Recombinant human monoclonal antibodies. Basic principles of the immune system transferred to E. coli.

To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction. Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity. For the selection of specific monoclonal antibodies from these libraries, we have developed two E. coli vector systems that facilitate the surface display of an antibody physically linked to its own gene. The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages. Specific antibody genes can therefore be enriched by antigen affinity chromatography. The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein. Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface. This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen. These vectors permit three major principles of the antibody response to be mimicked in E. coli: 1. Generation of a highly complex antibody repertoire; 2. Clonal selection procedures for library screening; and 3. The possibility of increasing a given affinity by repeated rounds of mutation and selection.     

Purification and partial characterization of 200 kDa oncofetal antigen from radiation induced murine lymphocytic lymphoma

• 1.1. A 200 kDa glycoprotein (gp200) oncofetal antigen was purified from solubilized membranes of a radiation-induced murine lymphomcytic lymphoma cell line (XR11-5T), grown in syngeneic RFM mice, by successive gel chromatography of the active fraction on lentil lectin agarose, Q- and S-Sepharose and Superose-12 using an FPLC system.
• 2.2. A murine monoclonal antibody 115, produced by the syngeneic immunization of adult male C57BL/6N mice with 12-day mouse fetal cells, was used in a slot blot antibody assay to follow up the active fractions.
• 3.3. The purified glycoprotein has a pi of 5.4.
• 4.4. Treatment of radiolabeled gp200 with neuraminidase caused a slight reduction in size due to the removal of sialic acid groups and a shift in pI to 6.3.
• 5.5. Treatment of gp200 with different glycosidases shows that gp200 is susceptible to N- and O-glycanase but not to endoglycosaminidase H.
• 6.6. On extraction of gp200 with Triton X-114 it partitions exclusively into the detergent-rich fraction consistent with being an integral membrane protein.

     

Seasonal variation in the biochemical composition of Mytilus viridis at Ratnagiri on the West Coast of India

1. The seasonal variation in the water, protein, fat and glycogen contents of the mussel, Mytilus viridis has been studied for the year March, 1974 to March, 1975.
2. The water level increased during the monsoon season and decreased in summer.
3. The level of protein, fat and glycogen showed correlation with the reproductive cycle of the mussel.
4. The protein level was high when the mussels were mature and dropped during the breeding period.
5. During sex change from male to female in May the protein level remained high whereas during sex change from female to male in October and November it was low.
6. The fat level was high in mature mussels and declined on spawning.
7. The glycogen level was at its peak in immature mussels and low in mature.

     

Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts

• 1.1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri).
• 2.2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots.
• 3.3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions.
• 4.4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

     

The utilisation of d-galactonate and d-2-oxo-3-deoxygalactonate by Escherichia coli K-12

1. Escherichia coli K-12 mutants unable to grow on d-galactonate have been isolated and found to be defective in either galactonate dehydratase, 2-oxo-3-deoxygalactonate 6-phosphate aldolase or devoid of both of these enzymes and of 2-oxo-3-deoxygalactonate kinase.
2. 2-Oxo-3-deoxygalactonate kinase and 2-oxo-3-deoxygalactonate 6-phosphate aldolase are still induced by galactonate in mutants lacking galactonate dehydratase, suggesting that galactonate rather than a catabolic product of galactonate is the inducer of the galactonate catabolic enzymes. Synthesis of the enzymes is subject to glucose catabolite repression.
3. Mutants defective in 2-oxo-3-deoxygalactonate 6-phosphate aldolase accumulate 2-oxo-3-deoxygalactonate 6-phosphate when exposed to galactonate and this compound causes general growth inhibition.
4. Secondary mutants that no longer show this inhibition fail to make 2-oxo-3-deoxygalactonate 6-phosphate due to additional defects in galactonate transport, galactonate dehydratase, 2-oxo-3-deoxygalactonate kinase or a putative promoter mutation that prevents formation of these enzymes.
5. A spontaneous mutant capable of growth on 2-oxo-3-deoxygalactonate has been isolated. It has two genetically distinct mutations. One permits constitutive formation of the galactonate catabolic enzymes and the other allows the uptake of 2-oxo-3-deoxygalactonate. Neither mutation on its own permitted growth on 2-oxo-3-deoxygalactonate.
6. Genes specifying the various galactonate catabolic enzymes have been located at min 81.7 on the E. coli K-12 linkage map and probably constitute an operon. The gene sequence in this region was shown to by: pyrE uhp dgo dnaA.

     

Studies on intestinal protease: Isolation,purification and effect of dietary proteins on alkaline protease activity of the air-breathing fish,Clarias batrachus (Linn.)

1. The effect of dietary protein levels on the proteolytic activity in the intestines of the air-breathing fish, Clarias batrachus (Linn.) has been studied
2. Activity of proteolytic enzymes increased significantly in fishes maintained with a 50% protein diet from those maintained with a 25% protein diet; still higher dietary protein percentage showed no further stimulation of enzyme activity.
3. In a study on the determination of sub-cellular localisation, it has been found that protease activity is more prominent in lysosomes than in other organelles of the cell.
4. A sixty fold purification of alkaline protease from the intestine of Clarias batrachus has been achieved by ion exchange chromatography on DEAE cellulose which has been further checked by polyacrylamide gel electrophoresis.

     

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

Background

Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein.

Results

In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody.

Conclusion

The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.     

Vitellogenesis in the shrimp,Penaeus vannamei: in vitro studies of the isolated hepatopancreas and ovary

• 1.1. Yolk proteins were isolated from ovaries of the shrimp Penaeus vannamei and used as an antigen for antibody production in rabbits.
• 2.2. Protein synthesis was measured for both the hepatopancreas and the ovary in vitro, and proteins present in both tissues were immunoreactive with the antibodies.
• 3.3. Extracts of shrimp eyestalks inhibited in vitro protein synthesis by both tissues. The inhibitory factor from the eyestalks was heat stable and had a molecular weight of 3300 daltons.

     

A study on the rate of water transport of the clam katelysia opima in relation to environmental conditions

1. The neutral red technique was employed to study the rate of filtration in Katelysia opima.
2. The weight specific water filtration was found to be greater for younger clams compared to the older ones.
3. The rate of water filtration increased with decreasing salinity.
4. Water filtration was found to increase as temperature increased, reaching a maximum at 35°C. but then sharply decreasing at 39°C.
5. Light had no significant effect on the rate of filtration.
6. Suspended matter was found to affect the rate of water filtration.
7. The rate of filtration was low at high pH and high in low pH.
8. The rate of water filtration was found to be faster during high tide than during low tide.
9. The presence of the parasitic crab, Pennotheris sp., in the mantle cavity of clams had a marked effect on the particle filtration.
10. Accidental cut of the siphon tips had no effect on the rate of filtration.

     

Seasonal variations in biochemical constituents of the clam,Paphia laterisulca

1. The total protein, fat and glycogen contents were estimated from the edible clam, P. laterisulca. Seasonal variations in these constituents along with the water content were studied.
2. The gonad index in P. laterisulca was found to increase during the ripe condition and in winter (December–January) and decrease on spawning.
3. A relatively high water content was obtained during monsoon (June to September). This might be due to the loss of salts and gain of water in low salinities.
4. Protein content varied with the reproductive cycle of the clam. The level reached its peak in the mature stage and declined on spawning. Immature clams showed less protein content than gravid ones.
5. Lipid content started to increase as the gametogenesis commenced, reached its peak in fully mature condition (August) and sharply declined due to the shedding of gametes during spawning.
6. Glycogen content was high during the period of active gameto-genesis (May–June). A sharp decrease took place when the clams were fully ripe (July). The glycogen might have been utilized in the formation of active ripe gametes.
7. After starvation for twelve days, total protein and fat contents remained constant, while glycogen content decreased by 66.82%. The water content increased by 4.67%.
8. Seasonal variation in the organic constituents are discussed in relation to the reproductive cycle of the clam.

     

Growth of a floating aquatic weed,Salvinia under standard conditions

1. Growth of the floating aquatic weed, Salvinia, in sterile culture was exponential for at least 2 weeks under standardized conditions.
2. Increase in light intensity or in CO₂ resulted in increases in growth rate, but did not extend the exponential period of growth.
3. This aquatic plant, like many others, discriminates against calcium relative to strontium.
4. In culture Salvinia exhibited luxury consumption of N and P.
5. Because of high C/N ratios, Salvinia may not be a favorable source of animal food, but might be useful in nutrient removal schemes.
6. In sterile culture, S. molesta produced fewer leaves than S. minima, but maintained a significant increase in leaf area and dry weight. This may be correlated with the ability of the first species to rapidly spread over tropical waterways.

     

The anaerobic metabolism of malate of Saccharomyces bailii and the partial purification and characterization of malic enzyme

1. The main pathway of the anaerobic metabolism of l-malate in Saccharomyces bailii is catalyzed by a l-malic enzyme.
2. The enzyme was purified more than 300-fold. During the purification procedure fumarase and pyruvate decarboxylase were removed completely, and malate dehydrogenase and oxalacetate decarboxylase were removed to a very large extent.
3. Manganese ions are not required for the reaction of malic enzyme of Saccharomyces bailii, but the activity of the enzyme is increased by manganese.
4. The reaction of l-malic enzyme proceeds with the coenzymes NAD and (to a lesser extent) NADP.
5. The K _m-values of the malic enzyme of Saccharomyces bailii were 10 mM for l-malate and 0.1 mM for NAD.
6. A model based on the activity and substrate affinity of malic enzyme, the intracellular concentration of malate and phosphate, and its action on fumarase, is proposed to explain the complete anaerobic degradation of malate in Saccharomyces bailii as compared with the partial decomposition of malate in Saccharomyces cerevisiae.

     

Diurnal variation in Sagar Lake,Sagar (India) I studies in the deep water area

1. A diurnal study of inland fresh water lake has been made with respect to physical and chemical properties and the plankton.
2. Chlorides have followed the total carbonates while dissolved oxygen and pH have shown no relation.
3. Microcystis has followed no definite pattern of diurnal movement.
4. All crustaceans, some of the rotifers andTrachelomonas perform considerable diurnal movement in the course of a twenty four hour period.

     

Effet des androgènes sur l’activité sportive

Androgenic hormones seem to be of beneficial effects on sports performance:

- they increase motivation, will, aggressiveness, resistance to the stress and to the fatigue, leading to an increase of the training quantity,

- they increase bone mineralization and probably mechanical resistance,

- they stimulate the bone marrow and so, with the erythropoietin, the erythropoiesis,

- they increase the tendancy to hyperglycemia, but with a decrease of the tolerance to the glucose,

- they stimulate the fatty acids mobilization from the adipose tissue, for their utilization in the muscle during the exercise,

- they participate, for the trained sportmen, to a better gestion of the muscle glycogen storage: their utilization during exercise is decreased,

- they increase the lean body mass, with an increase of the protein synthesis and a decrease of the protein catabolism, leading also to an increase of the muscle force under training. There is no beneficial effect upon the tendons,

- they have an immunomodulation action.

     

Effects of Environmental Perturbations on Short-Term Phytoplankton Production off Lawson's Bay,A Tropical Coastal Embayment

1. The phytoplankton cycle off Lawson's Bay, Waltair follows a bimodal pattern with a major peak during March–May; a minor peak during October–November months and with a low production during the summer months i.e., June–August.
2. During the summer months of 1957, 1958, 1960 and 1962 dumping of dredged spoil from the entrance channel of the harbour into the sea resulted in a natural enrichment of waters.
3. Following this enrichment, there was a qualitative and quantitative increase in the phytoplankters thus leading to the development of a bloom.
4. Only Thalassiosira subtilis and Chaetoceros curvisetus commonly bloomed during the four years.
5. The increase in gross production which varied from 3–13 fold and the high photosynthesis-respiration ratios 5.1 to 10.5 indicated that the bloom populations were in a healthy state.
6. The decrease of the populations to the initial levels suggests that some unknown factor, other than those investigated must have been operating.
7. Consequences of eutrophication of different origins on stimulation of phytoplankton production are briefly discussed.

     

Detection of 180 kDa proteins in electroplax sodium channel preparations

• 1.1. Co-isolating proteins (M_r 170,000–220,000) from sodium channel preparations made from the electric organ of the electric eel (Electrophorus electricus) were detected on Western blots using monoclonal a antibodies.
• 2.2. Similar protein patterns were seen on immunoblots containing immunoprecipitated protein from eel muscle and brain tissues but not heart.
• 3.3. These co-isolating proteins could be separated from the mature TTX-sensitive channel protein (M_r 280,000) using a lentil lectin-Sepharose column.
• 4.4. The 180 kDa proteins do not appear to be channel-related and can be detected as contaminants in electroplax sodium channel preparations using the monoclonal antibodies described here.

     

Subcellular distribution of enzymes involved in α-glucan utilization in Klebsiella pneumoniae

The double-isotopic labelling technique was used to identify comprehensively proteins involved in α-glucan catabolism in Klebsiella pneumoniae NCTC 9633. Cells were grown with either glycerol in the presence of ³H-leucine or with glycerol plus maltose in the presence of ¹⁴C-leucine. Each labelled culture was then fractionated into the main subcellular components, i.e. the cytoplasm, periplasm, cytoplasmic and outer membrane. Corresponding fractions derived from ³H-labelled and ¹⁴C-labelled cells were combined, and the proteins were analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Gel slices were then counted for ³H- and ¹⁴C-radioactivity, a positive deviation from the standard ¹⁴C/³H ratio being evidence for the presence of a protein specifically induced by maltose in the culture medium. The protein pattern thus obtained was compared with the properties of proteins comprising a similar pathway for maltodextrin utilization in Escherichia coli K-12. Ample information which has been obtained mainly by genetic analysis is available about maltodextrin-utilizing enzymes in E. coli K-12.

1. Cytoplasm. Neither amylomaltase nor maltodextrin phosphorylase, well-known soluble enzymes, were identifiable by the double-labelling technique, presumably because these enzymes constitute only a very minor portion of all soluble proteins in the cytoplasm.
2. Periplasm. A prominent protein with a mass of 43000 daltons (43 kD) was found similar to the maltose-binding protein of E. coli K-12 (44 kD).
3. Cytoplasmic membrane. At least 2 proteins with a mass between 40 and 50 kD were detected, minor proteins were seen at ≈ 15 and ≈ 20 kD. One or 2 of the proteins may function as a permease catalyzing the active transport of maltodextrins.
4. Outer membrane. The major protein had a mass of 55 kD, other proteins were found with ≈ 18, ≈48, and ≈140 kD. The major protein may have the same function as the maltodextrin pore protein in E. coli K-12 (55 kD), because K. pneumoniae could grow on 10 μM maltose at practically the same rate as on 10 mM maltose. The 140 kD protein is pullulanase.

     

An ecological study of the Ponggol Estuary in Singapore

1. An ecological study of the Ponggol Estuary was conducted from July 1965 to June 1966 and the seasonal data on physical, chemical and biological characteristics were presented.
2. The Ponggol River represents a short, narrow and shallow estuary in Singapore. The river mouth is open throughout the year and water from eastern Johore Straits drains in twice a day at high tide. The upper reach, however, is left exposed at low tide.
3. The Ponggol River was classified as a vertically and laterally homogeneous estuary and was found to exhibit a mesohaline to polyhaline environment.
4. Significant hydrological gradients from the river mouth 10 the upper reach were noted in the river system. Salinity, dissolved oxygen and pH increased towards the mouth of the river and other parameters such as nutrients, dissolved organic matter and turbidity increased towards the source.
5. Although the river received organic pollutants at the upper reach the estuary was able to discharge them fairly rapidly through regular flushing by the tides. The transient rise of organic matter did not appear to impart any serious affect on the biota in the estuary.
6. Over 98% of the phytoplankton consisted of diatoms, most of which were brought into the estuary from eastern Johore Straits. Freshwater forms were relatively few.
7. Phytoplankton biomass was considerably higher than the adjoining waters. and was reduced at the upper reach due to high turbidity of the water.
8. 80% of the zooplankton was composed of dinoflagellates,Difflugia, copepods and bivalve larvae dominating at all sections of the estuary.
9. Percentage composition of the zooplankton showed that dinoflagellates and copepod nauplii predominated at high tide whileDifflugia and bivalve larvae were abundant at low tide.
10. Zooplankton standing crop, in general, was higher towards the source at high tide but the reverse was found at low tide, i.e. standing crop increased towards the river mouth. This was attributed to the process of concentration.
11. Species composition of zooplankton was found to be more or less similar to that of the eastern Johore Straits.
12. The nekton consisted predominantly of small and juvenile fish. Close correlation of fish and copepods was found to be statistically valid and it was concluded that the fish entered the estuary to feed rather than to spawn.
13. The squids formed an important catch of the beach seine unit and were caught throughout the year.
14. The fish population could be grouped into four categories: estuarine components, euryhaline components, marine components and migratory components.
15. Benthic invertebrates were abundant. Commercially important species consisted of prawns,Metapenaeus andPenaeus, and crabs,Neptunus pelagicus andScylla serrata.
16. The river bed was inhabited predominantly by molluscs and the distribution resembled that of the sheltered shore of muddy-sand type.

     

Effects of the discharge of thermal effluent from a power station on Lake Wabamun,Alberta, Canada — The epipelic and epipsamic algal communities

1. The epipelic algal standing crops were increased by the discharge of thermal effluent into Lake Wabamun, particularly in the discharge canal at station (03–04) and 05.
2. The increase in the standing crop size of the epipelon was due to Oscillatoria amoena and O. borneti in the heated area, while the discharge canal provided the inoculum of the algae for the heated area of the lake.
3. At station (03–04) the increased standing crop size was also a function of increased light penetration to the sediment due to the heated effluent keeping an area of the lake free of ice during the winter.
4. The species composition of the diatoms was similar at all stations except in the discharge canal where there was a reduction in the number of diatom species.
5. Navicula cuspidate developed best in the discharge canal in the summer where water temperatures of 31°C were recorded.
6. Amphora ovalis var. pediculus was the dominant diatom species during the winter under ice-cover.
7. The heated effluent had no effect upon the standing crop or species composition of the epipsammon.
8. Results obtained from the sediment core study showed that the shallow littoral zone of the lake is very disturbed due to wind-induced wave action.