The enhancement of specific antibody production rate in glucose- and glutamine-controlled fed-batch culture

The concentration effects of certain amino acids (Asp, Ile, Leu, Lys, Met, Val, Phe and Gln which were highly consumed during cultivation), and glucose on cell growth and antibody productivity were investigated using dish culture. From these experiments, it was found that only glutamine enrichment enhanced the specific antibody production rate. The other amino acids described above did not affect either the specific growth rate or specific antibody production rate. Thus we investigated the quantitative effects of glutamine concentration in the range of 0.4∼33.3 mmol·1⁻¹ on kinetic parameters in fed-batch culture which kept both glucose and glutamine concentration constant. As a result the specific growth rate decreased with increase in glutamine concentration in the range larger than 20 mmol·1⁻¹. The specific antibody production rate had a maximum value at about 25 mmol·1⁻¹ glutamine concentration.     

Design,synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane derivatives as GPR119 agonists

The design and synthesis of a novel class of 7-azaspiro[3.5]nonane GPR119 agonists are described. In this series, optimization of the right piperidine N-capping group (R²) and the left aryl group (R³) led to the identification of compound 54g as a potent GPR119 agonist. Compound 54g showed a desirable PK profile in Sprague-Dawley (SD) rats and a favorable glucose lowering effect in diabetic rats.     

Maximum virginiamycin production by optimization of cultivation conditions in batch culture with autoregulator addition

A strategy for optimization of non-growth-associated production in batch culture employing an empirical approach was developed through the study of virginiamycin production. The strategy is formulated with two aims: attaining a high cell concentration at the beginning of the production phase without decrease in production activity; and enhancing the production activity during the production phase. As a practical example, the goal of a maximum virginiamycin (M and S) production in the batch culture of Streptomyces virginiae was set. To attain a high cell concentration in the production phase of the batch culture, that is, to extend the growth phase for as long as possible, the optimum composition and concentration of the complex medium, especially the yeast extract (YE) concentration, were first investigated. Dissolved oxygen (DO) concentration control was also a parameter considered in maintaining the production activity during the production phase. In addition, to enhance the production activity, an optimum addition strategy of an autoregulator, virginiae butanolide-C (VB-C), was investigated. Combining these measures, the optimum cultivation conditions were found to be an initial YE concentration in the complex medium of 45 g/L, the shot addition of 300 mug/L of VB-C 11.5 h after the start of the batch culture, and a DO concentration maintained above 2 mg/L. The maximum concentrations of virginiamycin M and S were about ninefold those obtained under nonoptimum cultivation conditions. Nonoptimum cultivation conditions consisted of an initial YE concentration one sixth (7.5 g/L) that of the optimum cultivation conditions, and no VB-C addition. These conditions were used as representative of the standard cultivation of virginiamycin in this study. The strategy developed here will be applicable to the production of other antibiotics, especially to the cultivation of Streptomyces species, in which a hormonelike signal material (an autoregulator) plays an important role in antibiotic production. (c) 1996 John Wiley & Sons, Inc.     

Phylogeny of Sym plasmids of rhizobia by PCR-based sequencing of a nodC segment.

To understand the host specificity of rhizobia and the relationship between the evolution of Sym plasmids and that of host plants, we determined partial nodC sequences of 10 representative rhizobium strains and then constructed an evolutionary tree for the deduced amino acid sequences with four published sequences. These coding sequences yield a phylogenetic tree similar to that for leghemoglobin of host plants, suggesting that the evolution of common nodulation genes may be linked to host legume evolution and speciation.     

Cranial anomaly of homozygous rSey rat is associated with a defect in the migration pathway of midbrain crest cells

Craniofacial development of vertebrates depends largely on neural crest contribution and each subdomain of the crest-derived ectomesenchyme follows its specific genetic control. The rat small eye ( rSey ) involves a mutation in the Pax-6 gene and the external feature of rSey homozygous embryos exhibits craniofacial defects in ocular and frontonasal regions. In order to identify the mechanism of craniofacial development, we examined the cranial morphology and migration of cephalic crest cells in rSey embryos. The chondrocranial defects of homozygous rSey embryos primarily consisted of spheno-orbital and ethmoidal anomalies. The former defects appeared to be brought about by the lack of the eye. In the ethmoid region, the nasal septum and the derivative of the medial nasal prominence were present, while the rest of the nasal capsule, as well as the nasal and lachrymal bones, were totally absent except for a pair of cartilaginous rods in place of the nasal capsule. This suggests that the primary cranial defect is restricted to the lateral nasal prominence derivatives. Dil labeling revealed the abnormal migration of crest cells specifically from the anterior midbrain to the lateral nasal prominence in homozygous rSey embryos. Pax-6 was not expressed in the crest cells but was strongly expressed in the frontonasal ectoderm. To determine whether or not this migratory defect actually resides in environmental cues, normal midbrain crest cells from wild-type embryos were labeled with Dil and were orthotopically injected into host rSey embryos. Migration of the donor crest cells into the lateral nasal prominence was abnormal in homozygous host embryos, while they migrated normally in wild-type or heterozygous embryos. Therefore, the cranial defects in rSey homozygous embryos are due to inappropriate substrate for crest cell migration towards the lateral nasal prominence, which consistently explains the cranial morphology of homozygous rSey embryos.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. III. Comparison of shikonin derivatives of cultured cells and ko-shikon

A method for quantitative analysis of shikonin derivatives using high pressure liquid chromatography (HPLC) was established. With this method the composition of shikonin derivatives in cultured cells and roots of Lithospermum erythrorhizon (ko-shikon) was compared. The composition of shikonin derivatives produced by cell suspension cultures was similar to that of the ko-shikon, and the composition in cultured cells was found to fluctuate less than that of the ko-shikon.     

Peripheral specialization for fine analysis of doppler-shifted echoes in the auditory system of the "CF-FM" bat Pteronotus parnellii.

Pteronotus parnellii uses the second harmonic (61-62 kHz) of the CF component in its orientation sounds for Doppler-shift compensation. The bat's inner ear is mechanically specialized for fine analysis of sounds at about 61-62 kHz. Because of this specialization, cochlear microphonics (CM) evoked by 61-62 kHz tone bursts exhibit prominent transients, slow increase and decrease in amplitude at the onset and cessation of these stimuli. CM-responses to 60-61 kHz tone bursts show a prominent input-output non-linearity and transients. Accordingly, a summated response of primary auditory neurones (N1) appears not only at the onset of the stimuli, but also at the cessation. N1-off is sharply tuned at 60-61 kHz, while N1-on is tuned at 63-64 kHz, which is 2 kHz higher than the best frequency of the auditory system because of the envelope-distortion originating from sharp mechanical tuning. Single peripheral neurones sensitive to 61-62 kHz sounds have an unusually sharp tuning curve and show phase-locked responses to beats of up to 3 kHz. Information about the frequencies of Doppler-shifted echoes is thus coded by a set of sharply tuned neurones and also discharges phase-locked to beats. Neurones with a best frequency between 55 and 64 kHz show not only tonic on-responses but also off-responses which are apparently related to the mechanical off-transient occuring in the inner ear and not to a rebound from neural inhibition.     

Changes in CoA pools in hepatic peroxisomes of the rat under various conditions

Changes in peroxisomal CoA pools in the liver of fasted, diabetic, high-fat diet-fed and clofibrate-treated rats were studied. Total-CoA increased slightly in the fasted group and markedly in the diabetic, high-fat and clofibrate-treated groups. Fractionation studies showed that changes in free CoA levels were much greater in peroxisomes than in mitochondria. The concentrations of CoAs were calculated from the contents of CoAs in organelles and the changes in volume of organelles under these conditions; the concentration of total CoA in peroxisomes was higher than that in cytosol, but lower than that in mitochondria. These changes were accompanied by an increase in the activity of peroxisomal beta-oxidation. The results obtained from these experiments indicate that the peroxisomal beta-oxidation system is controlled not only at the enzyme level but also at the substrate or co-factor level.     

Effect of long-term L-dopa administration on the dopaminergic and cholinergic (muscarinic) receptors of striatum in 6-hydroxydopamine lesioned rats

L-Dihydroxyphenylalanine (L-Dopa) (200 mg/kg/day) was administered for 30 days to the rats whose nigrostriatal dopamine pathway was lesioned unilaterally with 6-hydroxydopamine and the receptor binding of ³H-spiperone and ³H-quinuclidinyl benzilate (³HQNB) was measured in the dopaminergic and muscarinic cholinergic receptors of the striatum. ³H-spiperone binding increased by 73% and ³HQNB binding decreased by 14% in the lesioned side when compared to the control side of L-Dopa-non-treated rats. ³H-spiperone binding was measured in the lesioned sides of L-Dopa-treated and L-Dopa-non-treated rats and was found to have decreased by 21% in the former. In the control side of the L-Dopa-treated lesioned rats, however, ³H-spiperone binding increased by 27% when compared to the opposite striatum of the same rats. ³HQNB binding in the lesioned side of L-Dopa-treated rats was not significantly different from that of the control side statistically. These results suggest that changes in functional equilibrium between the dopaminergic and cholinergic mechanisms influence the muscarinic cholinergic receptors and that supersensitivity of dopamine receptors after lesion of the nigrostriatal pathway also remains after long-term L-Dopa treatment.