Chapter 3: Results

Section 1: Initial Growth Trend Analysis for Defined Media (CbMM):

Population Growth Trends: In order to determine the general growth patterns of C. elegans in CbMM and HS-YE-HLE media, populations were recorded in a set of 10 tubes over 24 days. The results (Figures 2 & 3) illustrate the standard population growth in a defined and an undefined culture medium. An assay to determine the most practical and accurate counting method was also constructed and followed. The laggards, cultures in which no growth was present, were removed from calculations. The population in CbMM both increased and decreased over the 24 day period (Figure 2).

Figure 2. Population Growth Trend

The average population growth of C. elegans cultures in CbMM. Ten replicates were observed.

Population Growth Rate in CbMM: The rates of nematode population growth in the cultures described above were examined. Special interest was given to the peaks of population production and the time at which they occur. The observed rates illustrate two time periods in which nematode population increases were maximized. The maximum rate of nematode production was 8 nematodes per day at day 7 and 9 nematodes per day at day 13. There was a minor peak of 2 nematodes per day at day 16. After day 16 the rate declined to a net loss of 4 nematodes per day at day 20. After day 20 the rate of production increased through the end of the 24 day recording period with a maximum production of 12 nematodes per day.

Population Growth Trends for An Undefined Stock Media: A set of 6 tubes of HY-YE-HLE were inoculated and one tube was opened immediately and the nematodes were counted to give a series of 7 time points over a 22 day period (Figure 3). Another tube was opened and the population of nematodes was determined on each of the subsequent sampling dates. There was a minor increase in population from day 3 to day 8 for nematodes grown in HS-YE-HLE media. At day 8, the population increased sharply

Figure 3. Population Growth Pattern of C. elegans in Stock HS-YE-HLE Media

until day 10 at which time the population remained constant for a period of 4 days. From day 14 the population increased rapidly from below 4,000 nematodes per ml to above 16,000 nematodes per ml within 8 days. By day 22, there was a 4-fold increase over the population present at day 8.

Counting Method Experiment: A counting method experiment was first performed to determine the most accurate and practical method of population determination with the current culture system. The first method utilized an inverted microscope to observe and count the entire nematode culture through the glass walls of the culture bottles. The second counting method utilized a dissecting microscope and a gridded petri dish. The population of the tube previously counted under the inverted microscope was poured into the petri dish and counted by a direct count of each grid on the dish.

Figure 4. Difference in Accuracy for the Different Counting Methods

Populations determined using two different methods. Dissecting and inverted identify the microscope and method used to determine the population.

A comparison of populations observed using both methods (Figure 4) illustrates a two-fold population difference between the dissecting microscope and the inverted microscope at days 9, 13, and 20.

Section 2: Preliminary Evaluation of the Methodology

In an effort to evaluate the proposed bioassay and improve its methodology, two groups of extracts were used as test substances. Extracts were tested for bioactivity using the previously described methods. The unavailability of a positive control made it necessary to test samples with previously undetermined activity. These extracts, which exhibited bioactivity toward other organisms, were under investigation in Dr. Schubert's laboratory. The initial extracts were provided by Dr. Reimann-Philipp (URP).

Effects of URP extracts Experiment # 1: In the initial test, crude extracts from three different plant species were tested. These included URPHLS, URPHLN and two different preparations of the URPPCM (URPPCM#28 and URPPCM#29). These extracts were added to the CbMM media according to described methods. Each extract was tested in 0.2 ml and 0.4 ml additions with 5 replicates of each concentration. The control was replicated eight times using 0.4 ml water per tube. The results of this experiment are presented in Table 1.

Cultures with added extract were the only cultures with populations lower than the control contained either URPPCM#28 or 0.2 ml of Tris-Cl. The additions of URPPCM#28 or URPPCM#29 to the cultures caused the culture media to gel. This gelling occurred within the first week of the experiment. Increased post-inoculation mortality was observed in these tubes. The gel was mildly persistent with URPPCM#28 but did not persist, after shaking the culture tubes, with URPPCM#29.

Table 1. Change in Nematode Populations in cultures containing of URP-Extracts Experiment # 1.

Sample

0.2 ml of Sample Used

0.4 ml of Sample Used

 

% Change in Population at Day

% Change in Population at Day

 

7

10

7

10

URPHLN

-94

-318

-119

-401

URPHLS

-271

-549

-115

-287

URPPCM #29

48

31

10

-13

URPPCM #28

-366

-484

21

30

0.1 M TRIS - Cl pH 8.5

26

34

1

-24

Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations were significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the extract.

The rate of population growth for CbMM cultures containing 0.2 ml of URPHLN was 60 nematodes per day and increased to 65 nematodes per day when the concentration was doubled. The rate of population growth in the cultures containing 0.2 ml URPHLS was 80 nematodes per day and decreased to 70 nematodes per day with the concentration was doubled.

The addition of 0.2 ml URPPCM#28's resulted in a rate of nematode production of 55 nematodes per day while cultures containing 0.4 ml of URPPCM#28 actually exhibited a much lower rate of production of 5 nematodes per day. The maximum population increase occurred sooner in cultures containing URPPCM#28 than in cultures with URPPCM#29. The maximum population increase associated with the additions of 0.2 ml or 0.4 ml of URPPCM#29 occurred on day 22. The rates were 35 and 30 nematodes per day, respectively. The lowest rate of population increase was present in cultures with 0.4 ml of URPPCM#28. The minimum rate of population increase occurred at the end of the experiment and was never greater than 9 nematodes per day in the controls as well as in cultures with 0.1 M Tris-Cl.

Experiment # 2: Effects of URP Extracts on Growth of C. elegans in a Non-nutrient Medium: A short-term toxicity test was setup to test for acute or chronic toxicity of the URP extracts. If any of the extracts were acutely or chronically toxic to C. elegans,

Figure 5. Experiment # 2 Average Population for C. elegans in M-9 Buffer with the Addition of URP-Extract

a significant increase in mortality would be observed within 24 to 48 hours after inoculation. In this assay, 0.2 ml of the sample and 2.3 ml of M-9 buffer were combined then the nematodes were added. URPHLN1-2x is twice the concentration of URPHLN2-1x. URPHLS3-1x is twice the concentration of URPHLS4-1x. URPPA5 was a purified isolate from the URPPCM extracts in experiment #1. Effects of the addition of 0.2 ml CbMM was also tested because the nematodes inoculum contained CbMM.

The averages populations declined throughout the experiment (Figure # 5).

No significant decline in population was occurred within the first 24 to 48 hours after inoculation. Populations declined sharply starting at day 5 and continuing to day 12. From day 12 to day 20 no significant decrease in population was observed. Survival of nematode inoculum was 2 to 3 times higher M-9 buffer than in tubes with a nutrient additive.

Section 3: KRS-Extracts

Experiment # 1: KRS-Extracts - Extracts and partially purified fractions were prepared from the same plant species and provided by Dr. Schubert. The high stimulatory activity of the URP extracts suggested that another group of extracts should be tested in an effort to identify a reproductive inhibitor. For this reason, extract KRS-1086 consecutive fractions (4, 5, 6 and 7) were tested. Fractions 1086-6 and 7 were pooled separately. Different volumes of the pooled materials were included in the culture medium. The volume of media was adjusted with CbMM. The populations of laggard tubes in these experiments were included in the calculations. The data is presented in Table 2.

The experimental volume of CbMM and extracts followed standard procedures unless otherwise noted. The standard culture volume was 2.5 ml. Extracts and partially purified fractions were tested in 0.2 ml, 0.4 ml and 0.5 ml additions. As a control test samples were replaced with 0.5 ml water. Five replicates were observed. Due to the limited availability of these extracts only 3 replicates of the 0.5 ml addition were observed. Six replicates of the control were observed.

Table 2. Change in Nematode Population in Cultures with KRS-1086 Extracts Experiment #1.

KRS-1086-4&5

% Change in Population at Day

Volume Used

7

8

9

14

17

21

24

0.5 ml

77

78

84

80

85

87

85

0.4 ml

60

64

57

63

69

65

80

0.2 ml

79

79

85

84

80

88

83

KRS-1086-6&7

% Change in Population at Day

Volume added

7

8

9

14

17

21

24

0.5 ml

11

5

-10

-71

-217

-448

-727

0.4 ml

-12

-44

-43

-148

-92

-442

-955

0.2 ml

62

52

64

44

56

47

60

KRS-1086-4&5 reduction data is ordered by the volume of extract added into the culture. Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations are significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the extract.

Population reduction was present in cultures containing both pooled fractions. Additions of KRS-1086-4&5 ( 0.2 ml, 0.4 ml and 0.5 ml) to cultures reduced population from 57 % to a maximum of 88 % of the control between days 7 and 24. The population in cultures containing KRS-1086-6&7 (0.4 ml and 0.5 ml) increased from day 9 to day 24. However, the 0.2 ml addition of KRS-1086-6&7 reduced population from day 7 to day 24. Data is presented Table 2.

The rate of nematode population increase in cultures containing KRS-1086-4&5 peaked at day 7 and day 9 with the maximum production of nematodes at day 9. The highest rate of nematode production in the control was 6 nematodes per day. Cultures containing 0.4 ml of KRS-1086-4&5 had a maximum growth rate of 4 nematodes per day at day 9. Cultures containing either 0.2 ml or 0.5 ml of KRS-1086-4&5 did not produce nematodes until after day 8 when their maximum rate of production was 1 nematode per day.

The nematode population growth rate of CbMM cultures containing KRS-1086-6&7 reached a maximum at day 9. Cultures with the 0.4 ml or 0.5 ml of sample produced 9 nematodes per day, compared to a nematode population production rate of 6 nematodes per day for the control. CbMM cultures to which 0.2 ml of KRS-1086-6&7 was added, reached a maximum nematode population growth rate of less than 4 nematodes per day between day 4 and 8.

Experiment # 2: Activity of KRS Fractions After Heat Treatment - Fractions similar to those tested in KRS Experiment # 1 were re-tested to verify their inhibitory activity. These fractions were prepared up to 1 year earlier and stored at 4 C. KRS-cdsp7518k80c1 was a crude heat-treated extract from which KRS-3316113 and KRS-13256 were isolated. The control for this series was of 0.5 ml of water. Laggard tube populations were removed from the data set prior to any calculations.

An increase in average population was seen in cultures where either 0.2 ml or 0.5 ml of KRS-13256 or 0.5 ml of KRS-3316113 were added. The 0.2 ml addition of KRS-3316113 and the 0.5 ml addition of KRS- cdsp7518k80c1 to cultures only increased population above the control levels at day 24. Prior to day 24, the presence of KRS-3316113 (0.2 ml) and KRS-cdsp7518k80c1 (0.5 ml) in cultures reduced average populations. The populations in cultures containing 0.2 ml of KRS-cdsp7518k80c1 were low (Table 3).

Table 3. Change in Nematode Population in Cultures Containing Fractions after Heat Treatment Experiment # 2.

0.2 ml Addition of

% Change in Population at Day

Sample

10

13

16

21

24

n

KRS-13256

-165.28

-252.33

-401.65

-206.45

-342.67

8

KRS-cdsp7518k80c1

25.00

65.70

75.58

91.73

87.23

6

KRS-3316113

22.22

31.98

14.52

15.01

-36.08

7

0.5 ml Additions of

% Change in Population at Day

Sample

10

13

16

21

24

n

KRS-13256

-143.06

-153.49

-182.18

-54.63

-115.49

8

KRS-cdsp7518k80c1

48.61

33.14

17.16

28.61

-49.26

7

KRS-3316113

52.78

-19.19

-170.63

-111.15

-161.20

7

Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations are significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the sample.

In all cultures with 0.2 ml of sample, a primary peak in nematode production was observed at day 13 and a second peak of production at day 21. The highest rate of population growth occurred in the water control with the rate in cultures containing KRS-13256 slightly lower at 118 nematodes per day. The next highest rate of population increase was observed in cultures containing KRS-3316113 at 45 nematodes per day. A rate of nematode population increase greater than 2.5 nematodes per day was not observed in cultures containing KRS-7518k80c1 at any point over the 24 day period.

With the 0.5 ml additions, the maximum rate of increase for nematode populations was 124 nematodes per day in the control. Additions of KRS-3316113 slightly decreased the reproduction rate of nematodes to 104 per day. The KRS-13256 decreased the reproduction of nematodes to 56 nematodes per day. The lowest rate of increase in nematode population was observed in cultures containing KRS-cdsp7518k80c1 with a maximum increase of 38 nematodes per day.

Experiment #3: KRS-Extracts - A third set of fractions, similar to those used in KRS Experiments # 2, were tested to confirm observations from Experiment # 1 and # 2. Four fractions (3, 4, 5 and 6) isolated from a crude, non-heat treated extract KRS-1297 were used in this experiment. Consecutive fractions 3 and 4 and fractions 5 and 6 were combined to create KRS-1297-34s and KRS-1297-56s, respectively. Extracts tested in this experiment were prepared two weeks prior to use. The laggard tubes were removed from the population data prior to any calculations. Individual treatments included five replicates except for the control where six replicates were observed. The controls for this experiment were 0.2 ml and 0.4 ml of 10 mM sodium phosphate buffer (pH 7.5). Water (0.5 ml) was also tested as a blank solution.

An increase in population was present in cultures with the 0.4 ml addition of water and the 0.4 ml KRS-s129770c2. The increase in population of cultures with water was low compared to control cultures. The increased in populations containing KRS-s129770c2 went from 8 % of the control at day 10 to 104 % at day 17. A reduced

Table 4. Change in Nematode Population in Cultures with KRS-Fractions Experiment # 3.

0.2 ml Additions of

 

 

 

Sample

10

13

17

Protein micrograms

 

KRS-1297-34s

16

18

28

8.2

 

KRS-s129770c2

63

3

-25

n.d.

 

KRS-1297-56s

80

79

78

11.0

 

0.4 ml Additions of

% Change in Population at Day

 

 

Sample

10

13

17

Protein micrograms

 

water

-8

-8

-23

0

 

KRS-1297-34s

40

39

38

20.5

 

KRS-1297-56s

69

54

-20

28.5

 

KRS-s129770c2

-8

-67

-104

n.d.

 

Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations are significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the fraction (n.d. is not determined).

population occurred in cultures with 0.2 ml or 0.4 ml KRS-1297-34s and with 0.2 ml KRS-1297-56s. Populations of cultures with 0.4 ml KRS-1297-56s remained below control levels from day 10 to day 13 but increased above control values by day 17. The changes in population compared to the control are presented in Table 4.

The highest rate of population growth was observed in cultures containing 0.2 ml of KRS-1297 fractions. For example, the rate of population growth of cultures containing KRS-1299770c2 were 135 nematodes per day. The control populations increased in population at a rate of 82 nematodes per day. In comparison , the rate for cultures with 0.5 ml water was only 53 nematodes per day. Cultures with KRS-1297-34s had a population growth rate of 46 nematodes per day which was 57 % of the control at day 10. The rate for cultures containing KRS-1297-56s never increased above 10 nematodes per day.

The highest rate of population increase for experiment # 3 was observed in cultures with 0.5 ml KRS-s129770c2 at 151 nematodes per day by day 13. The next highest rate of 92 nematodes per day was observed in cultures with KRS-129756s at day 17. Water or buffer controls exhibited rates of 60 and 55 nematodes per day, respectively. The maximum rate for cultures with 0.5 ml of KRS-129734s was 32 nematodes per day which was 21 % of the control.

Experiment # 4: KRS-Extracts - This experiment is presented in two parts. In the first part, fractions KRS-1297-5 and KRS-1297-6 were heat treated in an attempt to inactivate proteinaceous activity. The second part of this experiment measures the effects of individual fractions. Based on previous experimental results, which suggested that KRS-1297 contained a reproductive inhibitor, fractions #'s 4, 5, 6, & 7 were tested as individual fractions to identify the fractions with the highest activity. Fractions KRS-1297- 5 and KRS-1297- 6 were heated at 100 C for five minutes. Other substances tested in this experiment include CbMM (double strength), HS-YE, and water. The total culture volume was 2.5 ml, 2.3 ml CbMM and 0.2 ml test substance. Populations of laggard tubes were removed from data prior to calculations. All treatments were observed in six replicates except for the treatment with heat-treated KRS-1297-5 which was limited to five due to availability of this fraction.

Part 1: Heat Treatment of Factions - Although populations of cultures containing non-heat-treated KRS-1297-5 were above cultures with heat-treated fractions, the populations were still below control levels (Table 5). The rate of population increase was

Table 5. Change in Nematode Population in Cultures with KRS-1297 Fractions Experiment # 4.

Sample

% Change in Population at Day

 

 

8

11

15

protein micrograms

KRS-1297-6 heated 5 min. @ 100 C

36.65

33.79

17.75

n.d.

KRS-1297-6

71.10

53.20

24.62

10.06

KRS-1297-5 heated 5 min. @ 100C

41.93

36.07

14.00

n.d.

KRS-1297-5

70.67

59.38

28.43

10.14

CbMM 2X

51.35

40.78

34.15

6.2

HS-YE

-42.96

-5.60

0.00

6.0

water

0.00

0.00

0.00

0

Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations are significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the extract (n.d. = not determined).

significant in the heat-treated fractions for day 8 and 15. Growth rates observed in the heat-treated fractions decreased synchronously from 115 to 17 nematodes per day from day 4 to day 15. The peak rate for the heated fractions was observed at day 8. General growth rate of cultures declined from day 8 to day 15 for all fractions.

The maximum population growth rate in cultures containing HS-YE extract was 287 nematodes per day on day 8. The growth rate of cultures with water was 180 nematodes per day. Cultures with 0.2 ml 2X-CbMM exhibited a growth rate below both of the heat-treated fractions. The rates at days 8 and 11 were 82 and 73, respectively.

Part 2: Fractionation of Activity - The average population for cultures containing KRS-1297-6 and KRS-1297-5 were reduced to a higher degree and for a longer period of time than cultures with KRS-1297-3 and KRS-1297-4 (Table 6). Reduction of population

Table 6. Change in Nematode Population in Cultures with Individual Fractions From KRS-1297, Experiment # 4.

Sample

% Change in Population at Day

 

8

11

15

protein micrograms

KRS-1297-6

71.10

53.20

24.62

10.06

KRS-1297-5

70.67

59.38

28.43

10.14

KRS-1297-4

41.74

18.02

8.33

9.92

KRS-1297-3

28.85

24.32

11.67

6.3

Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations are significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the fraction.

declined from day eight to day fifteen for all cultures except for HS-YE and water.

Cultures with water exhibited maximum nematode growth rate of 180 nematodes per day at day 8. Cultures containing KRS-12973 increased in population at a peak rate of 122 nematodes per day at day 11. Cultures containing fraction KRS-1297-4 increased in population at a rate of 117 nematodes per day at day 8. Rates of population growth increased in cultures containing KRS-1297-5 or KRS-1297-6 following a similar pattern with a minimum rate of nematode production below 17 at day 4 to a maximum rate of nematode production near 110 by day 15. The population in control cultures reached 1,000 by day 15, for this reason, the experiment was terminated. Cultures with HS-YE had populations of 1,000 by day 8.

Experiment #5: KRS-Fractions - Further testing of KRS-1297-5 and KRS-1297-6 was needed. This experiment is presented in four parts. Size fractionation of the extract by ultrafiltration using a Centricon filtration apparatus to help determine the size of the active portion of the extracts. The percent population reduction as a function of the protein content in the added fractions was compared to demonstrate the concentration dependence of population reduction. A haemagglutination test was performed to measure the presence of lectin in these fractions. SDS-PAGE analysis of the Centricon fractionated samples was performed in an effort to determine protein purity and to provide preliminary identification of active components.

Based on a results of previous experiments with KRS-1297-5 and KRS-1297-6, these fractions were pooled and refractionated using Centricon-10 and Centricon-50 filter units.. The concentrated fractions KRS-1297-56 were tested for reproductive reduction. After ultrafiltration, the retentates was returned to original volume and both retentate and filtrates were tested by the standard CbMM assay for reduction of average population. The percent reduction of average population was compared to protein concentration of the extracts. The duration of the experiment was 16 days. A haemagglutination test was performed on the Centricon fractions since agglutination activity from similar preparations has been observed (Schubert Laboratory unpublished results). The fractions were subject to SDS-PAGE analysis to determine protein purity and to visualize protein banding patterns of the fractions.

Part 1: Size Determination - Neither of the Centricon filtrates reduced populations below control levels. A reduced population was observed in cultures containing both of the Centricon retentate fractions (Table 7). The rates of population change for all Centricon fractions were significant. Nematode population growth rates for cultures containing retentate fractions were significantly lower while those containing filtrate fractions were significantly higher for each observation. The highest reduction of population was seen in cultures with addition of the Centricon-10 retentate which reduced populations up to 77 % for day 12 but not going below 62 % for day 16. The addition of the Centricon-50 retentate reduced populations by 47% at day 9, but the reduction dropped by day 16 to 29 %.

The rates of nematode population increase for cultures containing KRS-1297-filtrate from the Centricon-50 was 134 nematodes per day at day 9. The maximum population increase observed after the addition of KRS-1297-filtrate from the Centricon 10 was 128 nematodes per day. The peak occurred 3 days later than the maximum rate for cultures with filtrate from the Centricon-50. Cultures with 0.2 ml of water had maximum nematode population growth rates of 117 nematodes per day by day 12. The rate of nematode production was never higher than 60 nematodes per day in cultures containing retentate fractions. This rate was reached by cultures with the retentate of Centricon-50 at day 12. The maximum rate was somewhat lower, 39 nematodes per day by day 16, in cultures with the retentate from the Centricon-10.

Table 7. Change in Nematode Population in Cultures Containing Fractions of KRS-1297-5&6, After Ultra-filtration Experiment #

Sample

% Change in Population at Day

 

9

12

16

n

Retentate (1x)-Centricon -10K

73.96

77.04

62.27

4

Filtrate (1x)-Centricon -10K

-67.36

-35.49

-24.72

3

Retentate (1x)- Centricon -50K

47.83

49.31

29.91

4

Filtrate (1x) Centricon -50K

-135.33

-32.56

-24.72

4

Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations are significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the fraction.

Part 2: Concentration Series Fraction KRS-1297-5&6 was concentrated 2-fold by Centricon-5- ultrafiltration. 2 x KRS1297-5&6 was tested at concentrations of (2, 1, 1/2, 1/4, and 1/16) times normal fraction concentration. The average population was reduced for all cultures containing KRS-1297-56 from day 9 to day 12 except for the lowest concentration (0.3725 micrograms). In the later case a stimulation of growth was observed (Figure 6). The percent reduction in population for cultures containing 2.7 microgram of KRS-1297-5&6 was between 53 % and 58%. The lowest concentration (0.3725 microgram) used in these experiments stimulated population growth by 54 % on day 9. This stimulation decreased to only 14 % by day 12. The percent reduction in population seen with the addition of 0.725 micrograms increased from 19 % on day 9 to 39 % on day 12. The addition of 1.45 micrograms reduced percent population reproduction by 42 % on day 9. The percent reduction increased at day 12 to 62% which was the highest reduction observed in this series. The highest amount of KRS-1297-5&6 added to the cultures was 5.4 micrograms but it only reduced populations by 29% for day 9 and 16 % for day 12. Average populations at day 12 were not significant for the 0.372, 1.45 and 5.9 microgram additions or the 0.725 microgram addition at day 9.

The maximum rate of population growth for the cultures to which only water was added at day 12 was 118 nematodes per day. This value was similar to the rate for 5.4 micrograms of the test fraction. The next highest rate of nematode population growth occurred in cultures with 0.3725 micrograms of concentrated sample. The rate in these cultures was 100 nematodes per day between days 9 and 12. The other additions, 0.725 micrograms and 0.3725 micrograms, increased the rate of population growth from 10 nematodes per day at day 5 to 120 and 30 nematodes per day at day 16, respectively.

Figure 6. Change in Nematode Population Growth Rate in CbMM Increasing Concentration of KRS-1297-5&6 Experiment #5.

Part 3: Haemagglutination - Because others have found haemagglutination activity in these fractions, an agglutination test was conducted on selected fractions of KRS-1297. KRS-1297-5&6 was fractionated by ultrafiltration in Centricon-50 filter units. The retentate was returned to its starting volume prior to use.

Table 8. Agglutination Activity and Protein Concentrations of KRS-1297-5&6 Fractionated by Ultrafiltration in Centricon-50 Filters Experiment # 5.

Centricon filtration of extract

KRS-1297-5&6

Agglutination

Protein

micrograms

0.2 ml-Retentate (1x)-Centricon-10

r, p, h

5.6

0.2 ml-Retentate (1x)-Centricon-50

r, p, h

5.4

0.2 ml-Filtrate (1x) Centricon-50

none

0

0.2 ml-Filtrate (1x) Centricon- 10

none

0

Agglutination activity was tested on red blood cells of rabbit, human and pig (r=Rabbit, p=Pig and h=Human).

The haemagglutination activity was observed in the biologically active supplement KRS-1297-5&6 (Table 9). Haemagglutination was present for Centricon samples from a concentration of 1.4 to 0.17 micrograms of protein.

Part 4: SDS-PAGE - 12.5% Acrylamide: The protein banding patterns were observed after SDS acrylamide gel electrophoresis. Several protein bands were noted in lanes containing KRS-1297-5 and 6 not present in fractions KRS-1297-3 and 4. The most intensely stained bands were sized around 200,000 molecular weight. In an effort to determine the approximate size of the active component, Centricon concentration filters were used to size fractionate the fraction. Centricon filters separate proteins by size through the use of a restrictive membrane while being centrifuged. Centricon-50 has a molecular weight cutoffs of 50,000 and Centricon-10 has a 10,000 cutoff.

Two gels were prepared. The first prepared gel showed supplement KRS # 1297-3 through 7 while the second contained Centricon-10 and 50 filter separations, both filtrate and retentate of pooled KRS-1297-5 and 6. The first and second gel included fractions from KRS extracts experiments # 4 and # 5 and in experiments # 6, respectively. Both gels were stained first with Coomassie Brilliant Blue and then destained to view the protein banding patterns. Due to the small amount of protein in these fractions, the gels were then silver stained.

Gel #1: Protein banding was distinctly visible in KRS-1297 fractions. Light banding was noticeable with silver staining in fractions 3 and 4. Darker banding was visible with fractions #'s 5, 6, and 7 with Coomassie staining. These bands became more distinct after silver staining.

Gel #2: After staining with Coomassie Brilliant Blue, there were very few distinct bands in either retentate or filtrate fractions. With silver staining, the retentate banding from both Centricon fractions were heavy and distinct. The filtrate fractions remained clear of protein bands even when the gel was over developed in the silver staining process.

Section 4: Anatomical Variations

Anatomical variations were seen in the C. elegans grown in axenic culture system. Under standard conditions, cuticular swellings occurred in frequently in small percentage of the population. These swellings looked like a blister on the cuticle of the nematode (Figure 7-c). Prolapsed vulva (Figure 7 b & d) and abnormal swellings around the midsection of the nematode occurred frequently. This swelling was not restricted to a specific supplement. Blistering has been seen with and without the post-anal swelling (Figure 7 a) or the swelling of the midsection, seen between the vulva and the posterior end of the second cuticle blister as seen in Figure 7. A post-anal bump was observed in the experimental tubes with supplement additions. The abnormality was observed in extracts # KRS-1297-5 and # KRS-1297-6. The swelling was always seen in connection with these supplements.

Figure 7. Anatomical Variations Seen in C. elegans Cultures

  1. post-anal bump, (b) prolapse of the vulva, (c) cuticle blister, (d) vulva. ~180 x actual size (HTML alteration for web publication only one picture was originally published but these are more detailed.)

 

 

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