Search

Many years ago, Engel pointed out that grief fulfills all the requirements of a disease process. Despite the current interest in grief, most studies have concentrated on the psychological results of grief and the dysfunction associated with it; the physiological consequences have been largely ignored. The persistent reports of infection and neoplasia following shortly after a grief experience, makes it mandatory that the relationship between grief as a stress mechanism and its consequences via the pituitary-adrenal axis on the depression of the immune response, be explored on a physiological level.

The branching isozymes of the red alga, Rhodymenia pertusa are of two types: Q , which can branch, via the synthesis of α-1,6-glucosyl linkages, linear amyloses to amylopectin; and b.e. , which can further branch the amylopectin formed to the more highly-branched floridean starch. Using the technique of tandem crossed-immunoelectrophoresis, it is shown that the Q branching isozyme of the red alga is more closely related to the b.e. type of branching isozymes of Anacystis nidulans and Cyanidium caldarium than it is to the exclusively Q types of branching isozymes found in Chlorella pyrenoidosa and other chlorophytes. The possibility of a biphyletic evolution of the red and the green algae from blue-green ancestral forms is discussed.

Extracts of the Cyanophyte, Oscillatoria princeps, when subjected to electrophoresis on polyacrylamide gels, normally show two phosphorylase isozymes. One of these has been shown to be a glycoprotein and can initiate polyglucan synthesis in the absence of exogenous primer (a2). The other isozyme, a1, is primer-dependent. The a2 isozyme can be converted to the a1 isozyme by digestion with alpha-amylase. By incubation with uridine 5′-diphosphoglucose (UDPG) in the presence of original extract, the a1 isozyme has now been converted to the a2, primer-independent isozyme. The transfer of glucosyl residues from the sugar nucleotide is probably mediated by a transcosylase in the extract, and may be the first step in the initiation of polyglucoside synthesis in the alga.

The branching isozymes (E.C. 2.4.1.18) of Cyanidium caldarium , isolated by polyacrylamide gel electrophoresis, showed important differences when compared with those of the chlorophyte, Chlorella pyrenoidosa , and great similarities with those of the cyanophyte, Anacystis nidulans : 1. 1.|The branching isozymes of Cyanidium and Anacystis were relatively inactive at temperatures below 20°C.; optimum formation of alpha-1,6-glucosyl linkages was at temperatures above 40°C. 2. 2.|The branching isozymes of Chlorella showed optimum activity at low temperatures and were inactive at temperatures above 40°C. 3. 3.|When the branching isozymes of Cyanidium were tested in incubation mixtures containing phosphorylase (E.C. 2.4.1.1) at 42°C, branching of the phosphorylase-formed alpha-1,4-glucan was immediate when the chain-length reached 5–6 residues; those of Chlorella required a minimum chain-legth of about 40 residues before active branching occurred.

Can the hospice “experience” alter the physiological (and particularly, the immunological) and psychological changes that occur among the bereaved following the death of a loved one? Does t...

The primer-dependent phosphorylase isozyme, a1, of algae has been covalently coupled to certain glucose polymers (glucose through maltoheptaose) by means of the controlled substitution of the halogens on 2,4,6- trichloro -s- triazine . The synthetic glycoproteins resulting from this procedure were active on glucose-1-phosphate, forming linear maltoglucosides without the need for exogenous primer. The “synthetic” enzymes behaved like the naturally occurring a2 phosphorylase isozyme, which had been shown to initiate glucan synthesis without added primer, and which was a glycoprotein. The synthetic enzymes also showed similar mobilities with the naturally occurring a2 isozyme when subjected to polyacrylamide gel electrophoresis. It was found that the minimum chain length of the covalently bonded glucan moiety necessary for initiation of polyglucan formation without added primer, was greater than five glucose residues. Bonded maltossacharides containing less than five glucose residues required primer maltosccharides before glucan synthesis could be started. It is suggested that these are the minimum chain lengths bonded to the a1 phosphorylase isozyme protein in vivo which are probably active in initiating primerless polyglucoside synthesis in algae.

The effect of acute grief on the pituitary-adrenal axis and the hypersecretion of cortisol is reviewed. Particular attention is devoted to the immunosuppressive effects of this hypersecretion. The increased susceptibility to infectious disease agents—bacterial, fungal and viral—is explored in terms of the altered biochemistry of the bereaved individual. The probable increased susceptibility towards oncogenic “passenger” viruses, such as the Epstein-Barr Virus and the Herpes Virus, is discussed as a possible mechanism for the increased incidence of malignancies in the bereaved. The use of dexamethasone in preventing hypersecretion of cortisol in the bereaved, as well as the use of L-dopa and clonidine to control A.C.T.H. levels in such individuals, has given rise to much conflicting data in recent studies. The use of antibiotics in a prophylactic sense, to bolster falling immunity during the grief period, remains a distinct possibility. However, until the basic reactions of grief are defined and the altered biochemistry established, it appears that psychological methods offer the best therapeutic means.

The first step in the metabolism of the phytotoxic compound, 3-amino-1,2,4-triazole in yeast is its linking with glucose to form an amine glucoside. This compound may then be phosphorylated by hexokinase and split by yeast aldolase. Isolation of the end-product of this metabolism has made possible the delineation of its physico-chemical properties and an interpretation of its molecular structure via infrared studies.

The increase in mortality reported in individuals undergoing the stress imposed by the grief situation, may be directly traced to the overstimulation of the pituitary resulting in the production of excess quantities of adrenocorticotropic hormone (ACTH), which in turn causes increased levels of corticosteroids to be produced by the adrenal cortex. The physiologic affects of these substances include the suppression of the inflammatory response. Since the inflammatory response is essential to the immune mechanism of the individual, the bereaved becomes more susceptible to common bacterial, fungal and viral pathogens. Indeed, because of the suppression of this response, the grief-stricken person may fall prey to overwhelming infections which are caused by “opportunistic” micro-organisms. It seems possible that the stress imposed by grief may be at the basis for the increase in death rates of individuals who are bereaved.

Purified phosphorylase preparations from representative members of three algal Groups were subjected to two-dimensional electrophoresis on polyacrylamide gel. Three groups of closely associated isozymes were found in each preparation. Two isozymes of the conventional phosphorylase type were present in preparations from blue-green and from green algae; only a single enzyme appeared in red algal preparations. Two isozymes of the glucosyltransferase type, utilizing either UDPG or ADPG as substrate, were present in all three groups of algae. Each preparation examined by this orthacryl gel technique also showed the presence of two or three isozymes capable of “branching” linear polyglucosides.

Algal phosphorylases require pyridoxal (vitamin B6) for the biosynthesis of active protein. When the B6 antimetabolite, 5-hydroxy-2-(hydroxymethyl)-4H-pyridone was added to cultures of Oscillatoria princeps, Chlorella pyrenoidosa and Cyanidium caldarium, the phosphorylase isozymes formed were devoid of enzyme activity. Activity was not restored after incubation of the proteins with pyridoxal. However, when the algae were cultured in the presence of the antimetabolite and vitamin B6, the isozymes were fully active. It appears that there is an absolute requirement for pyridoxal in the biosynthesis of algal phosphorylases, and that this requirement is more basic and probably occurred much earlier in the evolution of 4-glucosyltransferases than the requirement for adenosine monophosphate.

1. 1. The inhibitory effect of 3-AT on Oscillatoria princeps phosphorylase is probably due to the chelation by 3-AT of the essential metal required by this enzyme. 2. 2. The inhibition of this phosphorylase by 3-AT can be effectively reversed by the addition of manganous or ferric ions only in the proper concentrations for forming the bivalent and trivalent chelates. 3. 3. Evidence is presented for the 3:1 structure of the ferric chelate of 3-AT.

Both Oscillatoria princeps and Cyanidium caldarium contain phosphorylase isozymes that can cause the synthesis of polyglucan from glucose-1-phosphate in the absence of added maltodextrin ‘primer’. In addition, O. princeps contains a primer-dependent phosphorylase isozyme. When the phosphorylase fractions isolated from extracts of the algae were treated with α-amylase, the primer-independent isozyme became primer-dependent and shifted from the position it was normally found at after polyacrylamide gel electrophoresis. This primer-independent isozyme became less mobile towards the anode, and was found at the locus usually occupied by the primer-dependent isozyme. It was not possible to restore its mobility towards the anode and its primer-independent properties by preincubation with maltoheptaose. The indication is that this isozyme is a glucoprotein and that the glucan component is chemically bonded to the protein.

Three isozymes specifically concerned with the “branching” of linear polyglucosides have been delected in algae. These enzymes were detected using two-dimensional polyacrylamide gel electrophoresis, and were found to be present in blue-green, red and in green algae. Two isozynies were found in Oscillatoria princeps; three enzymes were present in Spirogyra setiformis, and two and three such enzymes were detected in red algae of the Rhodymenia type. The significance of the multiple forms of this branching enzyme was assessed in light of the type of storage poly-glucosides formed by these plants. The “degree of branching” of the storage sugar appeared to be related to the evolutionary status of these algae.

The branching isozymes of a Cyanophyte, Oscillatoria princeps , a Rhodophyte, Rhodymenia pertusa and a Chlorophyte, Spirogyra setiformis , when isolated by two-dimensional polyacrylamide gel electrophoresis, show an evolutionary progression. The isozymes were tested with various polyglucan substrates. Those of Oscillatoria were able to branch amylopectin and amylose, forming a phytoglycogenlike polymer which exhibited maximum absorption of its iodine complex at 550 nm. One of the three isozymes of Rhodymenia synthesized the identical polysaccharide, but the other two isozymes of this alga were without effect on amylopectin. They did branch amylose, however, forming a polyglucan which showed an absorption peak of its iodine complex at 580 nm. The green alga, Spirogyra , had three isozymes which were without effect on amylopectin, but which did branch amylose. The branched sugar formed by these algal enzymes showed an absorption peak of the iodine complex at 580 nm.

Biosynthesis of the slower anodic-moving phosphorylase isozyme of the blue-green alga, Oscillatoria princeps can be completely suppressed by the herbicide, amitrole. The compound is without effect on the faster anodic-moving isozyme. It has been found that amitrole also suppresses the formation of the isozyme in Chlorella pyrenoidosa, while having no effect on the faster isozyme of this alga. In Cyanidium caldarium, amitrole likewise does not affect the biosynthesis of the faster anodic-moving isozyme. The anomalous behaviour of the faster isozyme of Chlorella in utilizing both glucose-1-phosphate and ADPG is similar to that previously reported in the other green alga, Spirogyra. It may represent a transformation of phosphorylase into a synthetase type enzyme.

Chemical tests and infrared absorption spectra indicate that the d -glucose adduct of 3-amino-1,2,4-triazole is a definite chemical compound, an amine glucoside or secondary amine. The participation of this amine glucoside in phosphorylating reactions is discussed with respect to the observed effects on carbohydrate-metabolizing enzymes such as hexokinase and phosphorylase.

1. 1. The stability constant, log K , for the manganese chelate of 3-amino-1,2,4-tri-azole (3-AT) has been determined via ion-exchange methods to be 2.3–2.4. 2. 2. In light of the fact that this log K is ten times smaller than that of the enzyme, it is seriously doubted that the inhibition of Oscillatoria phosphorylase by 3-AT can be explained by chelation phenomenon. 3. 3. The possibility is advanced that a glucose complex formed by the interaction of 3-AT and glucose 1-phosphate causes a competitive type of inhibition of this enzyme.

Cyanidium caldarium is an enigmatic eukaryotic alga which is both acidophilic and thermophilic. Its taxonomic position has been in doubt and, hence

Cyanidioschyzon merolae , a primitive eukaryotic alga isolated from supposedly pure cultures of the thermoacidophilic alga, Cyanidium caldarium , has many of the characteristics of such prokaryotes as bacteria and the cyanobacteria. Cyanidioschyzon appears to have even more of these prokaryotic features than does Cyanidium . Cyanidioschyzon divides by binary fission as do most bacteria. Its thylakoids are arranged along the periphery of the cell, like the cyanobacteria. Its formation of storage glucan, as well as the type of sugar formed is more like that of the blue-green algae rather than that of the red algae. Cyanidioschyzon merolae may be much more primitive than Cyanidium caldarium , and could be the most primitive eukaryotic cell.

The storage glucans of Chlorella pyrenoidosa and Prototheca zopfii are identical and consist of a linear polyglucan akin to amylose and a branched amylopectin component. The branched glucans of these algae differ markedly from that formed by the hot-springs alga, Cyanidium caldarium. The more highly branched Cyanidium glucan appears to be formed by branching glucosyltransferases which are different from those of the other two algae. The relevance of the data to the possibility of Cyanidium being a Prototheca-like Chlorella that has acquired symbiotic Cyanobacteria as chloroplasts is discussed.

Gel electrophoresis of the glucosyltransferases of the blue-green alga, Oscillatoria princeps , followed by immunodiffusion against anti-phosphorylase rabbit serum, showed cross-reactions of the two phosphorylase isozymes and the two synthetase isozymes of the alga. Weak cross-reactions were also obtained with the branching isozymes. Apparent extensive similarities in the structure of the phosphorylases and the synthetases were indicated. However, only partial structural similarities between the two groups of α-1,4-glucosidic bond formers and the branching isozymes exist as indicated by the weak immunological reactions obtained and the formation of “spurs” on the immunoprecipitin lines. If the synthesis of α-1,4-glucosidic linkages and the formation of α-1,6 cross linkages were at one time due to the bifunctional action of a single catalytic protein, then the separation of these two enzymatic activities took place prior to the derivation of the synthetases from the phosphorylases.

Plectonema nostocorum , a thermophilic cyanophyte which lives under alkaline conditions at pHs approaching 13, forms a storage glucan showing a maximum absorption of its iodine complex almost identical with that of another thermophilic cyanophyte, Oscillatoria princeps , which exists at a more neutral pH, and with that of the acidophilic thermophile, Cyanidium caldarium . Gel electrophoretic patterns of the storage glucan-forming isozymes of Plectonema do not differ essentially from those of Oscillatoria . The a 2 phosphorylase isozyme appears to be primer-independent, and resembles the a 2 isozymes of both Oscillatoria and Cyanidium . The isozymes responsible for forming α-1,6-glucosidic branched linkages in Plectonema are of the b.e. type (able to further branch amylopectin), rather than of the Q type (able to branch amylose only to amylopectin).

Prochloron , a symbiont found associated with Lissoclinum patella (a marine colonial ascidian), was lyophilized and its glucans extracted. The glucans were complexed with s -triazine reactive dyes and separated by electrophoresis on cellulose acetate membranes. A highly branched glucan similar to phytoglycogen, and a linear unbranched glucan resembling a short-chain amylose were both detected. This unusual polysaccharide mixture suggests a possible mode of starch biosynthesis in algae in general.

The cyanelles of Glaucocystis nostochinearum were isolated after disruption of the algal cells by sonication. The aqueous extracts from these cyanelles were subjected to molecular filtration and electrophoresis on polyacrylamide gels. By comparison with extracts of a unicellular Chroococcalian alga, Anacystis nidulans treated in the same way only about half the number of protein bands were found. The proteins were water-soluble with a MW in excess of 10 000. Three protein-pigment complexes were detected in Anacystis. Two of these (phycoerythrin and phycocyanin) were not present in the cyanelles of Glaucocystis. Three branching glucosyltransferase isozymes capable of converting amylopectin to phytoglycogen were present in the Cyanophyte; only two branching isozymes with typical Chlorophycean ‘Q’ activity were present in the cyanelles of Glaucocystis. It seems improbable that the cyanelles of this alga are endosymbiotic blue-green algae; rather, they may represent some intermediate stage in the development of the chloroplast of green algae.

The α2 phosphorylase isoenzyme of Oscillatoria princeps, Rhodymenia pertusa and Chlorella pyrcnoidosa has been found to be capable of the synthesis of amylose-like polyglucans without the necessity for addition of primers. This enzyme differs in this respect from the a1 isoenzyme of blue-green and green algae. The a2 enzyme appears to be a glyco-protein and synthesis of linear polyglucans appears to occur by the apposition of glucosyl residues from glucose-1-phos-phate directly to the glycosyl moiety of its molecule.