Evaluation of feeding different forms of therapeutic diet on the feed intake, digestibility, feed efficiency, and growth of calves experimentally infected with foot-mouth disease virus.

Oral ulcers induce acute weight loss due to anorexia in foot-and-mouth disease virus (FMDV) infected cattle. We hypothesized that providing a palatable form of a therapeutic diet (TD) in different physical forms would increase the feed intake, digestibility and restoration of body weight. A TD was formulated with 19% CP and 2.9 Mcal ME/kg on dry matter basis. Bull calves of 10-12 months with mean body weight of 123 ± 1.3 kg were experimentally infected with FMDV (n = 18) and offered one of the following three forms of the TD (n = 6/group) for 6 weeks post-FMDV infection (WPI): (i) TD in mash form (TDM) (ii) TD in cooked form (TDC) and (iii) TDC + customised nutrient supplement (TDCNS) such as Zn, Cu, Cr, Mn, and Se. The CNS was fed before the TDC. A group of uninfected control (n = 4) was fed TDM. Green fodder was offered in the afternoon. Dry matter intake (DMI) of TD and green fodder were recorded at 24 h interval till WPI 6. Body weight (BW) was recorded at weekly interval. Digestibility trial was conducted at WPI 6. The palatability of the TD was scored from 1- 4 and healing of tongue ulcers was analyzed by Kaplan-Meier survival curve. The results indicated that the physical form of TD increased the total DMI by WPI 3, which was supported by the restoration of BW and higher palatability score. The digestibility of all the proximate principles except EE was significantly higher (P < 0.05) in the groups that were fed TDC. It was concluded that feeding TD irrespective of the physical form, restored the ADG and DMI in the calves by WPI 3. Further, feeding cooked form of TD increased the digestibility in the FMDV infected calves and supplementation of CNS hastened the healing of glossal ulcers.

Influence of boron supplementation on performance, immunity and antioxidant status of lambs fed diets with or without adequate level of calcium.

Little is known about biological significance of effects of dietary Boron (B) and Calcium (Ca) interaction on health and production of farm animals. This is a preliminary investigation to evaluate the effects of B supplementation in lambs fed diets with (normal) or without adequate (low) levels of Ca. Twenty-four crossbred ram lambs were randomly distributed into four groups with six animals each in a 2x2 factorial design namely, normal-Ca diet (NCa) and low-Ca diet (LCa) fed without or with 40 ppm B (NCaB-40 and LCaB-40). The lambs were fed paddy straw and hybrid napier hay-based total mixed ration (60 roughage: 40 concentrate) during 180 days experimental period. Compared to control, the LCa diet lowered (P<0.01) average daily gain of lambs, but B-supplementation (LCaB-40) of the same nullified the effect. The lowered (P<0.05) total antioxidant activity and humoral immune response in lambs fed LCa diet were restored (P>0.05) to become at par with the control (NCa) upon supplementation of B (LCaB-40). The mRNA expression of SOD1 was lowered (P<0.05) due to LCa diet feeding which too was normalized on B-supplementation to become at par (P>0.05) with the control (NCa). Further, B-supplementation restored lowered (P<0.05) SOD1 gene expression on LCa diet, but enhanced (P<0.05) that in NCaB-40 group, when compared to the control (NCa) diet fed animals. However, these variations were not reflected in the SOD activity in the erythrocytes. The cell-mediated immune response was higher (P<0.05) in lambs fed LCa and LCaB-40 groups and there was no significant interaction between the levels of either Ca or B in diets with the period of immune response measurement. B- supplementation of LCa diet ameliorated tissue degenerative changes in liver and kidney. It was concluded that feeding LCa diet to lambs resulted in reduced growth rate, total antioxidant activity, humoral immune response along with degenerative changes in liver and kidney tissues, but B-supplementation of such diet restored most of these changes and ameliorated histopathological alterations.

Effect of condensed tannins from Ficus infectoria and Psidium guajava leaf meal mixture on nutrient metabolism, methane emission and performance of lambs.

OBJECTIVE: The study examined the effect of condensed tannins (CT) containing Ficus infectoria and Psidium guajava leaf meal mixture (LMM) supplementation on nutrient metabolism, methane emission and performance of lambs. METHODS: Twenty four lambs of ~6 months age (average body weight 10.1±0.60 kg) were randomly divided into 4 dietary treatments (CT-0, CT-1, CT-1.5, and CT-2 containing 0, 1.0, 1.5, and 2.0 percent CT through LMM, respectively) consisting of 6 lambs each in a completely randomized design. All the lambs were offered a basal diet of wheat straw ad libitum, oat hay (100 g/d) along with required amount of concentrate mixture to meet their nutrient requirements for a period of 6 months. After 3 months of experimental feeding, a metabolism trial of 6 days duration was conducted on all 24 lambs to determine nutrient digestibility and nitrogen balance. Urinary excretion of purine derivatives and microbial protein synthesis were determined using high performance liquid chromatography. Respiration chamber study was started at the mid of 5th month of experimental feeding trial. Whole energy balance trials were conducted on individual lamb one after the other, in an open circuit respiration calorimeter. RESULTS: Intake of dry matter and organic matter (g/d) was significantly (p<0.05) higher in CT-1.5 than control. Digestibility of various nutrients did not differ irrespective of treatments. Nitrogen retention and microbial nitrogen synthesis (g/d) was significantly (p<0.01) higher in CT-1.5 and CT-2 groups relative to CT-0. Total body weight gain (kg) and average daily gain (g) were significantly (linear, p<0.01) higher in CT-1.5 followed by CT-1 and CT-0, respectively. Feed conversion ratio (FCR) by lambs was significantly (linear, p<0.01) better in CT-1.5 followed by CT-2 and CT-0, respectively. Total wool yield (g; g/d) was linearly (p<0.05) higher for CT-1.5 than CT-0. Methane emission was linearly decreased (p<0.05) in CT groups and reduction was highest (p<0.01) in CT-2 followed by CT-1.5 and CT-1. Methane energy (kcal/d) was linearly decreased (p<0.05) in CT groups. CONCLUSION: The CT supplementation at 1% to 2% of the diet through Ficus infectoria and Psidium guajava LMM significantly improved nitrogen metabolism, growth performance, wool yield, FCR and reduced methane emission by lambs.

Boron influences immune and antioxidant responses by modulating hepatic superoxide dismutase activity under calcium deficit abiotic stress in Wistar rats.

The influence of Boron (B) supplementation on immune and antioxidant status of rats with or without abiotic stress induced by dietary calcium (Ca) restriction was studied in a feeding trial of 90 days. Wistar strain rats (3-4 wk age, n=84) were divided into 7 dietary groups (4 replicates of 3 each) viz., normal-calcium (100%) basal diet alone (NC, control) or supplemented with B at 5 (NCB-5), 10 (NCB-10), 20 (NCB-20) and 40ppm (NCB-40) levels; low-calcium (50%) basal diet alone (LC) or supplemented with 40ppm B (LCB-40). After 75 days of experimental feeding, rats were challenged with intraperitoneal injection of sheep RBCs to assess their humoral immunity. At the end of the trial, cell-mediated immunity was assessed as foot pad reaction to sheep RBCs injected into the hind leg paws. Eight rats from each group were sacrificed to collect blood for estimation of minerals and total antioxidant activity, and liver for superoxide dismutase gene expression analysis. Supplementation of graded levels of B (5, 10, 20 and 40ppm) as borax in NC diets significantly increased (P<0.01) the footpad thickness and serum total antioxidant activity, hepatic expression levels of both Cu-Zn SOD (SOD1) and Mn-SOD (SOD2) mRNAs. The erythrocytic SOD activity and humoral response did not differ significantly among the dietary groups. In Ca restricted groups, humoral immune response was significantly decreased (P<0.01) compared to control but increased (P<0.05) with 40ppm B supplementation. Serum levels of copper (Cu) and zinc (Zn) remained similar among the dietary groups, while the manganese (Mn) content was significantly decreased (P<0.01) with increased levels of dietary B. In conclusion, B supplementation increased the hepatic mRNA expression levels of both SOD isoenzymes, thereby improving the immune and antioxidant status.

Effect of feeding Jerusalem artichoke (Helianthus tuberosus) root as prebiotic on nutrient utilization, fecal characteristics and serum metabolite profile of captive Indian leopard (Panthera pardus fusca) fed a meat-on-bone diet.

An experiment was conducted to determine the effect of incorporating Jerusalem artichoke (JA) as a prebiotic in the diet of Indian leopards (n = 11 adults) fed a meat-on-bone diet. The trial consisted of three periods (A1 , B, and A2 ). Each period comprised 17 days of adaptation and four days of collection. During the control periods (A1 and A2 ), the leopards were fed their normal zoo diets of 2.5-3 kg of buffalo meat-on-bone six days a week without any supplement. During trial B, meat-on-bone diets of the leopards were supplemented with JA at 2% of dietary dry matter (DM). Meat consumption was similar among the treatments. Supplementation of JA decreased the digestibility of crude protein (P < 0.01). Digestibilities of organic matter and ether extract were similar among the treatments. Serum concentrations of urea and triglycerides were lower (P < 0.05) when JA was added to the diet. Incorporation of JA to the basal diet increased fecal concentrations of acetate (P < 0.01), butyrate (P < 0.01), lactate (P < 0.01), Lactobacillus spp., and Bifidobacterium spp. (P < 0.01) with a simultaneous decrease in the concentration of ammonia (P < 0.01), Clostridia spp. (P < 0.01), and fecal pH (P < 0.01). Fecal microbial profiles and hind gut fermentation were improved, without any adverse effects on feed consumption, nutrient utilization, and serum metabolite profiles. Results of this experiment showed that feeding JA at 2% DM in the whole diet could be potentially beneficial for captive Indian leopards fed meat-on-bone diets.

Effects of Tinospora cordifolia supplementation on semen quality and hormonal profile in rams.

The present study was undertaken to assess the effect of dietary supplementation of Tinospora cordifolia on physico-morphological, biochemical, antioxidant profiles and serum testosterone concentration in Muzzafarnagari rams. Twelve rams were randomly divided into two groups, control (n=6) and supplemental (n=6) group. The control group was fed with a diet satisfying NRC recommendations whereas the supplemental group was fed with T. cordifolia at the rate of 1g/kg body weight for 6 months. The semen samples were collected 60 days post-feeding. The result revealed that T. cordifolia supplementation did not have a significant effect on physico-morphological, biochemical attributes of semen and serum testosterone concentrations in rams. The concentration of cholesterol, superoxide dismutase (SOD) and catalase were, however, increased (P<0.05) in seminal plasma. It was concluded that the possible protective effects of T. cordifolia supplementation were enhancing antioxidant enzymes and cholesterol concentrations in semen which may be protected the spermatozoa during cryopreservation and thus enhancing fertility in farm animals.

Influence of Dietary Supplementation of Condensed Tannins through Leaf Meal Mixture on Intake, Nutrient Utilization and Performance of Haemonchus contortus Infected Sheep.

The study assessed the effect of dietary supplementation of leaf meal mixture (LMM) containing condensed tannins (CT) on feed intake, nutrient utilization and performance of sheep infected with Haemonchus contortus. Eighteen adult sheep of similar age and body weight (25.03±1.52) were included in this study and out of these, 12 sheep were infected with single dose of infective third stage larvae of H. contortus at 2,000 larvae per sheep. The experimental sheep were allocated in three different groups' i.e. negative control (NC; no infection), control (C; H. contortus infected) and treatment (T; H. contortus infected+CT at 1.5% of the DM through LMM) and the experiment was conducted for a period of 90 d. The intake of dry matter (DM), organic matter (OM) and digestibility of DM, OM, neutral detergent fibre (NDF) and acid detergent fibre (ADF) were comparable among three animal groups. However, digestibility of crude protein (CP) and ether extract (EE) were significantly (p<0.05) higher in NC group as compared to both C and T groups. Nitrogen (N) retention (g/d or % of N intake) was significantly (p = 0.038) lower in C group as compared to T and NC groups. Daily intake (g/kg W(0.75)) of digestible crude protein (DCP), digestible organic matter (DOM) and total digestible nutrient (TDN) did not differ significantly (p<0.05) in the three groups. Haemoglobin (Hb) and packed cell volume (PCV) were significantly (p<0.001) higher in treatment group as compared to control. The level of Hb and PCV reduced (p<0.001) after 30 days of experimental feeding. CT significantly (p<0.001) reduced serum urea in T group as compared to NC and C groups. Serum proteins differed significantly (p<0.01) among the three groups. The activity of serum enzymes AST, ALT, ALP and LDH were also statistically non significant (p<0.05) among treatments. The weight of abomasal lymph nodes (ALN) in T group was higher (p<0.05) than in C group. Treatment group had lower (p<0.05) total worms and fecal egg count compared to control group. It may be concluded that dietary supplementation of CT through LMM significantly improved the N retention, and inhibited the different developmental stages of Haemonchus contortus in experimental sheep.

Effect of Varying the Energy Density of Protein-adequate Diets on Nutrient Metabolism, Clinical Chemistry, Immune Response and Growth of Muzaffarnagari Lambs.

Effects of varied dietary energy densities on immune response and performance of Muzzafarnagari lambs were ascertained in a 180-d study. Animals (n = 24), in three groups, were fed diets providing 100% (100E), 80% (80E) or 70% (70E) of their metabolizable energy requirement. Mean nutrient digestibilities varied significantly among treatments. Nitrogen intake was lower (p<0.01) in the 70E. Nitrogen retention, was reduced (p<0.001) in 80E and 70E vs 100E. The average daily gain (p<0.001) was 47.01±4.23, 13.54±1.72 and -16.67±8.24 g for 100E, 80E and 70E, respectively. Hemoglobin concentration, haematocrit, total and differential leukocyte counts were lower (p<0.001) for 80E and 70E than for 100E with a similar trend (p<0.05) for serum glucose and total protein. Serum cortisol was reduced (p<0.001) with decreased energy availability. Antibody titre to Brucella abortus S19 showed an initial reduction in 80E and 70E vs 100E. Delayed-type hypersensitivity response was lower (p<0.001) in 80E and 70E vs 100E, accompanying a lower (p<0.001) nitric oxide production by the peripheral lymphocytes. It is concluded that the reduced dietary energy density significantly affects the growth performance and immune response of lambs.

Nutrition-induced Changes of Growth from Birth to First Calving and Its Impact on Mammary Development and First-lactation Milk Yield in Dairy Heifers: A Review.

This review focuses on the nutritional effects from birth until age at first calving on growth, mammary developmental changes, and first-lactation milk yield in heifer calves. The advancement in the genetic potential and the nutritional requirements of the animals has hastened the growth rate. Genetic selection for high milk yield has suggested higher growth capacity and hence increasing nutritional inputs are required. Rapid rearing by feeding high energy or high concentrate diets not only reduces the age of sexual maturity but also lowers the time period of attaining the age of first calving. However, high energy diets may cause undesirable fat deposition thereby affecting future milk yield potential. Discrepancies exist whether overfed or overweight heifers at puberty can influence the mammary development and future milk yield potential and performance. The data on post-pubertal nutritional management suggested that body weight at calving and post-pubertal growth rate is important in first lactation milk yield. There is a continuous research need for strategic feeding that accelerates growth of dairy heifers without reduction in subsequent production. Nutritional management from birth, across puberty and during pregnancy is critical for mammary growth and for producing a successful cow. This review will mostly highlight studies carried out on dairy breeds and possible available opportunities to manipulate nutritional status from birth until age at first calving.

Immunobiochemical status of sheep exposed to periods of experimental protein deficit and realimentation.

Immunobiochemical status of sheep exposed to periods of experimental protein deficit and realimentation was studied in 12 sheep (15 mo) randomly distributed into 2 equal groups and fed individually 2 different concentrate supplements along with wheat straw to provide 100% (normal protein, NP) or 50% (low protein, LP) of CP requirements. The study was comprised of 3 periods; during period 1 (0 to 13 wk) and 2 (14 to 26 wk) animals in the 2 groups were fed NP and LP diets, respectively; during period 3 (27 to 44 wk), animals in the LP group were switched over to NP diet to allow realimentation, whereas animals in the NP group remained on the same NP diet. Blood was collected from all groups at end of each period, and serum glucose, total protein, albumin (sAlb), globulin (sGlb), urea (sU), creatinine (sCr), cholesterol, triiodothyronine, and thyroxine concentrations were determined. During the same periods, the cell-mediated immune (CMI) response was measured by a delayed type hypersensitivity (DTH) assay and in vitro nitrite production by lymphocytes. At the end of periods 2 and 3, humoral immune response (HIR) was measured by sensitizing the sheep with Brucella abortus S99 antigen and measuring antibody titers on 0, 7, 14, 21, and 28 d postinoculation by ELISA. Feed intake decreased with prolonged protein deficit and showed recovery during period 3. Blood chemistry revealed reduced sAlb concentration in the LP group resulting in narrow sAlb:sGlb ratio, increased sCr concentrations (P = 0.008) accompanying a decreased (P = 0.004) sU:sCr ratio, and decreased glucose concentrations (P = 0.05). Other variables did not change significantly between the NP and LP groups. The DTH response at the end of period 1 and 2 showed marked (P = 0.008) effect of protein restriction on CMI. Nitrite production, basal and after lipopolysaccharide stimulation, was greater (P = 0.04) in the NP group. The HIR was less (P = 0.04) in the LP group during period 3. Realimentation of protein in the LP group during period 3 showed recovery in CMI and HIR. In conclusion, protein deprivation induced a decline in CMI and HIR in sheep accompanying alterations in related metabolic profile. However, a marked recovery was observed after realimentation.

Effects of Nandrolone and TGF-beta1 in growing rabbits with osteopenia induced by over-supplementation of calcium and vitamin D3.

The study was undertaken to find out the effects of over supplementation of dietary calcium and vitamin D3 on the mineralization of growing skeleton, taking rabbit as an animal model; further to study the effects of Nandrolone deconoate and TGF-beta1 on the mineralization of osteopenic bones. Twenty four New Zealand White rabbits of either sex, 60 day old, were randomly divided in 4 equal groups, A, B, C and D. The animals of groups B, C and D were administered with oral supplementation of calcium (2000 mg/kg of standard rabbit feed) and vit-D3 (1000 IU/kg of standard feed) for 60 days. The animals of group A were given standard ration without any supplementation. After 60 days, the Ca-vit.D3 supplementation was discontinued; and the animals of group C were administered with TGF-beta1 (10 ng, i.m.) once in every three days and animals of group D were given Nandrolone deconoate (10 mg, i.m.) once every week for 30 days, whereas in animals of group B, no treatment was given. All the animals were evaluated based on different observations like body weight, radiographic observations, circulating biochemical and hormone profile (plasma Ca, IP, AP, OC and iPTH) every 15 days up to 60 days after initiation of treatment. The results indicated that the body weight of rabbits in different groups increased gradually and steadily at different intervals till the end of observation period, however, the increase was non-significantly more in group D. The CI in group A increased gradually at different intervals; whereas in groups B, C and D, there was no appreciable increase in the CI during the period of Ca-vit.D3 supplementation, suggesting development of osteopenia. Treatment with TGF-beta1 did not increase the CI significantly, whereas Nandrolone treatment resulted in significant increase in the CI on days 45 and 60. The plasma Ca levels showed slight but gradual increase from day 0 to 60 in almost all groups. Subsequently also, there was no marked change at different intervals in groups A and B; however, significant reduction in plasma Ca was noticed in group C on 15(th) day and in group D on 60(th) day after initiation of treatment. Plasma IP levels in groups B and C showed a decreasing trend up to day 60. After discontinuation of Ca-vit.D3 supplementation, in group B, it further decreased to remain significantly lower on 15(th) day, and in groups C and D, it increased significantly on 60(th) post-treatment day. There was no significant change in the AP activity during the entire period of study in group A; whereas significant reduction in AP activity was measured on 30(th) and 60(th) days of treatment in groups B and C, and on 15(th) day of treatment in group D. Plasma iPTH values did not show any significant change at any interval during the first 60 days in all groups. On 30(th) and 60(th) days of treatment, the mean iPTH level remained significantly lesser in group B. In all groups treated with over supplementation of Ca and vit.D3, there was a non-significant increase in the plasma OC levels up to day 60; however, there was no significant difference between the groups. It can be concluded that additional supplementation of Ca and vit.D3 results in osteopenia in growing rabbits. Administration of Nandrolone helps to increase the mineral density in osteopenic bones, whereas TGF-beta1 does not seem to have positive effect on the skeletal mineralization.

Enhanced erythrocytic lipid peroxides level in rabbits after repeated parental administration of iron.

An experiment was conducted in rabbits to evaluate the possible involvement of oxidative stress in iron-overload animals. Ten adult female New Zealand white rabbits were divided into 2 equal groups with 5 animals each. Group II animals received intramuscular iron dextran injections (120 mg/kg body wt/day) on alternate day for 14 days (8 injections), while Group I animals did not receive any iron supplementation to serve as negative controls. The blood samples were collected by cardiac puncture before the start of iron dosing and thereafter, at weekly intervals for 28 days. The samples were processed to measure blood iron concentration, packed cell volume, erythrocytic lipid peroxide (LPO) level, superoxide dismutase (SOD) and catalase (CAT) activities. The blood iron concentration showed a rising trend following repeated iron administration, and the mean level recorded on day 14 was significantly higher than respective day 0 value. LPO level remained significantly higher from day 14 onwards till the end of the observation period of 14 more days after cessation of iron adminstration. Erythrocytic superoxide dismutase activities showed a transient significant rise on day 7, and thereafter, showed a declining trend, but remained statistically comparable to respective day 0 or corresponding value of the control animals.

Replacement value of undecorticated sunflower meal as a supplement for milk production by crossbred cows and buffaloes in the northern plains of India.

The replacement value of undecorticated sunflower meal (SFM) in the diets of dairy animals was assessed on-station and on-farm. Eighteen primiparous crossbred (Bos taurus x Bos indicus) cows (350.4 +/- 8.84 kg), randomly allocated to three groups, were used in the on-station study. The animals were fed on either a conventional concentrate supplement (control) or on an experimental concentrate, in which SFM replaced 25% (SFM-25) or 50% (SFM-50) of the CP in the control supplement. Green oats (Avena sativa) were supplied ad libitum. A metabolism trial conducted following 60 days of experimental feeding revealed that the intakes of DM, DCP and TDN were similar among the groups. The digestibilities of OM, CP, EE, NDF and ADF were also without significant differences. All the groups were in positive nitrogen balance. Inclusion of SFM at either level had no effect on the intake, excretion or retention of nitrogen. The daily milk yield and its composition did not differ among the dietary treatments. Moreover, the efficiency of utilization of DOM and TDN for FCM production tended to reflect, although non-significantly, increasing levels of SFM inclusion. In the on-farm study, seven multiparous milking buffaloes belonging to six farmers were used to assess the effect of replacing 20% of the CP of the conventional supplement with SFM, in a predominantly crop residue-based diet. The study continued for 4 months and revealed that the average daily feed intake and milk production was similar in the control and SFM-fed groups. It was concluded that SFM can be effectively utilized as a cheaper replacement for costly oil cakes and wheat bran for economic milk production by smallholder farmers.

Effect of iodine level in mustard (Brassica juncea) cake-based concentrate supplement on nutrient utilisation and serum thyroid hormones of goats.

To assess the influence of dietary iodine (I) supplementation on nutritional performance and serum thyroid hormones of goats, 12 adult Barbari goats (average weight 18.8kg) were assigned randomly to three dietary treatments. The goats were fed a mustard (Brassica juncea) cake containing concentrate supplement along with either 0mg (control, I(0)), 0.050mg (I(50)), or 0.075mg (I(75)) I per animal per day for 180 days. Oat hay was given ad libitum as basal roughage. There was no difference in intake and digestibility of various nutrients or N retention among the three groups in a metabolism trial after 90 days of experimental feeding. A second metabolism trial conducted at 165 days post-feeding revealed that daily intake of DM, DCP and ME were 39.9, 41.1 and 44.8g, 2.64, 3.01 and 2.97g, and 366, 414 and 415kJ per unit metabolic body size, respectively, for the I(0), I(50) and I(75) groups. Retention of N by goats was 1.75, 2.58 and 2.56g per day (P>0.05) for the three groups, but one of the control animal was in negative balance. Mean live weight of I(75) animals was higher (P<0.05) at the end of the experimental feeding period with net live weight gains of 2.6, 4.8 and 5.4kg for the three groups. Mean serum concentration of triiodothyronine was 1.20, 1.23 and 1.65ngml(-1), being significantly higher in I(75) group. Mean level of thyroxine was 18.3, 24.9 and 27.4ngml(-1), significantly (P<0.05) higher in both I-supplemented groups. It is concluded that supplementing I at the tested levels positively influenced the live weight gain of goats with no significant impact on the utilisation of nutrients. Serum thyroid hormones also increased in response to I supplementation.

Elastic protein-based polymers in soft tissue augmentation and generation.

Five elastic protein-based polymers, designed as variations of polymer I, (GVGVP)251, elicited different responses when injected as subcutaneous implants in the guinea pig, a preclinical test used to evaluate materials for soft tissue augmentation and specifically for correction of urinary incontinence. All six polymers, prepared using recombinant DNA technology, expressed at good levels using transformed E. coli fermentation. These E. coli-produced polymers were purified for the first time to the exacting levels required for use as biomaterials where a large quantity could disperse into the tissues in a few days. Time periods of 2 and 4 weeks were used. Polymer I functioned as a bulking agent around which a fine fibrous capsule formed. Inclusion of (GVGVAP)8, a chemoattractant toward monocytes and elastin-synthesizing fibroblasts in the sequence of polymer I, resulted in an appropriate tissue response of invasion of macrophages. Inclusion of lysine residues, for lysyl oxidase cross-linking, suggested a possible remodeling of the implant toward fibers. Most promising however, when the cell attachment sequence, GRGDSP, was added to polymer I, the implant elicited tissue generation with a normal complement of collagen and elastic fibers, spindle-shaped histiocytes and angiogenesis. If this response is retained over time, the desired soft tissue augmentation and generation will have been achieved. Our working hypothesis is that on formation of elastin, with a half-life of the order of 70 years, a long lasting soft tissue augmentation would result rather than scar tissue as occurs with Contigen, the currently approved injectable implant for soft tissue augmentation.

Engineering protein-based machines to emulate key steps of metabolism (biological energy conversion)

Metabolism is the conversion of available energy sources to those energy forms required for sustaining and propagating living organisms; this is simply biological energy conversion. Proteins are the machines of metabolism; they are the engines of motility and the other machines that interconvert energy forms not involving motion. Accordingly, metabolic engineering becomes the use of natural protein-based machines for the good of society. In addition, metabolic engineering can utilize the principles, whereby proteins function, to design new protein-based machines to fulfill roles for society that proteins have never been called upon throughout evolution to fulfill. This article presents arguments for a universal mechanism whereby proteins perform their diverse energy conversions; it begins with background information, and then asserts a set of five axioms for protein folding, assembly, and function and for protein engineering. The key process is the hydrophobic folding and assembly transition exhibited by properly balanced amphiphilic protein sequences. The fundamental molecular process is the competition for hydration between hydrophobic and polar, e.g., charged, residues. This competition determines Tt, the onset temperature for the hydrophobic folding and assembly transition, Nhh, the numbers of waters of hydrophobic hydration, and the pKa of ionizable functions. Reported acid-base titrations and pH dependence of microwave dielectric relaxation data simultaneously demonstrate the interdependence of Tt, Nhh and the pKa using a series of microbially prepared protein-based poly(30mers) with one glutamic acid residue per 30mer and with an increasing number of more hydrophobic phenylalanine residues replacing valine residues. Also, reduction of nicotinamides and flavins is shown to lower Tt, i.e., to increase hydrophobicity. Furthermore, the argument is presented, and related to an extended Henderson-Hasselbalch equation, wherein reduction of nicotinamides represents an increase in hydrophobicity and resulting hydrophobic-induced pKa shifts become the basis for understanding a primary energy conversion (proton transport) process of mitochondria. Copyright 1998 John Wiley & Sons, Inc.

Evidence for altered proliferative ability of progenitors of urothelial cells in interstitial cystitis.

Secondary cultures of basal urothelial cells isolated from patients with stress incontinence (7 patients), neurogenic bladder (2 patients), interstitial cystitis (IC) (27 patients), bladder rupture (1 patient) and bacterial cystitis (3 patients) grew under growth restricting conditions. All groups displayed reproducible colony size distribution, reflecting the proliferative potential distribution in the population of progenitor cells seeded. The percentage of large colonies (> 6 cells/colony), progeny of basal cells with high proliferative potential, was low in cultures from control patients with stress incontinence, neurogenic bladder or bladder rupture. Exposure of cultures from control patients with stress incontinence to lipoteichoic acid from Streptococcus faecalis, in vitro, increased the percentage of large colonies to levels statistically indistinguishable from those in untreated IC cultures. This supported the possibility that exposure of progenitors of urothelial cells to infection in vivo may cause the persistent increase in the percentage of large colonies in 80% of the IC patients tested. Given these findings, it was not surprising that the percentage of large colonies was also high in cultures from patients with acute bacterial cystitis. In conclusion, the present findings support the theoretical model for the etiology of IC we proposed based on our studies in normal urothelial cells (Elgavish et al., Journal of Cellular Physiology 169: 42-51, 52-65, 66-77, 1996): (1) The proliferative ability of a subpopulation of progenitors of urothelial cells is increased in IC; and (2) This change may be the result of recurrent exposure of progenitors of urothelial cells to injury due, possibly but not exclusively, to infection and chronic inflammation. We propose to use this change as a diagnostic tool for IC.

Elastic protein-based materials in tissue reconstruction.

In natural tissues, cells form multiple attachment sites to their extracellular matrix. By means of those attachments, cells deform as the tissue deforms in response to the natural mechanical stresses and strains that the tissue must sustain during function. These mechanical forces are the energy input that instruct the cells to produce the extracellular matrix sufficient to sustain those forces. Thus, an ideal artificial material should have both the attachment sites for the natural cells and a compliance that matches the natural tissue. Elastic protein-based polymers have been designed to provide both cell attachment sites and to exhibit the required elastic modulus of the tissue to be replaced. Thus, this introduces the potential to design a temporary functional scaffolding that will be remodeled, while functioning, into a natural tissue. A feasibility study applies this concept to the problem of urinary bladder reconstruction in terms of the filling and emptying of a simulated bladder comprised of an elastic protein-based matrix containing cell attachment sites with human urothelial cells growing out onto the dynamic matrix. Furthermore, the elastic protein-based materials themselves have been designed to perform the set of energy conversions that occur in living organisms and, in particular, to convert mechanical energy into chemical energy with the result of chemical signals of the sort that could provide the stimuli to turn on the genes for producing the required extracellular proteins.

Long-term treatment with lipoteichoic acid from Streptococcus faecalis affects differentiation and expression and cellular distribution of beta 1 integrins in human urothelial cells.

Gram-positive bacteria are recognized pathogens in urinary tract infections. Cellular mechanisms triggered by lipoteichoic acids (LTs), cell well components of gram-positive bacteria, have not been completely defined. We have postulated that infection-induced altered function of progenitors of urothelial cells residing in the basal layer is likely to have long lasting effects on the architecture and function of the urothelium. Our recent studies in vitro showed that treatment of poorly differentiated urothelial cells of basal type with LT from Streptococcus faecalis (LT-2) stimulated rapid proliferation of a subpopulation of progenitors of urothelial cells, supporting this possibility (Elgavish et al., 1996, J. Cell. Physiol., 169:42-51). The hypothesis underlying the present studies was that, following LT-triggered increase in proliferation of progenitors, the rate of differentiation of the resulting progeny was also stimulated. We proposed that this mechanism may allow rapid removal of cells from the injured area and replacement by cells that have not been exposed to infection. To simulate in vitro conditions in the basal layer that inhibit terminal differentiation, cells grew on fibronectin or collagen-coated substrate, in medium containing low Ca2+ (0.2 mM) and low levels of growth factors (0.005% bovine pituitary extract [BPE]). During the last 3 days in culture, cells grew in the same low Ca2+ (0.2 mM) medium, but without BPE, with or without LT-2. In a positive control group, cells grew during their last 3 days in culture in medium without BPE and LT-2 but in which levels levels of Ca2+ were higher (2 mM), a condition known to stimulate differentiation in other cell types. Several lines of evidence supported the possibility that long-term treatment with LT-2 stimulated progression of large colonies (i.e., the progeny resulting from LT-triggered proliferation) to a more differentiated state: (1) the rate of their differentiation, determined by criterion of intense cytokeratin 8 expression, was increased; (2) steady-state level of beta mRNA and expression of beta 1 subunit of integrins at the protein level were inhibited; (3) in contrast to large colonies in control cultures, the entire population of LT-2-treated large colonies contained beta 1 integrins distributed at cell-cell contacts. Raising extracellular Ca2+ concentration to 2 mM induced similar effects, suggesting that LT-2 may act by stimulating an increase in intracellular levels of Ca2+. However, further studies will be needed to elucidate the molecular mechanisms underlying the stimulatory effect of LT-2 on proliferation of progenitors of urothelial cells in the basal layer of the urothelium and subsequent differentiation of their progeny. We propose that these processes may have a causative role in the pathological changes that occur in the aftermath of chronic or recurrent suburothelial infection in the urinary bladder.

Changing trends in dietetics.

Our body needs food everyday that supplies all the nutrients in required amount to keep the body healthy. But eating of food sometimes gets affected by socio- cultural factors that condition the dietary habits of the people which contribute significantly to nutritional deprivation in the communities, these faulty feeding habits arising from ignorance, superstitions, and social prejudices have bee so strongly entrenched that it is very difficult to dislodge them from the community in which malnutrition aggravates.