The inhibition of pepsin-catalysed reactions by products and product analogues. Kinetic evidence for ordered release of products

1. The inhibition of pepsin-catalysed hydrolysis of N-acetyl-l-phenylalanyl-l-phenylalanylglycine by products and product analogues was studied. 2. The non-competitive nature of the inhibition by the product N-acetyl-l-phenylalanine confirms an ordered release of products, and points to a common mechanism (involving an amino-enzyme) for pepsin-catalysed transpeptidation and hydrolysis reactions. 3. N-Acetyl-l-phenylalanine ethyl ester is also a non-competitive inhibitor, but here the inhibition is of the ;dead-end' type. No ethanol is detectable in reaction mixtures, indicating that this ester cannot act as an amino group acceptor in a transpeptidation process. 4. The same is true for N-methanesulphonyl-l-phenylalanine methyl and methyl thiol esters. No methanethiol is liberated when the methyl thiol ester is present as an inhibitor of the hydrolytic reaction, and the hope that such a thiol ester would effectively trap the amino-enzyme was not fulfilled.     

Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy

The family of a male with Duchenne muscular dystrophy (DMD) and a deletion within the dystrophin gene has been studied. Polymerase chain reaction analysis of ectopic mRNA from peripheral blood T+B lymphocytes and the use of (CA) _n repeat polymorphisms in and around the deleted region showed the proband's mother to be both a germline mosaic and a somatic mosaic for the deletion seen in her son. The mutation therefore occurred as a mitotic event early in embryogenesis.     

Crown alterations induced by decline: a study of relationships between growth rate and crown morphology in beech (Fagus sylvatica L.)

One of the first symptoms expressed by declining trees is reduced growth in stem diameter and length increment. The possibility of a relationship between length increment and crown thinning in beech (Fagus sylvatica L.) was investigated by developing a computer model to simulate first order branching patterns of the apical 2 m of monopodially branching beech trees, 70–100 years old, for a range of length increment rates. The model was based on values for branching angle, main axis and branch length increment, number of branches produced per year and branch mortality rates for six healthy and declining trees. Shoot growth rates in the apical 2 m of the sample trees ranged from about 5 cm/year (decline class 3) to 43 cm/ year (healthy). Simulations of branching patterns in the apical 2 m of trees growing at different rates indicated that, when growth rate exceeded about 20 cm/year, total first order branch length and area explored were independent of growth rate. When growth rates fell below this value there was a reduction in total area explored and first order branch length due primarily to the formation of fewer branches. More acute branching angles contributed to a reduction in the area explored. Growth rate-related crown thinning could increase the risk of bark necrosis and secondary pathogen infection during dry and/or hot spells.     

Interleukin-5, interleukin-3, and granulocyte-macrophage colony-stimulating factor cross-compete for binding to cell surface receptors on human eosinophils.

Human interleukin (IL)-5 receptors were characterized by means of binding studies using bioactive 125I-labeled IL-5. Of purified primary myeloid cells, eosinophils and basophils but not neutrophils or monocytes expressed surface receptors for IL-5. Binding studies showed that eosinophils expressed a single class of high affinity receptors (Ka = 1.2 x 10(10) M-1) with the number of receptors being small (less than 1000 receptors/cell) and varying between individuals. Among several cell lines examined only HL-60 cells showed detectable IL-5 receptors which were small in numbers (200 receptors/cell) and also bound 125I-IL-5 with high affinity. The binding of IL-5 was rapid at 37 degrees C while requiring several hours to reach equilibrium at 4 degrees C. Specificity studies revealed that the two other human eosinophilopoietic cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited the binding of 125I-IL-5 to eosinophils. No competition was observed by other eosinophil activating or nonactivating cytokines. The inhibition of 125I-IL-5 binding by IL-3 and GM-CSF was partial up to a concentration of competitor of 10(-7) M with GM-CSF consistently being the stronger competitor. Converse experiments using IL-5 as a competitor revealed that this cytokine inhibited the binding of 125I-IL-3 and of 125I-GM-CSF in some but not all the individuals tested, perhaps reflecting eosinophil heterogeneity in vivo. Cross-linking experiments on HL-60 cells demonstrated two IL-5-containing complexes of Mr 150,000 and Mr 80,000 both of which were inhibited by GM-CSF. The competition between IL-5, IL-3, and GM-CSF on the surface of mature eosinophils may represent a unifying mechanism that may help explain the common biological effects of these three eosinophilopoietic cytokines on eosinophil function. This unique pattern of competition may also be beneficial to the host by preventing excessive eosinophil stimulation.     

Evidence for a cerebral cholinergic system and suggested pharmacological patterns of neural organization in the prostomium of the polychaete Nereis virens (SARS)

The histological visualization of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) on frozen sections of prostomia of Nereis virens indicate a concentration of cholinergic activity in the anterior brain. Components are probably sensory epithelial cells with cholinergic axons entering the brain in cephalic nerves and efferent cholinergic axons to prostomial muscle leaving the brain in the same nerves. There are also subepidermal cholinergic cells that may be second order motor neurons serving epidermal mucous cells. The smaller, second lobe of the corpora pedunculata and its associated vertical fibre tract are CAT⁴ and appear continuous, on each side of the cerebral ganglion, with a dorsal and a ventral longitudinal bundle of AChE⁺ fibers. This system tapers to nothing at the level of the posterior eyes. There is a small AChE⁺ component to each optic nerve and AChE is present in the nuchal epithelium. These observations are discussed in relation to earlier studies on aminergic and neurosecretory activity in the same ganglion.     

Aberrant double replication of segments of chromosomal DNA following DNA synthesis inhibition by cytosine arabinoside.

Using two different cell lines growing logarithmically, it is demonstrated that inhibition of DNA synthesis by cytosine arabinoside leads to a disruption of the sequence of replication of the chromosomal DNA. After release of the inhibition, some DNA synthesis is reinitiated in DNA segments replicated earlier in that S phase, leading to double replication of some DNA segments. This was directly demonstrated by showing that, following removal of the inhibitor, DNA was synthesized off template strands which had themselves been synthesized only 1–2 h before the addition of the inhibitor.     

The human Ia system: Definition and characterization by xenogeneic antisera

The production of xenogeneic anti-Ia serum against Ia antigens in serum has been previously described in the mouse and we now describe the production of xenogeneic anti-human Ia antisera using similar methods. With an indirect resetting technique, Ia-like antibodies were shown to react with the majority of B cells (95%), a subpopulation of T cells, with carbonyl iron adherent cells, and with some E^–Ig^– null cells, but there was no reaction with red cells and platelets. These reactions were the same as those obtained with DRW antisera using cytotoxicity testing. In addition, antigens detected with xenogeneic antisera were also found in serum, where they were found to exist in a low molecular weight, dialyzable form. By the selective removal of different cell surface markers by cocapping, no association could be found with the specifities detected with the xenogeneic anti-Ia antisera and with surface Ig, ₂-microglobulin, or HLA-A and B specificities. Alloantibodies to DRW specificities (but not HLA-A, B specificities) were able to specifically block the binding of the rabbit anti-Ia antibodies to B cells, and reciprocal blocking of rabbit antisera by DRW antibodies was also observed. Several xenogenic antisera were produced by immunizing rabbits with the serum of different individuals. Each antiserum was shown to contain a number of different specificities, as they gave different reaction patterns with different individuals when testing was done both directly and by absorption. These xenogeneic anti-la sera also segregated in a family with HLA-A and B specificities. The detection of a polymorphic antigenic system segregating with the HLA complex, distinct from HLA-A and B specificities, and whose antigens occur predominantly on B cells is therefore described. Because of the similarity of the reactions of the xenogeneic antisera in man to those found in the mouse, and because of the close relationship to the DRW specificities, the system has been provisionally called the H.Ia system.Abbreviations used in this paper AET 2-aminoethyl isothiouronium bromide - ₂-M -2 microglobulin - BSA Bovine serum albumin - H.Ia Human Ia - HuRBC Human red blood cells - Ig Immunoglobulin - Ir Immune response - MHC Major histocompatibility complex - MLR Mixed lymphocyte reaction - NHS Normal human serum - NMS Normal mouse serum - PBL Peripheral blood lymphocytes - PBS Phosphate-buffered saline - RAHIg Rabbit anti-human immunoglobulin - RASIg Rabbit anti-sheep immunoglobulin - RFC Rosette-forming cells - SAHIg Sheep anti-human immunoglobulin - SARIy Sheep anti-rabbit immunoglobulin - SRBC Sheep red blood cells