Microcephaly, intractable seizures and developmental delay caused by biallelic variants in TBCD: further delineation of a new chaperone-mediated tubulinopathy.

Microtubule dynamics play a crucial role in neuronal development and function, and several neurodevelopmental disorders have been linked to mutations in genes encoding tubulins and functionally related proteins. Most recently, variants in the tubulin cofactor D (TBCD) gene, which encodes one of the five co-chaperones required for assembly and disassembly of α/ß-tubulin heterodimer, were reported to underlie a recessive neurodevelopmental/neurodegenerative disorder. We report on five patients from three unrelated families, who presented with microcephaly, intellectual disability, intractable seizures, optic nerve pallor/atrophy, and cortical atrophy with delayed myelination and thinned corpus callosum on brain imaging. Exome sequencing allowed the identification of biallelic variants in TBCD segregating with the disease in the three families. TBCD protein level was significantly reduced in cultured fibroblasts from one patient, supporting defective TBCD function as the event underlying the disorder. Such reduced expression was associated with accelerated microtubule re-polymerization. Morpholino-mediated TBCD knockdown in zebrafish recapitulated several key pathological features of the human disease, and TBCD overexpression in the same model confirmed previous studies documenting an obligate dependency on proper TBCD levels during development. Our findings confirm the link between inactivating TBCD variants and this newly described chaperone-associated tubulinopathy, and provide insights into the phenotype of this disorder.

Effect of nursing management and skeletal size at weaning on puberty, skeletal growth rate, and milk production during first lactation of dairy heifers.

Forty Israeli-Holstein 5-d-old calves were used to determine the effect of increasing calf body weight (BW) and skeletal size during the nursing period on age and skeletal size at puberty and on skeletal size and performance during first lactation. The calves were randomly allotted to 2 experimental groups as follows: milk replacer (MR) [calves were given 0.450 kg/d dry matter of milk replacer for the first 50 d of life] and milk-fed (MF) [calves had free access to milk in two 30-min meals/d]. From weaning to 180 d of age, all calves were fed the same diet. At 180 d of age, the MR and MF calves were each divided into 2 equal subgroups: one subgroup from each treatment was given only growing ration, and the other was given the same ration supplemented with fish meal to supply 2% crude protein (CP) (treatments MR + CP and MF + CP, respectively). Finally, at 270 d of age, all calves were housed together and fed a growing heifer's ration until first calving. During the entire nursing period, the MF calves consumed 9.8% more DM, 39.7% more CP, and 52.4% more metabolizable energy than the MR calves. At 60 d of age, BW and all skeletal parameters were higher in the MF calves than in the MR calves. During the entire rearing period (60 to 550 d), the average BW of the MF calves was greater by 16 kg than the BW of the MR calves. Nursing management did not affect differences in skeletal parameters at calving. Average age at puberty onset was 272 +/- 26.8 d; MF calves reached puberty 23 d earlier than MR calves. Yields of milk (kg/305 d) and fat-corrected milk (FCM, kg/d) were greater for the MF + CP heifers than for the MR heifers. It was concluded that nursing by ad libitum milk, as compared with milk replacer, affected BW but not skeletal size of the adult animal, decreased age of puberty onset, and increased FCM yield at first lactation. Supplementing the diet with 2% CP during the prepubertal period increased BW but not skeletal size of the adult animal and 305-d milk and FCM yields during first lactation.

Expression of albumin in nonhepatic tissues and its synthesis by the bovine mammary gland.

Albumin is a well-characterized product of the liver. In the present study, objectives were to determine if the albumin gene is also expressed in various nonhepatic tissues in the bovine; whether mammary gland epithelial cells synthesize albumin; and how its synthesis is affected by bovine mastitis. Albumin expression was monitored using reverse transcription-polymerase chain reaction. Tissues examined were: liver, mammary gland, tongue, intestine, lymph gland, testicle, ovary, and uterus. All tissues except the ovary expressed the albumin gene, albeit less so than the liver. The highest level of expression (other than liver) was found in the lymph nodes but expression was also found in the mammary gland. Incubation of mammary gland explants with the labeled amino acid L-[(35)S] methionine resulted in formation of labeled immunoprecipitable albumin, newly synthesized in the explant. Immunoprecipitable albumin in the medium verified that newly synthesized albumin was also secreted into the medium. This shows that the gland itself is a source of milk albumin. Albumin mRNA expression was approximately 4 times higher in mammary gland tissue from 6 mastitic cows compared with expression in mammary tissue from 6 healthy glands. Further, secretion of albumin was increased 3.5-fold from explants of mastitic mammary glands compared with secretion from explants of healthy mammary glands. Addition of lipopolysaccharide increased the synthesis and secretion of albumin in mammary gland cells in a dose-dependent manner. Exposure to lipopolysaccharide accelerated albumin synthesis in a time-dependent manner up to 48 h. These results lead us to suggest that the secretion of albumin by the mammary gland is part of the innate nonspecific defense system.

Interrelationships among ambient temperature, day length, and milk yield in dairy cows under a Mediterranean climate.

We examined the effect of calving month (CM) on the production of milk and milk protein by Israeli Holstein dairy cows located in the main climatic zone of Israel during their third and fourth lactations, and found it to be significant. Cows that calved in December produced the highest milk and milk protein yields, and those that calved in June produced the lowest, 92.8% of the maximum. The combined effect of the environmental average temperature and day length accounted for 0.96 of the variability in average milk production during lactation and 0.93 of that in average protein production during lactation. Average milk production was reduced by 0.38 kg/degree C and average protein production was reduced by 0.01 kg/degree C. Elongation of daylight increased average milk production by 1.2 kg/h and average protein production by 0.02 kg/h of daylight. Analysis of the temperature pattern effect on milk and protein yield during lactation indicated that cows at the second month (the pike of their milk yield) are more vulnerable to the negative temperature effect than cows on the ninth month of lactation.

Effects of low energy diets followed by a compensatory diet on body weight gain and plasma hormone concentrations in bull calves.

Holstein bull calves at 138.4 d of age were fed one of four diets that contained 2.28, 2.43, 2.61, or 2.8 Mcal of metabolizable energy/kg of dry matter and 10.8, 11.7, 12.8, or 13.9% crude protein, respectively, for 77 d followed by a diet that contained 2.80 Mcal of metabolizable energy/kg of dry matter and 13.9% CP. During the energy restriction period, the metabolizable energy of the diets was positively correlated with the plasma concentration of insulin-like growth factor-I, which was positively correlated with daily body weight (BW) gain during this period and the plasma concentration of total thyroxin. During the first 37 d of the realimentation period, compensatory growth occurred, and the rate of increase in plasma concentrations of insulin-like growth factor-I was positively correlated with that of daily BW gain. At d 37 of the compensatory period, the mean plasma concentration of total thyroxin in calves in three of the four groups did not differ significantly; only the concentration of total thyroxin in the plasma of calves fed the highest energy restricted diet was significantly higher. The mean BW of calves in groups fed the high energy diets during the restriction period tended to be heavier even after 158 d of the realimentation period.

Effect of season of birth on milk, fat, and protein production of Israeli Holsteins.

The effects of birth month on production of milk, fat, and protein and percentages of fat and protein were analyzed based on production records of 101,653 first parity, 77,541 second parity, and 51,856 third parity Israeli Holstein cows. Each parity was analyzed separately. The analysis model also included the effects of herd-year, DIM, calving age, and calving month. First parity Type III sums of squares for birth month were nearly as large as those for calving month but decreased for later parities. Similar results were obtained using multiplicative models in which the dependent variables were the logarithms of the production traits. The effects of calving month and birth month were not similar, but birth month had similar effects for milk, fat, and protein production. Production was lowest by cows born in the early spring and highest by cows born in the fall. Analyses of the log-transformed traits showed that the F values for calving month were greater than, and the F values for birth month were nearly identical to, the F values for the untransformed trait analyses. The physiological basis for these trends was not clear.

The effect of manipulation in energy allowance during the rearing period of heifers on hormone concentrations and milk production in first lactation cows.

Fifteen Holstein heifers that were 175 +/- 4.0 d old and at BW of 175 +/- 4.9 kg were used to determine the effect of three feeding regimens from 6 to 12 mo of age on growth, blood concentration of several hormones, and milk production during first lactation. The feeding regimens consisted of two periods, the first lasting for 4 mo and the other for the subsequent 2 mo. For group A (restricted) heifers, the diet during period 1 was restricted to 85% of NRC (1988) recommendations (a daily BW gain of .7 kg); during period 2, a high energy, high protein diet was provided for ad libitum intake. Group B (control) heifers received a diet that corresponded to 100 and 90% of the NRC (1988) recommendations in periods 1 and 2, respectively. Group C (ad libitum) intake heifers received a high energy, high protein diet throughout both periods. Daily BW gains of heifers of groups A, B, and C were, respectively, .625, .768, and 1.100 kg for period 1 and 1.162, .705, and .797 kg for period 2. The different feeding regimens influenced the age at which the heifers achieved puberty but did not affect BW at puberty. Milk production during 250 d of lactation was 7056, 6070, and 5975 kg for groups A, B, and C, respectively. A statistical model that included serum derived mitogenic activity and serum prolactin of period 2 accounted for 63% of the difference in milk production at first lactation.

Adverse effect of beta-carotene in diet on fertility of dairy cows.

A total of 155 high yielding dairy cows were used to study the effect of dietary beta-carotene (carotene) on fertility. The diet of group 1 was supplemented with 69 mg retinyl acetate per cow daily before calving and 96 mg retinyl acetate after calving: diet of group 2 was supplemented with 500 mg carotene per cow before calving and 96 mg retinyl acetate after calving; and that of group 3 with 500 mg carotene before and 700 mg after calving. Conception rates were 53.9, 42.2, and 27.9% for cows of groups 1, 2, and 3. Days open were 110 and 112 for young (second and third lactation) and old (fourth and later lactations) cows of the group 1; 130 and 129 for young and old cows of group 2; and 133 and 166 for young and old cows of group 3. Number of inseminations per conception was 1.8 and 1.8, 2.2 and 2.2, and 2.5 and 3.4 for the same groups, respectively. Within group 3 the percent of cows pregnant 120 d after calving was 57 and 20 for the young and old cows. Correlation coefficients between plasma carotene of 53 old cows of all groups 4 and 8 wk after calving, and the number of open days and the number of inseminations per conception were significant. The intake of high carotene may have adverse effects on the fertility of dairy cows.

Improved 63Ni radiochemical assay of free fatty acids in plasma.

We have modified the 63Ni radiochemical method of Ho (Anal Biochem 1970;36:105) for determination of free fatty acids (FFA) in plasma. Extracting 1 or 0.1 mL of plasma with Dole's mixture (J Biol Chem 1960;235:2595) and washing the "heptane" layer with two volumes of isopropanol/water/dilute (0.5 mol/L) H2SO4 (25/25/1, by vol) removes about 90% of the lipid phosphorus from the "heptane" layer without removing any FFA and is more convenient than treatment with silicic acid. The following modifications decrease background radioactivity and improve separation of the organic phase from the water phase containing the uncomplexed 63Ni: (a) use glassware instead of plastic test tubes; (b) evaporate the organic phase to dryness before adding the 63Ni (this removes the isopropanol, which interferes with the 63Ni assay); and (c) add anhydrous sodium sulfate before the final centrifugation step.

Differentiation of soluble proteins in cataracts caused by deficiencies of methionine, riboflavin or zinc in diets fed to Atlantic salmon, Salmo salar, rainbow trout, Salmo gairdneri, and lake trout, Salvelinus namaycush.

Water soluble extracts from cataractous and normal salmonid lenses were subjected to thin-layer isoelectric focusing. The cataracts resulted from deficiencies of specific nutrients in diets fed to three species of salmonids as follows: (1) rainbow trout (Salmo gairdneri) fed a diet deficient in riboflavin; (2) Atlantic salmon (Salmo salar) fed a diet deficient in methionine; and (3) rainbow trout and lake trout (Salvelinus namaycush) fed a diet deficient in zinc. In all tests, the protein pattern of cataractous lenses differed from that of normal lenses. Each cataract caused by the different dietary deficiencies had a specific pattern of soluble lens proteins. Even though the general patterns of the soluble lens proteins for all three salmonids though the general patterns of the soluble lens proteins for all three salmonids studied were similar, a specific pattern was unique for each of the species.

Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12.

Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).

Sodium and potassium requirements for active transport of glutamate by Escherichia coli K-12.

Active transport of glutamate by Escherichia coli K-12 requires both Na(+) and K(+) ions. Increasing the concentration of Na(+) in the medium results in a decrease in the K(m) of the uptake system for glutamate; the capacity is not affected. Glutamate uptake by untreated cells is not stimulated by K(+). K(+)-depleted cells show a greatly reduced capacity for glutamate uptake. Preincubation of such cells in the presence of K(+) fully restores their capacity for glutamate uptake when Na(+) ions are also present in the uptake medium. Addition of either K(+) or Na(+) alone restores glutamate uptake to only about 20% of its maximum capacity in the presence of both cations. Changes in K(+) concentration affect the capacity for glutamate uptake but have no effect on the K(m) of the glutamate transport system. Ouabain does not inhibit the (Na(+)-K(+))-stimulated glutamate uptake by intact cells or spheroplasts of E. coli K-12.

Glutamate transport in Escherichia coli K-12: nonidentity of carriers mediating entry and exit.

The exit of glutamate from Escherichia coli K-12 cells preloaded with the radioactive amino acid and its relation to the reaction of entry were studied. Experiments with cells preloaded to different intracellular concentrations of radioactive glutamate confirmed our earlier conclusion that glutamate exit was a first-order reaction. l-Glutamate, competitive inhibitors of glutamate uptake (d-glutamate and l-glutamate-gamma-methyl ester), noncompetitive inhibitors of glutamate uptake (l-serine and l-alanine), and the energy poison NaN(3) all accelerated glutamate exit 2.8-fold. No additive effect was observed in the presence of NaN(3) together with l-glutamate. Preloading with cold l-glutamate did not increase the rate of uptake of radioactive glutamate. It is concluded that the acceleration of glutamate exit in the presence of l-glutamate in the medium is not due to exchange diffusion and that l-glutamate and azide affect exit indirectly by preventing recapture. Sucrose, 25%, slowed down glutamate exit by a factor of about 4.7 and increased the steady-state level of glutamate accumulation to about the same extent. Increasing the intracellular K(+) concentration enhanced glutamate uptake but did not affect the half-time of exit. It is concluded that separate carriers are most probably involved in mediating the entry and exit reactions.