-stained two-dimensional gels are members of a B. cereus cold shock protein family. This hypothesis was corroborated by cloning and sequencing of the genes encoding five proteins of this family. The protein sequences deduced are highly similar and show homology to small procaryotic cold shock proteins and to the cold shock domain of eucaryotic Y-box proteins. Besides CspA, only one of the additional five CspA homologs was slightly cold inducible. In the presence of 100 mM NaCl, the two purified members of the protein family (CspA and CspE) elute as dimers at an apparent molecular mass of 15 kDa from a gel filtration column. At higher salt concentrations, they dissociate into their monomers. Their ability to bind to the ATTGG motif of single-stranded oligonucleotides was demonstrated by band shift analysis.     

Simulation of the effect of mixing,scale-up and pH-value regulation during glutamic acid fermentation

A mixing model is coupled with fermentation kinetics in order to simulate a fermentation as a function of mixing conditions and scale-up. The mixing model for a batch stirred tank with three stirrers consists of three regions, each of them characterized by an ideally mixed compartment around the stirrer and two macromixers, i.e. cascades of tank-in-series, describing the recirculation flow. The model contains four parameters — radial and axial circulation time, volume of the ideally mixed stirrer compartment and the number of tanks in each cascade. These values, determined by Mayr et al. in function of the operational conditions and scale-up, were choosen to simulate the fermentation of glutamic acid to show the pH-fluctuation at different control and scale conditions. By choosing optimal regulation properties, such as input flow rate and/or concentration of the base, regulation span, position of the pH-electrode and base input location, etc., fluctuations of the pH-value in the bio-reactor can be minimized. However, the negative effect of insufficient mixing conditions can be reduced only by an increasing number of the base input places. In large scale fermentors, the axial circulation time is rather high, about 5–10 times larger than the radial one. This might result in a large amplitude of the pH-fluctuation. As it is shown, using an input place for base in each stirrer region, the negative impact of the insufficient axial mixing on the fermentation can be diminished perfectly. In this case ammonia should be fed into the reactor as an aqueous solution.     

Investigation of the dithiocarbamate-induced coupling of allylidene and carbonyl ligands on a tungsten center

Reaction of the allylidene tungsten complex [W(CPhCHCHMe)Br₂(CO)₂(4-picoline)] (1) with the dithiocarbamates MS₂CNR₂ (a: M=Na, R=Et; b: M=Na, R=Me; c: M=Li, R=Ph) in THF at 50 °C affords the vinylketene tungsten complexes [W(S₂CNR₂)₂(OCCPhCHCHMe)(CO)] (2a–c). At lower temperatures, four reaction intermediates (3–6) may be discerned. Spectroscopic studies indicate that these compounds contain η⁴-allyldithiocarbamate ligands which are generated by addition of dithiocarbamate across the metal-carbon double bond of the allylidene-tungsten unit in 1. The structures of [W(S₂CNEt₂)₂(OCCPhCHCHMe)(CO)] (2a) and of one intermediate, [W(η⁴-Et₂NCS₂CPhCHCHMe)(S₂CNEt₂)(CO)₂] (5a) were elucidated by X-ray crystallography.     

Role of the Cys 2-Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1.

The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.     

Inositol tetrakisphosphate isomers and elevation of cytosolic Ca2+ in vasopressin-stimulated insulin-secreting RINm5F cells.

Signal generation during the stimulation of insulin secretion by arginine vasopressin (AVP) was investigated in RINm5F cells. AVP (0.1 microM) caused a biphasic cytosolic Ca2+ ([Ca2+]i) rise, namely a rapid transient marked elevation after stimulation followed by a series of oscillations. In the absence of extracellular Ca2+, the sustained oscillations were abolished, while the initial [Ca2+]i transient was only partly decreased, indicating that the former are due to Ca2+ influx and the latter due mainly to mobilization from internal Ca2+ stores. AVP also evoked a transient depolarization of the average membrane potential. AVP-induced Ca2+ influx during the sustained phase, which was strictly dependent on receptor occupancy, was attenuated by membrane hyperpolarization with diazoxide. However, blockade of Ca2+ channels of the L- or T-type was ineffective. AVP stimulated production of diacylglycerol and inositol phosphates; for the latter both [3H] inositol labeling and mass determinations were performed. A transient increase in Ins(1,4,5)P3 was followed by a marked enhancement of Ins(1,3,4,5)P4 (8-fold) peaking at 15 s and gradually returning to basal values. Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4 exhibited the most long-lasting augmentation (4- and 1.7-fold, respectively), and therefore correlated best with the period of sustained [Ca2+]i oscillations. InsP5 and InsP6 were not elevated. The effects of AVP, including the stimulation of insulin secretion from perifused cells, were obliterated by a V1 receptor antagonist. In conclusion, AVP induces protracted [Ca2+]i elevation in RINm5F cells which is associated with long-lasting increases in InsP4 isomers. The accumulation of InsP4 isomers reflects receptor occupancy and accelerated metabolism of the inositol phosphates. Activation of second messenger-operated Ca2+ channels is not necessarily implicated because of the attenuating effect of membrane hyperpolarization.     

Engineering approach to mixing quantification in bioreactors

Homogeneity-time is defined and introduced as the criterion for mixing quality in bioreactors. The criterion could replace the mixing time, in the case, when more than one measuring point (sensors) is included in the measuring system. Results based on the homogeneity-time and the temperature pulse method, achieved in stirred tank reactors under aerated conditions as well as in a jet-mixed tank, are presented.List of Symbols C _p,p kJ/kg K Heat capacity of the pulse medium - C _p,s kJ/kg K Heat capacity of the reactor-medium - F m³/s Flow rate of the pulse-input - i Inhomogeneity - I _N Inhomogeneity-number - M (t) °C Ideal response curve - m deNumber of combinations for certain number of sensors acc. to Table 1 - n Number of sensor - _p kg/m³ Density of the pulse medium - kg/m³ Density of the tank medium - s ₁ °C Mean absolute deviation of the sensor temperatures related on the ideal response curve s₂ s Mean absolute deviation of the homogeneity-times related on the time achieved with 6 sensors - t s Time - t (i) s Homogeneity-time - t _ps s Starting time of tracer injection - t _PE s End time of tracer injection - T _E °C Mean medium temperature at the end of experiment - T _k °C Temperature at k-th sensor position - T _p °C Pulse temperature - T _s °C Mean medium temperature before the tracer injection - V _s m³ Tank volume before pulse input     

Meiotic chromosome behaviour reflects levels of sequence divergence inSus scrofa domestica satellite DNA

We present a general model for the evolution of chromosome-specific satellite DNA subfamilies.Sus scrofa domestica has a bimodal karyotype with two autosomal subsets of 12 meta-/submetacentric (Mc) and 6 acrocentric (Ac) chromosome types (Mc and Ac subgenomes). We show that the centromeric heterochromatin is characterised by two distinct satellite DNA families designed Mc1 and Ac2. Mc1 is a diverse satellite family of the Mc subgenome of which certain members with a 100 bp repeat unit are found to occur at the pericentromeric regions of each Mc autosome, while others are chromosome-specific, e.g. clone Mc pAv1.5, a higher order repeat variant, which hybridises specifically to chromosome 1. Ac2 is a homogeneous satellite occurring at the subterminal pericentromeric regions of all Ac autosomes. DNA sequence analyses showed that all clones investigated are built up from a 14 bp repeat unit which is highly conserved. In situ hybridisation to meiotic pachytene nuclei revealed a distinct spatial arrangement of the Ac2 centromeric satellite.     

Further resolution of the quail karyotype and characterization of microchromosomes by counterstain-enhanced fluorescence

Sequential staining with a counterstain-contrasted fluorescent R-banding technique (chromomycin A3/distamycin A-DAPI) followed by DAPI-actinomycin D-induced quinacrine-fluorescence-Hoechst 33258 (QFH)-type banding allowed the identification of quail chromosomes up to chromosome 19. The chromomycin A3-positive staining behavior of the W chromosome and of the heterochromatic areas of most microchromosomes indicated their GC-rich nature.