A small family of nodule specific genes from soybean.

The primary structure of two nodule specific soybean genes are presented. The two genes code for primary products of 20.0 (nodulin 20) and 22.7 (nodulin 22) kdaltons, respectively. Both genes are related to the nodulin 23 and 44 genes. Alignment of the deduced amino acid sequences of all four genes revealed three domains of high homology interrupted by highly diverged regions due to numerous duplication and insertion events. The first conserved domain codes for a putative signal peptide, while the two others each contain four Cys residues that can be arranged in a way reminiscent of the metal binding domains present in some enzymes and in several DNA binding proteins.     

Clinical factors influencing the absorption of 125I-NPH insulin in diabetic patients

Clinical factors which might influence the absorption of subcutaneously injected 125I-NPH insulin were studied in 101 diabetics. The disappearance curve was monoexponential after a delay period of 1.5 +/- 0.8 h (mean +/- SD). Lipohypertrophy significantly prolonged insulin absorption (half life (T1/2) = 11.2 +/- 3.1 h, p = 0.0001). Low bicarbonate levels increased the absorption (T1/2 3.9 +/- 2.3 h, p less than 0.05). Lean diabetics had a faster absorption (6.2 +/- 1.9 h) than normal weight diabetics (7.5 +/- 2.0 h, p less than 0.02). Sex, age, diabetes duration and injection depth did not influence T1/2. The half life was significantly inversely correlated to the resting subcutaneous blood flow (r = 0.882, p less than 0.01). The overall interindividual coefficient of variation for insulin absorption in nonketotic diabetics was 27.4%. Also considerable intra-patient day-to-day variation was found (24.5%), and between different injection sites (30.2%). These variations emphasize the drawbacks of conventional insulin therapy in the management of insulin-requiring diabetics.     

A hydroxyproline-containing class IV chitinase of sugar beet is glycosylated with xylose

Two acidic chitinase isoforms, SP1 and SP2, have been purified to homogeneity from leaves of sugar beet (Beta vulgaris) infected with Cercospora beticola. SP1 and SP2 are extracellular proteins with an apparent molecular mass of 35 kDa and an approximate pI of 4.2. Since the only major difference was slightly diverging M _r's, only the SP2 chitinase was further characterized. Partial amino acid sequence data for SP2 was used to generate a polymerase chain reaction (PCR) clone employed for the isolation of a cDNA clone encoding SP2. SP2 exhibits significant structural identity with the class IV chitinases from sugar beet, rapeseed, bean and maize, but differs from the other members of this class in having a longer hinge region, comprising 22 amino acid residues, with a repeated TTP motif. Western blotting analyses, using antibody raised against SP2, demonstrated an induction of SP protein during infection with C. beticola. The induction was very local, with high protein accumulation found close to the infection site only. Amino acid compositional analysis of SP2 revealed that five out of fourteen prolines are hydroxylated. No glucosamine or galactosamine residues are present. Evidence was obtained that SP2 is glycosylated with a limited number (7) of xylose residues: (1) SP2 was stained with the periodic acid-Schiff (PAS) reagent, (2) electrospray mass spectrometry on SP2 gave a series of M _r's with a consistent increase between two molecular masses of 132 Da, (3) SP2 was recognized by an antibody specific for -1,4-D-xylopyranose. The vacuolar class I chitinases A and B in tobacco have recently been shown to comprise a new class of hydroxyproline-containing proteins (Sticher et al., Science 257 (1992) 655–657). The SP2 chitinase differs from these in being glycosylated and, thus, represents a novel type of hydroxyproline-containing glycoproteins in plants.     

Closed-loop system in evaluation of insulin infusion rates for an open-loop system.

The daily insulin requirements as calculated with a closed-loop system (Biostator), were used for insulin infusion with a portable, miniaturized pump in 6 juvenile-onset, insulin-requiring diabetics. Three diabetics were given insulin in a manner like that of the Biostator, e.g., maximal insulin infusion about 1 hour after start of meals. The MBG was 8.7 +/- 3.9 (SD) mmol/l. Three other diabetics had insulin in a fixed profile with peaks beginning simultaneously with meals. MBG of these patients was 4.4 +/- 1.9 mmol/l. Knowing the daily insulin dosage as calculated from the Biostator, normal blood glucose levels can be achieved with a fixed profile of insulin given by a portable pump.     

A group of α-1,4-glucan lyase genes from the fungi Morchella costata,M. vulgaris and Peziza ostracoderma. Cloning,complete sequencing and heterologous expression

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, -1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121530 and 121971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.     

A portable, Preprogrammable insulin infusion system with 16 rates.

An external, portable, programmable pump has been developed for long-term therapeutic i.v. insusion of drugs. The impulse-controlled system consists of a 250 g CMOS-electronic unit and an electromechanical infusor (200 g) containing a 10 ml disposable syringe. The infusion pattern during the 24-h cycle is manually programmed in 16 steps from 24 to 384 microliter per 0.5 h. The coefficient of variation of the infusate is less than 2% per 0.5 h. An alarm circuit monitors the pump speed and the reservoir volume. The infusion system can operate continuously for 7 days with a 1 Ah, 5.4 volt battery.     

A portable, pre-programmable insulin infusion system in diabetes mellitus.

A miniaturized portable pump has been developed in order to study the effects of pre-programmed insulin infusion patterns on the blood glucose levels in juvenile-onset, insulin-requiring diabetics. Six diabetics undergoing 7 insulin infusion periods of up to 4 days were studied. The mean blood glucose was 7.0 +/- 2.3 (SD) mmol/l and the mean amplitude of glycemic excursions was 5.0 +/- 1.4 mmol/l. Success in achieving normoglycemia will depend on choosing the correct dose of insulin for infusion.     

A group of alpha-1,4-glucan lyases and their genes from the red alga Gracilariopsis lemaneiformis: purification, cloning, and heterologous expression

We present here the first report of a group of alpha-1,4-glucan lyases (EC 4.2.2.13) and their genes. The lyases produce 1, 5-anhydro-D-fructose from starch and related oligomers and polymers. The enzymes were isolated from the red alga Gracilariopsis lemaneiformis from the Pacific coasts of China and USA, and the Atlantic Coast of Venezuela. Three lyase isozymes (GLq1, GLq2 and GLq3) from the Chinese subspecies, two lyase isozymes (GLs1 and GLs2) from the USA subspecies and one lyase (GLa1) from the Venezuelan subspecies were identified and investigated. GLq1, GLq3, GLs1 and GLa1 were purified and partially sequenced. Based on the amino acid sequences obtained, three lyase genes or their cDNAs (GLq1, GLq2 and GLs1) were cloned and completely sequenced and two other genes (GLq3 and GLs2) were partially sequenced. The coding sequences of the lyase genes GLq1, GLq2 and GLs1 are 3267, 3276 and 3279 bp, encoding lyases of 1088, 1091 and 1092 amino acids, respectively. The deduced molecular masses of the mature lyases from the coding sequences are 117030, 117667 and 117790 Da, respectively, close to those determined by mass spectrometry using purified lyases. The amino acid sequence identity is more than 70% among the six algal lyase isozymes. The algal GLq1 gene was expressed in Pichia pastoris and Aspergillus niger, and the expression product was identical to the wild-type enzyme.     

Diurnal variations in physiology and behaviour of the noble crayfish Astacus astacus and the signal crayfish Pacifastacus leniusculus

Heart rate, locomotor activity, and oxygen consumption were recorded simultaneously and continuously in seven individuals of the noble crayfish Astacus astacus (Linneus 1758) and seven individuals of the signal crayfish Pacifastacus leniusculus (Dana 1852). The recordings were made in the laboratory over 7 days at 15°C under a 12?:?12?h dark?:?light regime. Circadian rhythms in heart rate, locomotor activity and oxygen consumption were found both in A. astacus and P. leniusculus. Increased heart rate, locomotor activity, and oxygen consumption levels during night time in both A. astacus and P. leniusculus illustrated expression of nocturnal behaviour. No differences in oxygen consumption levels were observed between A. astacus and P. leniusculus. Also, no significant difference between heart rate levels or heart rate variances was found in A. astacus and P. leniusculus at night. During day, however, heart rate levels, heart rate variances and locomotor activity were higher in P. leniusculus than in A. astacus. The higher activity level in P. leniusculus than in A. astacus during daytime indicates that P. leniusculus is less strictly nocturnal than is A. astacus.